Annual Report

C/ Valderrebollo, 5 28031 Madrid GOBIERNO MINISTERIO 913 852 200 DE ESPAÑA DE ECONOMÍA www.ciberned.es Y COMPETITIVIDAD Instituto de Salud

Annual Report 2013 Annual Report Carlos III

Annual Report © 2014. CIBERNED Centro Investigación Biomédica en Red, Enfermedades Neurodegenerativas C/ Valderrebollo, 5 28031 Madrid www.ciberned.es

Diseño y Coordinación editorial: Comunicación y Ediciones Sanitarias, S.L. (CYESAN) Index

Letter From the Scientific Director...... 1

Overview...... 3

Aims...... 5

Directory of Research Groups and Associated Institutions...... 6

Organizational Structure...... 8

Organization Chart...... 9

External Scientific Advisory Committee...... 10

Consortium Members...... 11

Scientific Report...... 13

Program 1...... 17

Program 2...... 99

Program 3...... 197

Cooperative Research...... 229

International Relations...... 257

Scientific Productivity and Other Activities...... 269

Financial Report...... 281

Principal Investigators Index ...... 303

2013 Annual Report 1

Letter from the Scientific Director

The cooperative research program initiated in 2010 constitutes one of the cornerstones of the activity of CIBERNED, which aims to stimulate cooperative research between different research groups joining forces and exploiting synergies and complementary capabilities. Projects approved in the first two program callas have been completed during 2013, in which 49 research groups (85 % of the groups that make up CIBERNED) have been involved, along with a dozen external associated partner, and we have seen how the projects have been successfully completed, where more than one group of CIBERNED have participated. The success of this program has continued this year with the resolution of the third call for collaborative projects and the approval of 5 new biennial projects with the participation of 21 groups CIBERNED and an external partner group. In total, all three calls launched so​​ far in the cooperative program are allowed active involvement in such projects of 55 out of the 58 groups that make up CIBERNED (95%). This has led to increase the number of collaborative publications of high impact, a feature that is supposed to have a center like ours. As a consequence of the work by our groups, a total of 572 papers have been published, with a global impact factor of 2,741.12 and a mean impact factor of 5.51. Moreover, a new clinical guideline has been published and 13 clinical trials have been active during 2013. This represents the consolidation of the historical trend observed since the establishment of the Center towards an increased scientific productivity, not only in quantitative terms but particularly in qualitative terms, especially highlighting the significant increase in international collaborative publications as well as the traslational approach. We have had several meetings and collaborations with other CIBERs and we intend to keep increasing these relations through the newly created InterCIBER program from the Institute of Health Carlos III. On the other hand, we are strengthening our collaboration with CIEN Foundation, with which we share management and administration, and the Reina Sofia Foundation. Our aim is to cover the different types of research in neurodegeneration, from basic approaches, using simple models systems to monitoring of neurodegenerative processes in humans. As regards to the strengthening of the CIBERNED-CIEN Foundation -Reina Sofia Foundation association, a major development worth mentioning has been the combination of the 7th CIBERNED Scientific Forum, that used to bring together each year the over 500 researchers that constitute the CIBERNED, with the IX International Symposium on Advances in Alzheimer’s disease, which annually promoted the Reina Sofia Foundation and CIEN Foundation. Thus, on the occasion of World Alzheimer’s Day celebrated on September 21, the First International Congress for Research and Innovation in Neurodegenerative Diseases (CIIIEN) was held in Madrid, a format which is to be maintained in the future and whose second edition is scheduled to be held in Barcelona in September 2014. It is also worth noting that CIBERNED has joined the Technical Committee of the Strategy in Neurodegenerative Diseases of the National Health System, launched by the Ministry of Health, Social Services and Equality. With regard to international relations, this year CIBERNED continued to participate very actively in the activities of the JPND Joint Programme and the Network of Centres of Excellence in Neurodegeneration COEN. In particular, we participated in the call for international collaborative projects “Pathfinder”, in which applications from CIBERNED groups were considered of high level (by which we have been specially commended by the Call Evaluation Committee), having had a high success rate as a result. Of the five applications approved (out of 18), three involve CIBERNED groups, representing a success rate of 37.5 %. In addition, in 2013 CIBERNED has made ​​a significant effort to finance with funds from its own budget the project call for European Joint Programme JPND, thus demonstrating our responsibility and interest in maintaining international relations and commitments in this field. The resolution of this call resulted in the approval of a project involving CIBERNED. Finally, I wanted to express my gratitude and appreciation to the research community that make CIBERNED, whose work, dedication and talent have made this center a national and international reference for research in neurodegenerative diseases. We hope that the incipient economic upturn will allow us in rather short term to have the resources needed to continue progressing and consolidating this leading position through the incorporation of new groups and strengthening the existing structure, looking for synergies, bringing new ideas and renewing excitement, while we renew our commitment to quality research for the benefit of all those directly or indirectly affected by these diseases.

Jesús Ávila de Grado Scientific Director 2013 Annual Report 3

Overview

The Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED) emerges in 2006 as heir to the Center for Research in Neurological Diseases (CIEN), which together with the CNIO, CNIC or CNMVIS constituted the four centers whose mission is to combat the most prevalent health problems of Spanish society: cancer, cardiovascular diseases, infectious diseases and neurodegeneration. As for the other centers, a supporting foundation called CIEN Foundation was created in 2003 as the CIEN’s management authority, although unlike the other centers, it was created as the first Network Center in Spain, eventually being renamed as CIBERNED. Both CIEN and CIEN Foundation partnered with the Queen Sofia Foundation in creating the Alzheimer Center, located in Vallecas (Madrid) and where CIEN Foundation is headquartered since 2007. Currently, the CIBERNED-CIEN Foundation partnership is the only research center in Spain (and one of the few in the world) integrated into the international network of Centres of Excellence in Neurodegeneration (CoEN), an initiative arising from the European Union Joint Programme for Research in Neurodegenerative Diseases (JPND). This joint program constitutes an innovative collaborative research initiative created to address the growing challenges posed by this group of diseases. Its goal is to boost the impact of research by aligning existing national research programs and identifying the common objectives whose scope would benefit through joint action. CIBERNED is born with the idea of creating a Center for Translational Research, with a multidisciplinary and multi-institutional philosophy in which basic, clinical and population are integrated in order to develop a single joint research program, focusing on certain diseases of great importance for the National Health System that either for its prevalence or because of its social impact, are considered strategic. The genesis of CIBERNED was conducted through a coordination research strategy of exploiting synergies between the different groups involved in biomedical research in these areas. It seeks to promote quality research in neurodegenerative diseases being carried out within the National Health System and the System of Science and Technology through the development and promotion of a Network Research structure. The goal is to promote and fund, through the Institute of Health Carlos III, CIBERNED by combining research groups, through its association with the National Health System, that contribute to scientifically substantiate the programs and policies of the National Health System in the priority areas of the National R+D+I. CIBERNED was founded under the auspices of the Institute of Health Carlos III, on December 4th, 2006 in Madrid, under a cooperation agreement signed by the Government and other participating institutions, under the SCO/806/2006 Order of March 13th by which the regulatory basis for granting aid to fund stable structures of cooperative research in the area of biomedicine and health sciences within the framework of the Ingenio 2010 Consolider program, CIBER Actions and the RESOLUTION of March 30th, 2006 (BOE 07/04/2006) of the Institute of Health Carlos III, in virtue of which financial aids for stable structures of cooperative research in the area of biomedicine and health sciences are called, with the budgets of the Institute of Health Carlos III. CIBERNED is a research organism, endowed with legal status under the Article 6.5 of Law 30/1992 of November 26 on the Legal Regime of Public Administrations and Common Administrative Procedure, whose mission is the monographic research, broadly defined, on neurodegenerative diseases. It is formed by the association of research groups, with no physical contiguity, belonging to different administrations, institutions and regional governments, from public and private sectors with research lines and goals focused on the specific common area of neurodegenerative diseases and being coordinated to achieve scientific objectives that could hardly be considered in a more restricted execution context. CIBERNED is governed by its own statutes and its activity is currently structured round three Scientific Programs: - Program 1: Alzheimer’s disease and other degenerative dementias. - Program 2: Parkinson’s disease, Huntington’s chorea and other movement disorders. - Program 3: Neuromuscular Diseases. CIBERNED currently consists of 58 research groups supported by different universities, hospitals and the Research Council (CSIC), each led by a principal investigator or responsible. CIBERNED is a network research center, composed of research groups belonging to different Administrations and affiliated Institutions: researchers members physically work in their parent institutions they belong to while simultaneously and actively participating on CIBERNED’s own cooperative research agenda. Thus, is the result of a collaborative partnership between different institutions and the sum of various research groups. The Scientific Director Office has been set since October 2011 at the Center for Molecular Biology “Severo Ochoa” (CSIC) in Madrid. The headquarters, including the General Manager Office is located in the Alzheimer Center of the Queen Sofia Foundation, Valderrebollo street number 5 in Madrid.

4 4 2013 Annual Report 2013 Annual Report 5

Aims

CIBERNED’s primary goal is to foster scientific and technical research of excellence in the field of human health, with the overall goal of producing results quickly translatable into clinical practice to improve the health and wellbeing of patients with neurodegenerative diseases, as well as their families and caregivers, providing in turn economic and social benefits. Among its objectives the following should be highlighted:

• To develop groups in Spain carrying out significant, independent international upscale research on mechanisms underlying neurodegeneration. In addition to facilitate communication between these basic researcher-led groups and clinical services of excellence. • To strengthen, develop and create highly competitive research groups in clinical services of neurology and related medical specialties, led by scientists with clinical activity. • To encourage development of new therapies for neurodegenerative diseases, not only pharmacological but also physical therapies and cell- or gene-based ones, etc. • To implement an infrastructure of strategic and regional or national level for the development of research on prevention, diagnosis and treatment of neurodegenerative diseases. • To share with society the enormous socioeconomic and medical impact neurodegenerative diseases make and facilitate their involvement in the fight against neurodegeneration. • To foster the active involvement of CIBERNED’s research groups in international actions through cooperative activities that align national funding and programming of the different countries, in order to achieve greater impact as well as the provision of new funds. These goals are aligned with those established in the Research Strategy Agenda of the European Union Joint Programme for Research in Neurodegenerative Diseases (JPND), mentioned elsewhere in this document. Directory of Research Groups and Associated Institutions

Group Principal Investigator Institution

301 Alberch Vie, Jordi University of Barcelona 401 Ávila de Grado, Jesús Center for Molecular Biology " Severo Ochoa" CSIC-UAM, Madrid 601 Berciano Blanco, José Ángel Marqués de Valdecilla Foundation, Santander 502 Bermejo Pareja, Félix 12 de Octubre University Hospital, Madrid 510 Bullido Gómez-Heras, Mª Jesús§ Autonomous University, Madrid 402 Camins Espuny, Antonio University of Barcelona 201 Canela Campos, Enric Isidre University of Barcelona 511 Cantero Lorente, José Luis Pablo de Olavide University, Sevilla 106 Ceña Callejo, Valentín University of Castilla-La Mancha, Albacete 413 Comella Carnicé, Joan Xavier Vall d'Hebron University Hospital, Barcelona 101 Cuadrado Pastor, Antonio Autonomous University of Madrid 403 de Felipe Oroquieta, Javier Cajal Institute CSIC, Technical University, Madrid 509 de Pedro Cuesta, Jesús Institute of Health Carlos III, Madrid 114 del Río Fernández, José Antonio Catalonian Institute de Bioengineering, Barcelona 102 Fariñas Gómez, Isabel University of Valencia 606 Fernández Chacón, Rafael University of Seville 303 Fernández Ruiz, Javier Complutense University of Madrid 503 Ferrer Abizanda, Isidre Bellvitge Institute of Biomedical Research, Barcelona 103 Fuentes Rodríguez, José Manuel University of Extremadura, Cáceres 304 García de Yébenes Prous, Justo Ramón y Cajal University Hospital, Madrid 113 García Verdugo, José Manuel Cavanilles Institute, University of Valencia 104 González Castaño, José Autonomous University of Madrid 415 Gutiérrez Pérez, Antonia University of Málaga 305 Guzmán Pastor, Manuel Complutense University of Madrid 505 Herrero Ezquerro, María Trinidad University of Murcia 111 Iglesias Vacas, Teresa Institute of Biomedical Research CSIC-UAM, Madrid 603 Illa Sendra, Isabel Santa Creu i Sant Pau Hospital, Barcelona 202 Kulisevsky Bojarski, Jaime Santa Creu i Sant Pau Hospital, Barcelona 208 Labandeira García, José Luis University of Santiago de Compostela 203 Lanciego Pérez, José Luis Center of Applied Medical Research, Univ. Navarra, Pamplona 504 Lleó Bisa, Alberto Santa Creu i Sant Pau Hospital, Barcelona 105 López Barneo, José Virgen del Rocío University Hospital, University of Sevilla 506 López de Ceballos Lafarga, María Cajal Institute CSIC, Madrid 609 López de Munain Arregui, Adolfo Biodonostia Research Institute 306 Lucas Lozano, José Javier Center for Molecular Biology "Severo Ochoa" CSIC-UAM, Madrid 404 Matute Almau, Carlos University of the Basque Country, Bilbao 508 Mengod Los Arcos, Guadalupe Institute of Biomedical Research IDIBAPS-CSIC, Barcelona 204 Moratalla Villalba, Rosario Cajal Institute CSIC, Madrid

6 6 7

Group Principal Investigator Institution

604 Muñoz Cánoves, Pura Pompeu Fabra University, ICREA, Barcelona 307 Naranjo Orovio, José Ramón National Center of Biotechnology, CSIC, Madrid 607 Navarro Acebes, Xavier Autonomous University of Barcelona 205 Obeso Inchausti, José Ángel Center of Applied Medical Research, Univ. Navarra, Pamplona 507 Pastor Muñoz, María Asunción Center of Applied Medical Research, Univ. Navarra, Pamplona 110 Pérez Castillo, Ana María Institute of Biomedical Research CSIC-UAM, Madrid 209 Pérez Tur, Jordi Institute of Biomedicine of Valencia, CSIC 406 Rodríguez Álvarez, José Autonomous University of Barcelona 206 Rodríguez Díaz, Manuel University of La Laguna, Tenerife 407 Sáez Valero, Javier Miguel Hernández University, Elche 107 Sánchez Capelo, Amelia Ramón y Cajal University Hospital, Madrid 408 Soriano García, Eduardo University of Barcelona 207 Tolosa Sarró, Eduardo Hospital Clínic of Barcelona 409 Torres Alemán, Ignacio Cajal Institute CSIC, Madrid 410 Trullás Oliva, Ramón Institute of Biomedical Research IDIBAPS-CSIC, Barcelona 108 Vicario Abejón, Carlos Cajal Institute CSIC, Madrid 109 Vila Bover, Miquel Vall d'Hebron University Hospital, Barcelona 605 Vílchez Padilla, Juan Jesús La Fe University Hospital, Valencia 411 Vitorica Ferrández, Francisco Javier University of Seville 412 Wandosell Jurado, Francisco Center for Molecular Biology "Severo Ochoa" CSIC-UAM, Madrid

§ Takes over from Fernando Valdivieso Amate due to retirement

2 1 3 17 Geographic distribution 1 of CIBERNED research groups 20 5 1 1 1 5

1 Organizational Structure

CIBERNED governing bodies are the Governing Council, the Permanent Commission and the Scientific Director. The Governing Council, the highest body of CIBERNED, consists of three representatives of the Institute of Health Carlos III, that include the legal representative of the collaborating organization, the Foundation for Research in Neurological Diseases (CIEN) and one representative from each of the institutions participating in the comsortium. The President of the Executive Council is the Director of the Institute of Health Carlos III. The Secretary of the Governing Council will be the Manager of the consortium The Scientific Director and Manager of CIBERNED are also part of the Governing Council, without a right to vote. The Standing Committee is composed of the Vice President of the Executive Council or his delegate, and four members representing the consortium institutions in the Governing Council. The Scientific Director of the consortium and the Manager, who acts as secretary, are also part of the Permanent Commission, without a right to vote. The Scientific Director is appointed by the President of the Governing Council for a period of four years, renewable by agreement of the parties. The current Scientific Director of CIBERNED is Dr. Jesús Avila de Grado. CIBERNED Management is entrusted to the Managing Director of the Foundation CIEN, Ms. María Ángeles Pérez Muñoz. Support and advisory bodies to the organs of government: - The Steering Committee - The External Scientific Advisory Committee The Steering Committee consists of: - Dr. Jesús Ávila de Grado – Scientific Director - Ms. María Ángeles Pérez Muñoz – Manager - Dr. Miguel Medina Padilla – Deputy Scientific Director and the Program coordinators: - Dr. Isidre Ferrer Abizanda - Dr. Justo García de Yébenes - Dra. Isabel Illa Sendra - Dr. Albert Lleó Bisa - Dr. José Javier Lucas Lozano - Dr. Eduardo Soriano García - Dr. Eduardo Tolosa Sarró

8 8 2013 Annual Report 2013 Annual Report 9

CIBERNED Organization Chart

Institute of Health Carlos III

Governing Council

Associated Institutions

Standing Committee

Steering Committee Scientific Director Manager

Deputy Director

Research Program Coordinators

Research Groups External Scientific Advisory Committee

Dr. George Perry University of Texas, San Antonio, USA (Chairman)

Dr. Vincenzo Bonifati Erasmus University, Rotterdam, The Netherlands

Dr. Ángel Cedazo-Mínguez Karolinska Institute, Stockholm, Sweden

Dr. Mary Reilly National Hospital for Neurology and Neurosurgery and Institute of Neurology. London, United Kingdom

1010 2013 Annual Report 2013 Annual Report 11

Consortium Members (by December 2013)

The Central Government represented by: - The Institute of Health Carlos III. - The Spanish National Research Council (CSIC). The following Autonomous Regions (Comunidades Autónomas): - Autonomous Region of Andalusia, through the Andalusian Public Foundation for the Health Research Management at Seville. - Autonomous Region of Canarias, through the University of La Laguna. - Autonomous Region of Cantabria, through the Marqués de Valdecilla Foundation. - Autonomous Region of Castilla-La Mancha, through the University of Castilla-La Mancha. - Autonomous Region of Catalonia, through the Pompeu Fabra University, the Foundation Vall d’Hebron Research Institute, the Foundation Bellvitge Biomedical Research Institute, the University of Barcelona and the Autonomous University of Barcelona. - Autonomous Region of Valencia, through the La Fe Hospital Research Foundation, the Príncipe Felipe Research Centre, the University of Valencia and the Miguel Hernández University at Elche. - Autonomous Region of Extremadura, through the Foundation for Research and Training of Health Professionals (FUNDESALUD). - Autonomous Region of Madrid, through Madrid Health Services (12 de Octubre University Hospital and Ramón y Cajal University Hospital), the Autonomous University of Madrid and the Complutense University of Madrid. - Autonomous Region of Murcia, through the University of Murcia. - Autonomous Region of the Basque Country, through the University of the Basque Country and the Biodonostia Research Institute. - Autonomous Region of Galicia, through the University of Santiago de Compostela. Other centers and institutions that are not affiliated to the previously-cited public administrations: - Foundation Sant Pau Biomedical Research Institute. - August Pi i Sunyer Biomedical Research Institute. - Hospital Clínic of Barcelona. - Foundation for Applied Medical Research (FIMA).

Scientific

Scientific Report 15

Research Programs

The structure of CIBERNED research groups is organized through three scientific programs, whose goals, composition and structure is maintained as in previous years, and whose budget allocation is based on the scientific results of the internal scientific evaluation. The distribution of these three thematic programs shows CIBERNED commitment to encouraging translational research and obtaining scientific knowledge resulting in improving the prevention, diagnosis and treatment of neurodegenerative diseases. All this without detriment of fulfilling of transversal research projects of interest to different areas (see below). The aim of the structure described is to provide a coherent conceptual scheme embodying the real cooperative scientific activity conducting various research groups.

The programs are described below:

Program 1

Alzheimer’s Disease and other Degenerative Dementias

Research Groups

Ávila de Grado, Jesús______21 López de Ceballos Lafarga, María____ 65 Bermejo Pareja, Félix______25 Matute Almau, Carlos______67 Bullido Gómez-Heras, Mª Jesús______28 Mengod de los Arcos, Guadalupe_____ 70 Camins Espuny, Antonio______31 Pastor Muñoz, María Asunción______73 Cantero Lorente, José Luis______35 Rodríguez Álvarez, José______77 Comella Carnicé, Joan Xavier______38 Sáez Valero, Javier______80 de Felipe Oroquieta, Javier______41 Soriano García, Eduardo______85 de Pedro Cuesta, Jesús______44 Ferrer Abizanda, Isidre______49 Torres Alemán, Ignacio______87 Gutiérrez Pérez, Antonia______54 Trullás Oliva, Ramón______89 Herrero Ezquerro, María Trinidad______57 Vitorica Ferrández, Francisco Javier___ 92 Lleó Bisa, Alberto______61 Wandosell Jurado, Francisco______95

Program 1

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. Currently, there is no effective treatment to fight against it. In Spain, 500,000 people are estimated suffer from this disease and, owing to the increasing aging of our society, this figure it is expected to be quadruplicated in the next 50 years, bringing devastating consequences not only for affected individuals and their families but also for the stability of our healthcare system.

AD is characterized by the presence of two aberrant structures within the patients’ brains, senile plaques and neurofibrillary tangles, synapse loss (it is considered a synaptopathy), especially among hippocampal and cortical neurons, as well as a considerable neurodegeneration. Some aspects of these pathologies may be reproduced in cellular or animal models.

At present, dozens of laboratories around the world are actively working to identify new causative and risk genes involved in these pathologies, which could contribute to clarify its pathophysiological basis and identify new therapeutic targets. One of the main current issues in AD is that when diagnosed the brain has already suffered such an extensive and irreparable damage, so that finding biomarkers for an earlier detection becomes an urgent need, even at an asymptomatic stage, when any therapeutic strategy would have a greater chance of success.

Within this program, the 24 groups of clinical and basic scientists, engaged on Alzheimer’s diagnosis and care of patients as well as on basic research in the laboratory, will combine their expertise and effort to work in a coordinated way in the search for new genetic factors, disease-related biomarkers and new therapeutic strategies for AD and other degenerative dementias.

The main research lines are the following:

• Genetic Epidemiology • Research on disease-related biomarkers • Cellular and animal models of Alzheimer’s disease and other degenerative dementias • Molecular pathology of Alzheimer’s disease • Mechanisms of neurodegeneration, neuroprotection and design of new therapies.

19 Groups

Principal Investigator Institution

Ávila de Grado, Jesús Center of Molecular Biology “Severo Ochoa” (CSIC-UAM), Madrid Bermejo Pareja, Félix 12 de Octubre Hospital, Madrid Center of Molecular Biology “Severo Ochoa”, Bullido Gómez-Heras, Mª Jesús Autonomous University of Madrid Camins Espuny, Antonio University of Barcelona Cantero Lorente, José Luis Pablo de Olavide University, Seville Foundation Vall d’Hebron University Hospital, Research Institute, Comella Carnice, Joan Xavier Barcelona de Felipe Oroquieta, Javier Cajal Institute, CSIC / Center of Biomedical Technology, Technical University of Madrid de Pedro Cuesta, Jesús Institute of Health Carlos III, Madrid Ferrer Abizanda, Isidre IDIBELL, Bellvitge University Hospital / University of Barcelona Gutiérrez Pérez, Antonia University of Málaga Herrero Ezquerro, María Trinidad University of Murcia Lleó Bisa, Alberto Santa Creu i Sant Pau Hospital, Barcelona López de Ceballos Lafarga, María Cajal Institute, CSIC, Madrid Matute Almau, Carlos University of the Basque Country, Bilbao Mengod Los Arcos, Guadalupe CSIC, IDIBAPS, Barcelona Foundation of Applied Medical Research, University of Navarra, Pastor Muñoz, María Asunción Pamplona Rodríguez Álvarez, José Institute of de Neuroscience, Autonomous University of Barcelona Sáez Valero, Javier Miguel Hernández University, Elche Soriano García, Eduardo University of Barcelona Torres Alemán, Ignacio Cajal Institute, CSIC, Madrid Trullás Oliva, Ramón CSIC, IDIBAPS, Barcelona Vitorica Ferrández, Francisco Javier University of Seville Wandosell Jurado, Francisco Center of Molecular Biology “Severo Ochoa” (CSIC-UAM), Madrid

Program 1 is coordinated by Drs. Isidre Ferrer (IDIBELL-Bellvitge Hospital), Alberto Lleó (Santa Creu i San Pau Hospital) and Eduardo Soriano (University of Barcelona).

2020 Program 1 Group Jesús Ávila de Grado

Centro de Biología Molecular “Severo Ochoa” CSIC-UAM

Nicolás Cabrera, 1 (Laboratorio 208) 1 Campus de Cantoblanco 28049 Madrid (Spain)

Tel: +34 91 196 4564 Program Fax: +34 91 196 4420 [email protected]

Jesús Ávila de Grado Patricia Martín-Maestro Principal Investigator, Research Professor PhD student Félix Hernández Pérez Noemí Pallas Bazarra Associate Professor PhD student Laura Sayas Casanova Jesús Merchán Robira Researcher Student María Llorens-Martín Raquel Cuadros Catalán Researcher Technician Vega García-Escudero Elena Langa Gabriel Researcher Technician Alberto Gómez Ramos Esther García García Researcher Technician Almudena Fuster Matanzo Nuria de la Torre Alonso Postdoctoral Fellow Administrative Assistant Jerónimo Jurado Arjona Postdoctoral Fellow

21 Summary

During the last year, we have analyzed, in our group, the function of tau protein and GSK3 in processes related to the development of adult born neurons, like neurogenesis or dendritogenesis. Also, we have studied the function of those proteins in Alzheimer disease or other tauopathies. Mainly, we have focused on the role of tau on Alzheimer disease and on the overexpression of GSK3 on adult neurogenesis, although, in some works, we have analyzed the role of both proteins on the different processes. Also, we have looked for the function of other microtubule associated proteins (MAPs), different than tau, on axonogenesis. We have found that an overexpression of GSK3, on dentate gyrus, results in an impaired neurogenesis with morphological alterations (mainly at dendrite and dendrite spine levels) in the newborn neurons, in addition active gliosis was observed. About the role of tau in Alzheimer disease, we have mainly focused on the function of extracellular tau protein in the spreading of tau pathology. Finally, about the role of MAPs, different than tau, on axonogenesis, we have described that the process is regulated through the interaction of MAP1B with EB1/3 proteins.

On the other hand, we have carried out several collaborations with other groups of CIBERNED, mainly with those present at Program 1 (Alzheimer disease and other dementias). Also, we have collaborated with other external groups like, for example, that of Dr. Esteller in a project based on making a DNA metylation map in human brain to identify target genes in Alzheimer disease. Actually, in other project we have a collaboration with Dr. Soriano’s group to look for single nucleotide variations that could take place, specifically, in the exome of neurons from Alzheimer disease patients.

Keywords

Tau, GSK3, Alzheimer disease, Neurogenesis

Publications

• Medina M, Avila J. Understanding the relationship between GSK-3 and Alzheimer’s disease: a focus on how GSK-3 can modulate synaptic plasticity processes. Expert review of neurotherapeutics. 2013 May;13(5):495-503. • Garcia-Escudero V, Martin-Maestro P, Perry G, Avila J. Deconstructing mitochondrial dysfunction in Alzheimer disease. Oxidative medicine and cellular longevity. 2013;2013:162152. • Bidon-Chanal A, Fuertes A, Alonso D, Perez DI, Martinez A, Luque FJ, et al. Evidence for a new binding mode to GSK-3: Allosteric regulation by the marine compound palinurin. European journal of medicinal chemistry. 2013 Feb;60:479-89. • de Cristobal J, Garcia-Garcia L, Delgado M, Pozo MA, Medina M. A longitudinal FDG-PET study of transgenic mice overexpressing GSK-3β in the brain. Current Alzheimer research. 2013 Jul 8. • Medina M, Avila J, Villanueva N. Use of okadaic acid to identify relevant phosphoepitopes in pathology: a focus on neurodegeneration. Marine drugs. 2013 May 21;11(5):1656-68. • Medina M, Avila J. New insights into the role of glycogen synthase kinase-3 in Alzheimer’s disease. Expert Opin Ther Targets. 2014 Jan;18(1):69-77. • Avila J, de Barreda EG, Pallas-Bazarra N, Hernandez F. Tau and neuron aging. Aging and disease. 2013 Feb;4(1): 23-8. • Simon D, Hernandez F, Avila J. The involvement of cholinergic neurons in the spreading of tau pathology. Frontiers in neurology. 2013;4:74. • Fuster-Matanzo A, Llorens-Martin M, Hernandez F, Avila J. Role of neuroinflammation in adult neurogenesis and Alzheimer disease: therapeutic approaches. Mediators of inflammation. 2013;2013:260925. • Monroy-Ramirez HC, Basurto-Islas G, Mena R, Cisneros B, Binder LI, Avila J, et al. Alterations in the nuclear architecture produced by the overexpression of tau protein in neuroblastoma cells. Journal of Alzheimer’s disease: JAD. 2013 Jan 1;36(3):503-20. • Hernandez F, Garcia-Garcia E, Avila J. Microtubule depolymerization and tau phosphorylation. Journal of Alzheimer’s disease: JAD. 2013;37(3):507-13. • Perry G, Zhu X, Smith MA, Sorensen A, Avila J. Preface. Alzheimer’s disease: advances for a new century. Journal of Alzheimer’s disease: JAD. 2013;33(Suppl 1):S1. • Camero S, Benitez MJ, Barrantes A, Ayuso JM, Cuadros R, Avila J, et al. Tau Protein Provides DNA with Thermodynamic and Structural Features which are Similar to those Found in Histone-DNA Complex. Journal of Alzheimer’s disease: JAD. 2013 Nov 19.

2222 Jesús Ávila de Grado Group Jesús Ávila de Grado Group 23

• Del Ser T, Steinwachs KC, Gertz HJ, Andres MV, Gomez-Carrillo B, Medina M, et al. Treatment of Alzheimer’s Disease with the GSK-3 Inhibitor Tideglusib: A Pilot Study. Journal of Alzheimer’s disease: JAD. 2013 Jan 1;33(1):205-15. • Sanchez-Mut JV, Aso E, Panayotis N, Lott I, Dierssen M, Rabano A, et al. DNA methylation map of mouse and human brain identifies target genes in Alzheimer’s disease. Brain. 2013 Oct;136(Pt 10):3018-27. • Garcia-Cabrero AM, Guerrero-Lopez R, Giraldez BG, Llorens-Martin M, Avila J, Serratosa JM, et al. Hyperexcitability and epileptic seizures in a model of frontotemporal dementia. Neurobiology of disease. 2013 Oct;58:200-8. • Tortosa E, Galjart N, Avila J, Sayas CL. MAP1B regulates microtubule dynamics by sequestering EB1/3 in the cytosol of developing neuronal cells. The EMBO journal. 2013 May 2;32(9):1293-306. • Benoist M, Palenzuela R, Rozas C, Rojas P, Tortosa E, Morales B, et al. MAP1B-dependent Rac activation is required for AMPA receptor endocytosis during long-term depression. The EMBO journal. 2013 Aug 14;32(16):2287-99. • Fuster-Matanzo A, Llorens-Martin M, Sirerol-Piquer MS, Garcia-Verdugo JM, Avila J, Hernandez F. Dual effects of increased glycogen synthase kinase-3β activity on adult neurogenesis. Human molecular genetics. 2013 Jan 3;22(7):1300-15. • Merino-Serrais P, Benavides-Piccione R, Blazquez-Llorca L, Kastanauskaite A, Rabano A, Avila J, et al. The influence of phospho-τ on dendritic spines of cortical pyramidal neurons in patients with Alzheimer’s disease. Brain. 2013;136(6):1913-28. • Leon-Espinosa G, Garcia E, Garcia-Escudero V, Hernandez F, DeFelipe J, Avila, J. Changes in tau phosphorylation in hibernating rodents. Journal of Neuroscience Research. 2013;91(7):954-62. • Llorens-Martin M, Fuster-Matanzo A, Teixeira CM, Jurado-Arjona J, Ulloa F, Defelipe J, et al. GSK-3β overexpression causes reversible alterations on postsynaptic densities and dendritic morphology of hippocampal granule neurons in vivo. Molecular psychiatry. 2013 Apr;18(4):451-60. • Cuchillo-Ibanez I, Balmaceda V, Botella-Lopez A, Rabano A, Avila J, Saez-Valero J. Beta-amyloid impairs Reelin signaling. PLoS One. 2013;8(8):e72297. • Llorens-Martin M, Fuster-Matanzo A, Teixeira CM, Jurado-Arjona J, Ulloa F, Defelipe J, et al. Alzheimer disease-like cellular phenotype of newborn granule neurons can be reversed in GSK-3β-overexpressing mice. Molecular psychiatry. 2013 Apr;18(4):395.

Research Projects

• Code: SYL-DEN • Code: PI2010/07 Title: Análisis del efecto de siRNA sobre los niveles de la Title: Reelina y GSK3 como dianas terapéuticas en la enfer- proteína tau en un modelo transgénico que sobreexpresa medad de Alzheimer proteína tau humana con mutaciones presentes en enfer- Principal Investigator: Jesús Ávila de Grado mos de FTDP-17 CIBERNED’s collaboration: Yes Principal Investigator: Jesús Ávila de Grado CIBERNED’s groups: G 401; G 403; G 113; G 407; G 408; CIBERNED’s collaboration: No G 410 CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Intramurales Type: National Funding Agency: CIBERNED Funding Agency: SYLENTIS SA Funding: 540.000 € Funding: 120.000 € Duration: 2010-2013 Duration: 2010-2013 • Code: NOS-DEN • Code: SAF2011-24841 Title: Soluciones innovadoras para acelerar la identificación Title: Función de GSK3 y la proteina tau en neurogénesis y desarrollo de fármacos para patologías del Sistema Ner- y neurodegeneración. Las consecuencias en para enferme- vioso dad de Alzheimer Principal Investigator: Jesús Ávila de Grado Principal Investigator: Jesús Ávila de Grado CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: National Funding Agency: Noscira SA Funding Agency: MICINN Funding: 140.000 € Funding: 592.900 € Duration: 2010-2013 Duration: 2012-2014 • Code: S2010_BMD • Code: 2013/07A Title: Redes de señalización y vías efectoras en modelos Title: Papel de GSK-3 β en las alteraciones de los circuitos celulares y animales de enfermedades neurodegenerativas corticales que ocurren en la enfermedad de Alzheimer Principal Investigator: José Gonzalez Castaño Principal Investigator: Teresa Iglesias Vacas CIBERNED’s collaboration: Yes CIBERNED’s collaboration: Yes CIBERNED’s groups: G 401; G 104; G 111; G 307; G 409; CIBERNED’s groups: G 401; G 403; G 111; G 504; G 307 G 412 Other CIBER’s collaboration: No Other CIBER’s collaboration: Yes (CIBERER) Type: Intramurales Type: CCAA Funding Agency: CIBERNED Funding Agency: Comunidad de Madrid Funding: 158.000 € Funding: 17.250 € Duration: 2013-2015 Duration: 2012-2015

2424 Jesús Ávila de Grado Group Group Félix Bermejo Pareja

Instituto de Investigación Hospital 12 de Octubre

Avenida de Córdoba, s/n 1 28041 Madrid (Spain) Tel.: +34 913 908 765

Fax: +34 913 908 544 Program [email protected] / [email protected]

Félix Bermejo Pareja Julián Benito León Principal Investigator, Chief of Neurology Department Neurologist, Scientific staff Eva María Carro Díaz Jesús Hernández Gallego Principal Investigator Neurologist, Scientific staff Rocío Pérez-González Rocío Trincado Soriano PhD student Statistics Technician Teresa Díaz San Juan Alberto Villarejo Galende PhD student Neurologist, Scientific staff Agnieszka Krzyzanowska Álvaro Sánchez-Ferro Postdoctoral Fellow Río Hortega Fellow Consuelo Pascual Pérez Ignacio J Posada Lab Technician Neurologist, Scientific staff Desiree Antequera Tienda Fernando Sierra Research Technician Río Hortega Researcher José Antonio Molina Arjona ASP Neurology

25 Summary

The research group of Dr. Carro in 2013 has focused its researcher work on “Neuropathological mechanisms and searching for possible therapeutic targets for Alzheimer’s disease”. In this context two studies have been conducted, where it has been demonstrated therapeutic effects of trophic factors in an animal model of amyloidosis. In one of them, and due to the collaboration with the group of Dr. José Luis Pedraz from Basque Country University, and CIBERBBN, therapeutic capacity of nanoparticles of VEGF has been shown in double transgenic mice APP/Ps1, improving cognitive deficits, and reducing cerebral accumulation of beta-amyloid. In a second study in collaboration with the Fundación Eduardo Anitua, neurogenic capacity of intranasal delivery of plasma rich in growth factors (PRGF-Endoret) was demonstrated, opening new doors at clinical level. Also in collaboration with Dr. Ana Martinez of the Institute of Medical Chemistry of CSIC, neurorepair effect of a phosphodiesterase inhibitor 7 administered intraperitoneally in mice model of amyloidosis was evaluated. Also, and given previous studies of the group on the role of the choroid plexus in Alzheimer’s disease, two articles have been published regarding neurogenic effects of choroid plexus, and their possible therapeutic implications.

Dr. Felix Bermejo-Pareja group has continued the publication of the cohort NEDICES, primarily on mortality of neurological illnesses (dementia and Parkinson’s) and other works in this field are pending. In addition, the mortality of systemic diseases is being investigated. Also published other aspects of cognitive function and the parkinsonian patients in this cohort essential tremor and other work on cognitive aspects of the cohort and its evolution and mortality are assessed (primarily mild cognitive impariment).

Collaborations with Dra. MA Martín-Requero (Centro de Investigaciones Biológicas, CSIC) team and Drs. Toledano and Carmona (Instituto Cajal, Vibra Project) about biomarkers in Alzheimer disease, also with the Dr. I Contador from Psychology in Salaman- ca University about cognitive aspect of the NEDICES cohort.

Keywords

Alzheimer´disease, Dementia, Biomarkers, Transgenic mice, Choroid plexus, Therapeutic drugs, Amyloid, Neurogenesis, Morta- lity, Mild cognitive impairment

Publications

• Louis ED, Benito-Leon J, Moreno-Garcia S, Vega S, Romero JP, Bermejo-Pareja F, et al. Blood harmane (1-methyl-9H- pyrido[3,4-b]indole) concentration in essential tremor cases in Spain. Neurotoxicology. 2013 Jan;34:264-8. • Esteras N, Alquezar C, Bermejo-Pareja F, Bialopiotrowicz E, Wojda U, Martin-Requero A. Downregulation of extracellular signal-regulated kinase 1/2 activity by calmodulin KII modulates p21(Cip1) levels and survival of immortalized lymphocytes from Alzheimer's disease patients. Neurobiology of aging. 2013 Apr;34(4):1090-100. • Bolos M, Antequera D, Aldudo J, Kristen H, Bullido MJ, Carro E. Choroid plexus implants rescue Alzheimer's disease-like pathologies by modulating amyloid-β degradation. Cellular and molecular life sciences: CMLS. 2013 Dec 17.

Research Projects

• Code: 2009/01636 Duration: 2010-2013 Title: Búsqueda de nuevos factores de riesgo y marcadores biológicos en la enfermedad de Alzheimer y otras enferme- • Code: FIS PO 11-1508 dades neurodegenerativas: posible aplicación diagnóstica Title: Estudio basal de la cohorte NEDICES-2. Cohorte co- Principal Investigator: Eva Carro munitaria en el centro de España con informacion clínica CIBERNED’s collaboration: No y biobanco CIBERNED’s groups: Principal Investigator: Félix Bermejo-Pareja Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: National CIBERNED’s groups: Funding Agency: Instituto de Salud Carlos III Other CIBER’s collaboration: No Funding: 20.000 € Type: National Funding Agency: FIS

2626 Félix Bermejo Pareja / Eva María Carro Díaz Group Félix Bermejo Pareja / Eva María Carro Díaz Group 27

Funding: 104.665 € • Code: 2010/0004 Duration: 2013 Title: Neuroprotección basado en el implante intracerebral de células epiteliales de plexos coroideos en un modelo • Code: Fundación Ramón Areces 2012 murino de amiloidosis Title: Estudio de la capacidad neuroprotectora de las célu- Principal Investigator: Eva Carro las epiteliales de los plexos coroideos como potencial tera- CIBERNED’s collaboration: No pia regenerativa de la enfermedad de Alzheimer CIBERNED’s groups: Principal Investigator: Eva Carro Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: Privado CIBERNED’s groups: G 502; G 509 Funding Agency: Fundación Investigación Médica Mutua Other CIBER’s collaboration: No Madrileña Type: Privado Funding: 35.000 € Funding Agency: Fundación Ramón Areces Duration: 2010-2013 Funding: 30.000 € Duration: 2012-2015 • Code: Consejería de Salud. Junta de Andalucía Title: Obesidad y riesgo de enfermedades neurodegene- • Code: MUTUA 2012 rativas: búsqueda de nuevos marcadores biológicos en la Title: Estudio de las propiedades terapéuticas de fármacos enfermedad de Alzheimer. Implicación de la amino-procal- basados en Melatonina como tratamiento innovador para citonina enfermedades neurodegenerativas Principal Investigator: Eva Tavares Principal Investigator: Mª Isabel Rodriguez-Franco CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: National Funding Agency: Consejería de Salud. Junta de Andalucía Funding Agency: Fundación Investigación Biomédica de la Funding: 50.000 € MUTUA Duration: 2013 Funding: 20.000 € Duration: 2012-2015 • Code: PI2010/09 Title: BESAD-P: Biomarkers of Early Stages of Alzheimer • Code: Alzheimer´s Drug Discover Foundation Disease-Prevention Title: Evaluation of cognition and hippocampal neurogene- Principal Investigator: Isidre Ferrer Abizanda sis after oral administration of a phosphodiesterase 7 inhib- CIBERNED’s collaboration: Yes itor in an Alzheimer disease mice model CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; Principal Investigator: Ana Martínez G 506; G 508; G 412 CIBERNED’s collaboration: No Other CIBER’s collaboration: Yes (CIBERESP) CIBERNED’s groups: Type: Intramurales Other CIBER’s collaboration: No Funding Agency: CIBERNED Type: International Funding: 223500 € Funding Agency: Alzheimer´s Drug Discover Foundation Duration: 2011-2013 Funding: 140.000 € Duration: 2013 • Code: PI2011/04 Title: Estudio multicéntrico de biomarcadores en LCRy de • Code: AC655 neuroimagen en el continuum Enfermedad de Alzheimer Title: Neuroprotección basado en el implante intracerebral preclínica - prodrómica (Estudio SIGNAL) de células epiteliales de plexos coroideos en un modelo Principal Investigator: Alberto Lleó murino de amiloidosis CIBERNED’s collaboration: Yes Principal Investigator: Félix Bermejo Pareja CIBERNED’s groups: G 504; G 502; G 511; G 507; G 509 CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: Intramurales Other CIBER’s collaboration: No Funding Agency: CIBERNED Type: National Funding: 140.000 € Funding Agency: Fundación Investigación MUTUA 2010 Duration: 2011-2013 Funding: 35.000 € Duration: 2010-2013 Group María Jesús Bullido Gómez-Heras

Centro de Biología Molecular “Severo Ochoa”

1 Universidad Autónoma de Madrid Edificio CBM (Spain)

Program Tel.: +34 911 964 567 Fax: +34 911 964 420 [email protected]

María Jesús Bullido Henrike Kristen Principal Investigator PhD student Ana Frank García Iván Fernández Frías Researcher, Head of Department Master’s student Jesús Aldudo Soto Julia Terreros Roncal Postdoctoral Fellow Master’s student María Recuero Vicente Postdoctoral Fellow Isabel Sastre Merlín CSIC Advanced Technical

28 María Jesús Bullido Gómez-Heras Group 29

Summary

Using human neuroblastoma cell models that simulate aspects of the pathogenesis of sporadic and familial Alzheimer’s disease (AD), in combination with studies in patients our group aims to find new genetic markers and potential targets for disease. In recent years, our studies have focused on two processes closely related to aging: oxidative stress (OS) and infections, including herpes simplex virus 1 (HSV 1).

Genomic analysis of cell laboratory models has shown a group of genes whose expression changes in response to the OS when acting in the presence of the “Sweedish” mutation causing familial AD (APPswe) or of HSV 1 virus, but not in the control cultures. The functional annotation of this group indicates that the lysosomal function is altered as a result of the interaction between OS and both APPswe mutation as HSV 1, which suggests that this feature could be relevant in both familiar and sporadic forms of AD.

The functional analysis of the models also supports the possible involvement of lysosomal function in the process of neurodegeneration:

• HSV-1 induces a profound alteration of autophagy, the main lysosomal degradation pathway in the cell, which produces a massive accumulation of Aß peptide in immature autophagic vesicles, an effect heightened in the presence of OS. In addition, unpublished results of our laboratory suggest that HSV-1 may also alter the function of late endosomes/multivesicular bodies, which are intimately linked to the autophagy lysosome route, also favoring the accumulation of Aß. In summary, we have evidence that HSV-1 and its interaction with the OS, can alter the functionality of the endocytic-autophagic-lysosomal pathway acting at different levels of the route. • Oxidative stress by itself also modulates the autophagy lysosome and ubiquitin proteasome (UPS) degradatory machineries, and said modulation influences the traffic, metabolism and proteolytic processing of APP. In addition, the recently initiated study of these processes in the familial AD model- APPswe cells is showing us that the effect of OS on the two routes is different to that observed in control cells, and that OS induces a greater increase in the maturation and proteolytic processing of APP in the APPswe cells. In summary, the data collected so far suggest that the “Swedish” mutation modifies the links between oxidative stress, autophagy lysosome pathway and APP metabolism.

As for studies in patients, during 2013 we have continued to participate in various genetic association projects in collaboration with CIBERNED groups, as well as in relation with the EADI and IGAP consortia, that are showing new genetic risk factors for AD. This year, in collaboration with CIBERNED, it has been established a Spanish Group for Genetic Studies of Dementia (DEGESCO) whose early work has focused to the study of rare genetic variants in relation with AD and other dementias. We also continued the longitudinal study of patients with mild cognitive impairment (ELMO project) for which numerous neuropsychological, neuroimage, biochemical and genetic variables have been collected during three years. In 2013 we have started the analysis of the correlations of these variables among them and with clinical variables, mainly progression to dementia, and the first results have been sent for publication.

Keywords

Alzheimer’s, Herpes virus, Oxidative stress, APP metabolism, Functional genomics, Genetic association

Publications

• Di Deco J, Gonzalez AM, Diaz J, Mato V, Garcia-Frank D, Alvarez-Linera J, et al. Machine learning and social network analysis applied to Alzheimer’s disease biomarkers. Current topics in medicinal chemistry. 2013;13(5): 652-62. • Santana S, Sastre I, Recuero M, Bullido MJ, Aldudo J. Oxidative stress enhances neurodegeneration markers induced by herpes simplex virus type 1 infection in human neuroblastoma cells. PloS one. 2013;8(10):e75842. • Bolos M, Antequera D, Aldudo J, Kristen H, Bullido MJ, Carro E. Choroid plexus implants rescue Alzheimer’s disease-like pathologies by modulating amyloid-β degradation. Cellular and molecular life sciences: CMLS. 2013 Dec 17. • Casals-Coll M, Sanchez-Benavides G, Meza-Cavazos S, Manero RM, Aguilar M, Badenes D, et al. Spanish Multicenter Normative Studies (NEURONORMA Project): Normative Data and Equivalence of Four BNT Short-Form Versions. Archives of clinical neuropsychology. 2013 Nov 11. • Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Bellenguez C, et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer’s disease. Nature genetics. 2013 Dec;45(12):1452-8. • Ruiz A, Dols-Icardo O, Bullido MJ, Pastor P, Rodriguez-Rodriguez E, Lopez de Munain A, et al. Assessing the role of the TREM2 p.R47H variant as a risk factor for Alzheimer’s disease and frontotemporal dementia. Neurobiol Aging. 2014 Feb;35(2):444. e1-4. • Rubio-Moscardo F, Seto-Salvia N, Pera M, Bosch-Morato M, Plata C, Belbin O, et al. Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset Alzheimer’s disease patients alter calcium homeostasis. PloS one. 2013;8(9):e74203.

Research Projects • Code: SAF2010-15558 • Code: PI2010/09 Title: Identificación de genes asociados a la enfermedad de Title: BESAD-P: Biomarkers of Early Stages of Alzheimer Alzheimer mediante análisis genómico funcional de mode- Disease-Prevention los celulares patogénicos Principal Investigator: Isidre Ferrer Abizanda Principal Investigator: María Jesús Bullido Gómez-Heras CIBERNED’s collaboration: Yes CIBERNED’s collaboration: No CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; CIBERNED’s groups: G 506; G 508; G 412 Other CIBER’s collaboration: No Other CIBER’s collaboration: Yes (CIBERESP) Type: National Type: Intramurales Funding Agency: MICINN Funding Agency: CIBERNED Funding: 121.000 € Funding: 223.500 € Duration: 2011-2013 Duration: 2011-2013

PhD Dissertations

• Author: Genny Lubrini Title: Velocidad de procesamiento de la información en Es- clerosis Múltiple Date: 1 July 2013 Supervisor: Ana Frank García

3030 María Jesús Bullido Gómez-Heras Group Group Antonio Camins Espuny

Facultad de Farmacia

Departamento de Farmacología y Química Terapéutica 1 Universidad de Barcelona Avenida Joan XXIII, s/n 08028 Barcelona (Spain) Program Tel.: +34 934 024 531 [email protected]

Antoni Camins Espuny Dmitry Petrov Principal Investigator, Full Professor Research Fellow Merce Pallàs LLiberia Mirem Ettecheto Full Professor Research Fellow Jordi Vilaplana Mª Jesús Álvarez López Associate Professor Research Fellow Carme Pelegrí Marta Cosin Tomas Associate Professor Research Fellow Francesc Sureda Christian Grañe Farre Associate Professor Research Fellow Carme Auladell David Porquet Associate Professor Research Fellow Jaume Folch Gemma Manich Associate Professor Research Fellow Anna María Canudas Teixidó Itsaso Cabezón Rodríguez Assistant Professor Research Fellow Andrés Jiménez Iván Patraca Assistant Professor Research Fellow Ester Verdaguer Cardona Nacho Pedrós Lecturer Research Fellow Luisa de Lemos Nora Martínez López Portuguese government Fellow Research Fellow

31 Summary

1) Previous studies have shown that mice APPSwe/PS1dE9 that produce higher levels of β-amyloid and treated with a high- calorie diet developed a memory loss. Although the process of memory loss is unknown alterations in metabolism (metabolic syndrome) may contribute to the onset of Alzheimer’s disease. Our studies shown that obesity in mice causes’ glucose intolerance and insulin accompanying an alteration of gene IGF1 levels that hippocampal area of the brain related to memory and learning process. Alterations of memory genes c – fos, ARC, CBP is of interest for prevention of Alzheimer’s disease. So from this research study in cultured neuronal changes in insulin receptors, insulin growth factors, signaling pathways and altered the role of antidiabetic drugs as a strategy to prevent Alzheimer’s disease.

Hypothesis on the relationship between insulin system and Alzheimer’s disease.

Alzheimer’s disease is also associated with the decrease in insulin levels in the central nervous system and/or the ratio of the central nervous system insulin, in addition to the decrease in the expression/altered receptors insulin.

Here are some theories that link the two diseases:

• Assumptions based on PI3K pathway Defects in signaling insulina/IGF-1 affect PI3K/Akt causing a process of neurodegeneration. The decrease in the activation of glucose transporters at the brain mediated by PI3K/Akt and decreased expression in the brains of patients with Alzheimer’s disease can cause lower glucose metabolism in the brain and consequently a lower mitochondrial metabolism and reduced ATP production. Decreased phosphorylation of GSK-3β and subsequent increase in activity may enhance the activity β-secretase and amyloidogenic APP process, resulting in increased intracellular levels of beta-amyloid. • Hypothesis based in MAPK pathway The β-amyloid peptide competes with insulin for binding to the insulin receptor. This activates MAPK, which induces an increase in neuroinflammation (conducted by cytokines), hyperphosphorylation of Tau protein and increased accumulation of APP but otherwise, cause insulin resistance in not being able to join the receiver and exert their function. It is shown that Alzheimer’s patients the MAPK pathway is stimulated.

• Other alternative explanations The decrease in insulin levels as -1 IGF (insulin growth factor 1) suggests that there is a problem of the transport of insulin from the pancreas to the brain. This could happen through a defect in the blood-brain barrier permeability induced by prolonged peripheral hyperinsulinemia. Insulin and β-amyloid pro gradients are the same enzyme called IDE (insulin degrading enzyme), a zinc metalloproteases that degrade various extracellular substrates significant. In fact, recent studies have shown that reduced activity of IDE mRNA and protein levels in the hippocampus of patients with Alzheimer number is inversely related content Aβ1-42.

2) Aging: we are working with mice SAMP8 (senescence accelerated mouse). These mice are an excellent model to study the relationship between the aging process and the predisposition to develop a neurodegenerative disease, particularly Alzheimer’s disease. Our results have allowed us to demonstrate that SAMP8 mice compared with SAMR1 (control) a series of major changes: 1) an increase in oxidative stress in the brain; 2) an increase in the expression of CDK5 and GSK-3β proteins (key proteins in Alzheimer’s disease) and 3) changes in the degree of phosphorylation of tau protein (buds). Therefore, we are working with a compound specifically resveratrol and we show that this compound is able to improve some biological parameters in SAMP8. This is important because it directly relates changes in aging (improvement) with neurodegenerative diseases. The interest is based on resveratrol which is an activator of sirtuin 1. The interest of our group is to study the role of SIRT1 and other sirtuins 1 as SIRT3 in the process of aging and disease in mice Alzheimer APPSwe/PS1dE9.

Keywords

Apoptosis, Alzheimer’s, Aging SAMP8, APP/PS1, JNK3, JNK1

Publications

• Castro-Torres RD, Chaparro-Huerta V, Flores-Soto ME, Banuelos-Pineda J, Camins A, Orozco-Suarez SA, et al. A single dose of pirfenidone attenuates neuronal loss and reduces lipid peroxidation after kainic Acid-induced excitotoxicity in the pubescent rat hippocampus. J Mol Neurosci. 2014 Feb;52(2):193-201. • Ortuno-Sahagun D, Gonzalez RM, Verdaguer E, Huerta VC, Torres-Mendoza BM, Lemus L, et al. Glutamate Excitotoxicity

3232 Antonio Camins Espuny Group Antonio Camins Espuny Group 33

Activates the MAPK/ERK Signaling Pathway and Induces the Survival of Rat Hippocampal Neurons In Vivo. Journal of molecular neuroscience: MN. 2013 Nov 5. • Manich G, Cabezon I, del Valle J, Duran-Vilaregut J, Camins A, Pallàs M, et al. Study of the transcytosis of an anti-transferrin receptor antibody with a Fab' cargo across the blood-brain barrier in mice. European journal of pharmaceutical sciences. 2013 Jul 16;49(4):556-64. • Perez-Caceres D, Ciudad-Roberts A, Rodrigo MT, Pubill D, Camins A, Camarasa J, et al. Depression-like behavior is dependent on age in male SAMP8 mice. Biogerontology. 2013 Apr;14(2):165-76. • Vilar A, de Lemos L, Patraca I, Martinez N, Folch J, Junyent F, et al. Melatonin suppresses nitric oxide production in glial cultures by pro-inflammatory cytokines through p38 MAPK inhibition. Free Radic Res. 2014 Feb;48(2):119-28. • Camins A. Advances in the treatment of neurodegenerative diseases and epilepsy. Current pharmaceutical design. 2013;19(38):6699-700. • Folch J, Pedros I, Patraca I, Martinez N, Sureda F, Camins A. Metabolic basis of sporadic Alzeimer's disease. role of hormones related to energy metabolism. Current pharmaceutical design. 2013;19(38):6739-48. • Flores-Soto ME, Chaparro-Huerta V, Escoto-Delgadillo M, Urena-Guerrero ME, Camins A, Beas-Zarate C. Receptor to glutamate NMDA-type: the functional diversity of the nr1 isoforms and pharmacological properties. Current pharmaceutical design. 2013;19(38):6709-19. • Abad S, Fole A, Del Olmo N, Pubill D, Pallàs M, Junyent F, et al. MDMA enhances hippocampal-dependent learning and memory under restrictive conditions, and modifies hippocampal spine density. Psychopharmacologia. 2013 Oct 26. • Manich G, Del Valle J, Cabezon I, Camins A, Pallàs M, Pelegri C, et al. Presence of a neo-epitope and absence of amyloid beta and tau protein in degenerative hippocampal granules of aged mice. Age (Dordr). 2014 Feb;36(1):151-65. • de Lemos ML, de la Torre AV, Petrov D, Brox S, Folch J, Pallàs M, et al. Evaluation of hypoxia inducible factor expression in inflammatory and neurodegenerative brain models. The international journal of biochemistry & cell biology. 2013 Jul;45(7):1377-88. • Monica E, Urena-Guerrero AI, Feria-Velasco G. Modifications in the Seizures Susceptibility by Excitotoxic. 2013;59-76. • Auladell C, Junyent F, Vazquez de la Torre A. The Role of JNK Pathway in the Process of Excitotoxicity. 2013. • Bayod S, Menella I, Sanchez-Roige S, Lalanza JF, Escorihuela RM, Camins A, et al. Wnt pathway regulation by long-term moderate exercise in rat hippocampus. Brain Res. 2014 Jan 16;1543:38-48. • Rivera-Cervantes M, Feria-Velasco AI, Junyent F. Intracellular Pathways Associated with Neuronal Survival. Pharmacoresistance in Epilepsy: From Genes and Molecules to Promising Therapies. 2013. • Vazquez de la Torre A, Junyent F, Folch J, Pelegri C, Vilaplana J, Auladell C, et al. PI3 k/akt inhibition induces apoptosis through p38 activation in neurons. Pharmacological research. 2013 Apr;70(1):116-25. • Redondo-Castro E, Romero R, Torres-Espin A, Utrera J, Duque D, Junyent F, et al. Dithiocarb (N,N-diethyldithiocarbamate, DEDTC) decreases levels of biogenic monoamines in the adult mouse brain. Neuropathology and applied neurobiology. 2013 Aug 22. • Barroso E, del Valle J, Porquet D, Vieira Santos AM, Salvado L, Rodriguez-Rodriguez R, et al. Tau hyperphosphorylation and increased BACE1 and RAGE levels in the cortex of PPARβ/δ-null mice. Biochimica et Biophysica Acta-Molecular Basis Of Disease. 2013 Aug;1832(8):1241-8.

Research Projects

• Code: BFU2010-22149 en Red (GENIAR) Title: Caracterizacion de los agregados amiloides presentes Principal Investigator: Antoni Camins Espuny en el hipocampo de ratones CIBERNED’s collaboration: No Principal Investigator: Antoni Camins Espuny CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: International Other CIBER’s collaboration: No Funding Agency: CYTED Type: National Funding: 33.000 € Funding Agency: CYCYT Duration: 2010-2013 Funding: 25.000 € Duration: 2011-2013 • Code: BFU2010-19119 Title: Identificación de las moléculas de la vía JNK, claves • Code: P609RT0454 en la Title: Group de Estudio en Neurociencias Iberoamericano Principal Investigator: Antoni Camins Espuny CIBERNED’s collaboration: No de CIBERNED’s groups: Principal Investigator: Merce Pallàs LLiberia Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: National CIBERNED’s groups: Funding Agency: CYCYT Other CIBER’s collaboration: No Funding: 25.000 € Type: National Duration: 2011-2013 Funding Agency: Ministerio de Ciencia e Innovación Funding: 110.000 € • Code: SAF2011-23631 Duration: 2013 Title: Implicación de la isoforma Jnk3 en la enfermedad de Alzheimer. Evaluación de su potencial como diana terapéu- • Code: PI2010/09 tica Title: BESAD-P: Biomarkers of Early Stages of Alzheimer Principal Investigator: Antonio Camins Espuny Disease-Prevention CIBERNED’s collaboration: No Principal Investigator: Isidre Ferrer Abizanda CIBERNED’s groups: CIBERNED’s collaboration: Yes Other CIBER’s collaboration: No CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; Type: National G 506; G 508; G 412 Funding Agency: Ministerio de Ciencia e Innovación Other CIBER’s collaboration: Yes (CIBERESP) Funding: 33.000 € Type: Intramurales Duration: 2012-2014 Funding Agency: CIBERNED Funding: 223.500 € • Code: SAF2012-39852-C02-01 Duration: 2011-2013 Title: Interacción SIRTUINA1-AMPK-MTOR en los procesos

PhD Dissertations

• Author: Sergio Bayod Gimeno Date: 25 June 2013 Title: Efecto del ejercicio físico en el envejecimiento: Impli- Supervisor: Ester Verdaguer Cardona cación de la via de la sirtuina 1, el proceso de autofagia i la via de la Wnt en rata • Author: Laura Canadell Vilarrasa Date: 1 July 2013 Title: Análisis del grado de adherencia al tratamiento anti- Supervisor: Mercedes Pallàs Lliberia vírico durante la pandemia de gripe A (H1N1)del 2009 y el período postpandémico 2010-11 en las unidades de cuida- • Author: Aurelio Vazquez de la Torre dos intensivos españolas. Title: Estudio de la apoptosis inducida por la inhibición de Date: 2 July 2013 la vía PI3K/AKT en neuronas Supervisor: Francesc X. Sureda Batlle

3434 Antonio Camins Espuny Group Group José Luis Cantero Lorente

Laboratorio de Neurociencia Funcional

Servicios Centralizados de Investigación (Edificio 21) 1 Universidad Pablo de Olavide Carretera de Utrera, Km. 1 41013 Sevilla (Spain) Program Tel.: +34 954 977 433 Fax: +34 954 34151 [email protected]

José Luis Cantero Lorente Mayely Sánchez Espinosa Principal Investigator PhD student Mercedes Atienza Ruiz Olga Prian Serrano Researcher PhD student Maité Crespo García Eulogio Gil Néciga Postdoctoral Fellow Researcher Gabriel González Escamilla David García Solís PhD student Researcher

35 Summary

In 2013, our laboratory has carried out experiments to fully characterize sleep deficits in preclinical Alzheimer’s disease (AD). We found that elderly subjects with mild cognitive impairment (MCI) showed an altered sleep structure, reported more sleep complaints, and overestimated the sleep onset latency compared to healthy elderly subjects (Hita-Yáñez et al., 2013). We are currently exploring relationships between impaired sleep, plasma beta-amyloid levels, and thinning of cortical regions affected by AD.

We further studied cerebral oscillatory correlates underlying working memory load in young subjects. Results showed that changes in amplitude and phase of alpha oscillations between prefrontal and parietal regions accounted for different levels of memory load, revealing different neural mechanisms of top-down control critical for mediating working memory processes in young subjects (Crespo-García et al., 2013). These results will be considered as reference in experiments aimed to understand how cerebral mechanisms underlying working memory load are impaired in normal aging and incipient neurodegeneration.

Further experiments have allowed us to determine that asymmetric cortical atrophy, a common neuroimaging marker of frontotemporal dementia (FTD), does not predict progranulin-linked FTD unless the clinical phenotype is a well-defined frontotemporal syndrome (Gómez-Tortosa et al., 2013a). Moreover, we have systematically studied the clinical significance of some rare mutations in FTD, showing that 20-30 repetitions in the C9ORF72 gene are significantly related to frontotemporal cognitive deterioration (Gómez-Tortosa et al., 2013b).

Keywords

Cerebral aging, Neuroimaging and cerebral electrophysiology markers of prodromal stages of Alzheimer’s disease, Anatomical and functional connectivity of human neocortex, Computational neuroanatomy techniques applied to neurodegenerative diseases

Publications

• Palomar FJ, Suarez A, Franco E, Carrillo F, Gil-Neciga E, Mir P. Abnormal sensorimotor plasticity in CADASIL correlates with neuropsychological impairment. Journal of neurology, neurosurgery, and psychiatry. 2013;84(3):329-36. • Benitez-Rivero S, Marin-Oyaga VA, Garcia-Solis D, Huertas-Fernandez I, Garcia-Gomez FJ, Jesus S, et al. Clinical features and 123I-FP-CIT SPECT imaging in vascular parkinsonism and Parkinson's disease. Journal of Neurology, Neurosurgery, and Psychiatry. 2013;84(2):122-9. • Gomez-Tortosa E, Guerrero-Lopez R, Gil-Neciga E, Franco E, del Ser T, Jimenez Escrig A, et al. Plasma progranulin levels in cortical dementia phenotypes with asymmetric perisylvian atrophy. European Journal of Neurology. 2013;20(9):1319-24. • Crespo-Garcia M, Pinal D, Cantero JL, Diaz F, Zurron M, Atienza M. Working memory processes are mediated by local and long- range synchronization of alpha oscillations. Journal of Cognitive Neuroscience. 2013;25(8):1343-57. • Hita-Yanez E, Atienza M, Cantero JL. Polysomnographic and subjective sleep markers of mild cognitive impairment. Sleep. 2013;36(9):1327-34. • Garcia-Gomez FJ, Garcia-Solis D, Luis-Simon FJ, Marin-Oyaga VA, Carrillo F, Mir P, Vazquez-Albertino RJ. Elaboration of the SPM template for the standardization of SPECT images with 123I-Ioflupane. Revista Espanola de Medicina Nuclear e Imagen Molecular. 2013;32(6):350-6. • Gomez-Tortosa E, Gallego J, Guerrero-Lopez R, Marcos A, Gil-Neciga E, Sainz MJ, et al. C9ORF72 hexanucleotide expansions of 20-22 repeats are associated with frontotemporal deterioration. Neurology. 2013;80(4):366-70.

Research Projects

• Code: SAF2011-25463 CIBERNED’s collaboration: No Title: Combining beta-amyloid biomarkers with anato- CIBERNED’s groups: mo-functional neuroimaging to predict cognitive decline in Other CIBER’s collaboration: No presymptomatic stages of Alzheimer’s disease Type: National Principal Investigator: Jose Luis Cantero Lorente Funding Agency: Ministerio de Ciencia e Innovación, Plan

3636 José Luis Cantero Lorente Group José Luis Cantero Lorente Group 37

National I+D+I, Program National de Biomedicina Principal Investigator: Mercedes Atienza Ruiz Funding: 150.000 € CIBERNED’s collaboration: No Duration: 2012-2014 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: 2013 Type: National Title: Deterioro de la conectividad anatómica asociado a Funding Agency: MICINN, Plan National I+D+i las fases presintomáticas de la enfermedad de Alzheimer: Funding: 80.000 € De la microestructura de la sustancia blanca cerebral a los Duration: 2012-2014 cambios en los haces de fibras nerviosas Principal Investigator: Jose Luis Cantero Lorente • Code: PI2011/04 CIBERNED’s collaboration: No Title: Estudio multicéntrico de biomarcadores en LCR y de CIBERNED’s groups: neuroimagen en el continuum Enfermedad de Alzheimer Other CIBER’s collaboration: No preclínica - prodrómica (Estudio SIGNAL) Type: Privado Principal Investigator: Alberto Lleó Funding Agency: Instituto de Especialidades Neurológicas CIBERNED’s collaboration: Yes (IENSA) CIBERNED’s groups: G 504; G 502; G 511; G 507; G 509 Funding: 10.000 € Other CIBER’s collaboration: No Duration: 2013-2014 Type: Intramurales Funding Agency: CIBERNED • Code: PSI2011-24922 Funding: 140.000 € Title: Papel de las oscilaciones cerebrales del sueño no REM Duration: 2011-2013 en la codificación y consolidación de memorias asociativas

PhD Dissertations

• Author: Maité Crespo-García Title: Efectos del envejecimiento sobre la dinámica cortical que subyace a la formación de la memoria asociativa Date: 22 March 2013 Supervisor: José Luis Cantero Lorente Group Joan Xavier Comella Carnicé

VHIR – Vall d’Hebron Institut de Recerca

1 Passeig Vall d’Hebron, 119-129 08035 Barcelona (Spain) Tel.: +34 934 893 807 http://www.vhir.org Program [email protected]

Joan Xavier Comella Carnicé Jorge Urresti Ibáñez Principal Investigator PhD student Víctor Yuste Mateos Laura Planells Ferrer PhD PhD student Paulina Carriba Domínguez Koen Galenkamp PhD PhD student Joaquín López Soriano María Sánchez Osuna PhD PhD student Bruna Barneda Zahonero Elena Coccia PhD Master’s student Nuria Llecha Cano Sonia Moreno Grau PhD Master’s student

38 Joan Xavier Comella Carnicé Group 39

Summary

Our main interest is to characterize the mechanisms controlling neuronal death induced by a group of receptors collectively known as death receptors, and also the relevance of some of their intracellular antagonists (mainly Lifeguard, FAIM-L and FLIP). Our hypothesis is that Alzheimer’s disease (AD) has a neuroinflammatory component. In early stages, glial activation plays a neuroprotective role, executed by cytokines such as TNF, which induces survival pathways. In advanced stages, chronic neuroinflammation activates death receptors with a loss of the protective role of their functional antagonists (such as FAIM-L) and thus promoting neuronal death.

Thus, our main objective is the molecular characterization of these death receptor antagonists, their role in neuronal differentiation and physiology, and their involvement in different pathologies, particularly neurodegenerative diseases. In addition, we are characterizing molecular partners of these molecules that may explain their mechanism of action. In this sense, during 2013 we described in detail the interaction between FAIM-L and XIAP, in an article published at the Journal of Neuroscience. Finally, we are, in collaboration with different CIBERNED groups, characterizing the role of FAIM-L in both animal models (APP/PS1) and patients of Alzheimer’s disease.

The knowledge of the molecular mechanisms of action of these death receptor antagonists and how they induce survival signaling pathways could be highly relevant to a better understanding of the etiology and also to open new strategies to improve the treatment of some neurodegenerative illnesses, such as AD or Parkinson’s disease.

Our main research lines at present are:

• To study FAIM-L function in in vitro and in vivo models of AD, and its relation with the duality of TNF as a pro-apoptotic molecule and at the same time as a survival promoter. • To characterize FAIM-L functional partners. • To study Faim promoter and the functional differences of FAIM isoforms. • To characterize the function of Lifeguard in models of neuroblastoma, and its interaction with MycN.

• To characterize Lifeguard and its functional partners.

Keywords

Alzheimer, Neuroinflammation, Death receptors, FAIM, Lifeguard, TNF, Fas

Publications

• di Penta A, Moreno B, Reix S, Fernandez-Diez B, Villanueva M, Errea O, et al. Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation. PloS one. 2013;8(2):e54722. • Iglesias-Guimarais V, Gil-Guinon E, Sanchez-Osuna M, Casanelles E, Garcia-Belinchon M, Comella JX, et al. Chromatin collapse during caspase-dependent apoptotic cell death requires DNA fragmentation factor, 40-kDa subunit-/caspase-activated deoxyribonuclease-mediated 3’-OH single-strand DNA breaks. The Journal of biological chemistry. 2013 Mar 29;288(13):9200- 15. • Casanelles E, Gozzelino R, Marques-Fernandez F, Iglesias-Guimarais V, Garcia-Belinchon M, Sanchez-Osuna M, et al. NF-κB activation fails to protect cells to TNFα-induced apoptosis in the absence of Bcl-xL, but not Mcl-1, Bcl-2 or Bcl-w. Biochimica et Biophysica Acta-Molecular Cell Research. 2013 May;1833(5):1085-95. • Ye J, Llorian M, Cardona M, Rongvaux A, Moubarak RS, Comella JX, et al. A pathway involving HDAC5, cFLIP and caspases regulates expression of the splicing regulator polypyrimidine tract binding protein in the heart. Journal of Cell Science. 2013 Apr 1;126(Pt 7):1682-91. • Marques-Fernandez F, Planells-Ferrer L, Gozzelino R, Galenkamp KM, Reix S, Llecha-Cano N, et al. TNFα induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway. Cell death & disease. 2013 Feb 14;4:e493. • Moubarak RS, Planells-Ferrer L, Urresti J, Reix S, Segura MF, Carriba P, et al. FAIM-L Is an IAP-Binding Protein That Inhibits XIAP Ubiquitinylation and Protects from Fas-Induced Apoptosis. The Journal of neuroscience. 2013 Dec 4;33 (49):19262-75. • La Torre A, del Mar Masdeu M, Cotrufo T, Moubarak RS, del Rio JA, Comella JX, et al. A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching. Cell death & disease. 2013 Apr 18;4:e602. • Giralt A, Sanchis D, Cherubini M, Gines S, Canas X, Comella JX, et al. Neurobehavioral characterization of Endonuclease G knockout mice reveals a new putative molecular player in the regulation of anxiety. Experimental neurology. 2013 Sep;247:122-9. • Fado R, Moubarak RS, Minano-Molina AJ, Barneda-Zahonero B, Valero J, Saura CA, et al. X-linked inhibitor of apoptosis protein negatively regulates neuronal differentiation through interaction with cRAF and Trk. Scientific Reports. 2013;3:1-11.

Research Projects

• Code: SAF2010_19953 Title: Relevancia fisiológica y patológica de los antagonistas de los receptores de muerte (FAIM, Lifeguard y FLIP) en el sistema nervioso Principal Investigator: Joan Xavier Comella Carnicé CIBERNED’s collaboration: No CIBERNED’s groups: Other CIBER’s collaboration: No Type: National Funding Agency: Ministerio Ciencia e Innovación Funding: 400.000 € Duration: 2010-2013

PhD Dissertations

• Author: Fernando Marqués Fernández • Author: Elisenda Casanelles Abella Title: Activation of survival pathways triggered by death re- Title: Estudi de Ia rellevància dels membres anti-apoptòtics ceptors in the nervous system de la família de Bcl-2 en la resistència a la mort induïda per Date: 13 February 2013 la via extrínseca a través dels lligands de mort Supervisor: Joan Xavier Comella Carnicé Date: 17 July 2013 Supervisor: Víctor Yuste Mateos

4040 Joan Xavier Comella Carnicé Group Group Javier de Felipe Oroquieta

Laboratorio de Circuitos Corticales (CTB) Instituto Cajal (CSIC)

Universidad Politécnica de Madrid Avenida Doctor Arce, 37 1 Campus Montegancedo, s/n 28002 Madrid (Spain) 28223 Pozuelo de Alarcón, Madrid (Spain) Program Tel.: +34 913 364 639 [email protected]

Javier de Felipe Oroquieta Mari Carmen Álvarez Pérez Principal Investigator, Research Professor UPM Advanced Technician Lidia Alonso-Nanclares Silvia Tapia González CSIC Postdoctoral Fellow UPM Advanced Technician Ruth Benavides-Piccione Paloma Carrero Peña CSIC Postdoctoral Fellow UPM Advanced Technician (until June 2013) Rodrigo Rodríguez Sánchez Alejandro Antón Fernández CSIC Senior Scientist UPM PhD student Isabel Fernaud Espinosa Liulia Diana Furcila UPM Postdoctoral Fellow UPM PhD student Asta Kastanauskaite Andrea Santuy Muñoz UPM Postdoctoral Fellow UPM PhD student Ángel Merchán-Pérez Marta Álvarez Almazán Postdoctoral Fellow UPM UPM Grant project colaboration Alberto Muñoz Sandra Sáez Raspeño UPM Postdoctoral Fellow UPM Grant project colaboration Gonzalo León Espinosa Miguel Miguens Vázquez Postdoctoral Fellow, Collaborator UNED Visitant Investigator Lorena Valdés Lora Juncal González UPM Technician UCM Visitant Investigator Ana Isabel García Ramírez Pilar Flores Romero UPM Technician UPM R&D Manager Débora Cano Laiseca Montserrat Fernández Bouzo UPM Technician CSIC R&D Management Technician

41 Summary

The two main lines of research that we have developed are:

• Neurochemical and microanatomical analysis of the cerebral cortex in different mouse models for Alzheimer’s disease, with the idea of obtaining data about the substrate and temporal course of the microanatomical alterations that occur in Alzheimer’s disease. • Neurochemical and microanatomical analysis of the cerebral cortex of patients with Alzheimer’s disease. In particular, we are performing a quantitative and qualitative analysis of the synaptic circuits and neurochemical characteristics of the cerebral cortex of these patients. The aim of this objective is to try to know in detail the alterations of the neuronal circuits in relation with the senile plaques and the presence of neurofibrillary tangles.

Keywords

Cerebral cortex, Microorganization, Neuronal circuits, Electron microscopy, Alzheimer

Publications

• lopez-Cruz PL, Larranaga P, DeFelipe J, Bielza C. Bayesian network modeling of the consensus between experts: An application to neuron classification. International Journal of Approximate Reasoning. 2013;55(1):23-35. • LaTorre A, Alonso-Nanclares L, Muelas S, Pena JM, Defelipe J. Segmentation of neuronal nuclei based on clump splitting and a two-step binarization of images. Expert Systems with Applications. 2013;40:6521-30. • Montes J, Gomez E, Merchan-Perez A, DeFelipe J, Pena JM. A Machine Learning Method for the Prediction of Receptor Activation in the Simulation of Synapses. PloS One. 2013;8(7):e68888. • Armananzas R, Alonso-Nanclares L, Defelipe-Oroquieta J, Kastanauskaite A, de Sola RG, Defelipe J, et al. Machine learning approach for the outcome prediction of temporal lobe epilepsy surgery. Plos One. 2013;8(4):e62819. • Morales J, Rodriguez A, Rodriguez JR, Defelipe J, Merchan-Perez A. Characterization and extraction of the synaptic apposition surface for synaptic geometry analysis. Frontiers in neuroanatomy. 2013;7:20. • Brito JP, Mata S, Bayona S, Pastor L, DeFelipe J, Benavides-Piccione R. Neuronize: A tool for building realistic neuronal cell morphologies. Frontiers in Neuroanatomy. 2013;7:15. • Blazquez-Llorca L, Merchan-Perez A, Rodriguez JR, Gascon J, DeFelipe J. FIB/SEM technology and Alzheimer's disease: three- dimensional analysis of human cortical synapses. Journal of Alzheimer's disease: JAD. 2013 Jan 1;34(4):995-1013. • DeFelipe, J. Cajal and the discovery of a new artistic world: The neuronal forest. Progress in Brain Research. 2013;203:201-20. • DeFelipe J, Alonso-Nanclares L. The synapse: differences between men and women. In: Pfaff DW, Christen Y, Eds. Research and Perspectives in Endocrinology. Springer Berlin Heidelberg, 2013; pp 43-57. • DeFelipe J. Cajal and the discovery of a new artistic world: The neuronal forest. Progress in Brain Research. The Fine Arts, Neurology and Neuroscience: History and Modem Perspectives. 2013;203:201-20. • Alonso-Nanclares L, Merino-Serrais P, Gonzalez S, DeFelipe J. Synaptic changes in the dentate gyrus of app/ps1 transgenic mice revealed by electron microscopy. J Neuropathol Exp Neurol. 2013;72(5):386-95. • Leon-Espinosa G, Garcia E, Garcia-Escudero V, Hernandez F, DeFelipe J, Avila J. Changes in tau phosphorylation in hibernating rodents. Journal of Neuroscience Research. 2013;91(7):954-62. • Merino-Serrais P, Benavides-Piccione R, Blazquez-Llorca L, Kastanauskaite A, Rabano A, Avila J, DeFelipe J. The influence of phospho-τ on dendritic spines of cortical pyramidal neurons in patients with Alzheimer's disease. Brain. 2013;136(6):1913-28. • Llorens-Martin M, Fuster-Matanzo A, Teixeira CM, Jurado-Arjona J, Ulloa F, Defelipe J, et al. GSK-3β overexpression causes reversible alterations on postsynaptic densities and dendritic morphology of hippocampal granule neurons in vivo. Molecular psychiatry. 2013 Apr;18(4):451-60. • Miguens M, Kastanauskaite A, Coria SM, Selvas A, Ballesteros-Yanez I, Defelipe J, et al. The Effects of Cocaine Self-Administration on Dendritic Spine Density in the Rat Hippocampus Are Dependent on Genetic Background. Cerebral cortex (New York, N.Y. 1991). 2013 Aug 21. • Merchan-Perez A, Rodriguez JR, Gonzalez S, Robles V, Defelipe J, Larranaga P, et al. Three-Dimensional Spatial Distribution of Synapses in the Neocortex: A Dual-Beam Electron Microscopy Study. Cerebral cortex (New York, N.Y. 1991). 2013 Jan 30.

4242 Javier de Felipe Oroquieta Group Javier de Felipe Oroquieta Group 43

• Inan M, Blazquez-Llorca L, Merchan-Perez A, Anderson SA, DeFelipe J, Yuste R. Dense and overlapping innervation of pyramidal neurons by chandelier cells. The Journal of neuroscience. 2013 Jan 30;33(5):1907-14. • DeFelipe J, Lopez-Cruz PL, Benavides-Piccione R, Bielza C, Larranaga P, Anderson S, et al. New insights into the classification and nomenclature of cortical GABAergic interneurons. Nature reviews. Neuroscience. 2013 Mar;14(3):202-16. • Llorens-Martin M, Fuster-Matanzo A, Teixeira CM, Jurado-Arjona J, Ulloa F, Defelipe J, et al. Alzheimer disease-like cellular phenotype of newborn granule neurons can be reversed in GSK-3β-overexpressing mice. Molecular psychiatry. 2013 Apr;18(4):395. • Mellström B, Sahun I, Ruiz-Nuno A, Murtra P, Gomez-Villafuertes R, Savignac M, et al. DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways. Mol Cell Biol. 2014 Mar;34(5):877-87.

Research Projects

• Code: BFU2012-34963 • Code: PI2010/07 Title: Microanatomical and neurochemical alterations of Title: Reelina y GSK3 como dianas terapéuticas en la enfer- the cerebral cortex in alzheimer’s disease medad de Alzheimer Principal Investigator: Javier de Felipe Principal Investigator: Jesús Ávila de Grado CIBERNED’s collaboration: No CIBERNED’s collaboration: Yes CIBERNED’s groups: CIBERNED’s groups: G 401; G 403; G 113; G 407; G 408; Other CIBER’s collaboration: No G 410 Type: National Other CIBER’s collaboration: No Funding Agency: Ministerio de Economía y Competitividad Type: Intramurales Funding: 193.050 € Funding Agency: CIBERNED Duration: 2013 Funding: 540.000 € Duration: 2010-2013 • Code: Cajal Blue Brain Project Title: Cajal Blue Brain Project. International Blue Brain Pro- • Code: 2013/07A yect Title: Papel de GSK-3 β en las alteraciones de los circuitos Principal Investigator: Javier de Felipe corticales que ocurren en la enfermedad de Alzheimer CIBERNED’s collaboration: Yes Principal Investigator: Teresa Iglesias Vacas CIBERNED’s groups: G 204; G 403 CIBERNED’s collaboration: Yes Other CIBER’s collaboration: No CIBERNED’s groups: G 401; G 403; G 111; G 504; G 307 Type: International Other CIBER’s collaboration: No Funding Agency: Ecole Polytechnique Federale de Lausan- Type: Intramurales ne (Suiza), IBM, Universidad Politecnica de Madrid, Consejo Funding Agency: CIBERNED Superior de Investigaciones Cientificas, España Funding: 158.000 € Funding: 0 € Duration: 2013-2015 Duration: 2009-2019 Group Jesús de Pedro Cuesta

Centro Nacional de Epidemiología

1 Instituto de Salud Carlos III Monforte de Lemos, 5 28029 Madrid (Spain)

Program Tel.: +34 918 222 650 Fax: +34 913 877 815 [email protected]

Jesús de Pedro Cuesta Belén Frades Payo Principal Investigator, CNE Head of Unit CIBERNED Technician Jimena Aguilera Abad Alberto García Canals Technician (until October 15, 2013) Technician (until October 15, 2013) Enrique Alcalde Cabero Carmen Matesanz Blanco PhD student Technician (from October 1, 2013) Javier Almazán Isla Pablo Martínez Martín Technician OPIs Senior Investigator Fuencisla Avellanal Calzadilla María José Medrano Technician OPIs Senior Investigator Miguel Calero Lara María del Carmen Rodríguez Blázquez Researcher Lab Head Research Assistant Olga Calero Rueda María Ruiz Tovar Postdoctoral Fellow Postdoctoral Fellow Javier Damián Moreno Postdoctoral Fellow

44 Jesús de Pedro Cuesta Group 45

Summary

In collaboration with several CIBERNED groups we have continued on the study of genetic traits shared by different neurodegenerative disorders such as Alzheimer’s and prion diseases and their interaction with age. We established collaboration with several International groups for the study of biomarkers for rapid progressive Alzheimer’s disease with the frame of a research grant of the first transnational call for “optimization of biomarkers and harmonization of their use” under the EU Joint Programme-Neurodegenerative Disease Research (JPND). Moreover, we have started a Coordinated FIS grant in collaboration with CIBERNED groups from CIEN Foundation and 12 de Octubre Hospital focused on the study of vascular dysfunction associated to aging in Alzheimer´s disease. On June 2013, M Calero directly collaborates with the Vallecas Project and the study of the patients from the Alzheimer Research Centre CIEN Foundation-Queen Sofía Foundation.

The public health subgroup in collaboration with Nordic scientists published the theoretical bases to prevent the surgical transmission of Creutzfeldt-Jakob disease. However, most reports this year focused on aging and disability, such as those by first time describing in door-to-door surveys at Aragón the high prevalences of disability and the specific affected domains and their neurodegenerative and other causes, the determinants of mortality and worsening in primary care groups as well as those of falls in residents at Institutions at Madrid region. The group contributed to the elaboration within the frame of the Joint Programme on Research on Neurodegenerative Disorders (JNPD) of an Action Group access document http://www. neurodegenerationresearch.eu/initiatives/jpnd-alignment-actions/longitudinal-cohorts

The recruitment of participants in the “Proyecto Vallecas for Early Detection of Alzheimer’s Disease” was concluded on 31-12- 2013. Over 1,200 subjects were assessed and 1,174 were found valid for the longitudinal 5-year follow-up. In addition, were re-assessed 836 subjects into the first follow-up year, and 53 into the second follow-up. New contributions to knowledge and measurement of the health-related quality of life, apathy (in collaboration with CIBERSAM), and motor disorders in patients with advanced dementia were carried out. Furthermore, the non-motor symptoms of Parkinson’s disease, with emphasis in emotional and cognitive were studies into international collaborations.

Keywords

Alzheimer disease, Prion diseases, Creutzfeldt-Jakob, Neurodegenerative disorders, Conformational disorders, Biomarkers, Ge- netic susceptibility markers, Risk genes, Vascular factors, Aging

Publications

• Martínez-Martín P, Rodríguez-Blazquez C, Alvarez-Sanchez M, Arakaki T, Bergareche-Yarza A, et al. Expanded and independent validation of the Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS). Journal of Neurology. 2013;260(1):228-36. • Martinez-Martin P, Chaudhuri KR, Rojo-Abuin JM, Rodríguez-Blazquez C, Alvarez-Sanchez M, Arakaki T, et al. Assessing the non-motor symptoms of Parkinson’s disease: MDS-UPDRS and NMS Scale. European Journal of Neurology. 2013, [in press]. • Martín-García S, Rodríguez-Blázquez C, Martínez-López I, Martínez-Martín P, Forjaz MJ. Comorbidity, health status, and quality of life in institutionalized older people with and without dementia. International Psychogeriatrics. 2013;25(7):1077-84. • Diaz-Redondo A, Rodríguez-Blázquez C, Ayala A, Martínez-Martín P, Forjaz MJ. EQ-5D rated by proxy in institutionalized older adults with dementia: Psychometric pros and cons. Geriatrics & Gerontology International. 2013, [in press]. • Rodriguez-Blazquez C, Rojo-Abuin JM, Alvarez-Sanchez M, Arakaki T, Bergareche-Yarza A, Chade A, et al. The MDS-UPDRS Part II (motor experiences of daily living) resulted useful for assessment of disability in Parkinson's disease. Parkinsonism & Related Disorders. 2013;19:889-93. • Kurtis MM, Rodríguez-Blázquez C, Martínez-Martín P. Relationship between sleep disorders and other non-motor symptoms in Parkinson's disease. Parkinsonism & Related Disorders. 2013;19(12):1152-5. • Martinez-Martin P, Rodriguez-Blazquez C, Forjaz MJ, Alvarez-Sanchez M, Arakaki T, Bergareche A, et al. Relationship between the MDS-UPDRS domains and the health-related quality of life of Parkinson’s disease patients. European Journal of Neurology. 2013, [in press]. • Rivera-Navarro J, Cubo E, Almazán J. The Impact of Tourette’s Syndrome in the School and the Family: Perspectives from Three Stakeholder Groups. Int J Adv Counselling. Jul 2013. • Alcalde-Cabero E, Alberquilla A, Damián J, Bosca G, López-Rodríguez F, Carmona M, et al. Disability transitions after 30 months in three community-dwelling diagnostic groups in Spain. PLoS One. 2013 Oct 31;8(10):e77482. • Almazán-Isla J, Comín-Comín M, Damián J, Alcalde-Cabero E, Ruiz C, Franco E, et al.; DISCAP-ARAGON ResearchGroup. Analysis of disability using WHODAS 2.0 among the middle-aged and elderly in Cinco Villas, Spain. Disabil Health J. 2014 Jan;7(1):78-87. • Alcalde-Cabero E, Almazán-Isla J, García-Merino A, de Sá J, de Pedro-Cuesta J. Incidence of multiple sclerosis among European Economic Area populations, 1985-2009: the framework for monitoring. BMC Neurol. 2013 Jun 12;13:58. • Cruz M, Mahillo-Fernandez I, Rábano A, Siden A, Calero M, Laursen H, et al.; EUROSURGYCJD ResearchGroup. Late-in-life surgery associated with Creutzfeldt-Jakob disease: a methodological outline for evidence-based guidance. Emerg Themes Epidemiol. 2013 May 23;10(1):5. • Damián J, Pastor-Barriuso R, Valderrama-Gama E, de Pedro-Cuesta J. Factors associated with falls among older adults living in institutions. BMC Geriatr. 2013 Jan 15;13:6. • Damian J, de Pedro-Cuesta J, Almazán J, Comín-Comín M, Quintanilla MA, Lobo A. Depressive symptoms and associated factors in an older Spanish population positively screened for disability. Int J Geriatr Psychiatry. 2013 Jul;28(7):745-55. • Rodríguez-Rodríguez E, Vázquez-Higuera JL, Sánchez-Juan P, González-Aramburu I, Pozueta A, Mateo I, et al. Screening for progranulin mutations by serum protein dosage in common neurodegenerative disorders. Parkinsonism Relat Disord. 2013 Aug;19(8):768-9. • Mateo I, González-Aramburu I, Pozueta A, Vázquez-Higuera JL, Rodríguez-Rodríguez E, Sánchez-Juan P, et al. Reduced serum progranulin level might be associated with Parkinson's disease risk. Eur J Neurol. 2013 Dec;20(12):1571-3. • Kuijl C, Pilli M, Alahari SK, Janssen H, Khoo PS, Ervin KE, et al. Rac and RabGTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria. EMBO J. 2013 Mar 6;32(5):713-27. • Carmona P, Molina M, Calero M, Bermejo-Pareja F, Martínez-Martín P, Toledano A. Discrimination analysis of blood plasma associated with Alzheimer's disease using vibrational spectroscopy. J Alzheimers Dis. 2013 Jan 1;34(4):911-20. • Muñoz-Nieto M, Ramonet N, López-Gastón JI, Cuadrado-Corrales N, Calero O, Díaz-Hurtado M, et al. A novel mutation I215V in the PRNP gene associated with Creutzfeldt-Jakob and Alzheimer's diseases in three patients with divergent clinical phenotypes. J Neurol. 2013 Jan;260(1):77-84. • Gil-Ruiz N, Osorio RS, Cruz I, Agüera-Ortiz L, Olazarán J, Sacks H, et al.; The Alzheimer Center of The Queen Sofia Foundation Multidisciplinary Therapy Group. An effective environmental intervention for management of the 'mirror sign' in a case of probable Lewy body dementia. Neurocase. 2013;19:1-13. • Carod-Artal FJ, Martinez-Martin P. Independent validation of the Non motor Symptoms Scale for Parkinson's disease in Brazilian patients. Parkinsonism Relat Disord. 2013;19:115-9. • Olazarán J, Hernández-Tamames JA, Molina E, García-Polo P, Dobato JL, Alvarez-Linera J, et al. Clinical and Anatomical Correlates of Gait Dysfunction in Alzheimer's Disease. J Alzheimers Dis. 2013;33:495-505. • Rodríguez-Violante M, Cervantes-Arriaga A, Corona T, Martínez-Ramírez D, Morales-Briceño H, Martínez-Martín P. Clinical Determinants of Health-related Quality of Life in Mexican Patients with Parkinson's Disease. Arch Med Res. 2013;44:110-4. • Ray Chaudhuri K, Rojo JM, Schapira AHV, Brooks DJ, Stocchi F, Odin P, et al. A proposal for a comprehensive grading of Parkinson’s disease severity combining motor and non-motor assessments: meeting an unmet need. PLoS ONE. 2013;8:e57221. • Martín F, Agüero CE, Cañas JM, Valenti M, Martínez-Martín P. Robotherapy with Dementia patients. Int J Advan Robotic Systems. 2013;10:10. • Martinez-Martin P. Instruments for holistic assessment of Parkinson's Disease. J Neural Transm. 2013;120:559-64. • Forjaz MJ, Martinez-Martin P, Dujardin K, Marsh L, Richard IH, Starkstein SS, et al. Rasch analysis of anxiety scales in Parkinson's disease. J Psychosom Res. 2013;74:414-9. • Martín F, Agüero CE, Cañas JM, Abella G, Benítez R, Rivero S, et al. Robots in therapy for dementia patients. J Phys Agents. 2013;7:48-55. • Todorova A, Martinez-Martin P, Martin A, Robinson S, Rizos A, Reddy P, et al. Daytime Apomorphine infusion combined with transdermal Rotigotine patch therapy: a 24 hours treatment option in Parkinson’s disease. Basal Ganglia. 2013;3:127-30. • Olazarán J, González B, López J, Castagna A, Osa-Ruiz E, Herrero-Cano V, et al. Motor effects of REAC in advanced Alzheimer's disease: Results from a pilot trial. J Alzheimers Dis. 2013;36:297-302. • Ray Chaudhuri K, Martinez-Martin P, Antonini A, Brown RG, Friedman JH, Onofrj M, et al. Rotigotine and specific non-motor symptoms of Parkinson’s disease: post hoc analysis of RECOVER. Parkinsonism Relat Disord .2013;19:660-5. • Martinez-Martin P, Rojo-Abuin JM, Dujardin K, Pontone GM, Weintraub D, Forjaz MJ, et al. Designing a new scale to measure anxiety symptoms in Parkinson's disease: item selection based on canonical correlation analysis. Eur J Neurol. 2013;20:1198-203. • Armañanzas R, Bielza C, Chaudhuri KR, Martinez-Martin P, Larrañaga P. Unveiling relevant non-motor Parkinson's disease severity symptoms using a machine learning approach. Artif Intell Med. 2013;58:195-202.

4646 Jesús de Pedro Cuesta Group Jesús de Pedro Cuesta Group 47

• Albanese A, Del Sorbo F, Comella C, Jinnah HA, Mink JW, Post B, et al. Dystonia rating scales: Critique and recommendations. Mov Disord. 2013;28:874-83. • León-Salas B, Olazarán J, Cruz-Orduña I, Agüera Ortiz L, Dobato JL, Valentí-Soler M, et al. Quality of life in community-dwelling and institutionalized Alzheimer's disease patients. Arch Gerontol Geriatrics. 2013;57:257-62. • Martinez-Martin P. Is AD a homogeneous nosologic entity? Yes. J Neural Transm. 2013;120:1467-73. • Leentjens AFG, Moonen AJH, Dujardin K, Marsh L, Martinez-Martin P, Richard IH, et al. Modeling depression in Parkinson's disease: disease-specific and non-specific risk factors. Neurology. 2013;81:1036-43. • Carod-Artal FJ, Mourão Mesquita H, Ziomkowski S, Martinez-Martin P. Burden and health-related quality of life among caregivers of Brazilian Parkinson’s disease patients. Parkinsonism Relat Disord. 2013;19:943-8. • Elble R, Bain P, Forjaz JM, Haubenberger D, Testa C, Goetz CG, et al. Task force report: Scales for screening and evaluating tremor: Critique and recommendations. Mov Disord. 2013;28:1793-800. • Martínez-Martín P. Dementia in Parkinson’s disease: Usefulness of the Pill Questionnaire. Mov Disord. 2013;28:1832-37. • Zesiewicz TA, Martinez-Martin P. Effects of rotigotine transdermal system on non-motor symptoms in Parkinson’s disease: an overview. Expert Rev Neurother. 2013;13:1329-42. • Heerink M, Albo-Canals J, Valenti-Soler M, Martinez-Martin P, Zondag J, Smits C, et al. Exploring requirements and alternative pet robots for robot assisted therapy with older adults with dementia. Social Robotics Lect Notes Comput Sci. 2013;8239:104-15. • Martínez-Martín P, Hernández B, Ricart J; Group de Trabajo del Estudio FAST. Factores determinantes del inicio de tratamiento con levodopa/carbidopa/entacapona en pacientes españoles con enfermedad de Parkinson. Neurología. 2013. PMID: 23465686 • Martinez-Martin P, Chaudhuri RK, Rojo-Abuin JM, Rodriguez-Blazquez C, Alvarez-Sanchez M, Arakaki T, et al. Assessing the non-motor symptoms of Parkinson’s disease: MDS-UPDRS and NMS Scale. Eur J Neurol. 2013. PMID: 23607783. • Dodel R, Jönsson B, Reese JP, Winter Y, Martinez-Martin P, Holloway R, et al. Measurement of costs and scales for outcome evaluation in health economic studies of Parkinson's disease. Mov Disord. 2013. PMID: 23861335. • Agüera-Ortiz L, Gil-Ruiz N, Cruz-Orduña I, Ramos-García I, Osorio RS, Valentí-Soler M, et al. A Novel Rating Scale for the Measurement of Apathy in Institutionalized Persons with Dementia: The APADEM-NH. Am J Geriatr Psychiatry. 2013. PMID: 23871117. • Starkstein SE, Dragovic M, Dujardin K, Marsh L, Martinez-Martin P, Pontone GM, et al. Anxiety Has Specific Syndromal Profiles in Parkinson Disease: A Data-Driven Approach. Am J Geriatr Psychiatry. 2013. PMID: 24200594. • Martínez-Martín P. Non-motor symptoms and health-related quality of life in early Parkinson’s disease (Editorial). Mov Disord. 2013. PMID: 24375626

Research Projects

• Code: PI 008/09 CIBERNED’s groups: Title: Activación de la ruta calpaina/GSK-3/ de CDK-5 en la Other CIBER’s collaboration: No enfermedad Alzheimer Type: International Principal Investigator: Felix Hernandez Perez Funding Agency: Michael J. Fox Foundation - USA CIBERNED’s collaboration: No Funding: 63.000 € CIBERNED’s groups: Duration: 2013 Other CIBER’s collaboration: No Type: Privado • Code: DEMTEST-MPY 990/12. Funding Agency: Fundación CIEN-ISCIII. II Ayudas para Re- Title: Biomarker based diagnosis of rapid progressive de- search Projects en Enfermedades de Alzheimer y Enferme- mentias - optimisation of diagnostic protocols (DemTest) dades Relacionadas Principal Investigator: Miguel Calero Funding: 66.000 € CIBERNED’s collaboration: No Duration: 2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: METC 11-4-057 Maastricht Type: International Title: Assessing and diagnosing anxiety in patients with Funding Agency: Unión Europea – FIS. Joint Programming Parkinson’s disease Neurodegenerative Disease Call for transnational research Principal Investigator: Prof. A.J. Leentjens (International) proposals 2011 "Neurodegenerative Diseases - a call for Eu- and Dr. P. Martinez Martin (for Spain) ropean research projects for the optimisation of biomark- CIBERNED’s collaboration: No ers and harmonisation of their use between c Funding: 117.370 € Igualdad - IMSERSO Duration: 2013 Funding: 33.000 € Duration: 2013 • Code: MVP 1052/08. Title: Contrato de Colaboración para I+D sobre “Determina- • Code: FIS PI10/02567 ción de la actividad de diversas Quinasas”. Tipo de contra- Title: Roboterapia en Demencia to: Apoyo Tecnológico e Investigación Principal Investigator: Pablo Martínez Martín Principal Investigator: Miguel Calero CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: Yes (CIBERSAM) Other CIBER’s collaboration: No Type: National Type: Privado Funding Agency: Fondo de Investigación Sanitaria – Institu- Funding Agency: Noscira – CDTI (Proyecto CENIT). Partici- to de Salud Carlos III pantes: Instituto de Salud Carlos III (OPI) – Noscira. Funding: 33.112 € Funding: 350.000 € Duration: 2013 Duration: 2013 • Code: INN-12D-01 • Code: PI 019/09 Title: The pilot clinical validation study of a novel pain scale Title: Genes relacionados con la fosforilación de la proteína for Parkinson's TAU y su influencia en el riesgo de desarrollar Enfermedad Principal Investigator: Prof. K. R. Chaudhuri (UK) and Dr. P. de Alzheimer esporádica Martinez-Martin (Spain) Principal Investigator: Jose Ignacio Fernandez Mateo CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: International Type: Privado Funding Agency: Parkinson UK – Innovation Grant Funding Agency: Fundación CIEN-ISCIII. II Ayudas para Re- Funding: 40.500 € search Projects en Enfermedades de Alzheimer y Enferme- Duration: 2013 dades Relacionadas Funding: 120.000 € • Code: Fundación Ramón Areces 2012 Duration: 2013 Title: Estudio de la capacidad neuroprotectora de las células epiteliales de los plexos coroideos como potencial • Code: N.A terapia regenerativa de la enfermedad de Alzheimer Title: Non-Motor Symptoms Longitudinal International Principal Investigator: Eva Carro Study (NILS) CIBERNED’s collaboration: Yes Principal Investigator: Prof. K.R. Chaudhuri (UK) and Dr. P. CIBERNED’s groups: G 502; G 509 Martinez-Martin (Spain) Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: Privado CIBERNED’s groups: Funding Agency: Fundación Ramón Areces Other CIBER’s collaboration: No Funding: 30.000 € Type: International Duration: 2012-2015 Funding Agency: International Collaboration – UK NHS Portfolio – UK National Health System • Code: PI2011/04 Funding: 0 € Title: Estudio multicéntrico de biomarcadores en LCR y de Duration: 2013 neuroimagen en el continuum Enfermedad de Alzheimer preclínica - prodrómica (Estudio SIGNAL) • Code: IMSERSO 231/2011 Principal Investigator: Alberto Lleó Title: Roboterapia en Demencia CIBERNED’s collaboration: Yes Principal Investigator: Pablo Martínez Martín CIBERNED’s groups: G 504; G 502; G 511; G 507; G 509 CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: Intramurales Other CIBER’s collaboration: Yes (CIBERSAM) Funding Agency: CIBERNED Type: National Funding: 140.000 € Funding Agency: Ministerio de Sanidad, Política Social e Duration: 2011-2013

4848 Jesús de Pedro Cuesta Group Group Isidre Ferrer Abizanda

Institut de Neuropatologia ·Servei d’Anatomia Patológica

IDIBELL-Hospital Universitari de Bellvitge 1 Carrer Feixa LLarga, s/n 08907 Hospitalet de LLobregat (Spain)

Tel.: +34 932 607 452 / +34 934 035 808 Program Fax: +34 932 045 065 [email protected]

Isidre Ferrer Abizanda Irene López González Principal Investigator PhD student Ester Aso Pérez Izaskun Villar Menéndez Scienific staff PhD student Franc Llorens Paula García-Esparcia Scienific staff PhD student Karina Hernández Ortega Anusha Koneti Scienific staff PhD student Marta Barrachina Castillo Rosa Blanco Soto Scienific staff Technician Marta Blanch Lozano Margarita Carmona Murillo Scienific staff Technician Mayelin Domínguez González Dolores Moreno León Scienific staff Technician Mercedes Armand-Ugon Jesús Moreno Castro Scienific staff Technician Montserrat Olivé Plana Lourdes Castillo Valentín Scienific staff Technician Xavier Altafaj Tadío M. José Zújar Rubio Scienific staff Technician Noemí Vidal Sarró Sara Berjaoui Assistant Researcher PhD student Belén Ansoleaga Ávila Ester Bergés Pujol PhD student Administrative Assistant

49 Summary

Study of molecular alterations at early stages of neurodegenerative diseases with abnormal protein aggregates, particularly Alzheimer’s disease (AD) and Parkinson’s disease (PD), from optimal post-mortem tissue samples obtained at the Institute of Neuropathology Brain Bank a branch of the HUB-ICO-IDIBELL Biobank. The main achievements in the last year can be summarized as follows: 1. Identification of olfactory and taste receptors and downstream functional effectors in the human and murine brain, and their deregulation in the cerebral cortex and substantia nigra in PD, cerebral neocortex and entorhinal cortex in AD, cerebral cortex in APP/PS1 transgenic mouse used as a model of AD, frontal cortex in progressive supranuclear palsy, and cerebral cortex and cerebellum in Creutzfeldt-Jakob’s disease subtypes MM1 and VV2; abnormal epigenetic regulation of selected mRNA expression in the cerebral cortex in AD, and striatum in Huntington’s disease; 2. Identification and description of new entities and mechanisms of disease, including tauopathies and prionopathies, and AD, respectively; 3. Treatments geared to delay or curb memory and learning impairment and molecular alterations in APP/PS1 transgenic mice including triheptanoin, rapamycin, small synthetic molecules, carabamylated erythropoietin, phosphodiestarase 7 inhibitors and cannabinoids; and 4. Identification of alterations in the mitochondrial respiratory chain, oxidative damage, and impaired proteasome ubiquitin system in X-linked adrenoleukodystrophy (X-ALD), and treatments in X-ALD murine models with cyclophilin and pioglitazone, in collaboration with Dr. Aurora Pujol (CIBERER).

The most innovative aspect is the utilisation of different “-omics” and bioinformatic processing of systems biology in the very same tissue samples, in different regions and at different stages of disease progression (mainly early preclinical stages) of neurodegenerative disease with abnormal protein aggregates which can be considered biological processes with long- lasting silent periods that offer suitable “windows” for selected combined treatment aimed at preventing and delaying the neurodegenerative process. This approach has generated a large amount of raw data that must be analysed in detail during the next years. The other important aspect is the close collaboration with other centres and groups worldwide in order to optimize resources in front of the budget shortcuts, and to improve experiences and a multidisciplinary approach to disease pathogenesis. Finally, the identification of abnormal molecular pathways detected in human brain samples have been translated to murine and cellular models to klearn about mechanistic aspects, and to validate putative therapeutic targets. All these data are then used to design possible therapies in animal models, and to prepare acquired knowledge to human pilot studies.

Keywords

Neuropathology, Epigenetics, Transcriptomics, Proteomics, Metabolomics, Lipidomics, Alzheimer’s, Parkinson’s, Creutzfeldt- Jakob disease, Frontotemporal lobar degeneration, Amyotrophic lateral sclerosis, Chromosome X-linked adrenoleukodystrophy, Muscle diseases

Publications

• lopez-Erauskin J, Ferrer I, Galea E, Pujol A. Cyclophilin D as a potential target for antioxidants in neurodegeneration: the X-ALD case. Biological chemistry. 2013 May,394(5):621-9. • Ansoleaga B, Garcia-Esparcia P, Llorens F, Moreno J, Aso E, Ferrer I. Dysregulation of brain olfactory and taste receptors in AD, PSP and CJD, and AD-related model. Neuroscience. 2013 Jun 28;248C:369-82. • Robledo P, Elena MG, Aso E, Maldonado R. Genetically Modified Mice as Tools to Understand the Neurobiological Substrates of Depression. Current pharmaceutical design. 2013 Oct 29. • Aso E, Semakova J, Joda L, Semak V, Halbaut L, Calpena A, et al. Triheptanoin supplementation to ketogenic diet curbs cognitive impairment in APP/PS1 mice used as a model of familial Alzheimer's disease. Current Alzheimer research. 2013 Mar;10(3):290-7. • Rosenkranz SC, Geissen M, Härter K, Szalay B, Ferrer I, Vogel J, et al. Amyloid-precursor-protein-lowering small molecules for disease modifying therapy of Alzheimer's disease. PloS one. 2013 Dec 18;8(12):e82255. • Pinacho R, Villalmanzo N, Roca M, Iniesta R, Monje A, Haro JM, et al. Analysis of Sp transcription factors in the postmortem brain of chronic schizophrenia: a pilot study of relationship to negative symptoms. Journal of psychiatric research. 2013 Jul;47(7):926-34. • Maerkens A, Kley RA, Olive M, Theis V, van der Ven PF, Reimann J, et al. Differential proteomic analysis of abnormal intramyoplasmic aggregates in desminopathy. Journal of proteomics. 2013 Sep 2;90:14-27. • Villar-Menendez I, Blanch M, Tyebji S, Pereira-Veiga T, Albasanz JL, Martin M, et al. Increased 5-methylcytosine and decreased

5050 Isidre Ferrer Abizanda Group Isidre Ferrer Abizanda Group 51

5-hydroxymethylcytosine levels are associated with reduced striatal A2AR levels in Huntington's disease. Neuromolecular medicine. 2013 Jun;15(2):295-309. • Aso E, Juves S, Maldonado R, Ferrer I. CB2 cannabinoid receptor agonist ameliorates Alzheimer-like phenotype in AβPP/PS1 mice. Journal of Alzheimer's disease: JAD. 2013;35(4):847-58. • Garcia-Esparcia P, Schlüter A, Carmona M, Moreno J, Ansoleaga B, Torrejon-Escribano B, et al. Functional genomics reveals dysregulation of cortical olfactory receptors in Parkinson disease: novel putative chemoreceptors in the human brain. Journal of neuropathology and experimental neurology. 2013 Jun;72(6):524-39. • Malfatti E, Olive M, Taratuto AL, Richard P, Brochier G, Bitoun M, et al. Skeletal muscle biopsy analysis in reducing body myopathy and other FHL1-related disorders. Journal of neuropathology and experimental neurology. 2013 Sep;72(9):833-45. • Aso E, Ferrer I. It may be possible to delay the onset of neurodegenerative diseases with an immunosuppressive drug (rapamycin). Expert opinion on biological therapy. 2013 Sep;13(9):1215-9. • Lopez-Gonzalez I, Carmona M, Blanco R, Luna-Munoz J, Martinez-Mandonado A, Mena R, et al. Characterization of thorn- shaped astrocytes in white matter of temporal lobe in Alzheimer's disease brains. Brain pathology (Zurich, Switzerland). 2013 Mar;23(2):144-53. • Pla V, Paco S, Ghezali G, Ciria V, Pozas E, Ferrer I, et al. Secretory sorting receptors carboxypeptidase E and secretogranin III in amyloid β-associated neural degeneration in Alzheimer's disease. Brain pathology (Zurich, Switzerland). 2013 May;23(3):274-84. • Sanmillan JL, Plans G, Vidal N, Acebes JJ. Supratentorial brain schwannomas: An uncommon location for a common tumour. Br J Neurosurg. 2014 Jan;28(1):25-8. • Gelpi E, Soler Insa JM, Parchi P, Saverioni D, Yagüe J, Nos C, et al. Atypical neuropathological sCJD-MM phenotype with abundant white matter Kuru-type plaques sparing the cerebellar cortex. Neuropathology. 2013 Apr;33(2):204-8. • Kwon KH, Kim JY, Kim SY, Min HK, Lee HJ, Ji IJ, et al. Chromosome 11-centric human proteome analysis of human brain hippocampus tissue. Journal of proteome research. 2013 Jan 4;12(1):97-105. • Klementieva O, Aso E, Filippini D, Benseny-Cases N, Carmona M, Juves S, et al. Effect of poly(propylene imine) glycodendrimers on β-amyloid aggregation in vitro and in APP/PS1 transgenic mice, as a model of brain amyloid deposition and Alzheimer's disease. Biomacromolecules. 2013 Oct 14;14(10):3570-80. • Bolos M, Spuch C, Ordonez-Gutierrez L F, Ferrer I, Carro E. Neurogenic effects of β-amyloid in the choroid plexus epithelial cells in Alzheimer's disease. Cellular and molecular life sciences: CMLS. 2013 Aug;70(15):2787-97. • Olive M, Kley RA, Goldfarb LG. Myofibrillar myopathies: new developments. Current opinion in neurology. 2013 Oct;26(5):527-35. • Torres JM, Castilla J, Pintado B, Gutierrez-Adan A, Andreoletti O, Aguilar-Calvo P, et al. Spontaneous generation of infectious prion disease in transgenic mice. Emerging infectious diseases. 2013 Dec;19(12):1938-47. • Antonell A, Llado A, Altirriba J, Botta-Orfila T, Balasa M, Fernandez M, et al. A preliminary study of the whole-genome expression profile of sporadic and monogenic early-onset Alzheimer's disease. Neurobiology of aging. 2013 Jul;34(7):1772-8. • Perez-Gonzalez R, Pascual C, Antequera D, Bolos M, Redondo M, Perez DI, et al. Phosphodiesterase 7 inhibitor reduced cognitive impairment and pathological hallmarks in a mouse model of Alzheimer's disease. Neurobiology of aging. 2013 Sep;34(9):2133-45. • Lopez-Erauskin J, Galino J, Ruiz M, Cuezva JM, Fabregat I, Cacabelos D, et al. Impaired mitochondrial oxidative phosphorylation in the peroxisomal disease X-linked adrenoleukodystrophy. Human molecular genetics. 2013 Aug 15;22(16):3296-305. • Fürst DO, Goldfarb LG, Kley RA, Vorgerd M, Olive M, van der Ven PF. Filamin C-related myopathies: pathology and mechanisms. Acta neuropathologica. 2013 Jan;125(1):33-46. • Ahmed Z, Bigio EH, Budka H, Dickson DW, Ferrer I, Ghetti B, et al. Globular glial tauopathies (GGT): consensus recommendations. Acta neuropathologica. 2013 Oct;126(4):537-44. • Launay N, Ruiz M, Fourcade S, Schlüter A, Guilera C, Ferrer I, et al. Oxidative stress regulates the ubiquitin-proteasome system and immunoproteasome functioning in a mouse model of X-adrenoleukodystrophy. Brain. 2013 Mar;136(Pt 3):891-904. • Morato L, Galino J, Ruiz M, Calingasan NY, Starkov AA, Dumont M, et al. Pioglitazone halts axonal degeneration in a mouse model of X-linked adrenoleukodystrophy. Brain. 2013 Aug;136(Pt 8):2432-43. • Llorens F, Banez-Coronel M, Pantano L, Del Rio JA, Ferrer I, Estivill X, et al. A highly expressed miR-101 isomiR is a functional silencing small RNA. BMC genomics. 2013 Feb 15;14(1):104. • Llorens F, Carulla P, Villa A, Torres JM, Fortes P, Ferrer I, et al. PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells. Journal of neurochemistry. 2013 Oct;127(1):124-38. • Llorens F, Ferrer I, Del Rio JA. Gene Expression Resulting from PrPC Ablation and PrP C Overexpression in Murine and Cellular Models. Molecular neurobiology. 2013 Aug 16;49(1):413-23. • Llorens F, Ansoleaga B, Garcia-Esparcia P, Zafar S, Grau-Rivera O, Lopez-Gonzalez I, et al. PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2. Prion. 2013 Sep 18;7(5). • Sanchez-Mut JV, Aso E, Panayotis N, Lott I, Dierssen M, Rabano A, et al. DNA methylation map of mouse and human brain identifies target genes in Alzheimer's disease. Brain. 2013 Oct;136(Pt 10):3018-27. • Suarez-Calvet M, Dols-Icardo O, Llado A, Sanchez-Valle R, Hernandez I, Amer G, et al. Plasma phosphorylated TDP-43 levels are elevated in patients with frontotemporal dementia carrying a C9orf72 repeat expansion or a GRN mutation. Journal of neurology, neurosurgery, and psychiatry. 2013 Dec 4.

Research Projects

• Code: CD10/00121 • Code: 278486 Title: Ayudas para contratos postdoctorales de perfeccio- Title: DEVELAGE namiento Sara Borrell Principal Investigator: Ferrer I. Medizinische Universität Principal Investigator: Isidre Ferrer Wien CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: International Funding Agency: Instituto Carlos III Funding Agency: Comisión Europea Funding: 36.000 € Funding: 348.852 € Duration: 2011-2014 Duration: 2012-2014

• Code: CAD-BIO-04 • Code: Contracte La Caixa- IDIBELL - ALTHIA Title: Banc Teixits Neurològics de Catalunya Title: Estudi de glioblastomas Principal Investigator: Isidre Ferrer Principal Investigator: Isidre Ferrer CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Privado Type: Privado Funding Agency: Farmaindustria Funding Agency: La Caixa / IDIBELL / ALTHIA Funding: 149.200 € Funding: 105.700 € Duration: 2012-2014 Duration: 2011-2013

• Code: PI2010/09 • Code: PI1100150 Title: BESAD-P: Biomarkers of Early Stages of Alzheimer Title: Estudio clínico, patológico y genético en miopatías Disease-Prevention cardioesqueléticas hereditarias Principal Investigator: Isidre Ferrer Abizanda Principal Investigator: Montserrat Olive Plana CIBERNED’s collaboration: Yes CIBERNED’s collaboration: No CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; CIBERNED’s groups: G 506; G 508; G 412 Other CIBER’s collaboration: No Other CIBER’s collaboration: Yes (CIBERESP) Type: National Type: Intramurales Funding Agency: Instituto de Salud Carlos III Funding Agency: CIBERNED Funding: 100.320 € Funding: 223.500 € Duration: 2012-2014 Duration: 2011-2013 • Code: AP94492011 • Code: CD09/00019 Title: Estudio de las potenciales propiedades terapéuticas Title: Contratos postdoctorales de perfeccionamiento Sara de los fármacos basados en cannabinoides en un modelo Borrell animal de la enfermedad de Alzheimer Principal Investigator: Isidre Ferrer Principal Investigator: Isidre Ferrer CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: National Funding Agency: Instituto de Salud Carlos III Funding Agency: Fundación Investigación Médica de Bell- Funding: 72.000 € vitge Duration: 2010-2013 Funding: 9.523 € Duration: 2011-2013

5252 Isidre Ferrer Abizanda Group Isidre Ferrer Abizanda Group 53

• Code: PI1100968 CIBERNED’s groups: Title: Identificación de un transcriptoma, proteoma y lipi- Other CIBER’s collaboration: No doma de riesgo en los primeros estadios de la enfermedad Type: Privado de Parkinson Funding Agency: Fundació ELA Principal Investigator: Isidre Ferrer Funding: 95.000 € CIBERNED’s collaboration: No Duration: 2010-2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: Proyecto Colaboración GW Pharma Ltd Type: National Title: Study of the molecular mechanisms underlying the Funding Agency: Instituto Carlos III, Fondo de investigación therapeutic effect of Sativex in APP/PS1, an animal model Sanitaria of Alzheimer’s Disease Funding: 141.734 € Principal Investigator: Isidre Ferrer Duration: 2012-2014 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: ESRF-European Synchrotron Radiation, Grenoble Other CIBER’s collaboration: No Title: Multimodal microspectrocopy study of human brain Type: Privado tissue affected by Alzheimer's disease using FTIR(SOLEIL) Funding Agency: Convenio de Colaboración and NEXAFS(BESSY) spectromicroscopy Funding: 0 € Principal Investigator: Isidre Ferrer Duration: 2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI11/03028 Other CIBER’s collaboration: No Title: DEMTEST: Biomarker based diagnosis of rapid pro- Type: International gressive dementias Funding Agency: ESRF-European Synchrotron Radiation, Principal Investigator: Pascual Sánchez Grenoble CIBERNED’s collaboration: Yes Funding: 0 € CIBERNED’s groups: G 114; G 601; G 503; G 609 Duration: 2013 Other CIBER’s collaboration: No Type: International • Code: Fundació ELA Miquel Valls Funding Agency: Instituto de Salud Carlos III- Comunidad Title: Proyecto de investigación sobre RNAs no codificantes Económica Europea (microRNAs) en pacientes con esclerosis lateral amiotrófica Funding: 10.000 € Principal Investigator: Isidre Ferrer Duration: 2012-2014 CIBERNED’s collaboration: No Group Antonia Gutiérrez Pérez

Departamento Biología Celular, Genética y Fisiología (Área de Biología Celular)

1 Facultad de Ciencias, Universidad de Málaga Campus de Teatinos 29071 Málaga (Spain)

Program Tel.: +34 952 133 344 [email protected]

Antonia Gutiérrez Pérez Manuel Francisco López Aranda Principal Investigator, Full Professor Postdoctoral Fellow José Carlos Dávila Cansino Raquel Sánchez Varo Full Professor Postdoctoral Fellow Zafaruddin Khan Elisabeth Sánchez Mejías Lecturer FPU PhD student Manuel Romero Acebal Laura Trujillo Estrada Chief of Neurology Department and Assistant Professor FPU PhD student David Baglietto Vargas Vanessa De Castro Carratalá Postdoctoral Fellow CIBERNED Staff Researcher Inés Moreno González Mercedes Aneiros Ferrer Postdoctoral Fellow CIBERNED Staff Technician Juan Félix López Tellez Researcher

54 Antonia Gutiérrez Pérez Group 55

Summary

In 2013, we have continued to work on the neuropathological characterization of transgenic models of Alzheimer’s disease (AD) and in the validation of the results with these models in postmortem samples from AD patients. In particular, and in collaboration with the group of Dr. J. Vitorica, this year we have focused on the following activities:

1) The study of the neuroinflammatory process in AD samples (Braak II to Braak V-VI), paying special attention to the nature (functional phenotypes) and the causative agent of the microglial activation. This activity is part of the objectives of a collaborative CIBERNED project, which we have been developing during 2010-2013 and that has been coordinated by Dr. Vitorica. In addition, in late 2013 we started a new collaborative project (coordinated by Dr. Torres-Aleman) on the glia- neuron relationship in both experimental models and AD patient samples, in order to determine whether a dysfunction in the glia-neuron homeostasis is the basis of the neurodegenerative process underlying dementia. 2) The study of the neuroprotective capacity of lithium in transgenic models of AD. We have determined that the oral treatment with lithium carbonate (for 6 months and starting before the onset of the amyloid pathology) induced a clear neuroprotective effect and cognitive improvement in our PS1/APP model (Trujillo-Estrada et al., Acta Neuropathol Commun 2013). Lithium produced a qualitative change in the Abeta plaques, so that they were smaller, more compact and less toxic. This reduced toxicity of the plaques resulted in the formation of less dystrophic neurites around them, and in a marked neuroprotection of the most vulnerable neuronal populations (SOM/NPY interneurons). This lithium-mediated neuroprotective action was driven by reactive astrocytes (novel cellular target of lithium) and the production of chaperones. These results show the possibility to modulate in vivo the toxic effect of the plaques during the disease progression. This Abeta plaque-modifying mechanism may represent a promising therapeutic approach to the, so far, continuing negative outcomes of AD clinical trials, aimed to clear Abeta plaques once they have already formed, and to the current inability to prevent plaques from forming in the first place. 3) We have completed two applied research projects of high traslational value. The first one, with the pharmaceutical company Sanofi (France), led to the identification of functional synaptic potential biomarkers in various in vivo models of AD and aging. The second project, with the company Neuron Biopharma, consisted of a preclinical study to evaluate the efficacy of a new neuroprotective therapeutic strategy in transgenic mouse models of AD.

Finally, we have collaborated with the group of Dr. Soto at the Health Science Center (University of Texas, USA) in a study, published in Nature Communications, on the effects of tobacco smoke in transgenic models of AD. The results of this study suggest that smoking exacerbates the pathological lesions (plaque burden, neuroinflammation and tau phosphorylation) in these models, and therefore constitutes an important environmental risk factor for this disease.

Keywords

Alzheimer, Neurodegeneration, Neuroprotection, Inflammation, Abeta, Oligomers, Transgenic models, Neuropathology

Publications

• Khan ZU, Martin-Montanez E, Muly EC. Schizophrenia: causes and treatments. Current pharmaceutical design. 2013;19(36):6451-61. • Trujillo-Estrada L, Jimenez S, De Castro V, Torres M, Baglietto-Vargas D, Moreno-Gonzalez I, et al. In vivo modification of Abeta plaque toxicity as a novel neuroprotective lithium-mediated therapy for Alzheimer's disease pathology. Acta neuropathologica communications. 2013 Nov 12;1(1):73. • Moreno-Gonzalez I, Estrada LD, Sanchez-Mejias E, Soto C. Smoking exacerbates amyloid pathology in a mouse model of Alzheimer's disease. Nature communications. 2013;4:1495.

Research Projects • Code: 8.06/5.02.3621 CDTI Principal Investigator: Antonia Gutiérrez Pérez Title: Evaluación in vivo de compuesto de interés (Desarro- CIBERNED’s collaboration: Yes llo preclínico y estudio previo de entrada a fases clínicas de CIBERNED’s groups: G 415; G 411 un nuevo fármaco) Other CIBER’s collaboration: No Type: Privado Funding Agency: Instituto de Salud Carlos III Funding Agency: Industria Neuron Biopharma SA Funding: 224.999 € Funding: 39.468 € Duration: 2013-2015 Duration: 2010-2013 • Code: PI2010/08 • Code: 8.06/5.02.3771 Title: Activación glial en el proceso neuroinflamatorio: Title: Identification, setting-up and validation, using histo- una potencial diana terapéutica para la enfermedad de logical and/or biochemical techniques, of functional syn- Alzheimer aptic/neuronal markers in several in vivo models of Alzhei- Principal Investigator: Francisco Javier Vitorica Ferrández mer’s disease, neurodegeneration and aging CIBERNED’s collaboration: Yes Principal Investigator: Antonia Gutiérrez Pérez CIBERNED’s groups: G 411; G 415; G 406; G 409 CIBERNED’s collaboration: Yes Other CIBER’s collaboration: No CIBERNED’s groups: G 415; G 411 Type: Intramurales Other CIBER’s collaboration: No Funding Agency: CIBERNED Type: Privado Funding: 375.000 € Funding Agency: Compañía Farmacéutica Sanofi Aventis Duration: 2010-2013 (Francia) Funding: 147.000 € • Code: AC652 Duration: 2011-2013 Title: Evaluación del efecto neuroprotector de un compues- to • Code: BFU2010-16500 Principal Investigator: Javier Vitorica Title: Mecanismos biológicos de la memoria visual: estudio CIBERNED’s collaboration: Yes de los reguladores de la señalización de proteínas G CIBERNED’s groups: G 411; G 415 Principal Investigator: Zafaruddin Khan Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: Privado CIBERNED’s groups: Funding Agency: Empresa Neuron Biopharm Other CIBER’s collaboration: No Funding: 57.060 € Type: National Duration: 2010-2013 Funding Agency: Ministerio de Ciencia e Innovación Funding: 356.950 € • Code: PI12/01439 Duration: 2011-2013 Title: Oligómeros tóxicos del Abeta como agentes causan- tes de la disfunción del citoesqueleto y los procesos pro- • Code: PI12/01431 teolíticos en la Title: Oligómeros tóxicos del Abeta como agentes causan- Principal Investigator: Javier Vitorica tes de la disfunción del citoesqueleto y los procesos pro- CIBERNED’s collaboration: Yes teolíticos en la CIBERNED’s groups: G 415; 411 Principal Investigator: Antonia Gutiérrez Pérez Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: National CIBERNED’s groups: G 411; 415 Funding Agency: Instituto de Salud Carlos III Other CIBER’s collaboration: No Funding: 228.629 € Type: National Duration: 2013-2015

5656 Antonia Gutiérrez Pérez Group Group María Trinidad Herrero Ezquerro

Universidad de Murcia

Neurociencia Clínica y Experimental (NiCE-CIBERNED) 1 2ª planta Facultad de Medicina Campus de Espinardo 30100 Murcia (Spain) Program Tel.: +34 868 884 683 / 8484 / 8468

María Trinidad Herrero Ezquerro Virginia Izura Azanza Principal Investigator Neurophysiologist, PhD Carmen Antúnez Almagro Dolores López López Neurologist, Dementia Unit Director Lab Technician Berta Claramonte Clausell Beatriz Maestre Sánchez Neurologist, PhD student Nurse, PhD student Teresa Cristina Costa José Javier Martín Fernández Biologist, PhD student (PhD Sandwich, Universidade Neurologist, PhD, Head of Deparment de Salvador de Bahia, Brazil) María Dolores Martínez Lozano Cristina Estrada Esteban Neurologist, PhD Veterinary, PhD student Víctor Ortiz Cullera Emiliano Fernández Villalba Biologist, PhD student Neurologist, Phd, Assistant Professor Carmen Mª Ros Gómez Aurora Gómez López Lab Technician Pathology Technician Ana González Cuello Assistant Professor, Pharmacist PhD

57 Summary

Neuroinflammation and parkinsonism:

i) We have finished the first part of the study on the role of the methalloproteínase-9 (MMP-9) in glial activation and neuronal death (Annese et al., 2014) as well as the role of oligondendrocytes in a context of experimental Parkinsonism (Annese et al., 2012; De Stefano and Herrero, 2014, in press); ii) It has been analyzed the positive effect of methalloproteínase’s modulation in the prevention of dopaminergic cell death; iii) We have demonstrated the fagocytic role of the microglia and its mobility (Barcia et al., 2013); as well as the role of astrocytes (Carrillo-de Sauvage et al., 2012) in a progressive and chronic model in MPTP-monkeys; iv) We have determined the specific role of the glucocorticoid receptor (GR) in microglia as balance and control of the inflammatory response in the central nervous system (Carrillo-de Sauvage et al, 2013); v) We have studied the expression of HO-1 and NRF-2 in the Parkinsonian and dyskinetic monkey brain as part of the collaborative project (Dr. Antonio Cuadrado, 2012-2013); vi) We have described the molecular and proteomic changes implicated in the energetic metabolism and in the signal transduction in the MPTP-treated monkeys retina (Campello et al., 2013); vii) We have studied different molecular changes in the heart of chronic MPTP-treated monkeys as part of the non-motor symptoms (this study is still in development); viii) We have analyzed the summatory effect of neurodegeneration, neuroinflammation and cerebral aging in different rodent’s models; ix) We have studied the location and role of the Barrington nucleus and its relevance in the deep brain stimulation in Parkinson’s disease (Blanco et al., 2013; 2014); x) We are analyzing the changes in the olfactory human mucosa with age and with Parkinson’s disease’ progression (in collaboration with Dr. Francisco Grandas, Hospital Gregorio Marañón, Proyecto FIS PI12/02970); xi) We are developing a comprehensive study of the characteristics and potential applicability of nanovectors obtained from Silkworm´s (Bombyx mori) cocoon, from fabrication and characterization to the ability to use them as nanocarries for suitable drugs useful in treating diseases that concur with an important neuroinflamatory component; and xii) We are collaborating with the study “Prionic propagation of synuclein pathology: study in the non-human primate (Papio papio)” (Dr. Bezard y Prof. Obeso) at the Universidad of Murcia.

Cerebral aging and cognitive impairment:

We have implemented an animal model for cerebral aging since 2009, the Octodon degus. Different behavioral paradigms for the assessment of memory and cognitive impairment have been validated under sleep deprivation procedures (Tarragon et al., 2013; 2014, in press). i) In order to evaluate the effect of different therapeutic drugs on brain activity, several experiments are being currently performed with an electroencephalography telemetry system; ii) We have evaluated the positive effect of TMS (Transcranial Magnetic Stimulation) and physical exercise on cognitive enhancement after sleep deprivation, as a putative non-invasive therapeutic alternative; iii) Collaboration with European partners within the PharmaCog project (IMI-under Grant Agreement n°115009) has contributed to the analysis of pathological markers in dementia (Babiloni et al., 2012; Deguil et al., 2013); iv) The pharmacological profile of different agents is now being analyzed in order to maximize the potential of such pharmacological trials; v) We have obtained and started a bilateral DAAD project with the Republic of Germany (Prof. Schwarting) in order to analyze the cognitive processes in experimental Parkinsonism associated with cerebral aging; vi) We have studied the aging effect in Stroop test combined with quantitative EEG analysis of cerebral Alpha-Theta waves (Nombela et al., in press); vii) We have participated in the clinical collaborative project (Signal study, Dr. Lleó) on CSF biomarkers and neuroimaging in alzheimer’s disease (2012-2013).

All these results generated scientific communications in neuroscience congresses (both national and international), and original research and review papers in peer-reviewed journals.

Keywords

Neurodegeneration, Dopamine, Neuroinflammation, Cytokines, Glucocorticoids, Brain aging, Memory, Sleep deprivation, Cog- nitive tests

Publications

• Barcia C, Ros CM, Ros-Bernal F, Gomez A, Annese V, Carrillo-de Sauvage MA, et al. Persistent phagocytic characteristics of microglia in the substantia nigra of long-term Parkinsonian macaques. Journal of neuroimmunology. 2013 Jun 5;261(1-2):60-6. • Blanco L, Yuste JE, Carrillo-de Sauvage MA, Gomez A, Fernandez-Villalba E, Aviles-Olmos I, et al. Critical evaluation of the anatomical location of the Barrington nucleus: Relevance for deep brain stimulation surgery of pedunculopontine tegmental

5858 María Trinidad Herrero Ezquerro Group María Trinidad Herrero Ezquerro Group 59

nucleus. Neuroscience. 2013 Jun 1;247:351-63. • Babiloni C, Infarinato F, Aujard F, Bastlund JF, Bentivoglio M, Bertini G, et al. Effects of pharmacological agents, sleep deprivation, hypoxia and transcranial magnetic stimulation on electroencephalographic rhythms in rodents: towards translational challenge models for drug discovery in Alzheimer's disease. Clinical neurophysiology. 2013 Mar;124(3):437-51. • Campello L, Esteve-Rudd J, Bru-Martinez R, Herrero MT, Fernandez-Villalba E, Cuenca N, et al. Alterations in energy metabolism, neuroprotection and visual signal transduction in the retina of Parkinsonian, MPTP-treated monkeys. PloS one. 2013 Sep 5;8(9):e74439. • Tarragon E, Lopez D, Estrada C, Ana GC, Schenker E, Pifferi F, et al. Octodon degus: A Model for the Cognitive Impairment Associated with Alzheimer's Disease. CNS neuroscience & therapeutics. 2013 May 27;19(9):643-8. • Carta A, Barcia C, Herrero MT. Experimental models of Parkinson's disease. Handbook of Neurotoxicity. 2013. • Sampedro A, Tarragon E, Yuste JE, Ros-Bernal F, Ortiz V, Campuzano CM, Gomez A, Ros C, Herrero MT. Glutamatergic receptors in Parkinson`s Disease. Handbook of Neurotoxicity. 2013. • Ros-Bernal F, Yuste JE, Tarragon E, Ortiz V, Gomez A, Ros C, Herrero MT. Neuroinflammation and Parkinson´s Disease. Handbook of Neurotoxicity. 2013. • Deguil J, Ravasi L, Auffret A, Babiloni C, Bartres Faz D, Bragulat V, et al. Evaluation of symptomatic drug effects in Alzheimer's disease: strategies for prediction of efficacy in humans. Drug discovery today. Technologies. 2013 Sep;10(3):e329-42. • Carrillo-de Sauvage MA, Maatouk L, Arnoux I, Pasco M, Sanz Diez A, Delahaye M, et al. Potent and multiple regulatory actions of microglial glucocorticoid receptors during CNS inflammation. Cell death and differentiation. 2013 Nov;20(11):1546-57.

Research Projects • Code: PI10/02827 Funding Agency: Fundación Séneca Title: Acción de las citoquinas pro-inflamatorias y linfocitos Funding: 62.500 € en el mantenimiento de la activación glial en el parkinso- Duration: 2011-2013 nismo Principal Investigator: Mª Trinidad Herrero Ezquerro • Code: IMI-115009 CIBERNED’s collaboration: No Title: Prediction of cognitive properties of new drug candi- CIBERNED’s groups: dates for neurodegenerative diseases Other CIBER’s collaboration: No Principal Investigator: Mª Trinidad Herrero Ezquerro Type: National CIBERNED’s collaboration: No Funding Agency: Fondo de Investigación Sanitaria (FIS) CIBERNED’s groups: Funding: 82.800 € Other CIBER’s collaboration: No Duration: 2011-2013 Type: International Funding Agency: Unión Europea – 7 Program Marco - IMI • Code: SAF2010-21274 Funding: 360.000 € Title: Evaluación de los mecanismos y efecto neuro-protec- Duration: 2010-2014 tor del inhibidor de la rho kinasa ha1077 en modelos expe- rimentales de enfermedad de parkinson • Code: PI2011/01 Principal Investigator: Mª Trinidad Herrero Ezquerro Title: El factor de transcripción Nrf2 como nueva diana te- CIBERNED’s collaboration: No rapéutica para la enfermedad de Parkinson CIBERNED’s groups: Principal Investigator: Antonio Cuadrado Pastor Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: National CIBERNED’s groups: G 101; G102; G103; G 505; G 209 Funding Agency: Ministerio de Ciencia e Innovación Other CIBER’s collaboration: No Funding: 96.800 € Type: Intramurales Duration: 2011-2013 Funding Agency: CIBERNED Funding: 390.000 € • Code: 15329/PI/10 Duration: 2011-2013 Title: Modulación farmacológica de la activación glial en el parkinsonismo. Estudio de citocinas y enzimas inflamato- • Code: MJFF Staff Initiated 2013 rias en el mono y en el ratón Title: Prion-like dissemination of synuclein pathology: a Principal Investigator: Mª Trinidad Herrero Ezquerro non-human primate study CIBERNED’s collaboration: No Principal Investigator: Erwan Bezard (Universite de Bor- CIBERNED’s groups: deaux, France) Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: CCAA CIBERNED’s groups: G 505; G 205; G 109 Other CIBER’s collaboration: No Funding: 33.000 € Type: International Duration: 2013-2016 Funding Agency: Michael J. Fox Foundation

6060 María Trinidad Herrero Ezquerro Group Group Alberto Lleó Bisa

Unidad de Memoria

Servicio de Neurología 1 Hospital de la Santa Creu i Sant Pau Sant Antoni Maria Claret, 167 08025 Barcelona (Spain) Program Tel.: +34 935 565 986 Fax: +34 935 565 602 [email protected]

Alberto Lleó Bisa Daniel Alcolea Rodríguez Principal Investigator, Clinical Head, Neurology PhD student Rafael Blesa González Isabel Sala Matavera Head of Department Postdoctoral Fellow Jordi Clarimón Echavarria Mª Belén Sánchez Saudinós “Miguel Servet” Postdoctoral Fellow PhD student Juan Fortea Ormaechea María Carmona Iragui Scienific staff PhD student Olivia Belvin Sofía Antón Aguirre “Juan de la Cierva” Postdoctoral Fellow PhD student Oriol Dols Icardo Laia Muñoz Llahuma PhD student Lab Technician Martí Colom Cadena Eduard Vilaplana Martínez PhD student PhD student Marc Suárez Calvet Inés Martín Matas PhD student Lab Technician

61 Summary

During 2013 our group has continued to work on different clinical and translational research projects in the field of neurodegenerative dementias. In particular, we have conducted different studies on the genetics and molecular basis of neurodegenerative dementias. We have investigated the frequency and role of C9ORF72 expansion in patients with frontotemporal dementia and amyotrophic lateral sclerosis, as well as novel genetic variants implicated in Alzheimer’s disease (AD). We have continued our work on familial AD and dementia with Lewy bodies and have investigated the interaction of amyloid, tau and alpha-synuclein in the brain. We have made progress in our CSF and plasma biomarker program along the AD continuum and frontotemporal dementia. Some of this work was performed as part of a European Project under the Joint Programme for neurodegenerative diseases. We also completed the CIBERNED-funded multicentre cooperative study on novel CSF biomarkers in AD (SIGNAL) and generated the first data. Finally, we have continued our work on magnetic resonance imaging (MRI) and amyloid positron-emission tomography (PET) imaging along the AD continuum. These projects are all translational in nature and have yielded several publications in biomedical journals. Furthermore, our translational profile allows a close interaction between neurologists, neuropsychologists, biologists and engineers, which provides a unique environment to investigate neurodegenerative diseases.

Keywords

Alzheimer, Dementias, Imaging, Biomarkers, Cerebrospinal fluid, Disease-modifying therapies

Publications

• Bufill E, Blesa R, Augusti J. Alzheimer's disease: an evolutionary approach. Journal of anthropological sciences: JASs. 2013 Dec 1;91:135-57. • Bufill E, Roura-Poch P, Sala-Matavera I, Anton S, Lleo A, Sanchez-Saudinos B, et al. Reelin Signaling Pathway Genotypes and Alzheimer Disease in a Spanish Population. Alzheimer disease and associated disorders. 2013 Dec 31. • Badiola N, Alcalde V, Pujol A, Münter LM, Multhaup G, Lleo A, et al. The proton-pump inhibitor lansoprazole enhances amyloid beta production. PloS one. 2013;8(3):e58837. • Suarez-Calvet M, Belbin O, Pera M, Badiola N, Magrane J, Guardia-Laguarta C, et al. Autosomal-dominant Alzheimer's disease mutations at the same codon of amyloid precursor protein differentially alter Aβ production. J Neurochem. 2014 Jan;128(2):330-9. • Schapira AH, Stocchi F, Borgohain R, Onofrj M, Bhatt M, Lorenzana P, et al. Long-term efficacy and safety of safinamide as add-on therapy in early Parkinson's disease. European journal of neurology. 2013 Feb;20(2):271-80. • Alcolea D, Martinez-Lage P, Izagirre A, Clerigue M, Carmona-Iragui M, Alvarez RM, et al. Feasibility of Lumbar Puncture in the Study of Cerebrospinal Fluid Biomarkers for Alzheimer's Disease: A Multicenter Study in Spain. Journal of Alzheimer's disease: JAD. 2013 Nov 19. • Molinuevo JL, Gispert JD, Dubois B, Heneka MT, Lleo A, Engelborghs S, et al. The AD-CSF-index discriminates Alzheimer's disease patients from healthy controls: a validation study. Journal of Alzheimer's disease: JAD. 2013 Jan 1;36(1):67-77. • Podlesniy P, Figueiro-Silva J, Llado A, Antonell A, Sanchez-Valle R, Alcolea D, et al. Low cerebrospinal fluid concentration of mitochondrial DNA in preclinical Alzheimer disease. Annals of neurology. 2013 Nov;74(5):655-68. • Costa S, Suarez-Calvet M, Anton S, Dols-Icardo O, Clarimon J, Alcolea D, et al. Comparison of 2 diagnostic criteria for the behavioral variant of frontotemporal dementia. American journal of Alzheimer's disease and other dementias. 2013 Aug;28 (5):469-76. • Inzitari M, Gine-Garriga M, Martinez B, Perez-Fernandez M, Barranco-Rubia E, Lleo A, et al. Cerebrovascular disease and gait and balance impairment in mild to moderate Alzheimer's disease. The journal of nutrition, health & aging. 2013;17(1):45-8. • Suarez-Calvet M, Dols-Icardo O, Llado A, Sanchez-Valle R, Hernandez I, Amer G, et al. Plasma phosphorylated TDP-43 levels are elevated in patients with frontotemporal dementia carrying a C9orf72 repeat expansion or a GRN mutation. Journal of neurology, neurosurgery, and psychiatry. 2013 Dec 4. • Colom-Cadena M, Gelpi E, Charif S, Belbin O, Blesa R, Marti MJ, et al. Confluence of α-synuclein, tau, and β-amyloid pathologies in dementia with Lewy bodies. Journal of neuropathology and experimental neurology. 2013 Dec;72(12):1203-12. • Colom-Cadena M, Gelpi E, Marti MJ, Charif S, Dols-Icardo O, Blesa R, et al. MAPT H1 haplotype is associated with enhanced α-synuclein deposition in dementia with Lewy bodies. Neurobiology of aging. 2013 Mar;34(3):936-42.

6262 Alberto Lleó Bisa Group Alberto Lleó Bisa Group 63

• Ortega-Cubero S, Lorenzo-Betancor O, Lorenzo E, Alonso E, Coria F, Pastor MA, et al. Fused in Sarcoma (FUS) gene mutations are not a frequent cause of essential tremor in Europeans. Neurobiology of aging. 2013 Oct;34(10):2441.e9-2441.e11. • Casals-Coll M, Sanchez-Benavides G, Meza-Cavazos S, Manero RM, Aguilar M, Badenes D, et al. Spanish Multicenter Normative Studies (NEURONORMA Project): Normative Data and Equivalence of Four BNT Short-Form Versions. Archives of clinical neuropsychology. 2013 Nov 11. • Ruiz A, Dols-Icardo O, Bullido MJ, Pastor P, Rodriguez-Rodriguez E, Lopez de Munain A, et al. Assessing the role of the TREM2 p.R47H variant as a risk factor for Alzheimer's disease and frontotemporal dementia. Neurobiol Aging. 2014 Feb;35(2):444. e1-4. • Rubio-Moscardo F, Seto-Salvia N, Pera M, Bosch-Morato M, Plata C, Belbin O, et al. Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset Alzheimer's disease patients alter calcium homeostasis. PloS one. 2013;8(9):e74203. • Katsouri L, Vizcaychipi MP, McArthur S, Harrison I, Suarez-Calvet M, Lleo A, et al. Prazosin, an α(1)-adrenoceptor antagonist, prevents memory deterioration in the APP23 transgenic mouse model of Alzheimer's disease. Neurobiology of aging. 2013 Apr;34(4):1105-15. • Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Bellenguez C, et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease. Nature genetics. 2013 Dec;45(12):1452-8. • Trueba-Saiz A, Cavada C, Fernandez AM, Leon T, Gonzalez DA, Fortea Ormaechea J, et al. Loss of serum IGF-I input to the brain as an early biomarker of disease onset in Alzheimer mice. Translational psychiatry. 2013 Dec 3;3:e330. • Dols-Icardo O, Garcia-Redondo A, Rojas-Garcia R, Sanchez-Valle R, Noguera A, Gomez-Tortosa E, et al. Characterization of the repeat expansion size in C9orf72 in amyotrophic lateral sclerosis and frontotemporal dementia. Human molecular genetics. 2013 Oct 8.

Research Projects

• Code: PI2011/04 Type: Intramurales Title: Estudio multicéntrico de biomarcadores en LCR y de Funding Agency: CIBERNED neuroimagen en el continuum Enfermedad de Alzheimer Funding: 158.000 € preclínica - prodrómica (Estudio SIGNAL) Duration: 2013-2015 Principal Investigator: Alberto Lleó CIBERNED’s collaboration: Yes • Code: PI12/01311 CIBERNED’s groups: G 504; G 502; G 511; G 507; G 509 Title: Variantes genéticas raras y su implicación en la en- Other CIBER’s collaboration: No fermedad de Alzheimer: uso de nuevas tecnologías de ul- Type: Intramurales trasecuenciación para el estudio de genes implicados en la Funding Agency: CIBERNED enfermedad Funding: 140.000 € Principal Investigator: Jordi Clarimon Duration: 2011-2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI11/02425 Other CIBER’s collaboration: No Title: Estudios multimodales de líquido cefalorraquídeo y Type: National resonancia magnética en la enfermedad de Alzheimer pre- Funding Agency: ISCIII clínica Funding: 110.110 € Principal Investigator: Juan Fortea Duration: 2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI11/030206 Other CIBER’s collaboration: No Title: Biomarkers for Alzheimer’s disease and Parkinson’s Type: National disease (BIOMARKAPD) Funding Agency: ISCIII Principal Investigator: Javier Saez Valero Funding: 112.540 € CIBERNED’s collaboration: Yes Duration: 2012-2014 CIBERNED’s groups: G 407; G 504 Other CIBER’s collaboration: No • Code: 2013/07A Type: International Title: Papel de GSK-3 β en las alteraciones de los circuitos Funding Agency: ISC-III corticales que ocurren en la enfermedad de Alzheimer Funding: 117.370 € Principal Investigator: Teresa Iglesias Vacas Duration: 2012-2014 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 401; G 403; G 111; G 504; G 307 • Code: 10/02410 Other CIBER’s collaboration: No Title: Descripción de un nuevo fenotipo de distrofia mus- cular de cinturas autosómica recesiva e identificación del Other CIBER’s collaboration: No gen responsable mediante análisis genómico de ultima ge- Type: National neración Funding Agency: Fondo de Investigación Sanitaria (FIS) Principal Investigator: Rafael Fernández Chacón Funding: 106.480 € CIBERNED’s collaboration: Yes Duration: 2011-2013 CIBERNED’s groups: G 606; G 603; G 504

6464 Alberto Lleó Bisa Group Group María López de Ceballos Lafarga

Instituto Cajal, CSIC

Avenida Doctor Arce, 37 1 28002 Madrid (Spain) Tel.: +34 915 854 716

Fax: +34 915 854 754 Program [email protected]

María López de Ceballos Lafarga Zayra Elisa Hernández Núñez Principal Investigator, Tenure Scientist, CSIC Master’s student, Escuela Superior de Medicina del Instituto Silvia de Vidania Ballesteros PoliTechnician Nacional, México (from October 2013) Researcher CM P2010/BMD-2349 Nathaniel Wilson Paloma Pérez-Domper Campos International student, Gitai Lab in Princeton Department of Research CIBERNED staff (until October 2013) Molecular Biology. Princeton University (from October 2013)

65 Summary

Neuroinflammation is an invariant and early event in Alzheimer’s disease (AD), but its possible relationship with the pathology or its contribution to patient symptoms is far from clear. Our working hypothesis is that there may exist an unbalance between inflammatory parameters, that would be increased, and anti-inflammatory parameters, that would be decreased in AD. We have studied different parameters related to glial activation, and to inflammatory or anti-inflammatory parameters in orbital cortex of patients at different Braak stages (AD I-IV). The protein expression, as measured by Western blotting, of GFAP and Iba- 1, characteristic of astroyctes and microglial cells respectively, were significantly increased in all groups in comparison with the AD I group, COX-2 was similar in all groups, in contrast, the anti-inflammatory cytokine IL-4 was diminished in group AD III. These changes will be correlated with the presence of amyloid-β peptide (Aβ), apoE genotype and the presence of herpes simplex virus type -1. The activity of Arginase-1 (Arg-1), an enzyme characteristic of M2 microglial phenotype and involved in tissue repair, was studied in temporal cortex samples from another series of patients showing dementia and pathologically defined as AD patients, and their corresponding controls matched for sex, age and post-mortem delay. Arg-1 activity did not change in AD or female Tg APP mice (12 months old) in comparison with their respective controls. In another work we have investigated the effect of estrogenic compounds, that have neuroprotective and anti-inflammatory properties, and is a requisite to phagocytise Aβ. Moreover we have studied a possible sexual dimorphism in the migratory response. Migration of cultured astrocytes was assessed following a scratch wound of the confluent monolayer by live cell-imaging (Incucyte). We have observed that female derived astrocytes showed increased migration compared to male cultures. Both estradiol (2h post-injury) and an aromatase inhibitor (2 h before injury) increased male astrocyte migration, while being ineffective in female astrocyte wounded cultures. The results suggest that astrocyte migration and the effect of estrogenic compounds are sexually dimorphic.

Keywords

Alzheimer’s disease, β-amyloid, Glia, Estrogenic compounds, Migration, M2 phenotype, Neuroinflammation

Publications

• Perez-Gonzalez R, Alvira-Botero MX, Robayo O, Antequera D, Garzon M, Martin Moreno AM, et al. Leptin gene therapy attenuates neuronal damages evoked by amyloid-β and rescues memory deficits in APP/PS1 mice. Gene therapy. 2014;21(3):298-308.

Research Projects • Code: S2010/BMD-2349 Type: National Title: Imagen Molecular Multimodal de la Inflamación Funding Agency: MCINN Principal Investigator: S Cerdan Funding: 75.000 € CIBERNED’s collaboration: No Duration: 2012-2014 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: PI2010/09 Type: CCAA Title: BESAD-P: Biomarkers of Early Stages of Alzheimer Funding Agency: CAM Disease-Prevention Funding: 85.000 € Principal Investigator: Isidre Ferrer Abizanda Duration: 2012-2015 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; • Code: BFU2011-30217-C03-01 G 506; G 508; G 412 Title: Señalización de notch/ngn3 en las acciones neuro- Other CIBER’s collaboration: Yes (CIBERESP) protectoras de compuestos Type: Intramurales Principal Investigator: Luis Miguel García Segura Funding Agency: CIBERNED CIBERNED’s collaboration: No Funding: 223.500 € CIBERNED’s groups: Duration: 2011-2013 Other CIBER’s collaboration: No

6666 María López de Ceballos Lafarga Group Group Carlos Matute Almau

Departamento de Neurociencias

Facultad de Medicina y Odontología 1 Universidad del País Vasco 48940 Leioa (Spain)

Tel.: +34 946 013 244 Program Fax: +34 946 015 055 [email protected]

Carlos Matute Almau Fabio Cavaliere Principal Investigator, Full Professor Postdoctoral Fellow Alberto Pérez Samartín Asier Ruiz Núñez Associate Professor Postdoctoral Fellow Fernando Pérez Cerdá Ane Wissenbach Associate Professor PhD student Elena Alberdi Alfonso Tania Quintela Assistant Professor PhD student María Domercq García Hazel Gómez Manrique Assistant Professor Technician María Victoria Sánchez Gómez Saioa Marcos Ezquerro Assistant Professor Technician Susana Mato Santos Ramón y Cajal Researcher

67 Summary

Our research is focused on the study of the signalling mechanisms and damage caused by β-amyloid 1-42 oligomers (Aβ1-42) in neurons, astrocytes, and oligodendrocytes. In this year we have observed that in astrocytes Aβ1-42 produces ROS and astrogliosis in vitro and in vivo. ROS production is prevented by inhibitors of NAPDH oxidase (NOX) whose expression in these cells is increased upon incubation with Aβ1- 42. The signalling cascade initiated by Aβ1-42 also includes activation of β1 integrin, PIK3 and PKC kinases, as well as Rac. These findings were reproduced in vivo in mice injected with Aβ1-42 into the hippocampus, and in triple transgenic mice that model Alzheimer´s disease. Moreover, the increase in GFAP, NOX2 and β1 integrin in these mice correlates with the amyloid load. Together, these results indicate that β1 integrin mediates Aβ1-42-mediated astrogliosis via NOX activation, and suggest that astrocytes may contribute directly to the pathophysiology of Alzheimer´s disease. On the other hand, we have observed that Aβ1-42 has a dual efect of the oligodendroglial lineage: i) it increases the levels of myelin proteins (MBP, CNPase and PLP); and ii) it elevates PDGF receptor expression in progenitors. The effects on the production of myelin are mediated by MAPK/ERK and PI3K/AKT signalling pathways. Finally, in the intramural project PI2011/02 we have investigated the incorporation of α-synuclein in primary cultures of neurons and glia, as well as in an organotypic culture that preserves the nigro-striatal pathway. We have found that human α-synuclein enters all cell types in primary cultures, and that it only accumulates into astrocytes and neurons in the organotypic culture after injection into the substancia nigra and striatum. We did not observed anterograde axonal transport of α-synucleinin this latter preparation nor in primary neurons cultured in microfluidic plates.

Keywords

Beta-amyloid toxicity, Neurons, Astrocytes, Oligodendrocytes, Calcium homeostasis, Neuroinflammation

Publications

• Palomino A, Gonzalez-Pinto A, Martinez-Cengotitabengoa M, Ruiz de Azua S, Alberich S, Mosquera F, et al. Relationship between negative symptoms and plasma levels of insulin-like growth factor 1 in first-episode schizophrenia and bipolar disorder patients. Progress in neuro-psychopharmacology & biological psychiatry. 2013 Jan 18;44C:29-33. • Lutz SE, Gonzalez-Fernandez E, Ventura JC, Perez-Samartin A, Tarassishin L, Negoro H, et al. Contribution of pannexin1 to experimental autoimmune encephalomyelitis. PloS one. 2013;8(6): e66657. • Sanchez-Gomez MV, Gonzalez-Fernandez E, Arellano RO, Matute C. Adenosine and Multiple Sclerosis. In Masino S and Boison D (Eds). Adenosine: A key link between metabolism and brain activity. Springer, New York. 2013; Chapter 21, pp. 435-58. • de Azua SR, Matute C, Stertz L, Mosquera F, Palomino A, de la Rosa I, et al. Plasma brain-derived neurotrophic factor levels, learning capacity and cognition in patients with first episode psychosis. BMC psychiatry. 2013 Jan 15;13:27. • Manterola L, Hernando-Rodriguez M, Ruiz A, Apraiz A, Arrizabalaga O, Vellon L, et al. 1-42 β-Amyloid peptide requires PDK1/ nPKC/Rac 1 pathway to induce neuronal death. Translational psychiatry. 2013 Jan 22;3:e219. • Domercq M, Vazquez-Villoldo N, Matute C. Neurotransmitter signaling in the pathophysiology of microglia. Frontiers in cellular neuroscience. 2013;7:49. • Cavaliere F, Benito-Munoz M, Panicker M, Matute C. NMDA modulates oligodendrocyte differentiation of subventricular zone cells through PKC activation. Frontiers in cellular neuroscience. 2013;7:261. • Gonzalez-Fernandez E, Sanchez-Gomez MV, Perez-Samartin A, Arellano RO, Matute C. A3 Adenosine receptors mediate oligodendrocyte death and ischemic damage to optic nerve. Glia. 2014 Feb;62(2):199-216. • Mato S, Sanchez-Gomez MV, Bernal-Chico A, Matute C. Cytosolic zinc accumulation contributes to excitotoxic oligodendroglial death. Glia. 2013 May;61(5):750-64. • Vazquez-Villoldo N, Domercq M, Martin A, Llop J, Gomez-Vallejo V, Matute C. P2X4 receptors control the fate and survival of activated microglia. Glia. 2013 Nov 19. • Domercq M, Mato S, Soria FN, Sanchez-Gomez MV, Alberdi E, Matute C. Zn(2+) -induced ERK activation mediates PARP-1- dependent ischemic-reoxygenation damage to oligodendrocytes. Glia. 2013 Mar;61(3):383-93. • Alberdi E, Wyssenbach A, Alberdi M, Sanchez-Gomez MV, Cavaliere F, Rodriguez JJ, et al. Ca(2+) -dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid β-treated astrocytes and in a model of Alzheimer's disease. Aging cell. 2013 Feb 15;12(2):292-302. • Busquets-Garcia A, Gomis-Gonzalez M, Guegan T, Agustin-Pavon C, Pastor A, Mato S, et al. Targeting the endocannabinoid system in the treatment of fragile X syndrome. Nature medicine. 2013 May;19(5):603-7.

6868 Carlos Matute Almau Group Carlos Matute Almau Group 69

Research Projects

• Code: 2012.0177 • Code: PRI-PIMNEU-2011-1378 Title: Assessment of the neuroprotective activity of FTY720 Title: Nanostroke (fingolimod) in experimental models of brain damage Principal Investigator: Carlos Matute Principal Investigator: Carlos Matute Almau CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: International Type: Privado Funding Agency: ERANET Neuron Funding Agency: Novartis Funding: 149.000 € Funding: 75.000 € Duration: 2011-2014 Duration: 2012-2014 • Code: ARSEP2013 • Code: IT702-13 Title: Therapeutic potential of monoacylglycerol lipase in- Title: Bases moleculares y celulares de la neurodegenera- hibition for the treatment of multiple sclerosis ción Principal Investigator: Carlos Matute Almau Principal Investigator: Carlos Matute Almau CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: International Type: CCAA Funding Agency: ARSEP Foundation (France) Funding Agency: Gobierno Vasco Funding: 20.000 € Funding: 444.000 € Duration: 2013-2014 Duration: 2013-2018 • Code: PI2011/02 • Code: SAF2010-21547 Title: Inicio y progresión de la Enfermedad de Parkinson. Title: Mecanismos del daño y neuroprotección de la sus- Vulnerabilidad de la vía nigroestriada, eventos en origen y tancia blanca destino Principal Investigator: Carlos Matute Principal Investigator: Jose Angel Obeso Inchausti CIBERNED’s collaboration: No CIBERNED’s collaboration: Yes CIBERNED’s groups: CIBERNED’s groups: G 205; G 201; G 104; G 208; G 203; Other CIBER’s collaboration: No G 404; G 206 Type: National Other CIBER’s collaboration: No Funding Agency: MICINN Type: Intramurales Funding: 380.000 € Funding Agency: CIBERNED Duration: 2010-2013 Funding: 362.000 € Duration: 2011-2013

PhD Dissertations

• Author: Estibaliz González Fernández Date: 8 March 2013 Title: Señalización y muerte mediada por los receptores de Supervisor: María Domercq García adenosina en oligodendrocitos Date: 14 February 2013 • Author: Federico Soria Lannes Supervisor: María Victoria Sánchez Gómez Title: Papel del intercambiador cistina-glutamato en la muerte neuronal tras isquemia • Author: Nuria Vázquez Villoldo. Date: 17 October 2013 Title: Papel del receptor P2X4 en la muerte microglial indu- Supervisor: María Domercq García cida por activación Group Guadalupe Mengod de los Arcos

IIBB-CSIC-IDIBAPS-CIBERNED

1 (Spain) Tel.: +34 933 638 323 Fax: +34 933 638 301 [email protected] Program

Guadalupe Mengod Giuseppe Mocci Principal Investigator, Research Professor, CSIC PhD student Roser Cortés Núria Paúl-Fernández CSIC Senior Scientist PhD student Mª Teresa Vilaró Rocío Martín-Álvarez Tenure Scientist, CSIC CIBERNED Lab Technician Emily Johansson Silvia Serrano Postdoctoral Fellow CIBERNED Advanced Technician Cristina Sanabra Irene Porcar Postdoctoral Fellow Advanced Technician, JAE-Tec CSIC Anna García-Miralles Cristina Villacampa PhD student Master’s student

70 Guadalupe Mengod de los Arcos Group 71

Summary

Our group is composed of three research sublines, which have the main and common research theme of neurodegeneration. In one of the sublines, we are studying the expression of neuronal markers in dementia, argyrophilic grain disease and Alzheimer’s disease by histochemical methods and RT-PCR, and analyzing the differential expression of proteins in these two diseases by proteomics. On the other hand, we are investigating the activation of serotonin 5-HT4 receptors as an approach to interfere with the deposition of Aß amyloid peptide in a triple transgenic mouse model of Alzheimer’s disease. In this work we are using both behavioral and histochemical techniques. And in the third subline of work, neuroinflammation, we are analyzing the molecular regulation of cAMP signaling in the murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and looking for innovative drugs for multiple sclerosis treatment through the selective inhibition of cAMP-specific phosphodiesterases.

Proteomic studies have led to the identification of a number of brain proteins that are differentially altered in argyrophilic grain disease and/or Alzheimer’s disease as compared to controls. Among them we found proteins related to oxidative stress, signal transduction, calcium mobilization, inflammatory events or cytoskeleton. Some of them, such as annexin 5 and gelsolin are plasma proteins and thus have an interesting potential as future biomarkers for argyrophilic grain disease. We are currently validating the alterations of these proteins.

In the 3xTg-AD triple-transgenic mouse model of Alzheimer’s disease we have observed that in aged animals, which have a profound cognitive impairment and extended amyloid neuropathology, continued stimulation of 5-HT4 serotonin receptors produces strong improvements in learning and memory as measured in the Morris water maze test, accompanied by a significant decrease of intraneuronal levels of Aß amyloid peptide as well as decreased extracellular amyloid load. This work was presented as a doctoral thesis in 2013.

We analyzed the expression of PDE4B mRNA splice variants and found an upregulation of PDE4B2 in brainstem and spinal cord of EAE mice which correlated with FoxP3 and TGF-beta mRNAs in a score-dependent manner. The increase observed for PDE4B protein was mainly found in antigen-presenting cells (APCs) (dendritic cells and microglia/macrophages) in areas with high cellular infiltration. The results point to a possible role of the PDE4B enzyme and in particular PDE4B2 splice variant during EAE pathogenesis, probably by modulating cAMP levels in APCs (Sanabra C et al., 2013). This work was part of PhD thesis defended by the first author and directed by the PI.

In collaboration with the group of F. Artigas (IIBB-CSIC-IDIBAPS, CIBERSAM) and the group of J. Sallés (University of the Basque Country, CIBERSAM) we have participated in several neuropharmacological studies in the field of depression and schizophrenia (Santana et al., 2013; Saenz del Burgo et al., 2013). In collaboration with C. Marín (IDIBAPS-CIBERNED we participated in a work with 6-OHDA lesioned rats in the subthalamic nucleus (Marín et al., 2013).

Keywords

Neurodegeneration, Neuroinflammation, Argyrophilic gran disease, Human brain, Molecular Neuropharmacology

Publications

• Ferres-Coy A, Santana N, Castane A, Cortes R, Carmona MC, Toth M, et al. Acute 5-HT(1A) autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions. Psychopharmacology. 2013 Jan;225(1):61-74. • Mocci G, Jimenez-Sanchez L, Adell A, Cortes R, Artigas F. Expression of 5-HT2A receptors in prefrontal cortex pyramidal neurons projecting to nucleus accumbens. Potential relevance for atypical antipsychotic action. Neuropharmacology. 2013 Nov 7;79C:49-58. • Marin C, Bonastre M, Mengod G, Cortes R, Giralt A, Obeso JA ,et al. Early L-dopa, but not pramipexole, restores basal ganglia activity in partially 6-OHDA-lesioned rats. Neurobiology of disease. 2013 Dec 24. • Lopez-Gimenez JF, Vilaro MT, Palacios JM, Mengod G. Multiple conformations of 5-HT2A and 5-HT 2C receptors in rat brain: an autoradiographic study with [125I](±)DOI. Experimental brain research. 2013 Oct;230(4):395-406. • Ferres-Coy A, Pilar-Cuellar F, Vidal R, Paz V, Masana M, Cortes R, et al. RNAi-mediated serotonin transporter suppression rapidly increases serotonergic neurotransmission and hippocampal neurogenesis. Translational psychiatry. 2013 Jan 15;3:e211. • Marin C, Bonastre M, Mengod G, Cortes R, Rodriguez-Oroz MC, Obeso JA. Subthalamic 6-OHDA-induced lesion attenuates levodopa-induced dyskinesias in the rat model of Parkinson's disease. Experimental neurology. 2013 Dec;250:304-12. Research Projects

• Code: PS09/00468 CIBERNED’s groups: Title: Activación de receptores 5-HT4 de serotonina como Other CIBER’s collaboration: No aproximación para interferir en la deposición del peptido Type: National amiloide Aß: evaluación en un modelo de ratón triple trans- Funding Agency: Instituto de Salud Carlos III, FIS génico de la enfermedad de Alzheimer Funding: 295.550 € Principal Investigator: María Teresa Vilaró Comas Duration: 2011-2014 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI2010/09 Other CIBER’s collaboration: No Title: BESAD-P: Biomarkers of Early Stages of Alzheimer Type: National Disease-Prevention Funding Agency: Ministerio de Ciencia e Innovación – Insti- Principal Investigator: Isidre Ferrer Abizanda tuto de Salud Carlos III - Acción Estrategica en Salud (AES) CIBERNED’s collaboration: Yes Funding: 71.995 € CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; Duration: 2010-2013 G 506; G 508; G 412 Other CIBER’s collaboration: Yes (CIBERESP) • Code: PI10/01874 Type: Intramurales Title: El papel de las fosfodiesterasas del AMPc en la evolu- Funding Agency: CIBERNED ción y tratamiento de la esclerosis múltiple en el modelo de Funding: 223.500 € EAE en ratón. Nuevas aproximaciones terapéuticas Duration: 2011-2013 Principal Investigator: Guadalupe Mengod CIBERNED’s collaboration: No

PhD Dissertations

• Author: Anna García Miralles Title: Efecto de la activación de receptores de serotonina 5-HT4 sobre la patología amiloide y el deterioro cognitivo en el modelo triple transgénico 3xTg-AD de la enfermedad de Alzheimer Date: 18 July 2013 Supervisor: María Teresa Vilaró Comas

7272 Guadalupe Mengod de los Arcos Group Group María Asunción Pastor Muñoz

Laboratorio de Neuroimagen · Área de Neurociencias

Centro de Investigación Médica Aplicada 1 Universidad de Navarra (CIMA) 31008 Pamplona (Spain)

Tel.: +34 948 255 400 Program Fax: +34 948 296 500 [email protected]

María Asunción Pastor Muñoz Gonzalo Arrondo Principal Investigator Postdoctoral Fellow Pau Pastor Muñoz Marta Vidorreta Díaz de Cario Co-Principal Investigator, PhD PhD student María Asunción Fernández Seara Elkin Luis García PhD PhD student Elisa Mengual Poza Martin Martínez Villar PhD PhD student José Luis Zubieta Zárraga Gabriel García Castellanos PhD PhD student Sara Ortega Cubero PhD student

73 Summary

The group of Neuroimaging/Neurogenetics at the Center for Applied Medical Research is focused on the study of genetic markers of sporadic and familial neurodegenerative diseases with dementia or Parkinsonism. Currently, we are performing projects of next generation sequencing to analyze the whole exome and transcriptome to identify new genes and risk factors involved in the aetiology of such diseases. The diseases we are studying are Parkinson disease, Alzheimer disease and other dementias. As a result of this work our laboratory and thanks to the collaborations established internationally and with other national groups including CIBERNED groups, in the part of genomics we have published 14 peer-reviewed articles, most of them in international journal in the first quartile. Some of the publications had made discoveries regarding the role of genetic rare variants in the risk for dementia and parkinsonisms. I want to highlight the setup of a national genetics research groups on dementia in which CIBERNED is fully involved, that has given place to one international peer-reviewed article and two on- going articles. Whit regard to the neuroimaging core to highlight the publication of 7 articles, in which we have defined new sequences of perfusion magnetic resonance Arterial Spin Labeling type working for the COST program of the EU, develop the application of fractal dimension techniques to brain cortical structural studies and defined new methods to study motor control in lower limb movements using functional magnetic resonance imaging. Through neuroimaging techniques: structural and functional magnetic resonance imaging and positron emission tomography we analyzed the patterns of brain regional neuronal loss associated with certain mutations or genetic risk factors such as APOE4 allele and H1 MAPT haplotype. These genetic markers will serve as a reliable diagnostic tool in initial stages of these diseases and they will allow identifying subjects with a common etiologic cause and subsequently discovering efficient therapies.

Keywords

Diagnostic markers, Genetics, Functional neuroimaging, Neuroimaging, Genetic mapping

Publications

• Jimenez-Jimenez FJ, Alonso-Navarro H, Garcia-Martin E, Lorenzo-Betancor O, Pastor P, Agundez JA. Update on genetics of essential tremor. Acta neurologica Scandinavica. 2013 Dec;128(6):359-71. • Palma JA, Urrestarazu E, Alegre M, Pastor MA, Valencia M, Artieda J, et al. Cardiac autonomic impairment during sleep is linked with disease severity in Parkinson’s disease. Clinical Neurophysiology. 2013;124(6):1163-8. • Gasca-Salas C, Arcocha J, Artieda J, Pastor P. Orthostatic myoclonus: an underrecognized cause of unsteadiness? Parkinsonism & related disorders. 2013 Nov;19(11):1013-7. • Garcia-Martin E, Martinez C, Alonso-Navarro H, Benito-Leon J, Lorenzo-Betancor O, Pastor P, et al. No association of the SLC1A2 rs3794087 allele with risk for essential tremor in the Spanish population. Pharmacogenetics and genomics. 2013 Nov;23(11):587-90. • Dobson-Stone C, Hallupp M, Loy CT, Thompson EM, Haan E, Sue CM, et al. C9ORF72 repeat expansion in Australian and Spanish frontotemporal dementia patients. PloS one. 2013;8(2):e56899. • McNeill A, Wu RM, Tzen KY, Aguiar PC, Arbelo JM, Barone P, et al. Dopaminergic neuronal imaging in genetic Parkinson's disease: insights into pathogenesis. PloS one. 2013;8(7):e69190. • Goni J, Cervantes S, Arrondo G, Lamet I, Pastor P, Pastor MA. Selective Brain Gray Matter Atrophy Associated with APOE ε4 and MAPT H1 in Subjects with Mild Cognitive Impairment. J Alzheimers Dis. 2013;33(4):1009-19. • Arbizu J, Prieto E, Martinez-Lage P, Marti-Climent JM, Garcia-Granero M, Lamet I, et al. Automated analysis of FDG PET as a tool for single-subject probabilistic prediction and detection of Alzheimer's disease dementia. European journal of nuclear medicine and molecular imaging. 2013 Sep;40(9):1394-405. • Garcia-Martin E, Lorenzo-Betancor O, Martinez C, Pastor P, Benito-Leon J, Millan-Pascual J, et al. LINGO1 rs9652490 and rs11856808 polymorphisms are not associated with risk for multiple sclerosis. BMC neurology. 2013 Apr 10;13:34. • Heckman MG, Elbaz A, Soto-Ortolaza AI, Serie DJ, Aasly JO, Annesi G, et al. The protective effect of LRRK2 p.R1398H on risk of Parkinson's disease is independent of MAPT and SNCA variants. Neurobiol Aging. 2014 Jan;35(1):266.e5-14. • Benitez BA, Cooper B, Pastor P, Jin SC, Lorenzo E, Cervantes S, et al. TREM2 is associated with the risk of Alzheimer's disease in Spanish population. Neurobiology of aging. 2013 Jun;34(6):1711.e15-7. • Goñi J, Sporns O, Cheng H, Aznárez-Sanado M, Wang Y, Josa S, et al. Robust estimation of fractal measures for characterizing the structural complexity of the human brain. NeuroImage. 2013;83:646-57.

7474 María Asunción Pastor Muñoz Group María Asunción Pastor Muñoz Group 75

• Fontes-Villalba A, Palma JA, Fernández-Seara MA, Pastor MA, de Castro P. A 47-year-old man with progressive gait disturbance and stiffness in his legs. Neurology. 2013;80(21):e224-e227. • Pastor P. Comment: double mutants of frontotemporal dementia genes--Simple co-occurrence? Neurology. 2013 Oct 8;81(15):1338. • Cruchaga C, Karch CM, Jin SC, Benitez BA, Cai Y, Guerreiro R, et al. Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease. Nature. 2013 Dec 11. • Benitez BA, Cruchaga C; United States-Spain Parkinson’s Disease Research Group. TREM2 and neurodegenerative disease. The New England journal of medicine. 2013 Oct 17;369(16):1567-8. • Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Bellenguez C, et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease. Nature genetics. 2013 Dec;45(12):1452-8. • Ruiz A, Dols-Icardo O, Bullido MJ, Pastor P, Rodriguez-Rodriguez E, Lopez de Munain A, et al. Assessing the role of the TREM2 p.R47H variant as a risk factor for Alzheimer's disease and frontotemporal dementia. Neurobiol Aging. 2014 Feb;35(2):444. e1-4. • Ortega-Cubero S, Lorenzo-Betancor O, Lorenzo E, Alonso E, Coria F, Pastor MA, et al. Fused in Sarcoma (FUS) gene mutations are not a frequent cause of essential tremor in Europeans. Neurobiology of aging. 2013 Oct;34(10):2441.e9-2441.e11. • Rubio-Moscardo F, Seto-Salvia N, Pera M, Bosch-Morato M, Plata C, Belbin O, et al. Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset Alzheimer's disease patients alter calcium homeostasis. PloS one. 2013;8(9):e74203. • Dols-Icardo O, Garcia-Redondo A, Rojas-Garcia R, Sanchez-Valle R, Noguera A, Gomez-Tortosa E, et al. Characterization of the repeat expansion size in C9orf72 in amyotrophic lateral sclerosis and frontotemporal dementia. Human molecular genetics. 2013 Oct 8.

Research Projects

• Code: COST Action BM1103 Funding: 54.532 € Title: Arterial spin labelling Initiative in Dementia (AID) Duration: 2013-2014 Principal Investigator: Xavier Golay (Action Chair) CIBERNED’s collaboration: No • Code: SAF2010-22329-C02-01 CIBERNED’s groups: Title: Identificación de genes implicados en el espectro fe- Other CIBER’s collaboration: No notípico de la enfermedad de Parkinson Familiar Type: International Principal Investigator: Pau Pastor Munoz Funding Agency: European Commission CIBERNED’s collaboration: No Funding: 10.000 € CIBERNED’s groups: Duration: 2013 Other CIBER’s collaboration: No Type: National • Code: SAF2011-29344 Funding Agency: Ministerio de Educacion y Ciencia Title: Evaluación de la conectividad cerebral usando la téc- Funding: 70.000 € nica de imagen funcional basada en perfusión “Arterial spin Duration: 2010-2013 labeling" Aplicación a la enfermedad de Parkinson Principal Investigator: Maria A. Fernández Seara • Code: RYC-2010-07161 CIBERNED’s collaboration: No Title: Medida de perfusión cerebral en patología usando la CIBERNED’s groups: técnica de imagen por resonancia magnética Other CIBER’s collaboration: No Principal Investigator: Maria A. Fernández Seara Type: National CIBERNED’s collaboration: No Funding Agency: Plan National - MICINN CIBERNED’s groups: Funding: 72.600 € Other CIBER’s collaboration: No Duration: 2013 Type: National Funding Agency: MICCIN - Ramón y Cajal • Code: 515-14 Funding: 243.626 € Title: Follow-up genotyping and functional analisis of PSP Duration: 2010-2015 H1 haplotype variants Principal Investigator: Pau Pastor • Code: PI2011/04 CIBERNED’s collaboration: No Title: Estudio multicéntrico de biomarcadores en LCR y de CIBERNED’s groups: neuroimagen en el continuum Enfermedad de Alzheimer Other CIBER’s collaboration: No preclínica - prodrómica (Estudio SIGNAL) Type: Privado Principal Investigator: Alberto Lleó Funding Agency: CurePSP's Research Program CIBERNED’s collaboration: Yes CIBERNED’s groups: G 504; G 502; G 511; G 507; G 509 Funding Agency: CIBERNED Other CIBER’s collaboration: No Funding: 140.000 € Type: Intramurales Duration: 2011-2013

7676 María Asunción Pastor Muñoz Group Group José Rodríguez Álvarez

Instituto de Neurociencias

Universidad Autónoma de Barcelona 1 Edificio M Campus de Bellaterra 08193 Cerdanyola del Vallès, Barcelona (Spain) Program Tel.: +34 935 813 861 [email protected]

José Rodríguez Álvarez Guillem Sánchez Opazo Principal Investigator, Associate Professor PhD student Carlos A. Saura Antolín Meng Cheng Associate Professor PhD student José Aguilera Ávila Arnaldo Parra Associate Professor PhD student Alfredo J. Miñano Molina Wen Wen Cheng CIBERNED Postdoctoral Fellow PhD student Rut Fadó Andrés Elsa Martín Capella Postdoctoral Fellow CIBERNED Technician Sergi Marco Capella PhD student

77 Summary

During 2013 we have continued to focus our research attention to the molecular events that could be related to cognitive impairment associated to early stages of Alzheimer disease (AD). In particular we are investigating the effect of Aß oligomers (oAβ) on the molecular mechanisms that regulate changes in gene expression involved in cognitive deficits in AD and the mechanisms that are involved in the reduction of synaptic AMPA receptors by oAβ. Gene expression analyses revealed specific downregulation of a subset of well-known activity-induced CREB-dependent genes, including c-fos, Bdnf and Nr4a2, in the hippocampus of memory-impaired APPSw, Ind transgenic mice. Activity-dependent CREB transcription induced by calcium/ cAMP signals is disrupted through a mechanism involving deregulation of calcium/calcineurin-mediated dephosphorylation and activation of CRTC1. On the hand we have studied the role of diffrent anchoring proteins in the post-synaptic terminal that are related to glutamate receptor trafficking in the synapse. Finally we have also demonstrated a novel function of X-linked Inhibitor of apoptosis protein (XIAP) as a regulator of neurite outgrowth in neuronal cells since XIAP is a negative regulator of neurotrophin-induced neurite outgrowth and neuronal differentiation in developing neurons through binding to Trk receptors.

Keywords

Alzheimer disease, Memory, Intracelular signaling, Gene regulation, Glutamate receptors, CRTC1, Early synaptic dysfunction

Publications

• Cubi R, Matas LA, Pou M, Aguilera J, Gil C. Differential sensitivity to detergents of actin cytoskeleton from nerve endings. Biochimica et Biophysica Acta-Biomembranes. 2013;1828(11):2385-93. • Cubi R, Candalija A, Ortega A, Gil C, Aguilera J. Tetanus Toxin Hc Fragment Induces the Formation of Ceramide Platforms and Protects Neuronal Cells against Oxidative Stress. PLos One. 2013.;8(6):e68055. • Fado R, Moubarak RS, Minano-Molina AJ, Barneda-Zahonero B, Valero J, Saura CA, et al. X-linked inhibitor of apoptosis protein negatively regulates neuronal differentiation through interaction with cRAF and Trk. Scientific Reports. 2013;3:1-11. • Jurado S, Goswami D, Zhang Y, Molina AJ, Südhof TC, Malenka RC. LTP requires a unique postsynaptic SNARE fusion machinery. Neuron. 2013 Feb 6;77(3):542-58. • Garcia-Ayllon MS, Campanari ML, Brinkmalm G, Rabano A, Alom J, Saura CA, et al. Cerebrospinal fluid Presenilin-1 complexes are increased in Alzheimer’s disease. Acta Neuropathologica Communications. 2013;1(1):1-13. • Balmaceda V, Cuchillo-Ibanez I, Pujadas L, Garcia-Ayllon MS, Saura CA, Nimpf J, et al. ApoER2 processing by presenilin-1 modulates reelin expression. FASEB journal. 2013 Dec 16. • Herrando-Grabulosa M, Casas C, Aguilera J. The C-terminal domain of tetanus toxin protects motoneurons against acute excitotoxic damage on spinal cord organotypic cultures. Journal of neurochemistry. 2013 Jan;124(1):36-44.

Research Projects

• Code: SAF2010-20925 como estrategia protectora en la isquemia cerebral Title: Análisis del transcriptoma en ratones transgénicos en Principal Investigator: José Rodríguez Álvarez la enfermedad de Alzheimer CIBERNED’s collaboration: No Principal Investigator: José Rodríguez Álvarez CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: Ministerio de Ciencia e Innovación Type: National Funding: 169.400 € Funding Agency: MCIN Duration: 2012-2015 Funding: 205.700 € Duration: 2011-2013 • Code: PI2010/08 Title: Activación glial en el proceso neuroinflamatorio: • Code: SAF2011-30281 una potencial diana terapéutica para la enfermedad de Title: Regulación de la señalización mediada por los recep- Alzheimer tores con dominios de muerte por antagonistas y miRNAs Principal Investigator: Francisco Javier Vitorica Ferrández

7878 José Rodríguez Álvarez Group José Rodríguez Álvarez Group 79

CIBERNED’s collaboration: Yes Funding Agency: CIBERNED CIBERNED’s groups: G 411; G 415; G 406; G 409 Funding: 375.000 € Other CIBER’s collaboration: No Duration: 2010-2013 Type: Intramurales

PhD Dissertations

• Author: Sergi Marco Martin Title: Regulation of axon elongation by presenilin-1/gamma secretes: role of EphA3 receptor Date: 11 October 2013 Supervisor: Carlos A. Saura Antolín Group Javier Sáez Valero

Instituto de Neurociencias de Alicante

1 Universidad Miguel Hernández-CSIC Avenida Ramón y Cajal, s/n 03550 Sant Joan d’Alacant (Spain)

Program Tel.: +34 965 919 580 Fax: +34 965 919 561 [email protected]

Javier Sáez Valero Maria Letizia Campanari Principal Investigator, Associate Professor PhD student, Consolider, CSIC Inmaculada Cuchillo Ibáñez Esther Llorens Álvarez Postdoctoral Fellow, CIBERNED Technician, project CP11/00067 María Salud García Ayllón Trinidad Mata Balaguer Postdoctoral Fellow, Miguel Servet, FISABIO Postdoctoral Fellow, project PI12/00593 Jordi Alom Poveda Inmaculada López Font Head of Neurology Department Postdoctoral Fellow, project PI11/03026 Valeria Balmaceda Valdez PhD student, JAE-Predoc, CSIC

80 Javier Sáez Valero Group 81

Summary

The aim of our research group stills focus into Alzheimer’s disease (AD) from a basic point of view, but also regarding translational benefits including clinical-diagnostic applications. Recently, we have demonstrated the presence in cerebrospinal fluid (CSF) of the amyloid-related secretase, presenilin 1 (PS1), and its potential as a new AD biomarker (García-Ayllón et al., 2013). Our finding are also including in a patent application (P201330230).

Reelin is a signaling protein increasingly associated with the pathogenesis of AD that relevantly modulates tau phosphorylation. We have previously demonstrated that β-amyloid peptide (Aβ) alters Reelin expression. Now, we demonstrated that abnormal Reelin triggered by Aβ results in signaling malfunction, which potentially contribute to the pathogenic process (Cuchillo-Ibáñez et al., 2013). Moreover, we showed that the Reelin receptor ApoER2 is a substrate of PS1. ApoER2 processing, induced by the binding of Reelin, suppresses Reelin expression at a transcriptional level. Our studies suggest that PS1/γ-secretase-dependent processing of the Reelin receptor ApoER2 inhibits Reelin expression and may regulate Reelin signaling (Balmaceda et al., Epub 2013 Dec 16).

In collaboration, we have identified a naturally occurring RELN mutation in a commercial flock of Spanish Churra sheep, which provide a genetically characterized large animal model for the study of Reelin functions in the developing and mature brain, including AD and other neuropsychiatric pathological process (Suárez-Vega et al., 2013).

Finally, we present evidence that, in the AD brain, the level of the acetylcholinesterase (AChE) protein does not accord with the decrease in enzymatic activity level. AChE is a key enzyme in the cholinergic nervous system, and is one of the most studied proteins in the AD field. Until now, the majority of investigation on AChE in AD pathology has been focus in the determination of its enzymatic activity level, which is depleted in the AD brain, but despite this overall decrease, AChE activity increases at the vicinity of the amyloid plaques and the neurofibrillary tangles. We demonstrated by Western blotting and immunohistochemistry that a prominent pool of enzymatically inactive AChE protein was existed in the AD brain. The potential significance of these unexpected levels of inactive AChE protein in the AD brain is discussed, especially in the context of protein-protein interactions with Aβ peptide and PS1 (Campanari et al., Epub 2013 Dec 7).

Keywords

Alzheimer, Reelin, P-tau, β-amyloid, Acetylcholinesterase, Presenilin 1, Glycosylation, Biomarker

Publications

• Campanari ML, Garcia-Ayllon MS, Blazquez-Llorca L, Luk WKW, Tsim K, Saez-Valero J. Acetylcholinesterase protein level is preserved in the Alzheimer’s brain. J Mol Neurosci. 2013;1-8. • Suarez-Vega A, Gutierrez-Gil B, Cuchillo-Ibanez I, Saez-Valero J, Perez V, Garcia-Gamez E, et al. Identification of a 31-bp deletion in the RELN gene causing lissencephaly with cerebellar hypoplasia in sheep. PLoS One. 2013;8(1-12):e81072. • Garcia-Ayllon MS, Campanari ML, Brinkmalm G, Rabano A, Alom J, Saura CA, et al. Cerebrospinal fluid Presenilin-1 complexes are increased in Alzheimer’s disease. Acta Neuropathologica Communications. 2013.;1(1):1-13. • Cuchillo-Ibanez I, Balmaceda V, Botella-Lopez A, Rabano A, Avila J, Saez-Valero J. Beta-amyloid impairs Reelin signaling. PLoS One. 2013;8(8):e72297. • Balmaceda V, Cuchillo-Ibanez I, Pujadas L, Garcia-Ayllon MS, Saura CA, Nimpf J, et al. ApoER2 processing by presenilin-1 modulates reelin expression. FASEB journal. 2013 Dec 16. Research Projects

• Code: PI12/00593 Title: Regulación epigenética de Reelina en la enfermedad Title: Alteración en la vía de señalización de Reelina en la de Alzheimer enfermedad de Alzheimer: implicaciones patológicas Principal Investigator: Javier Sáez Valero Principal Investigator: Javier Sáez Valero CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Privado Type: National Funding Agency: Fundación Ramón Areces (XVI Concurso Funding Agency: ISC-III National para la Adjudicación de Ayudas a la Investigación Funding: 222.035 € Científica y Técnica) Duration: 2013-2015 Funding: 95.804 € Duration: 2012-2015 • Code: PI11/030206 Title: Biomarkers for Alzheimer’s disease and Parkinson’s • Code: RGC-663012 disease (BIOMARKAPD) Title: The trafficking of PRiMA-linked tetrameric AChE: Principal Investigator: Javier Sáez Valero Probing roles of glycosylation in determining the degrada- CIBERNED’s collaboration: Yes tion of AChE in the brain CIBERNED’s groups: G 407; G 504 Principal Investigator: Karl Tsim Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: International CIBERNED’s groups: Funding Agency: ISC-III Other CIBER’s collaboration: No Funding: 117.370 € Type: International Duration: 2012-2014 Funding Agency: Resarch Grant Council, Hong Kong Funding: 3.000 € • Code: CP11/00067 Duration: 2012-2015 Title: Conexión entre tau y péptido β-amiloide en la enfer- medad de Alzheimer: Papel de GSK-3β, presenilina 1 y ace- • Code: PI2010/07 tilcolinesterasa Title: Reelina y GSK3 como dianas terapéuticas en la enfer- Principal Investigator: María Salud García Ayllón medad de Alzheimer CIBERNED’s collaboration: No Principal Investigator: Jesús Ávila de Grado CIBERNED’s groups: CIBERNED’s collaboration: Yes Other CIBER’s collaboration: No CIBERNED’s groups: G 401; G 403; G 113; G 407; G 408; Type: National G 410 Funding Agency: ISC-III Other CIBER’s collaboration: No Funding: 121.500 € Type: Intramurales Duration: 2013 Funding Agency: CIBERNED Funding: 540.000 € • Code: Fundacion Ramón Areces Duration: 2010-2013

8282 Javier Sáez Valero Group Group Eduardo Soriano García

Universitat de Barcelona

Parc Científic de Barcelona 1 Baldiri Reixac, 10-12 08028 Barcelona (Spain)

Tel.: +34 934 037 117 Program [email protected]

Eduardo Soriano García Sara Esmeralda Rubio Abejón Principal Investigator, Full Professor PhD student Marta Pascual Sánchez Daniela Rossi Associate Professor, Researcher PhD student Jesús Mariano Ureña Bares Núria Masachs Janoher Associate Professor, Researcher PhD student Ferrán Burgaya Márquez Beatriz García García Associate Professor, Researcher PhD student Lluís Pujadas Puigdomènech Pablo Barrecheguren Manero Postdoctoral Fellow PhD student Fausto Alexánder Ulloa Darquea Antoni Parcerisas Mosqueda Postdoctoral Fellow PhD student Ramon Martínez Mármol Carles Bosch Piñol Postdoctoral Fellow PhD student Oriol Ros Torres Ashraf Muhaisen PhD student Technician Román Serrat Reñe Maria Rosa Andrés Ventura PhD student Lab Technician María del Mar Masdeu Camara Jan Vara PhD student Administrative Serena Mirra PhD student

83 Summary

Synaptic remodeling and adult neurogenesis plasticity processes are well characterized, and are similar to those ocurring during development. Our group has hypothesized that the processes of plasticity in adults may be controlled by genes crucial to development. Reelin is an extracellular protein essential for neuronal migration and synaptic maturation. Here we propose Reelin signaling models in vitro and in vivo to determine the roles of this protein in adult neuronal plasticity and to study its role in the pathogenesis of neurological diseases and mental. To date we have carried out the following achievements: a) We have determined the role of Reelin in adult neurogenesis and functional integration of neurons generated in adults. b) We have determined the role of Reelin in synaptic plasticity and synaptic remodeling in the adult. c) We have identified the specific genetic and molecular networks underlying these events dependent on Reelin. d) We have established the role of Reelin in the formation of two pathological features of Alzheimer disease (AD) amyloid deposits and intracellular neurofibrillary tangles. e) We have determined that the Reelin has a protective role in mood disorders and addiction.

Keywords

Reelin, Synaptic plasticity, Synaptic remodeling, Adult neurogenesis

Publications

• Serrat R, Lopez-Domenech G, Mirra S, Quevedo M, Garcia-Fernàndez J, Ulloa F, et al. The non-canonical Wnt/PKC pathway regulates mitochondrial dynamics through degradation of the arm-like domain-containing protein Alex3. PloS one. 2013;8(7):e67773. • Llorens-Martin M, Fuster-Matanzo A, Teixeira CM, Jurado-Arjona J, Ulloa F, Defelipe J, et al. Alzheimer disease-like cellular phenotype of newborn granule neurons can be reversed in GSK-3β-overexpressing mice. Molecular psychiatry. 2013 Apr;18(4):395. • Medina-Pulido L, Molina-Arcas M, Justicia C, Soriano E, Burgaya F, Planas AM, et al. Hypoxia and P1 receptor activation regulate the high-affinity concentrative adenosine transporter CNT2 in differentiated neuronal PC12 cells. The Biochemical journal. 2013 Sep 15;454(3):437-45. • Balmaceda V, Cuchillo-Ibanez I, Pujadas L, Garcia-Ayllon MS, Saura CA, Nimpf J, et al. ApoER2 processing by presenilin-1 modulates reelin expression. FASEB journal. 2013 Dec 16. • Vega-Flores G, Rubio SE, Jurado-Parras MT, Gomez-Climent MA, Hampe CS, Manto M, et al. The GABAergic Septohippocampal Pathway Is Directly Involved in Internal Processes Related to Operant Reward Learning. Cerebral cortex (New York, N.Y. 1991). 2013 Mar 10. • Teixeira CM, Masachs N, Muhaisen A, Bosch C, Perez-Martinez J, Howell B, et al. Transient Downregulation of Dab1 Protein Levels during Development Leads to Behavioral and Structural Deficits: Relevance for Psychiatric Disorders. Neuropsychopharmacology. 2013 Sep 13. • Defourny J, Poirrier AL, Lallemend F, Mateo Sanchez S, Neef J, Vanderhaeghen P, et al. Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells. Nature communications. 2013;4:1438. • La Torre A, del Mar Masdeu M, Cotrufo T, Moubarak RS, del Rio JA, Comella JX, et al. A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching. Cell death & disease. 2013 Apr 18;4:e602. • Llorens-Martin M, Fuster-Matanzo A, Teixeira CM, Jurado-Arjona J, Ulloa F, Defelipe J, et al. GSK-3β overexpression causes reversible alterations on postsynaptic densities and dendritic morphology of hippocampal granule neurons in vivo. Molecular psychiatry. 2013 Apr;18(4):451-60.

Research Projects • Code: PI11/0704 CIBERNED’s collaboration: No Title: Alteraciones en la conexión GABAérgica septohipo- CIBERNED’s groups: cámpica en la enfermedad de Alzheimer. Implicaciones te- Other CIBER’s collaboration: No rapéuticas Type: National Principal Investigator: Marta Pascual Funding Agency: Ministerio de Sanidad y Consumo (ISCIII)

8484 Eduardo Soriano García Group Eduardo Soriano García Group 85

Funding: 70.281 € CIBERNED’s collaboration: No Duration: 2012-2014 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: BFU2008-03980 Type: National Title: Identificación y función de nuevos genes implicados Funding Agency: Instituto de Salud Carlos III en desarrollo y plasticidad neuronal Funding: 93.000 € Principal Investigator: Eduardo Soriano Duration: 2011-2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI12/02108 Other CIBER’s collaboration: No Title: Participación de las Semaforinas transmembranales y Type: National la cinasa Fak en patologías relacionadas con la sinapsis y la Funding Agency: Ministerio de Ciencia y Tecnología actividad neuronal Funding: 875.000 € Principal Investigator: Ferrán Burgaya Márquez Duration: 2009-2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI2013_08-1 Other CIBER’s collaboration: No Title: La dinámica mitocondrial y mitofagia como dianas te- Type: National rapéuticas en las Enfermedades de Parkinson y Huntington Funding Agency: Instituto de Salud Carlos III Principal Investigator: Eduardo Soriano Funding: 56.500 € CIBERNED’s collaboration: Yes Duration: 2013-2015 CIBERNED’s groups: G 301; G 207; G 410; G 109; G 408 Other CIBER’s collaboration: No • Code: 2009 SGR 1017 Type: Intramurales Title: Support to Research Groups Established in Catalonia Funding Agency: CIBERNED Principal Investigator: Eduardo Soriano Funding: 300.000 € CIBERNED’s collaboration: No Duration: 2013-2015 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: 2010/149 Type: CCAA Title: Papel Protector de la Reelina en trastornos de adic- Funding Agency: AGAUR ción: Identificación de los mecanismos moleculares involu- Funding: 49.920 € crados en la protección de la Reelina Duration: 2009-2013 Principal Investigator: Eduardo Soriano CIBERNED’s collaboration: No • Code: PI2010/07 CIBERNED’s groups: Title: Reelina y GSK3 como dianas terapéuticas en la enfer- Other CIBER’s collaboration: No medad de Alzheimer Type: National Principal Investigator: Jesús Ávila de Grado Funding Agency: Ministerio de Sanidad y Política Social e CIBERNED’s collaboration: Yes Igualdad CIBERNED’s groups: G 401; G 403; G 113; G 407; G 408; Funding: 86.086 € G 410 Duration: 2010-2013 Other CIBER’s collaboration: No Type: Intramurales • Code: PI10/01750 Funding Agency: CIBERNED Title: Participación de la tirosina quinasa Ack1 en patolo- Funding: 540.000 € gías neurodegenerativas y sinaptogénesis Duration: 2010-2013 Principal Investigator: Jesús Ureña Bares

PhD Dissertations

• Author: Beatriz García García Date: 27 June 2013 Title: Estudio de procesos de migración y plasticidad en el Supervisor: Eduardo Soriano García Sistema Nervioso Central: papel de semaforina 4F y kinasa de adhesión focal (FAK) • Author: Serena Mirra Date: 15 February 2013 Title: Characterization of Armcx/Armc10 gene family func- Supervisor: Eduardo Soriano García tion in mitochondrial dynamics and neural development Date: 26 July 2013 • Author: Oriol Ros i Torres Supervisor: Eduardo Soriano García Title: Paper de les proteínes sinàptiques i l’exocitosi en els processos de glia axonal i migració • Author: Daniela Rossi Title: Estudio de la función de la proteína Reelina en los mecanismos moleculares implicados en la enfermedad de Date: 17 October 2013 Alzheimer Supervisor: Eduardo Soriano García

8686 Eduardo Soriano García Group Group Ignacio Torres Alemán

Instituto Cajal

Avenida Doctor Arce, 37 1 28002 Madrid (Spain) Tel.: +34 915 854 723

Fax: +34 915 854 754 Program [email protected]

Ignacio Torres Alemán Edwin Hernández Principal Investigator PhD student Ana M Fernández García Ángel Trueba Postdoctoral Fellow PhD student Miguel García Manuel Domínguez Technician Víctor Munive Laura Genis Andrea Santi Postdoctoral Fellow Dolores Guinea Esquerdo Lab Technician

87 Summary

The lab is focused on the role of insulin factors in the healthy and diseased brain. In 2013 we have approached a little explored concept: the role of insulin and IGF-I in regulating brain glucose metabolism and underlying mechanisms. This new role of these hormones will in all probability modify our understanding of the biological significance of insulin peptides in the adult brain. Also within a physiological context we are analyzing the role of IGF-I in neuroprotection by physical exercise and the remarkable gender differences observed. In addition, we are interested in unveiling mechanisms of serum-to-brain traffic of circulating IGF-I under physiological and pathological conditions. We are currently investigating the effect of diet on this traffic and plan to incorporate the analysis of stress in the near future. Regarding pathology we are interested in neuro-inflammation and its links with Alzheimer´s disease within a Ciberned collaborative program. We are also analyzing the role of IGF-I in the responses to oxidative stress by astrocytes. In this regard, we are now determining a new ligand-independent role of the IGF-I receptor. Recently we have incorporated posttraumatic stress syndrome as a pathology of great interest because of its current clinical impact and because it possibly involves many of the known neuroprotective actions of IGF-, from protection against physicial injury and oxidative stress to modulation of mood and cognition.

Keywords

Role of Insulin like growth factors in neuronal plasticity and degenerative diseases, Therapeutic targets in neurodegeneration, The aging brain and cognition, Blood brain barriers

Publications

• Nishijima T, Llorens-Martin M, Tejeda GS, Inoue K, Yamamura Y, Soya H, et al. Cessation of voluntary wheel running increases anxiety-like behavior and impairs adult hippocampal neurogenesis in mice. Behavioural brain research. 2013 May 15;245:34-41. • Trueba-Saiz A, Cavada C, Fernandez AM, Leon T, Gonzalez DA, Fortea Ormaechea J, et al. Loss of serum IGF-I input to the brain as an early biomarker of disease onset in Alzheimer mice. Translational psychiatry. 2013 Dec 3;3:e330. • Fernandez S, Genis L, Torres-Aleman I. A phosphatase-independent gain-of-function mutation in PTEN triggers aberrant cell growth in astrocytes through an autocrine IGF-1 loop. Oncogene. 2013 Sep 23.

Research Projects • Code: SAF2010-17036 Other CIBER’s collaboration: Sií (CIBERER) Title: Regulación de la entrada de IGF-I al cerebro Type: CCAA Principal Investigator: Ignacio Torres Alemán Funding Agency: Comunidad de Madrid CIBERNED’s collaboration: No Funding: 17.250 € CIBERNED’s groups: Duration: 2012-2015 Other CIBER’s collaboration: No Type: National • Code: PI2010/08 Funding Agency: Ministerio Ciencia. Plan National Title: Activación glial en el proceso neuroinflamatorio: Funding: 312.000 € una potencial diana terapéutica para la enfermedad de Duration: 2011-2013 Alzheimer Principal Investigator: Francisco Javier Vitorica Ferrández • Code: S2010_BMD CIBERNED’s collaboration: Yes Title: Redes de señalización y vías efectoras en modelos CIBERNED’s groups: G 411; G 415; G 406; G 409 celulares y animales de enfermedades neurodegenerativas Other CIBER’s collaboration: No Principal Investigator: José González Castaño Type: Intramurales CIBERNED’s collaboration: Yes Funding Agency: CIBERNED CIBERNED’s groups: G 401; G 104; G 111; G 307; G 409; Funding: 375.000 € G 412 Duration: 2010-2013

8888 Ignacio Torres Alemán Group Group Ramón Trullás Oliva

Unidad de Neurobiología

IIBB-CSIC 1 IDIBAPS Rosselló 161, 7ª Planta, Lab. 711 08036 Barcelona (Spain) Program Tel.: +34 659 696 187 Fax: +34 933 638 301

Ramón Trullás Oliva Petar Podlesniy Principal Investigator, CSIC Research Professor Research Nicole Mahy Nuria Serra Full Professor of Biochemistry Technician Manuel Rodríguez Mónica Romo Assistant Professor Technician

89 Summary

During the last year we have confirmed that a decrease in the content of cell free circulating mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) is associated with both sporadic and familial forms of Alzheimer’s disease (AD), but not with Fronto- temporal Lobe Degeneration (FTLD). The drop in mtDNA content in CSF occurs in asymptomatic carriers of pathogenic AD mutations at least one decade before the expected emergence of clinical signs of dementia and well before any alteration in currently known AD biomarkers, indicating that mtDNA depletion is an early event in the neurodegenerative process of AD. The discovery that depletion of mtDNA precedes the appearance of clinical signs both in familial and sporadic AD subjects indicates that, independently of the etiology, regulation of mtDNA copy number is a common mechanism onto which different pathways causing AD converge. Because neurons are highly dependent on aerobic energy provided by mitochondria, a drop in the amount of mtDNA of the magnitude found in CSF is likely to cause neurodegeneration by cellular energy deprivation. Based on these previous findings, we hypothesize that mtDNA depletion is a fundamental pathophysiological mechanism of AD and that restoring mtDNA copy number to normal levels should have a therapeutic effect in preventing neurodegeneration and cognitive disorders associated with AD.

Keywords

Synaptic Neurodegeneration, Alzheimer’s disease Biomarkers, Apoptosis, Mitochondrial dynamics, Mitochondrial transport in neurodegeneration, Neuronal Death, Neuronal Pentraxins, Molecular mechanisms of Alzheimer’s disease

Publications

• Podlesniy P, Figueiro-Silva J, Llado A, Antonell A, Sanchez-Valle R, Alcolea D, et al. Low cerebrospinal fluid concentration of mitochondrial DNA in preclinical Alzheimer disease. Annals of Neurology. 2013;74(5):655-68. • Redondo-Castro E, Hernández J, Mahy N, Navarro X. Phagocytic microglial phenotype induced by glibenclamide improves functional recovery but worsens hyperalgesia after spinal cord injury in adult rats. Eur J Neurosci. 2013 Dec;38(12):3786-98. • Rodríguez MJ, Martínez-Moreno M, Ortega FJ, Mahy N. Targeting microglial K(ATP) channels to treat neurodegenerative diseases: a mitochondrial issue. Oxid Med Cell Longev. 2013;2013:194546. • Ortega FJ, Jolkkonen J, Mahy N, Rodríguez MJ. Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia. J Cereb Blood Flow Metab. 2013 Mar;33(3):356-64.

Research Projects

• Code: SAF2011-23550 CIBERNED’s groups: G 401; G 403; G 113; G 407; G 408; Title: Mechanisms of synapse elimination and mitochondri- G 410 al dynamics in neurodegeneration: role of Neuronal Pen- Other CIBER’s collaboration: No traxin 1 Type: Intramurales Principal Investigator: Ramón Trullás Oliva Funding Agency: CIBERNED CIBERNED’s collaboration: No Funding: 540.000 € CIBERNED’s groups: Duration: 2010-2013 Other CIBER’s collaboration: No Type: National • Code: PI2013_08-1 Funding Agency: MICINN Title: La dinámica mitocondrial y mitofagia como dianas te- Funding: 150.000 € rapéuticas en las Enfermedades de Parkinson y Huntington Duration: 2011-2014 Principal Investigator: Eduardo Soriano CIBERNED’s collaboration: Yes • Code: PI2010/07 CIBERNED’s groups: G 301; G 207; G 410; G 109; G 408 Title: Reelina y GSK3 como dianas terapéuticas en la enfer- Other CIBER’s collaboration: No medad de Alzheimer Type: Intramurales Principal Investigator: Jesús Ávila de Grado Funding Agency: CIBERNED CIBERNED’s collaboration: Yes Funding: 300.000 € Duration: 2013-2015

9090 Ramón Trullás Oliva Group Ramón Trullás Oliva Group 91

PhD Dissertations

• Author: Joana Figueiró-Silva Title: Mecanismos de regulación sináptica por Pentraxina neuronal 1 Date: 17 September 2013 Supervisor: Ramón Trullás Oliva Group Francisco Javier Vitorica Ferrández

Dpto. Bioquímica y Biología Molecular Instituto de Biomedicina de Sevilla

1 Facultad de Farmacia Hospital Universitario Virgen Universidad de Sevilla del Rocío de Sevilla (Spain) (Spain) Program Tel.: +34 954 556 770 Fax: +34 955 923 053 [email protected]

Francisco Javier Vitorica Ferrández Francisco Javier Moyano Principal Investigator, Full Professor FPU Fellow Marisa Vizuete Chacón Sebastián Jiménez Muñoz Associate Professor CIBERNED Advanced Technician Victoria Navarro Garrido Mª Luisa García-Calvo FPI Fellow FIS Technician

92 Francisco Javier Vitorica Ferrández Group 93

Summary

During the year 2013, our group has been mainly dedicated to the characterization of the inflammatory response in human postmortem samples from different Braak stages; from BraakII to Braak V -VI. The latter are patients diagnosed as Alzheimer’s dementia.

To perform this characterization, we have determined the mRNA expression, protein levels and, by immunohistochemistry (in collaboration with the group of Dr. A. Gutierrez, University of Málaga) of genes considered markers for the activation and / or microglial differentiation. This project is part of the objectives of the collaborative project we have been developing CIBERNED during 2010-2013.

Also within this context, we isolated the so-called soluble fractions (cytosolic plus extracellular) of the different human samples. The accumulation of soluble forms of Abeta and/or tau was analyzed by western blots, immunoprecipitations and ELISAs. Furthermore, we have also analyzed, in vitro, the toxicity and/or immunogenicity of the soluble fractions. Finally, these soluble fractions have been characterized using proteomic techniques (in collaboration with Dr. J. Vazquez, CNIC). Following this project, in late 2013, we began a new collaborative project (coordinated by Dr. Torres-Alemán) on glial-neuronal interactions in both, experimental models and in AD patient samples; in order to determine whether a dysfunction homeostasis in glia-neuron is the basis of the neurodegenerative process underlying dementia.

With regard to the characterization of transgenic models PS1/APP, we determined that chronic oral treatment with lithium carbonate, in early stages of amyloid pathology, induces a clear neuroprotective effect and cognitive enhancement. Lithium produces a qualitative change in compaction Abeta plate so that they are smaller, more compact and less toxic. This reduced toxicity of the plates results in a reduced formation of dystrophic neurites around and in a marked neuroprotection from the most vulnerable neuronal populations (SOM/NPY).

We are currently determined the possible implication of astrocytes in the protective action of lithium. These results show the possibility to modulate in vivo the toxic effect of the plates during the progression of the disease and may represent a promising therapeutic strategy to reduce nerve damage in contrast to the so far unsuccessful attempts to prevent or eliminate plaques.

Finally, we have completed two research projects applied high translational value. The first company to Sanofi (France) led to the identification of functional synaptic potential biomarkers in various in vivo models of AD and aging. The second project has been with the company Neuron Biopharma and consisted of a preclinical study to evaluate the efficacy of a new therapeutic strategy neuroprotective in transgenic models of AD

Keywords

Alzheimer, Neurodegeneration, Neuroprotection, Inflammation, Abeta, Oligomers, Transgenic models, Neuropathology

Publications

• Torres M, Price SL, Fiol-Deroque MA, Marcilla-Etxenike A, Ahyayauch H, Barceló-Coblijn G, et al. Membrane lipid modifications and therapeutic effects mediated by hydroxydocosahexaenoic acid on Alzheimer’s disease. Biochimica et Biophysica Acta- Biomembranes. 2013. • Manterola L, Hernando-Rodriguez M, Ruiz A, Apraiz A, Arrizabalaga O, Vellon L, et al. 1-42 β-Amyloid peptide requires PDK1/ nPKC/Rac 1 pathway to induce neuronal death. Translational psychiatry. 2013 Jan 22;3:e219. • Trujillo-Estrada L, Jimenez S, De Castro V, Torres M, Baglietto-Vargas D, Moreno-Gonzalez I, et al. In vivo modification of Abeta plaque toxicity as a novel neuroprotective lithium-mediated therapy for Alzheimer’s disease pathology. Acta neuropathologica communications. 2013 Nov 12;1(1):73. Research Projects

• Code: PI2010/08 preclínico y estudio previo de entrada a fases clínicas de un Title: Activacion glial en el proceso neuroinflamatorio: una nuevo fármaco) potencial diana terapéutica para la enfermedad de Alzheimer Principal Investigator: Antonia Gutiérrez Pérez Principal Investigator: Francisco Javier Vitorica Ferrández CIBERNED’s collaboration: Yes CIBERNED’s collaboration: Yes CIBERNED’s groups: G 415; G 411 CIBERNED’s groups: G 411; G 415; G 406; G 409 Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Privado Type: Intramurales Funding Agency: Industria Neuron Biopharma SA Funding Agency: CIBERNED Funding: 39.468 € Funding: 375.000 € Duration: 2010-2013 Duration: 2010-2013 • Code: 8.06/5.02.3771 • Code: AC652 Title: Identification, setting-up and validation, using histolog- Title: Evaluación del efecto neuroprotector de un compues- ical and/or biochemical techniques, of functional synaptic/ to neuronal markers in several in vivo models of Alzheimer’s Principal Investigator: Javier Vitorica disease, neurodegeneration and aging CIBERNED’s collaboration: Yes Principal Investigator: Antonia Gutiérrez Pérez CIBERNED’s groups: G 411; G 415 CIBERNED’s collaboration: Yes Other CIBER’s collaboration: No CIBERNED’s groups: G 415; G 411 Type: Privado Other CIBER’s collaboration: No Funding Agency: Empresa Neuron Biopharm Type: Privado Funding: 57.060 € Funding Agency: Compañía Farmacéutica Sanofi Aventis Duration: 2010-2013 (Francia) Funding: 147.000 € • Code: PI12/01439 Duration: 2011-2013 Title: Oligómeros tóxicos del Abeta como agentes causan- tes de la disfunción del citoesqueleto y los procesos pro- • Code: PI12/01431 teolíticos en la Title: Oligómeros tóxicos del Abeta como agentes causan- Principal Investigator: Javier Vitorica tes de la disfunción del citoesqueleto y los procesos pro- CIBERNED’s collaboration: Yes teolíticos en la CIBERNED’s groups: G 415; 411 Principal Investigator: Antonia Gutiérrez Pérez Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: National CIBERNED’s groups: G 411; 415 Funding Agency: Instituto de Salud Carlos III Other CIBER’s collaboration: No Funding: 228.629 € Type: National Duration: 2013-2015 Funding Agency: Instituto de Salud Carlos III Funding: 224.999 € • Code: 8.06/5.02.3621 CDTI Duration: 2013-2015 Title: Evaluación in vivo de compueto de interes (Desarrollo

9494 Francisco Javier Vitorica Ferrández Group Group Francisco Wandosell Jurado

Centro de Biología Molecular “Severo Ochoa”, CSIC-UAM

Universidad Autónoma de Madrid 1 Nicolás Cabrera, nº 1, Cantoblanco 28049 Madrid (Spain)

Tel.: +34 911 964 561 / 4591 Program Fax: +34 911 964 420 [email protected]

Francisco Wandosell Jurado Ricardo Gargini Principal Investigator, CSIC Research Professor Postdoctoral Fellow Inés M. Antón Laura Mateos-Montejo CSIC Staff Scientist-Researcher Postdoctoral Fellow Mª José Benítez Moreno Maria Isabel Escoll Associate Professor PhD student María José Pérez Alvarez Irene Benito UAM Assistant Professor PhD student Juan José Garrido Jurado Marta Antón CSIC Staff Scientist-Researcher CIBERNED Technician Lara Ordóñez Gutiérrez CIBERNED Postdoctoral Fellow

95 Summary

Our group, “Molecular mechanisms of Neurodegeneration “, is generated from an established line in the CBM over several years. At present, this CIBERNED line is formally composed by three sub-groups (PIs: F. Wandosell (CBM), I.M. Antón (CNB) and J.J. Garrido, Cajal Institute).

We are interested in the analysis of the molecular mechanisms fired by processes neurodegenerative, trying to understand the key points of these processes; for a second attempt to design new regenerative alternatives, or to propose new therapeutic targets.

First, we are interested in the molecular mechanisms that regulate the generation and maintenance of the axonal polarity. This morphological polarity appears during development when the neuron differentiates and begins to extend an axon. Subsequently they “mature” and form their initial segment of the axon, key to generate neural action potentials.

Studies of several laboratories, including ours, have helped to identify some of the elements that control polarity, such that GSK3, control the polarity. Our work continues in this line, trying to identify upstream and downstream elements that essentials for this “morphogenetic process”. More recently we characterized the role of HSP90 in axonogenesis. (PI: Dr. Garrido).

In addition we wish to understand the molecular basis of the mechanism that regulates actin polymerization, an essential process underlying neuronal morphology and functions. We are interested in regulatory elements controlling actin polymerization such as WASP-WIP. We recently demonstrated that WIP regulated neurite extension, and this process is in part due to the regulation of mTORC1-S6K signaling (PI: Dr. I.M. Antón).

In addition, our studies of molecular mechanisms of degeneration we saw that the deregulation of GSK3 activity correlates with neuronal death in some models of neuronal apoptosis. We have seen that some neuroprotective elements as estradiol modulates elements, such as GSK3 and β-catenin, proteins shared by other signaling pathways like Wnt. We have verified recently in a model of Isquemia (pMCAO) that the administration of estradiol protects, used post-injury and that does recovering the PI3K-Akt-GSK3 signaling. In summary, we are focusing on track PI3K-Akt signalling.

In a second complementary analysis of neurodegeneración, we are studying the amiloidosis (Alzheimer type) using the double APP/PS1 transgenic mice; besides we have verified the genetic interaction that exists between the amyloid accumulation of and the cellular protein of prion, PrPc (PI: Dr. Wandosell).

Keywords

Neurodegeneration, Cellular Signaling, Neuroprotection, Estrogen, Alzheimer Disease, Neuritogenesis-Axonal Polarity , Cytos- keletal proteins, Actin

Publications

• Simon D, Herva ME, Benitez MJ, Garrido JJ, Rojo AI, Cuadrado A, et al. Dysfunction of the PI3K-Akt-GSK-3 pathway is a common feature in cell culture and in vivo models of prion disease. Neuropathology and applied neurobiology. 2013 Jun 6. • Del Puerto A, Wandosell F, Garrido JJ. Neuronal and glial purinergic receptors functions in neuron development and brain disease. Frontiers in cellular neuroscience. 2013;7:197. • Tapia M, del Puerto A, Pallas-Bazarra N, Puime A, Sanchez-Ponce D, Fronzaroli-Molinieres L, et al. GSK3 and β-catenin determines functional expression of sodium channels at the axon initial segment. Cellular and molecular life sciences. 2013;70(1):105-20. • Ordonez-Gutierrez L, Torres JM, Gavin R, Anton M, Arroba-Espinosa AI, Espinosa JC, et al. Cellular prion protein modulates β-amyloid deposition in aged APP/PS1 transgenic mice. Neurobiology of aging. 2013 Dec;34(12):2793-804. • Banon-Rodriguez I, Saez de Guinoa J, Bernardini A, Ragazzini C, Fernandez E, Carrasco YR, et al. WIP regulates persistence of cell migration and ruffle formation in both mesenchymal and amoeboid modes of motility. PloS one. 2013;8(8):e70364. • Benitez MJ, Sanchez-Ponce D, Garrido JJ, Wandosell F. Hsp90 activity is necessary to acquire a proper neuronal polarization. Biochim Biophys Acta. 2014 Feb;1843(2):245-52. • Varea O, Escoll M, Diez H, Garrido JJ, Wandosell F. Oestradiol signalling through the Akt-mTORC1-S6K1. Biochimica et

9696 Francisco Wandosell Jurado Group Francisco Wandosell Jurado Group 97

Biophysica Acta-Molecular Cell Research. 2013 May;1833(5):1052-64. • Bolos M, Spuch C, Ordonez-Gutierrez L, Wandosell F, Ferrer I, Carro E. Neurogenic effects of β-amyloid in the choroid plexus epithelial cells in Alzheimer’s disease. Cellular and molecular life sciences: CMLS. 2013 Aug;70(15):2787-97.

Research Projects • Code: AC130 CIBERNED’s groups: G 401; G 104; G 111; G 307; G 409; Title: Nanoparticles for therapy and diagnosis of Alzheimer G 412 disease Other CIBER’s collaboration: Yes (CIBERER) Principal Investigator: Massimo Masserini Type: CCAA CIBERNED’s collaboration: No Funding Agency: Comunidad de Madrid CIBERNED’s groups: Funding: 17.250 € Other CIBER’s collaboration: No Duration: 2012-2015 Type: International Funding Agency: Seventh Framework Programme (2008- • Code: PI2010/09 2012) NMP: Nanosciences, Nanotechnologies, Materials Title: BESAD-P: Biomarkers of Early Stages of Alzheimer and new Production Technologies:“Grant ref: CP-IP 212043- Disease-Prevention 2 NAD Principal Investigator: Isidre Ferrer Abizanda Funding: 760.000 € CIBERNED’s collaboration: Yes Duration: 2009-2013 CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; G 506; G 508; G 412 • Code: S2010_BMD Other CIBER’s collaboration: Yes (CIBERESP) Title: Redes de señalización y vías efectoras en modelos Type: Intramurales celulares y animales de enfermedades neurodegenerativas Funding Agency: CIBERNED Principal Investigator: José González Castaño Funding: 223.500 € CIBERNED’s collaboration: Yes Duration: 2011-2013

Program 2

Parkinson’s Disease, Huntignton’s Chorea and other Movement Disorders

Research Groups

Alberch Vie, Jordi______103 Lanciego Pérez, José Luis______154 Canela Campos, Enric Isidre______107 López Barneo, José______157 Ceña Callejo, Valentín______111 Lucas Lozano, José Javier______162 Cuadrado Pastor, Antonio______114 Moratalla Villalba, Rosario______165 del Río Fernández, José Antonio____ 117 Naranjo Orovio, José Ramón______168 Fariñas Gómez, Isabel______120 Obeso Inchausti, José Ángel______171 Fernández Ruiz, Javier______123 Pérez Castillo, Ana María______175 Fuentes Rodríguez, José Manuel____ 127 Pérez Tur, Jordi______178 García de Yébenes Prous, Justo_____ 130 Rodríguez Díaz, Manuel______180 García Verdugo, José Manuel______134 González Castaño, José______138 Sánchez Capelo, Amelia______183 Guzmán Pastor, Manuel______141 Tolosa Sarró, Eduardo______185 Iglesias Vacas, Teresa______145 Vicario Abejón, Carlos______191 Kulisevsky Bojarski, Jaime______148 Vila Bover, Miquel______194 Labandeira García, José Luis______151

Program 2

CIBERNED’s Program 2 brings together basic and clinical research groups with a mainly translational character, joining forces to study neurodegenerative diseases of different etiology that cause important problems in patient’s mobility. Among this group of diseases we can find, by decreasing prevalence: Parkinson’s disease, Huntington’s chorea, and different kinds of ataxias among other movement disorders.

Research Line 1: Parkinson’s Disease In Spain, 70,000 persons are affected by Parkinson’s disease (PD), which is characterized by neuronal loss, the formation of Lewy bodies and neurites in the substantia nigra and the consequent loss of striatal dopamine (DA). However, it is currently well known that PD is a multisystem neurodegenerative process, in which, as the neurodegenerative process evolves, many areas of the nervous system are affected and there exists a deficit in several neurotransmission and neuromodulation systems. Preventing and correcting DA deficit are still important goals, but nowadays they are not considered to be the ultimate challenge in PD. Within this area, it is considered of vital importance to make progress on defining the following: a) Key aspects related to the ethiopathogenesis in PD. b) Physiopatological mechanisms related to disease onset and progression. c) Development of symptomatic treatments, especially neuroprotective and curative. Thus, a truly translational research is sought, having the disease and the patients as the main targets. The main research topics in this line are the following: • Cognitive impairment and non-motor problems in PD • Biomarkers in Parkinson’s disease • Problems related to symptomatic treatment: diskynesias • New targets and novel therapeutic strategies in Parkinson’s disease • Circuits and physiopathology of basal ganglia • Neuronal stress, cell protection and death in Parkinson’s disease • Neurogenesis and cell therapy in Parkinson’s disease • Early biomarkers in Parkinson’s disease.

Research Line 2: Huntington’s Disease and ataxias This program also focuses on research into other movement disorders such as Huntington’s disease (HD) and ataxias. The prevalence of HD is much lower (about 4,500 patients in Spain) and it is characterized by the initial loss of spiny interneurons of the striatum. It is an autosomal dominant neurodegenerative pathology with complete penetrance produced by polyglutamine expansion in the huntingtin N-terminus. HD has no treatment and leads to death in around 10-20 years depending on the number of polyglutamines, the age of onset, some unknown environmental factors and the modulation of some genes, some of which have been located but remain unidentified. The estimated prevalence of HD is 10 cases for every 100,000 persons, which means 4,500 patients in Spain and about 50,000 in the European Union. Its social health cost is considerable because of the importance of cognitive and motor deficits, as well as the severe behavioral problems that patients present. There is a large number of studies with neuroprotective drugs which modify the supposed pathogenic mechanisms or that are used in other neurodegenerative diseases. These drugs include inhibitors of neuronal excitation, coenzymes of the respiratory chain, vitamins, antioxidants, co-adjuvants in energy production, etc. Some of these products offer encouraging results in experimental models of the disease but, unfortunately, they are not later confirmed in the clinic. The study of HD is important because is the best studied and most prevalent model of neurodegenerative diseases caused by triplet expansions, which also includes some ataxias. Discovering the pathogenic mechanisms of HD and finding an effective, either neuroprotective or curative, would have immediate implications on any of the other neurodegenerative pathologies caused by triplet expansions. The main research topics in this line are the following: • Identification of molecular and cellular basis of Huntington’s disease • Experimental studies in animal models of Huntington’s disease • Clinic, genetics and neuropathology of Huntington’s disease

101 Groups

Principal Investigator Institution

Alberch Vie, Jordi University of Barcelona Canela Campos, Enric Isidre University of Barcelona Ceña Callejo, Valentín University of Castilla-La Mancha, Albacete Cuadrado Pastor, Antonio Autonomous University of Madrid del Río Fernández, José Antonio Catalonian Institute of Bioengineering (IBEC), Barcelona Fariñas Gómez, Isabel University of Valencia Fernández Ruiz, Javier Complutense University of Madrid Fuentes Rodríguez, José Manuel University of Extremadura, Cáceres García de Yébenes Prous, Justo Ramón y Cajal University Hospital, Madrid* García Verdugo, José Manuel Cavanilles Institute, University of Valencia González Castaño, José Autonomous University of Madrid Guzmán Pastor, Manuel Complutense University of Madrid Iglesias Vacas, Teresa Institute of Biomedical Research “Alberto Sols” (CSIC-UAM), Madrid Kulisevsky Bojarski, Jaime Santa Creu i Sant Pau Hospital, Barcelona Labandeira García, José Luis University of Santiago de Compostela Lanciego Pérez, José Luis Foundation for Applied Medical Research, University of Navarra, Pamplona Andalusian Public Foundation for the Health Research Management, López Barneo, José Institute of Biomedicine of Seville (IBIS) Lucas Lozano, José Javier Center for Molecular Biology “Severo Ochoa” (CSIC-UAM), Madrid Moratalla Villalba, Rosario Cajal Institute CSIC, Madrid Naranjo Orovio, José Ramón National Center of Biotechnology, CSIC, Madrid Obeso Inchausti, José Ángel Foundation for Applied Medical Research, University of Navarra, Pamplona Pérez Castillo, Ana María Institute of Biomedical Research “Alberto Sols” (CSIC-UAM), Madrid Pérez Tur, Jordi Institute of Biomedicine of Valencia, CSIC Rodríguez Díaz, Manuel University of La Laguna, Tenerife Sánchez Capelo, Amelia Ramón y Cajal University Hospital, Madrid Tolosa Sarró, Eduardo Hospital Clínic of Barcelona Vicario Abejón, Carlos Cajal Institute CSIC, Madrid Vila Bover, Miquel Vall d’Hebron, Barcelona

* In the year 2013 has ceased her activity by retirement Justo García de Yébenes, being replaced as IP by Mª Angeles Mena

Program 2 is coordinated by Drs. José J. Lucas (Center for Molecular Biology, CSIC, Madrid) and Eduardo Tolosa (Hospital Clínic, Barcelona).

102102 Program 2 Group Jordi Alberch Vie

Departament de Biologia Cel·lular, Immunologia i Neurociències

Facultat de Medicina, Universitat de Barcelona 2 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS)

(Spain) Program

Tel.: +34 934 035 258 / 37252 Fax: +34 934 021 907 [email protected]

Jordi Alberch Gerardo García Principal Investigator, Full Professor University of Barcelona PhD student Josep M Canals Inés Guardia Associate Professor University of Barcelona PhD student Sílvia Ginés Andreas Mezger Assistant Professor University of Barcelona PhD student Esther Pérez Mónica Pardo Associate Professor University of Barcelona PhD student Ana Saavedra Mar Puigdellívol Juan de la Cierva Researcher IDIBAPS-CHDI PhD student Xavier Xifró Shiraz Tyebji University of Girona Lecturer University of Barcelona PhD student Raquel Martín Laura Vidal Postdoctoral Fellow, University of Barcelona University of Girona PhD student Verónica Brito Elena Álvarez IDIBAPS-CHDI Postdoctoral Fellow Master’s student Albert Giralt Andrea Comella University of Barcelona Postdoctoral Fellow Master’s student Andrés Míguez Jordi Creus IDIBAPS Postdoctoral Fellow Master’s student Phil Sanders Núria Suelves IDIBAPS-CHDI Postdoctoral Fellow Master’s student Marco Straccia Georgina Bombau IDIBAPS-CHDI Postdoctoral Fellow IDIBAPS-CHDI Technician Garikoitz Azkona Jordi Carrere IDIBAPS Postdoctoral Fellow IDIBAPS-CHDI Technician Rafael Alcalá Cristina Herranz University of Barcelona PhD student University of Barcelona Technician Marta Anglada Ana López University of Barcelona PhD student CIBERNED Technician Marta Cherubini Mª Teresa Muñoz IDIBAPS-CHDI PhD student University of Barcelona Technician

103 Summary

Our goal is to identify new molecular mechanisms affected by mutant huntingtin (mhtt) to develop treatments that stop or slow the progression of Huntington’s disease (HD). We have described different intracellular pathways that are altered in striatal and hippocampal neurons expressing mhtt, the modulation of which provides new therapeutic approaches. We have observed that an imbalance of p75(NTR)/TrkB levels occurs in striatal cells expressing mhtt, which likely contributes to increase their vulnerability (Brito et al., 2013). Autophagy levels are also differentially altered in mhtt expressing neurons. In addition, p62, a cargo receptor, accumulates in neuronal nuclei, where it co-localizes with mhtt inclusions, both in mouse models and HD patients (Rue et al., 2013,). A new mechanism that we have described in the pathophysiology of HD is the regulation of the NMDA receptor GluN3A. Mutant huntingtin modifies the expression of the levels of GluN3A. Thus the genetic deletion of GluN3A prevents synaptic degeneration and ameliorates motor deficits in HD mouse models (Marco et al., 2013). We have also studied the involvement of the activity of mitocondrial complex II in the neurodegenerative process activated by mhtt (Damiano et al., 2013).

Mouse models of HD, as occurs in patients, show cognitive deficits. We have shown that treatment with papaverine or sildenafil (inhibitors of phosphodiesterases 10 and 5, respectively) improves cognitive function in an animal model of HD (Giralt et al., 2013; Saavedra et al., 2013). We have also participated in other studies in collaboration with other groups of CIBERNED: (1) We have shown that methylation of ADORA2A gene, encoding the A2A adenosine receptor, is altered in striatal neurons expressing mhtt contributing to the decrease in receptor protein levels (Villar-Menendez et al., 2013); (2) The study of neuroprotective mechanism of the induction of ATF5 by endoplasmic reticulum stress (Torres-Peraza et al., 2013).

Keywords

BDNF, BDNF receptors, Cdk5, Cognition, Motor control, PKA/STEP, Neurogenesis, Regenerative medicine

Publications

• Rue L, Lopez-Soop G, Gelpi E, Martinez-Vicente M, Alberch J,Perez-Navarro E. Brain region- and age-dependent dysregulation of p62 and NBR1 in a mouse model of Huntington’s disease. Neurobiology of Disease. 2013;52:219-28. • Saavedra A, Giralt A, Arumi H, Alberch J, Perez-Navarro E. Regulation of hippocampal cGMP levels as a candidate to treat cognitive deficits in Huntington’s disease. PloS one. 2013 Sep 5;8(9):e73664. • Torres-Peraza JF, Engel T, Martin-Ibanez R, Sanz-Rodriguez A, Fernandez-Fernandez MR, Esgleas M, et al. Protective neuronal induction of ATF5 in endoplasmic reticulum stress induced by status epilepticus. Brain. 2013 Apr;136(Pt 4):1161-76. • Barroso E, del Valle J, Porquet D, Vieira Santos AM, Salvado L, Rodriguez-Rodriguez R, et al. Tau hyperphosphorylation and increased BACE1 and RAGE levels in the cortex of PPARβ/δ-null mice. Biochimica et Biophysica Acta-Molecular Basis Of Disease. 2013 Aug;1832(8):1241-8. • Azkona G, Sagarduy A, Aristieta A, Vazquez N, Zubillaga V, Ruiz-Ortega JA, et al. Buspirone anti-dyskinetic effect is correlated with temporal normalization of dysregulated striatal DRD1 signalling in L-DOPA-treated rats. Neuropharmacology. 2014 Apr;79:726-37. • Giralt A, Sanchis D, Cherubini M, Gines S, Canas X, Comella JX, et al. Neurobehavioral characterization of Endonuclease G knockout mice reveals a new putative molecular player in the regulation of anxiety. Experimental neurology. 2013 Sep;247:122-9. • Rue L, Alcala-Vida R, Lopez-Soop G, Creus-Muncunill J, Alberch J, Perez-Navarro E. Early Down-Regulation of PKCδ as a Pro- Survival Mechanism in Huntington’s Disease. Neuromolecular medicine. 2013 Jul 30. • Anglada-Huguet M, Xifro X, Giralt A, Zamora-Moratalla A, Martin ED, Alberch J. Prostaglandin E2 EP1 Receptor Antagonist Improves Motor Deficits and Rescues Memory Decline in R6/1 Mouse Model of Huntington’s Disease. Molecular neurobiology. 2013 Nov 7. • Giralt A, Saavedra A, Carreton O, Arumi H, Tyebji S, Alberch J, et al. PDE10 inhibition increases GluA1 and CREB phosphorylation and improves spatial and recognition memories in a Huntington’s disease mouse model. Hippocampus. 2013 Aug;23(8):684-95. • Pascual-Garcia M, Rue L, Leon T, Julve J, Carbo JM, Matalonga J, et al. Reciprocal negative cross-talk between liver X receptors (LXRs) and STAT1: effects on IFN-γ-induced inflammatory responses and LXR-dependent gene expression. J Immunol.

104104 Jordi Alberch Vie Group Jordi Alberch Vie Group 105

2013;190(12):6520-32. • Brito V, Puigdellivol M, Giralt A, del Toro D, Alberch J, Gines S. Imbalance of p75(NTR)/TrkB protein expression in Huntington’s disease: implication for neuroprotective therapies. Cell death & disease. 2013 Apr 184:e595. • Damiano M, Diguet E, Malgorn C, D’Aurelio M, Galvan L, Petit F, et al. A role of mitochondrial complex II defects in genetic models of Huntington’s disease expressing N-terminal fragments of mutant huntingtin. Human molecular genetics. 2013 Oct 1;22(19):3869-82. • Marco S, Giralt A, Petrovic MM, Pouladi MA, Martinez-Turrillas R, Martinez-Hernandez J, et al. Suppressing aberrant GluN3A expression rescues synaptic and behavioral impairments in Huntington’s disease models. Nature medicine. 2013 Aug;19(8):1030-8. • Villar-Menendez I, Blanch M, Tyebji S, Pereira-Veiga T, Albasanz JL, Martin M, et al. Increased 5-methylcytosine and decreased 5-hydroxymethylcytosine levels are associated with reduced striatal A2AR levels in Huntington’s disease. Neuromolecular medicine. 2013 Jun;15(2):295-309. • Marin C, Bonastre M, Mengod G, Cortes R, Giralt A, Obeso JA, et al. Early L-dopa, but not pramipexole, restores basal ganglia activity in partially 6-OHDA-lesioned rats. Neurobiology of disease. 2013 Dec 24.

Research Projects

• Code: SAF2012-39142 neuronal por nuevos métodos de liberación de BDNF en Title: Cdk5 como diana terapéutica para el tratamiento modelos de enfermedad de Huntington de los déficits sinápticos y cognitivos en la enfermedad de Principal Investigator: Jordi Alberch Huntington CIBERNED’s collaboration: No Principal Investigator: Yeslvia Ginés CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: Ministerio de Ciencia e Innovación Type: National Funding: 230.000 € Funding Agency: Ministerio de Economia y Competitividad Duration: 2012-2014 Funding: 152.100 € Duration: 2013 • Code: PI10/01072 Title: Regulación de la vía PKA como estrategia terapéutica • Code: CIVP16A1842 para la enfermedad de Huntington Title: Cdk5: Una diana terapéutica para el tratamiento Principal Investigator: Esther Pérez Navarro de los déficits motores y cognitivos en la enfermedad de CIBERNED’s collaboration: No Huntington CIBERNED’s groups: Principal Investigator: Yeslvia Ginés Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: National CIBERNED’s groups: Funding Agency: Ministerio de Ciencia e Innovación, Insti- Other CIBER’s collaboration: No tuto de Salud Carlos III Type: National Funding: 148.830 € Funding Agency: Fundación Ramón Areces Duration: 2011-2013 Funding: 84.500 € Duration: 2013 • Code: A4530 Title: Stem Cell Differentiation JSC – Joint collaborative ef- • Code: 2009SGR-00326 fort between the IDIBAPS (Barcelona), the University of Mi- Title: Group de Fisiopatología de enfermedades neurode- lano and the University of Cardiff generativas Principal Investigator: Josep M. Canals Principal Investigator: Jordi Alberch CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: International Type: National Funding Agency: CHDI Foundation Inc. Funding Agency: AGAUR, Generalitat de Catalunya Funding: 750.000 € Funding: 45.760 € Duration: 2012-2013 Duration: 2009-2014 • Code: RD12/0019/0008 • Code: SAF2011-29507 Title: RETIC de Terapia Celular Title: Regulación de la plasticidad sináptica y la disfunción Principal Investigator: Isabel Fariñas (coordinador de red: Jose Maria Moraleda) • Code: PI2013_08-1 CIBERNED’s collaboration: Yes Title: La dinámica mitocondrial y mitofagia como dianas te- CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; rapéuticas en las Enfermedades de Parkinson y Huntington G 607 Principal Investigator: Eduardo Soriano Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: National CIBERNED’s groups: G 301; G 207; G 410; G 109; G 408 Funding Agency: Fondo de Investigaciones Sanitarias Other CIBER’s collaboration: No Funding: 296.700 € Type: Intramurales Duration: 2013-2016 Funding Agency: CIBERNED Funding: 300.000 € • Code: PI2010/05 Duration: 2013-2015 Title: Generación de un modelo neuronal dopaminérgico a • Code: RD12/0019/0011 partir de células madre pluripotentes inducidas de pacien- Title: RETIC de Terapia Celular tes con enfermedad de Parkinson asociada a mutaciones Principal Investigator:Xavier Navarro Acebes (coordinador en el gen LRRK2 de red: Jose Maria Moraleda) Principal Investigator: Eduardo Tolosa Sarró CIBERNED’s collaboration: Yes CIBERNED’s collaboration: Yes CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; CIBERNED’s groups: G 207; G 301; G 105; G 109 G 607 Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Intramurales Type: National Funding Agency: CIBERNED Funding Agency: Instituto de Salud Carlos III Funding: 262.500 € Funding: 180.000 € Duration: 2010-2013 Duration: 2013-2016

PhD Dissertations

• Author: Marta Anglada Huguet Title: Characterization of mechanisms underlying neuronal survival and plasticity in Huntington’s disease Date: 22 July 2013 Supervisor: Jordi Alberch Vie

106106 Jordi Alberch Vie Group Group Enric Isidre Canela Campos

Departamento de Bioquímica y Biología Molecular

Facultad de Biología. Universidad de Barcelona 2 Diagonal 643, planta -2

08028 Barcelona (Spain) Program Tel.: +34 934 021 211 Fax: +34 934 021 559 [email protected]

Enric Isidre Canela Campos Jasmina Jiménez Cano Principal Investigator Technician Antonio Cortés Tejedor Isaac Naval Macabuhay Scienific staff PhD student Carmen Lluís Biset Daniel Farré Pérez Scienific staff PhD student Josefa Mallol Montero Berta Lorenzo Boluda Scienific staff PhD student Vicent Casadó Burillo Mireia Medrano Moya Scienific staff PhD student Peter J. McCormick David Aguinaga Andrés Postdoctoral Fellow PhD student Gemma Navarro Brugal Mar Rodríguez Ruiz Postdoctoral Fellow PhD student Estefanía Moreno Guillén Verònica Casadó Anguera Postdoctoral Fellow PhD student David Moreno Delgado Edgar Angelats Canals Postdoctoral Fellow Master’s student Paola Gasperini Postdoctoral Fellow

107 Summary

Our area of interest has been the G-protein coupled receptors (GPCR) in the CNS, specially their ability to form homo- and heterooligomers. We have focused on discovering receptor heteromers as new targets for treatment of CNS disorders.

Most relevant results in this period are:

• We have identified regions of the enzyme adenosine deaminase (ADA) responsible for modulating both its catalytic activity and its ability to modulate agonist binding to A1 and A2A adenosine receptors (A1R and A2AR), using alanine scanning mutagenesis of various amino acid stretches, selected through in silico docking experiments. The combination of computational and in vitro experiments shows that the structural gate to the catalytic site is important to maintain both functions of ADA. We propose that it is the ADA open form, but not the closed one, that is responsible for the functional interaction with A1R and A2AR. • Using bioluminescence resonance energy transfer and proximity ligation assays, we obtained the first direct evidence that A1Rs form homomers not only in cell cultures but also in brain cortex. By radioligand binding experiments in the absence or in the presence of the A1Rs allosteric modulator, adenosine deaminase, and by using the two-state dimer receptor model to fit binding data, we demonstrated that caffeine binding to one protomer increases the agonist affinity for the other protomer in the A1R homomer. This pharmacological property can explain the biphasic effects reported at low and high concentration of caffeine on locomotor activity. • We demonstrated that inhibition of cGMP-preferring PDE is involved in the locomotor activating effects of the acute administration of paraxanthine. These results demonstrate a unique psychostimulant profile of paraxanthine, which might contribute to the reinforcing effects of caffeine in humans. • Using primary cultures of rat astrocytes, we showed that a further level of regulation of GABA uptake occurs via modulation of the GATs by A1R and A2AR. This regulation occurs through A1R–A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron–glia–neuron) synapse. • We described how, by combining BRET and FRET in the Sequential BRET-FRET (SRET) technique, heteromers formed by three different proteins can be identified. Using this methodology we described the heteromerization between adenosine A2A, dopamine D2, and cannabinoids CB1 receptors in living cells. • Exploring the effect of cocaine on dopamine D2 receptors function, we presented evidence of sigma1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors can complex with sigma1 receptors. We demonstrated that cocaine, by binding to sigma1-D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. These data shed light on the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance between the direct (D1 containing) and indirect (D2 containing) pathways in basal ganglia. • We also studied the mechanisms underlying the neuroprotective effects of cannabidiol using a brain injury model in newborn pigs. By bioluminescence resonance energy transfer we demonstrated that CB2 and 5HT1A receptors may form heteromers in living HEK-293T cells. Our findings demonstrated that cannabidiol exerts robust neuroprotective effects in vivo in this model, modulating excitotoxicity, oxidative stress and inflammation, and that both CB2 and 5HT1A receptors are implicated in these effects.

Keywords

Allosteric interactions, Caffeine, Cocaine, G-protein coupled receptors, Multifunctional proteins, Receptor oligomerization, Sigma-1 receptor, Two-state dimer receptor model

108108 Enric Isidre Canela Campos Group Enric Isidre Canela Campos Group 109

Publications

• Cristovão-Ferreira S, Navarro G, Brugarolas M, Perez-Capote K, Vaz SH, Fattorini G, et al. A1R-A2AR heteromers coupled to Gs and G i/0 proteins modulate GABA transport into astrocytes. Purinergic signalling. 2013 Sep;9(3):433-49. • Navarro G, Moreno E, Bonaventura J, Brugarolas M, Farre D, Aguinaga D, et al. Cocaine inhibits dopamine D2 receptor signaling via sigma-1-D2 receptor heteromers. PloS one. 2013;8(4):e61245. • Gracia E, Moreno E, Cortes A, Lluis C, Mallol J, McCormick PJ, et al. Homodimerization of adenosine A₁ receptors in brain cortex explains the biphasic effects of caffeine. Neuropharmacology. 2013 Aug;71:56-69. • Pazos MR, Mohammed N, Lafuente H, Santos M, Martinez-Pinilla E, Moreno E, et al. Mechanisms of cannabidiol neuroprotection in hypoxic-ischemic newborn pigs: role of 5HT(1A) and CB2 receptors. Neuropharmacology. 2013 Aug;71:282-91. • Orru M, Guitart X, Karcz-Kubicha M, Solinas M, Justinova Z, Barodia SK, et al. Psychostimulant pharmacological profile of paraxanthine, the main metabolite of caffeine in humans. Neuropharmacology. 2013 Apr;67:476-84. • Bonaventura J, Rico AJ, Moreno E, Sierra S, Sanchez M, Luquin N, et al. l-DOPA-treatment in primates disrupts the expression of A2A adenosine-CB1 cannabinoid-D2 dopamine receptor heteromers in the caudate nucleus. Neuropharmacology. 2013 Nov 11;79C:90-100. • Pinna A, Bonaventura J, Farre D, Sanchez M, Simola N, Mallol J, et al. l-DOPA disrupts adenosine A2A-cannabinoid CB1- dopamine D2 receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: Biochemical and behavioral studies. Exp Neurol. 2014 Jan 9;253C:180-91.

Research Projects

• Code: SAF2011-23813 Funding: 44.720 € Title: Heterómeros de receptores acoplados a proteína g Duration: 2010-2013 como dianas terapéuticas de la adicción a drogas Principal Investigator: Enric Isidre Canela Campos • Code: HHSN271201300384P CIBERNED’s collaboration: No Title: Performance of BRET and signaling experiments to CIBERNED’s groups: detect dopamine D4 and adrenergic receptors heteromer- ization Other CIBER’s collaboration: No Principal Investigator: Enric I. Canela Campos Type: National CIBERNED’s collaboration: No Funding Agency: Ministerio de Ciencia e Innovación CIBERNED’s groups: Funding: 242.000 € Other CIBER’s collaboration: No Duration: 2012-2014 Type: International • Code: SAF2010-18472 Funding Agency: NIDA/NIH (National Institute on Drug Title: La modulación de los heterómeros entre receptores Abuse) de dopamina y sigma-1 como nuevos mecanismos de ac- Funding: 12.000 € ción de la cocaína Duration: 2013 Principal Investigator: Peter McCormick • Code: FJL-01/01/2013 CIBERNED’s collaboration: No Title: Validation of mGluR1/5-Histamine 3 receptor het- CIBERNED’s groups: erodimers as novel targets to treat Fragile X Syndrome Other CIBER’s collaboration: No Principal Investigator: Peter Joseph McCormick Type: National CIBERNED’s collaboration: No Funding Agency: Ministerio de Ciencia e Investigación CIBERNED’s groups: Funding: 121.000 € Other CIBER’s collaboration: No Duration: 2011-2013 Type: International Funding Agency: Fondation Jerôme Lejeune • Code: 2009-SGR-12 Funding: 20.000 € Title: Neurobiología Molecular Duration: 2013-2015 Principal Investigator: Enric I. Canela Campos CIBERNED’s collaboration: No • Code: PI2011/02 CIBERNED’s groups: Title: Inicio y progresión de la Enfermedad de Parkinson. Other CIBER’s collaboration: No Vulnerabilidad de la vía nigroestriada, eventos en origen y Type: CCAA destino Funding Agency: Generalitat de Catalunya: Ajuts a Grups Principal Investigator: Jose Angel Obeso Inchausti de recerca consolidats CIBERNED’s collaboration: Yes CIBERNED’s groups: G 205; G 201; G 104; G 208; G 203; ciones de receptores cannabinoides en poliglutaminopatías G 404; G 206 Principal Investigator: Manuel Guzmán Pastor Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: Intramurales CIBERNED’s groups: G 201; G 303; G 304; G 305 Funding Agency: CIBERNED Other CIBER’s collaboration: No Funding: 362.000 € Type: Intramurales Duration: 2011-2013 Funding Agency: CIBERNED Funding: 50.000 € • Code: CIBERNED 2013/05 Duration: 2013-2015 Title: Identificación y caracterización molecular de subpobla-

PhD Dissertations

• Author: Víctor Casanova Güell na terapéutica del VIH Title: La adenosina desaminasa y las alfa defensinas como Date: 2 December 2013 moduladores de respuestas inmunitarias contra el VIH Supervisor: Josefa Mallol Montero Date: 1 March 2013 Supervisor: Enric Isidre Canela Campos • Author: Marc Brugarolas Campillos Title: Identification and characterization of A2A hetero- • Author: Isaac Naval Macabuhay mers in the Central Nervous System Title: Evaluación de la adenosina desaminasa como molé- Date: 20 December 2013 cula coestimuladora de la actividad linfocitaria en una vacu- Supervisor: Vicente Casadó Burillo

110110 Enric Isidre Canela Campos Group Group Valentín Ceña Callejo

Unidad Asociada Neurodeath

Universidad de Castilla-La Mancha 2 Facultad de Medicina

Avenida Almansa, 14 Program 02006 Albacete (Spain) Tel.: +34 680 222 322 [email protected]

Valentín Ceña Callejo Maricarmen Mateo Gómez Principal Investigator, Full Professor Lab Technician Inmaculada Posadas Vanesa Guijarro Assistan Professor, PhD Lab Technician Beatriz López Hernández Elena Galera PhD student Lab Technician Ana Belén García Sáez Lab Technician

111 Summary

The research of the Neurodeath group is focused on the use of nanoparticles to transfect siRNA into different cell types. Most drug targets are proteins that are inhibited or modulated by drugs. One limitation to drug effectivity is the lack of selectivity on similar proteins (receptor types and subtypes, ionic channels, isoenzymes, etc.) producing an important number of side effects that limit treatment efficiency. An innovative therapeutic approach, that solves this limitation, is the use of siRNA to remove selectively certain proteins whose function is modified in several illness which are difficult to treat because the lack of effective therapies, drug toxicity and/or the presence of resistance to treatment (cancer, TB, etc.). This approach will produce, in a short period of time, new generations of drugs, based on siRNA, with the potential to revolutionize the therapeutics. Our group has focused during this year in the development of dendrimers to transfect neuronal cultures with the idea of transfecting in vivo in a near future.

Keywords

Gene therapy, Dendrimers, Neurodegeneration, siRNA, Transfection

Publications

• Klajnert B, Peng L, Cena V. Dendrimers in Biomedical Applications. RSC Publishing, 2013. • Posadas I, Lopez-Hernandez B, Cena V. Nicotinic receptors in neurodegeneration. Current neuropharmacology. 2013 May;11(3): 298-314. • Perez-Martinez FC, Ocana AV, Pavan GM, Danani A, Cena V. Dendrimers as vectors for small interfering RNA transfection in the nervous system. In: Klajnert B, Peng L, Cena V, Eds. Dendrimers in Biomedical Applications. RSC Publishing, 2013. • Lopez-Herradon A, Portal-Nunez S, Garcia-Martin A, Lozano D, Perez-Martinez FC, Cena V, et al. Inhibition of the canonical Wnt pathway by high glucose can be reversed by parathyroid hormone-related protein in osteoblastic cells. Journal of cellular biochemistry. 2013 Aug;114(8):1908-16. • Perez-Carrion MD, Cena V. Knocking down HMGB1 using dendrimer-delivered siRNA unveils its key role in nmda-induced autophagy in rat cortical neurons. Pharmaceutical research. 2013 Oct;30(10):2584-95. • Guerra J, Rodrigo AC, Merino S, Tejeda J, Garcia-Martinez JC, Sanchez-verdu P, et al. PPV-PAMAM Hybrid dendrimers: Self Assembly and stabilization of gold nanoparticles. Macromolecules. 2013;46(18):7316-24.

Research Projects • Code: POII10-0274-3182 Type: Europeo Title: Dendrímeros como base de la terapia génica en la te- Funding Agency: EURONANOMED-EU rapéutica de la osteopenia asociada a la diabetes mellitus Funding: 0 € Principal Investigator: Valentín Ceña Duration: 2010-2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: BFU2011-30161-C02-01 Other CIBER’s collaboration: No Title: Nanoparticulas no virales como vectores para siRNA Type: CCAA en células tumorales y neuronas Funding Agency: JCCM-Educacion Principal Investigator: Valentín Ceña Funding: 45.000 € CIBERNED’s collaboration: No Duration: 2010-2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: DENANORNA Type: National Title: Dendrimers as nanovectors for siRNA delivery in gene Funding Agency: MINECO therapy Funding: 156.370 € Principal Investigator: Ling Peng Duration: 2012-2014 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI2011/03 Other CIBER’s collaboration: No Title: Generación de neuronas dopaminérgicas a partir de

112112 Valentín Ceña Callejo Group Valentín Ceña Callejo Group 113

células somáticas de pacientes parkinsonianos con fallo Other CIBER’s collaboration: No cognitivo Type: Intramurales Principal Investigator: Rosario Moratalla Villalba Funding Agency: CIBERNED CIBERNED’s collaboration: Yes Funding: 300.000 € CIBERNED’s groups: G 204; G 106; G 202; G 110; G 108 Duration: 2011-2013

PhD Dissertations

• Author: Maria Dolores Pérez Carrión Date: 19 June 2013 Title: Papel de la autofagia en la muerte excitotóxica Supervisor: Valentín Ceña Callejo Date: 12 June 2013 Supervisor: Valentín Ceña Callejo • Author: Blanca Carrión Tébar Title: Protección por atorvastatina y aliskiren de las células • Author: Silvia Monteagudo Abietar mesoteliales de rata tratadas con soluciones de diálisis Title: Uso de nanopartículas terapéuticas en Cáncer de Date: 13 November 2013 Próstata y en la Osteoporosis Diabética Supervisor: Valentín Ceña Callejo Group Antonio Cuadrado Pastor

Departamento de Bioquímica e Instituto de Investigaciones Biomédicas

2 Facultad de Medicina, Universidad Autónoma de Madrid Arzobispo Morcillo, 4 28029 Madrid (Spain) Program Tel.: +34 915 854 383 Fax: +34 915 854 401 [email protected]

Antonio Cuadrado Pastor Luis Miguel Gutiérrez González Principal Investigator, UAM Molecular Biosciences Master’s student, UAM Isabel Lastres Becker Anna Nelke Ramón y Cajal Researcher, UAM Molecular Biosciences Master’s student, UAM Ana Isabel Rojo Sanchís Abel Santamaría del Ángel CIBERNED Postdoctoral Fellow Research visitor, Patricia Rada Llano Instituto Nacional de Neurología de México CIBERNED Postdoctoral Fellow Marta Pajares Cabetas Ángel Juan García Yagüe Final Year of Biochemistry Student, UAM CIBERNED Postdoctoral Fellow Natalia Jiménez Moreno Julio César Tobón Velasco Student in business practices PhD student, Chair Issac Costero UAM/UNAM

114 Antonio Cuadrado Pastor Group 115

Summary

Oxidative and inflammatory stress and proteinopathy are key elements in the pathology of several neurodegenerative diseases, including Parkinson’s (PD) and Alzheimer’s disease (AD). Our group is studying the regulation of cellular defenses represented by the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) and one of its target genes encoding HO-1 (heme oxygenase-1). During this year, we have analyzed the protective role of NRF2 in brain degeneration at three levels:

1) Characterization of the connection between GSK-3 and NRF2 in response to WNT signaling. Signaling by members of the WNT family is central to the acquisition and maintenance of dopaminergic neuronal differentiation. We observed that NRF2 is part of a protein complex with Axin1, which marks NRF2 to degradation by the GSK-3/β-TrCP pathway, similarly to β-catenin. Treatment with WNT-3A disrupts this complex and releases NRF2 to exert its transcriptional function. These observations have been very important to obtaining an international grant from CoEN (Centres of Excellence in Neurodegeneration) that we coordinate. 2) Development of a new mouse model of tauopathy based on stereotaxic delivery in hippocampus of an adeno-associated vector expressing human TAU P301L protein (rAAV6-TAUP301L). An advantage of this model is that it can be combined with different transgenic backgrounds, in our case NRF2-deficiency. We have described a communication between damaged neurons and microglia that serves to repair the brain parenchyma and is based on the release of the chemokine Fractalkina by the neuron and activation of a signaling pathway leading to microglial activation of NRF2. The main results of the study in mice have been validated in postmortem samples from patients with AD and progressive supranuclear palsy. 3) We have continued with the development of a new mouse model of AD carrying the deficiency in NRF2 together with APPV717I and TAUP301L and have obtained all bigenic mice (TAU/Nrf2-KO; APP/Nrf2-KO) and trigenic mice (TAU/APP/Nrf2- KO) which will be a very powerful tool to study the neuroprotective role of NRF2 and its therapeutic validation. 4) The transcription factor NURR1 is fundamental in acquisition and maintenance of the dopaminergic phenotype, in modulating inflammation and in cytoprotection. In this year, we reported that NURR1 contains a nuclear localization signal (NLS) within its DNA binding domain and two nuclear export signals rich in leucine (NES) in its ligand binding pseudo-domain. These signals jointly regulate NURR1 movement between cytoplasm and nucleus. Oxidative stress, caused by hydrogen peroxide or sodium arsenite, promotes NURR1 export and decreases the expression of the genes that NURR1 regulates, with the consequent loss of dopaminergic function.

Together these results identify NURR1 and NRF2 as new therapeutic targets to slow progression of neurodegenerative diseases associated with chronic low-grade inflammation, oxidative stress and proteinopathy. In line with our trajectory on the study of NRF2, we have initiated collaborations with Biopharmaceutical companies to validate novel compounds that activate NRF2 in preclinical models of Parkinson’s disease.

Keywords

Oxidative stress, Neuroinflammation, Proteinopathy, Neuroprotection, NRF2, WNT, GSK-3, TAU

Publications

• Drost J, Nonis D, Eich F, Leske O, Damrath E, Brunt ER, et al. Ataxin-2 modulates the levels of Grb2 and SRC but not ras signaling. Journal of molecular neuroscience: MN. 2013 Sep;51(1):68-81. • Garcia-Yague AJ, Rada P, Rojo AI, Lastres-Becker I, Cuadrado A. Nuclear import and export signals control the subcellular localization of Nurr1 in response to oxidative stress. The Journal of biological chemistry. 2013 Jan 2;288(8):5506-17. • Martin-de-Saavedra MD, Budni J, Cunha MP, Gomez-Rangel V, Lorrio S, Del Barrio L, et al. Nrf2 participates in depressive disorders through an anti-inflammatory mechanism. Psychoneuroendocrinology. 2013 Oct;38(10):2010-22. • Mobasher MA, Gonzalez-Rodriguez A, Santamaria B, Ramos S, Martin MA, Goya L, et al. Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity. Cell death & disease. 2013 May 9;4:e626. • Parada E, Buendia I, Leon R, Negredo P, Romero A, Cuadrado A, et al. Neuroprotective effect of melatonin against ischemia is partially mediated by alfa-7 nicotinic receptor modulation and HO-1 overexpression. Journal of pineal research. 2013 Dec 18. • Lastres-Becker I, Innamorato NG, Jaworski T, Rabano A, Kügler S, Van Leuven F, et al. Fractalkine activates NRF2/NFE2L2 and heme oxygenase 1 to restrain tauopathy-induced microgliosis. Brain. 2013 Nov 25. • Simon D, Herva ME, Benitez MJ, Garrido JJ, Rojo AI, Cuadrado A, et al. Dysfunction of the PI3K-Akt-GSK-3 pathway is a common feature in cell culture and in vivo models of prion disease. Neuropathology and applied neurobiology. 2013 Jun 6.

Research Projects • Code: PI2011/01 • Code: SAF2010-17822 Title: El factor de transcripción Nrf2 como nueva diana ter- Title: Papel del factor de transcripción Nrf2 en diferencia- apéutica para la enfermedad de Parkinson ción neuronal, neuroprotección y neuroinflamación Principal Investigator: Antonio Cuadrado Pastor Principal Investigator: Antonio Cuadrado Pastor CIBERNED’s collaboration: Yes CIBERNED’s collaboration: No CIBERNED’s groups: G 101; G102; G103; G 505; G 209 CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Intramurales Type: National Funding Agency: CIBERNED Funding Agency: MICINN Funding: 390.000 € Funding: 314.601 € Duration: 2011-2013 Duration: 2011-2013

116116 Antonio Cuadrado Pastor Group Group José Antonio del Río Fernández

Molecular and Cellular Neurobiotechnology

Institute for Bioengineering of Catalonia (IBEC) 2 Baldiri Reixac, 10-15

08028 Barcelona (Spain) Program Tel.: +34 934 035 923 / 20296 [email protected] [email protected]

José Antonio del Río Fernández Sara Nocentini Principal Investigator PhD student Rosalina Gavín Marín Cristina Vergara Paños Senior Researcher PhD student Vanessa Gil Fernández Diego Reginensi Espinoza Postdoctoral Fellow PhD student Ariadna Pérez Balaguer Ágata Mata Rodríguez Postdoctoral Fellow PhD student Oscar Seira Oriach Miriam Segura Feliú Postdoctoral Fellow Lab Technician Patricia Carulla Martí PhD student

117 Summary

Role of PrPc in the nervous system: control of stem cells proliferation and differentiation. We have demonstrated, in collaboration with other groups, the role of the cellular prion protein (PrPc) in neural stem cell proliferation and differentiation, in particular hippocampal neuronal progenitors (Bribian et al., 2012) and during the last year neuroblastoma cells using -omics techniques. We determined the PrPC-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrPC over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrPC silencing slows down cell cycle progression. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) activity by PrPC in the plasma membrane, where the two proteins interact in a multimeric complex. We also described how PrPC over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway (Llorens et al., 2013a). This data has also been reviewed in (Llorens et al., 2013b). In addition, we investigated whether β-amyloid accumulation may affect prion infectivity and, conversely, whether different amounts of PrPc may affect β-amyloid accumulation (funded by BESAD-P). For this purpose, we used the APPswe/PS1dE9 mouse line, a common model of Alzheimer’s disease, crossed with mice that either overexpress (Tga20) or that lack prion protein (knock-out) to generate mice that express varying amounts of prion protein and deposit β-amyloid. On these mouse lines, we investigated the influence of each protein on the evolution of both diseases. Our results indicated that although the presence of APP/PS1 and β-amyloid accumulation had no effect on prion infectivity, the accumulation of β-amyloid deposits was dependent on PrP(c), whereby increasing levels of prion protein were accompanied by a significant increase in β-amyloid aggregation associated with aging. (Ordóñez-Gutierrez et al., 2013). Lastly, we determined that central domain (CD) peptide of the PrPc is neurotoxic. In our studies we used Biochemical, Transmission Electron Microscopy and Atomic Force Microscopy experiments to demonstrate that the CD peptide is able to activate caspase-3 and disrupt the cell membrane, leading to neuronal death (Vilches et al., 2013). Lastly, we have determined the different expresión of PrPc in CJD patients and Parkinson disease and FTD (Llorens et al., 2013e). In addition, the group described in an invited review the role of GSK3 during neural development and regeneration (Seira and Del Río, 2013).

Finally, group members have collaborated on the study that identificates Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells (La Torre et al., 2013); the function of isoMIRs as functional variants of particular miRNAs. These isoMIRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression (Llo- rens et al., 2013c). In addition, from our studies we propose that the use of global genomic miRNA cross-validation derived from high throughput technologies can be used to generate more reliable datasets inferring more robust networks of co-regulated predicted miRNA target genes (Llorens et al., 2013d). Furthermore, we also collaborate to determine that the endogenous phosphodiesterase 7 is involved in multiple sclerosis (Bribian et al., 2013).

Keywords

Cell proliferation, PrPc-dependent genomic signature, Beta-amyloid, miRNA

Publications

• Riggio C, Nocentini S, Catalayud MP, Goya GF, Cuschieri A, Raffa V, et al. Generation of magnetized olfactory ensheathing cells for regenerative studies in the central and peripheral nervous tissue. International journal of molecular sciences. 2013 May 24;14(6):10852-68. • Llorens F, Ansoleaga B, Garcia-Esparcia P, Zafar S, Grau-Rivera O, Lopez-Gonzalez I, et al. PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2. Prion. 2013 Sep 18.;7(5):383-93. • Llorens F, Carulla P, Villa A, Torres JM, Fortes P, Ferrer I, et al. PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells. Journal of neurochemistry. 2013 Oct;127(1):124-38. • Llorens F, Banez-Coronel M, Pantano L, Del Rio JA, Ferrer I, Estivill X, et al. A highly expressed miR-101 isomiR is a functional silencing small RNA. BMC genomics. 2013 Feb 15;14(1):104. • Llorens F, Ferrer I, Del Rio JA. Gene Expression Resulting from PrPC Ablation and PrP C Overexpression in Murine and Cellular Models. Molecular neurobiology. 2013 Aug 16;49(1):413-23. • Vilches S, Vergara C, Nicolas O, Sanclimens G, Merino S, Varon S, et al. Neurotoxicity of prion peptides mimicking the central domain of the cellular prion protein. PloS one. 2013;8(8):e70881. • Llorens F, Hummel M, Pantano L, Pastor X, Vivancos A, Castillo E, et al. Microarray and deep sequencing cross-platform

118118 José Antonio del Río Fernández Group José Antonio del Río Fernández Group 119

analysis of the mirRNome and isomiR variation in response to epidermal growth factor. BMC genomics. 2013 Jun 1;14:371. • La Torre A, del Mar Masdeu M, Cotrufo T, Moubarak RS, del Rio JA, Comella JX, et al. A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching. Cell death & disease. 2013 Apr 18;4:e602. • Seira O, Del Rio JA. Glycogen Synthase Kinase 3 Beta (GSK3β) at the Tip of Neuronal Development and Regeneration. Molecular neurobiology. 2013 Oct 25. • Ordonez-Gutierrez L, Torres JM, Gavin R, Anton M, Arroba-Espinosa AI, Espinosa JC, et al. Cellular prion protein modulates β-amyloid deposition in aged APP/PS1 transgenic mice. Neurobiology of aging. 2013 Dec;34(12):2793-804.

Research Projects

• Code: PI11/03028 Other CIBER’s collaboration: No Title: DEMTEST: Biomarker based diagnosis of rapid pro- Type: Privado gressive dementias Funding Agency: Fundación Obra Social la Caixa. Program Principal Investigator: Pascual Sanchez de valorización y Transferencia de tecnología CIBERNED’s collaboration: Yes Funding: 40.000 € CIBERNED’s groups: G 114; G 601; G 503; G 609 Duration: 2012-2013 Other CIBER’s collaboration: No Type: International • Code: BFU2012-32617 Funding Agency: Instituto de Salud Carlos III- Comunidad Title: Nuevas funciones de PLEXIND1/SEMA3E, PRPC y las Económica Europea proteínas asociadas a la mielina durante el desarrollo de la Funding: 10.000 € corteza cerebral de roedores y en neurodegeneración Duration: 2012-2014 Principal Investigator: Jose Antonio del Río CIBERNED’s collaboration: No • Code: BIO12/AL/004 CIBERNED’s groups: Title: Estudio de la replicación “tipo prión” en modelos Other CIBER’s collaboration: No celulares obtenidos mediante la reProgramción de células Type: National somáticas de pacientes geneticamente susceptibles a la en- Funding Agency: MINECO fermedad de Alzheimer Funding: 304.200 € Principal Investigator: Joaquín Castilla Duration: 2013-2015 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: AC50 Other CIBER’s collaboration: No Title: Protecting the food chain of prions: Shaping Euro- Type: Privado pean priorities through basic and applied research Funding Agency: Fundacion Vasca de Innovación e Investi- Principal Investigator: Jesus Requena (USC) gación Sanitarias CIBERNED’s collaboration: No Funding: 28.080 € CIBERNED’s groups: Duration: 2013-2014 Other CIBER’s collaboration: No Type: International • Code: SGR2009-0366 Funding Agency: FP/ - KBBE-2007-2-4-06 Title: Group de investigación consolidado de la Generalitat Funding: 217.000 € de Catalunya Duration: 2009-2014 Principal Investigator: Jose A del Río CIBERNED’s collaboration: No • Code: PI2010/09 CIBERNED’s groups: Title: BESAD-P: Biomarkers of Early Stages of Alzheimer Other CIBER’s collaboration: No Disease-Prevention Type: CCAA Principal Investigator: Isidre Ferrer Abizanda Funding Agency: AGAUR CIBERNED’s collaboration: Yes Funding: 70.720 € CIBERNED’s groups: G 503; G 502; G 510; G 402; G 114; Duration: 2009-2013 G 506; G 508; G 412 Other CIBER’s collaboration: Yes (CIBERESP) • Code: P1-L14-SRI-NEURO Type: Intramurales Title: NeuroBiotechnology for regenerative Neuroscience Funding Agency: CIBERNED Principal Investigator: Jose Antonio del Río Funding: 223.500 € CIBERNED’s collaboration: No Duration: 2011-2013 CIBERNED’s groups: Group Isabel Fariñas Gómez

Departamento de Biología Celular

2 Universidad de Valencia Doctor Moliner, 50 46100 Burjassot (Spain) Program Tel.: +34 963 543 784 (despacho/office) 3246 (lab) Fax: +34 963 543 404 [email protected]

Isabel Fariñas Ana C. Delgado Fumero Principal Investigator Postdoctoral Fellow Francisco Pérez Sánchez Beatriz Martí Scienific staff PhD student Martina Kirstein Raúl Soriano Cantón Scienific staff PhD student Sacramento Rodríguez Ferrón Germán Belenguer Scienific staff PhD student Ana Pérez Villalba María José Palop Postdoctoral Fellow Technician José Manuel Morante Fabrice Durupt Postdoctoral Fellow Technician

120 Isabel Fariñas Gómez Group 121

Summary

Our laboratory studies two topics related to neurodegeneration and cell therapy: 1) The study of cellular and molecular alte- rations that underlie degeneration of dopaminergic neurons, and 2) The study of neural stem cell (NSC) self-renewal. In recent years, we have also been working in combining both aspects by studying the regulation of adult neurogenesis in parkinsonism and during aging. In 2013, we have worked with several groups of CIBERNED in three different topics. On the one hand, we have collaborated with the groups of Drs. Vila and Obeso in demonstrating that pathological forms of alpha-synuclein present in patients who died with Parkinson’s disease are able to initiate the neurodegenerative process in normal brains of mice and monkeys. Furthermore, in collaboration with the groups of Drs. Toledo and Moratalla, we have discovered that dopamine not only controls the proliferation of the progenitors of the subependymal zone but also regulates the potential status of NSCs and their maintenance over time and that alpha-synuclein, present in the dopaminergic terminals that innervate the subependymal zone, is necessary for the action of dopamine. Another line that we have been exploring this year, in collaboration with the group of Dr. Cuadrado in the context of an intramural collaborative project, has been the role of the transcription factor Nrf2 in the behavior of NSCs. Our results indicate that this factor regulates the balance between neurogenesis and oligodendrogénesis. We have also observed an interaction between neurogenesis and aging in mouse models of accelerated senescence and the effect of alpha-synuclein on dopaminergic neurodegeneration in such genetic background. As with regards to the molecular regulation of the property of self-renewal in the population of NSCs of the adult subependymal zone we have reported this year that the cyclin-dependent kinase inhibitor p21, which regulates the cycle of NSCs, also helps to maintain their status and their properties through transcriptional actions on the Sox2 gene and the Bmp2 gene..

Keywords

Adult neurogenesis and cell therapy, Neural stem cell, Neurogenic niches, Neurotrophic factors and cell death, Murine models of neurodegenerative diseases, Parkinson disease and alpha-synuclein

Publications

• Recasens A, Dehay B, Bove J, Carballo-Carbajal I, Dovero S, Perez A, et al. Lewy body extracts from parkinson’s disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. Annals of neurology. 2013. • Eguren M, Porlan E, Manchado E, Garcia-Higuera I, Canamero M, Farinas I, et al. The APC/C cofactor Cdh1 prevents replicative stress and p53-dependent cell death in neural progenitors. Nature Communicatios. 2013;4:2880. • Porlan E, Morante-Redolat JM, Marques-Torrejon MA, Andreu-Agullo C, Carneiro C, Gomez-Ibarlucea E, et al. Transcriptional repression of Bmp2 by p21(Waf1/Cip1) links quiescence to neural stem cell maintenance. Nature neuroscience. 2013 Nov;16(11):1567-75.

Research Projects • Code: ISIC2012/010 Jose Maria Moraleda) Title: ISIC STEMT CIBERNED’s collaboration: Yes Principal Investigator: Isabel Fariñas (IP coordinado: Ange- CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; la Nieto) G 607 CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: Fondo de Investigaciones Sanitarias Type: National Funding: 296.700 € Funding Agency: Conselleria de Educacion Generalitat Va- Duration: 2013-2016 lenciana Funding: 90.000 € • Code: PI2011/01 Duration: 2013 Title: El factor de transcripción Nrf2 como nueva diana te- rapéutica para la enfermedad de Parkinson • Code: RD12/0019/0008 Principal Investigator: Antonio Cuadrado Pastor Title: RETIC de Terapia Celular CIBERNED’s collaboration: Yes Principal Investigator: Isabel Fariñas (coordinador de red: CIBERNED’s groups: G 101; G102; G103; G 505; G 209 Other CIBER’s collaboration: No dor de red: Jose Maria Moraleda) Type: Intramurales CIBERNED’s collaboration: Yes Funding Agency: CIBERNED CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; Funding: 390.000 € G 607 Duration: 2011-2013 Other CIBER’s collaboration: No Type: National • Code: RD12/0019/0011 Funding Agency: Instituto de Salud Carlos III Title: RETIC de Terapia Celular Funding: 180.000 € Principal Investigator: Xavier Navarro Acebes, (coordina- Duration: 2013-2016

PhD Dissertations

• Author: Natividad Pozo Title: Caracterización de la función de dyrk1a como modu- lador de las células madre neurales, normales y cancerosas Date: 15 May 2013 Supervisor: Pilar Sánchez

122122 Isabel Fariñas Gómez Group Group Javier Fernández Ruiz

Departamento de Bioquímica y Biología Molecular III

Facultad de Medicina 2 Universidad Complutense

Avenida Complutense, s/n Program 28040 Madrid (Spain) Tel.: +34 913 941 450 Fax: +34 913 941 691 [email protected] / [email protected]

Javier Fernández Ruiz Miguel Moreno Martet Principal Investigator, Full Professor FPU PhD student José Antonio Ramos Atance Carmen Rodríguez Cueto Full Professor FPI PhD student Mariluz Hernández Gálvez Cristina Palomo Garo Associate Professor PhD student María Gómez Ruiz María Gómez Cañas Doctor Assistant Professor PhD student Eva de Lago Femia Sara Valdeolivas Rojas Doctor Assistant Professor UCM PhD student Onintza Sagredo Ezkioga Francisco Espejo Porras Assistant Professor Doctor FPI PhD student Concepción García García Yolanda García Movellán CIBERNED Postdoctoral Fellow CIBERNED Administrative Assistant Moisés García Arencibia PICATA–Campus de Excelencia Moncloa Postdoctoral Fellow

123 Summary

The work during 2013 has been concentrated in the study of the neuroprotective potential of cannabinoids in several chronic neurodegenerative disorders with motor symptoms, and, in particular, in the identification of cellular and molecular mechanisms that underlie these effects.

In the case of Huntington’s disease, the work has addressed four objectives:

• To finish the evaluation of different combinations of Δ9-THC and cannabidiol, similar to those present in GW Pharmaceuticals medicine Sativex®, in relation with their neuroprotective effect in R6/2 mice (manuscript in preparation). • To finish the clinical trial conducted in the “Hospital Ramón y Cajal” in Madrid in patients using Sativex®, trial in which our group has participated in the analysis of different biomarkers in collaboration with other CIBERNED groups (manuscript in preparation). • To extend the studies with phytocannabinoids to cannabigerol and its derivatives, also in R6/2 mice and in collaboration with the company VivaCell Biotechnology (manuscript in evaluation in Neurotherapeutics). • To investigate the role of COX-2-dependent metabolism of the two major endocannabinoids in the pathogenesis of this disease, as well as to study the potential of the pharmacological manipulation of this process (Valdeolivas et al., 2013).

In the case of Parkinson’s disease, the work has addressed three objectives:

• To investigate the advantage of the combination of cannabidiol and Δ9-THCV (GW Pharmaceuticals) for the symptomatic and neuroprotective treatment of this disease, using rodents lesioned with 6-hydroxydopamine or LPS (manuscript in preparation), and to extend these studies to cannabigerol derivatives developed by the company VivaCell Biotechnology. • To investigate the role of CB2 receptors in neuroprotective effects of Δ9-THCV in rodents lesioned with 6-hydroxydopamine or LPS (manuscript in preparation). • To study the changes in CB2 receptors as well as the neuroprotective effects derived from their activation in LRRK2 mutant mice, a genetic model of Parkinson’s disease. This study will be conducted within a project funded by the Michael J. Fox Foundation.

In the case of amyotrophic lateral sclerosis, the work has addressed three objectives:

• To finish the experiments addressed to identify the changes that the disease provokes in the endocannabinoid system and to complete the evaluation of Sativex®-like combination of phytocannabinoids in SOD-1 mutant mice (Moreno-Martet et al., 2014). • To extend these biochemical and pharmacological studies to dogs with degenerative myelopathy, an ALS-like disorder also produced by SOD-1 mutations. • To repeat similar studies in TDP-43 mutant mice, one of the new genes identified in relation with amyotrophic lateral sclerosis, which also serves for the experimental study of frontotemporal dementia.

In the case of spinocerebellar ataxias, the work has addressed two objectives:

• To investigate the changes that the disease produces in the endocannabinoid system (ligands, receptors and enzymes) in the cerebellum of SCA3 transgenic mice (manuscript in preparation). • To conduct pharmacological studies with different cannabinoids in SCA3 transgenic mice addressed to evaluate the neuroprotective potential of these compounds.

Keywords

Cannabinoids, CB1 receptors, CB2 receptors, Neuroprotection, Huntington’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, Spinocerebellar ataxias

124124 Javier Fernández Ruiz Group Javier Fernández Ruiz Group 125

Publications

• Fernandez-Fernandez C, Decara J, Bermudez-Silva FJ, Sanchez E, Morales P, Gomez-Canas M, et al. Description of a bivalent cannabinoid ligand with hypophagic properties. Archiv der Pharmazie. 2013;346(3):171-9. • Alvarado M, Decara J, Luque MJ, Hernandez-Folgado L, Gomez-Canas M, Gomez-Ruiz M, et al. Novel antiobesity agents: synthesis and pharmacological evaluation of analogues of Rimonabant and LH21. Bioorganic & Medicinal Chemistry. 2013;21(7):1708-16. • Gonzalez-Naranjo P, Perez N, Campillo NE, Perez C, Aran VJ, Giron R, et al. Cannabinoid agonists showing BuChE inhibition as potential therapeutic agents for Alzheimer´s disease. European Journal of Medicinal Chemistry. 2013. • Morales P, Vara D, Gomez-Canas M, Zuniga MC, Olea-Azar C, Goya P, et al. Synthetic cannabinoid quinones: preparation, in vitro antiproliferative effects and in vivo prostate antitumor activity. European Journal of Medicinal Chemistry. 2013;70C:111-9. • Fernandez-Ruiz J, Sagredo O, Pazos MR, Garcia C, Pertwee RG, Mechoulam R, et al. Cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid? British Journal of Clinical Pharmacology. 2013;75(2):323-33. • Gomez del Rio MA, Sanchez-Reus MI, Iglesias I, Pozo MA, Garcia-Arencibia M, Fernandez-Ruiz J, et al. Neuroprotective properties of standardized extracts of Hypericum perforatum on rotenone model of Parkinson’s disease. CNS Neurological Disorders and Drug Targets. 2013;12(5):665-79. • Szafrańskia P, Dyducha K, Koscioleka T, Wrobela T, Gomez-Canas M, Gomez-Ruiz M, et al. Design and synthesis of novel cannabinoid ligands based on a 1,2,3-triazole scaffold. Letters in Drug Design & Discovery. 2013;10:169-72. • Espejo-Porras F, Fernandez-Ruiz J, Pertwee RG, Mechoulam R, Garcia MC. Motor effects of the non-psychotropic phytocannabinoid cannabidiol that are mediated by 5-HT1A receptors. Neuropharmacology. 2013;75C:155-63. • Casarejos MJ, Perucho J, Gomez A, Munoz MP, Fernandez-Estevez M, Sagredo O, et al. Natural cannabinoids improve dopamine neurotransmission and tau and amyloid pathology in a mouse model of tauopathy. Journal of Alzheimer´s disease. 2013;35(3):525-39. • Pryce G, Cabranes A, Fernandez-Ruiz J, Bisogno T, Di Marzo V, Long L, et al. Control of experimental spasticity by targeting the degradation of endocannabinoids using selective fatty acid amide hydrolase inhibitors. Multiple Sclerosis Journal. 2013;19(14):1896-904. • Valhondo M, Marco I, Martin-Fontecha M, Vazquez-Villa H, Ramos JA, Berkels R, et al. New Serotonin 5-HT1A Receptor Agonists Endowed with Antinociceptive Activity in Vivo. Journal of Medicinal Chemistry. 2013;56(20):7851-61. • Valdeolivas S, Pazos MR, Bisogno T, Piscitelli F, Iannotti F, Allara M, et al. The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis, rather than enhancing striatal damage, protects striatal neurons from malonate-induced death: potential role of ciclooxygenase-2-dependent metabolism of 2-AG. Cell Death & Disease. 2013;4:e862.

Research Projects

• Code: MJFOXF Other CIBER’s collaboration: No Title: Cannabinoid CB2 receptors as a new target for the Type: National treatment of disease progression in Parkinson’s disease: Funding Agency: Ministerio de Economía y Competitividad. studies in LRRK2-transgenic mice Direccion Gral. de Investigación y Gestión Plan Nacional Principal Investigator: Javier Fernández Ruiz / Mª Concep- I+D+i ción García García Funding: 222.300 € CIBERNED’s collaboration: No Duration: 2013-2015 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: GW2012 Type: International Title: Evaluation of phytocannabinoids as disease-modi- Funding Agency: Michael J. Fox Foundation fying agents in chronic neurodegenerative disorders Funding: 107.781 € Principal Investigator: Javier Fernández-Ruiz Duration: 2012-2014 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: SAF2012-39173 Other CIBER’s collaboration: No Title: El sistema cannabinoide como diana para una terapia Type: Privado neuroprotectora en la esclerosis lateral amiotrófica Funding Agency: GW Pharmaceluticals Ltd. (UK) Principal Investigator: Javier Fernández Ruiz Funding: 37.500 € CIBERNED’s collaboration: No Duration: 2012-2013 CIBERNED’s groups: • Code: ALZH (ELF) Type: Intramurales Title: Role of the endocannabinoid system in TDP43-relat- Funding Agency: CIBERNED ed dementia Funding: 417.600 € Principal Investigator: Eva de Lago Femia Duration: 2010-2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: CIBERNED 2013/05 Other CIBER’s collaboration: No Title: Identificación y caracterización molecular de subpo- Type: International blaciones de receptores cannabinoides en poliglutamino- Funding Agency: Alzheimer´s Association patías Funding: 66.941 € Principal Investigator: Manuel Guzmán Pastor Duration: 2013-2014 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 201; G 303; G 304; G 305 • Code: GW2013 Other CIBER’s collaboration: No Title: Studies with phytocannabinoids as disease modifying Type: Intramurales agents in different neurodegenerative disorders Funding Agency: CIBERNED Principal Investigator: Javier Fernández Ruiz Funding: 50.000 € CIBERNED’s collaboration: No Duration: 2013-2015 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: S2010/BMD-2308 Type: Privado Title: Neurofarmacología del sistema endocannabinoide: Funding Agency: GW Pharmaceuticals, Ltd del laboratorio a la clínica Funding: 34.000 € Principal Investigator: Manuel Guzmán Pastor Duration: 2013-2015 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 305; G 303; G 304 • Code: PI2010/06 Other CIBER’s collaboration: Yes (CIBERSAM) Title: Neuroprotección en enfermedad de Huntington Type: CCAA Principal Investigator: Justo García de Yébenes Prous Funding Agency: Comunidad de Madrid CIBERNED’s collaboration: Yes Funding: 177.500 € CIBERNED’s groups: G 304; G 303; G 305; G 306; G 307 Duration: 2012-2015 Other CIBER’s collaboration: No

126126 Javier Fernández Ruiz Group Group José Manuel Fuentes Rodríguez

Group PARK

Dpto. Bioquímica y Biología Molecular y Genética 2 F. Enfermería y T.O.

Universidad de Extremadura Program Avenida de la Universidad, s/n 10003 Cáceres (Spain) Tel.: +34 927 257 450 (ext 51286) Fax: +34 927 257 451 [email protected]

José Manuel Fuentes Rodríguez Sokhna Maryama Seydina Yakhine DIOP Principal Investigator, Full Professor PhD student Rosa Ana González Polo Mario Rodríguez Arribas “Miguel Servet” Researcher PhD student Mireia Niso Santano Ignacio Casado Naranjo Postdoctoral Fellow Chief of Neurology Department José Manuel Bravo San Pedro Guadalupe Martínez Chacón Postdoctoral Fellow CIBERNED Technician Rubén Gómez Sánchez María Pura Delgado Luceño Postdoctoral Fellow Technician Elisa Pizarro Estrella Raquel Ronco Barrantes CIBERNED PhD student Technician

127 Summary

Our efforts during 2013 have continued focusing on the study of basic molecular mechanisms involved in the pathogenesis of Parkinson’s disease, autophagy regulation and role of PARK genes derived proteins in both processes. Moreover we have also continued our collaboration and service delivery to Extremadura Health Service in molecular diagnosis of Parkinson’s disease.

As part of a collaborative project CIBERNED (PI2011/01) led by Dr. Antonio Cuadrado continue studying the role Nrf2/keap1 axis in the regulation of the autophagic response. For this we used MEFs from WT, Nrf2 and Keap1-deficient mice. All lines are sensitive to the effect of PQ and MPP+, succumbing by a mechanism compatible cell death mediated apoptosis by oxidative stress, with the line Nrf2-/- more sensitive than the WT and the Keap1-/- which, in addition show a slight resistance and protection against these agents. This greater sensitivity shown by the Nrf2-/line-, against oxidative stress caused by MPP+ and PQ, is due to the inability of these cells to remove reactive oxygen species by inducing cytoprotective genes. The induction and/ or inhibition of the autophagic process in this model induces death in the three lines, suggesting a protective effect of this mechanism. In this sense MEFs Nrf2-/- have higher levels of basal autophagy induction relative to the WT and Keap1-line /line- and after induction process, but this process is less efficient, showing lower levels of degradation. The Keap1-/line- have a lower induction of autophagy but exhibit a stronger degradation line compared to WT, also seem to show some kind of disturbance in the ATPase proton of the lysosomal membrane, since the effects of the Baf.A1 are attenuated by this line. SFN treatment in line WT induced the expression of antioxidant defenses, cytosolic levels decreases oxidative stress produced by MPP+ and PQ and further promotes a degradative autophagic mechanism. However, these events are not sufficient to rescue this line of apoptotic cell death mediated by oxidative stress.

Another line of work culminating in 2013 has been to determine the role of PINK1 in mitochondrial dysfunction mediated uncoupler CCCP and its role in mitophagy. So we found that mitochondrial uncoupler CCCP produces mitochondrial dysfunction and fission, as well as a slight neurotoxic effect, in the neuroblastoma human cell line SH-SY5Y in a time-dependent manner. Also, CCCP generates an initial increase in the intracellular calcium levels mainly through L- and N-type voltage-dependent calcium channels increasing the FL-PINK1 protein levels and its mitochondrial localization, being mainly due to a higher calcium-dependent gene expression rates and not to the FLPINK1stabilization. Finally we have shown that CCCP activates the transcription factor c-Fos in a calcium-dependent manner and induces its nuclear recruitment, being a separate event to the observed PINK1 increase after the uncoupler exposure

Following the work we carried out since 2009 in collaboration with the group of Dr. Adolfo López de Munain (CIBERNED), have continued to characterize the basal macroautophagy deregulation in fibroblasts from Parkinson patients carriers of pathogenic G2019S mutation in LRRK2. Fibroblasts from individuals with G2019S LRRK2 mutation presents levels of autophagy basally higher than those observed in fibroblasts from individuals without the mutation, this flare is accompanied by an increased sensitivity of the cells to toxic substances, such as MPP+, regarding Parkinson ‘s disease. After a molecular dissection we found that the MEK/ERK pathway could play an active role in the deregulation of basal autophagy and consequent increased sensitivity presented in individuals with LRRK2 G2019S mutation. Finally we are moving in functional studies using fibroblasts from individuals with the mutation of LRRK2 R1441G. In this case we have also seen an accelerated basal autophagy, however no higher degradation than fibroblasts from individuals without this mutation.

Finally we have followed interested in the study of general mechanisms of regulation of autophagy, this part of our business is still carried out in collaboration with the laboratory of Dr. Guido Kroemer of Apoptosis, Cancer & Immunity Laboratory, INSERM Cordeliers Research Center, Paris, Francia.

Regarding our collaboration with the Extremadura Health Service should be noted that we have signed several agreements to provide services in relation to the molecular diagnosis of Parkinson’s. It has been increased to twenty the number of products in our portfolio of services, and participate in research training for healthcare professionals.

Keywords

Pesticides and Parkinson’s Disease, Roles of apoptosis and autophagy in Parkinson’s Disease, Detection of mutations in PARK genes, Functional roles of PARK gene-derived proteins (synuclein, parkin, DJ-1, PINK1 and LRRK2), Nrf2/keap1 axis, ASK1

128128 José Manuel Fuentes Rodríguez Group José Manuel Fuentes Rodríguez Group 129

Publications

• Bravo-San Pedro JM, Senovilla L. Immunostimulatory activity of lifespan-extending agents. Aging. 2013 Nov;5(11):793-801. • Gonzalez-Polo RA, Gomez-Sanchez R, Alvarez-Erviti L, Bravo-San Pedro JM, Pizarro-Estrella E, Niso-Santano M, et al. Neuroprotective effect of autophagy in pesticide toxicity. New Insights into Toxicity and Drug Testing. 2013;177-96. • Gonzalez-Polo RA, Fuentes JM, Niso-Santano M, Alvarez-Erviti L. Implication of autophagy in Parkinson’s disease. Parkinson’s disease. 2013;2013:436481. • Gomez-Sanchez R, Gegg ME, Bravo-San Pedro JM, Niso-Santano M, Alvarez-Erviti L, Pizarro-Estrella E, et al. Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression. Neurobiology of disease. 2013 Oct 29;62C:426-40. • Pietrocola F, Izzo V, Niso-Santano M, Vacchelli E, Galluzzi L, Maiuri MC, et al. Regulation of autophagy by stress-responsive transcription factors. Seminars in cancer biology. 2013 Oct;23(5):310-22.

Research Projects

• Code: GR10054 CIBERNED’s collaboration: No Title: Ayudas para la consolidación y apoyo a los Groups de CIBERNED’s groups: investigación inscritos en el Catálogo de Groups de Investi- Other CIBER’s collaboration: No gación de Extremadura Type: National Principal Investigator: José Manuel Fuentes Rodríguez Funding Agency: FIS. ISCIII. MICINN CIBERNED’s collaboration: No Funding: 86.515 € CIBERNED’s groups: Duration: 2012-2014 Other CIBER’s collaboration: No Type: CCAA • Code: PI2011/01 Funding Agency: Consejeria Infraestructura y Desarrollo Title: El factor de transcripción Nrf2 como nueva diana te- Tecnológico de la Junta de Extremadura rapéutica para la enfermedad de Parkinson Funding: 74.811 € Principal Investigator: Antonio Cuadrado Pastor Duration: 2011-2014 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 101; G102; G103; G 505; G 209 • Code: PI11/00040 Other CIBER’s collaboration: No Title: Estudio de la importancia de la autofagia en células Type: Intramurales procedentes de enfermos de Parkinson con mutaciones en Funding Agency: CIBERNED genes park Funding: 390.000 € Principal Investigator: Rosa Ana González Polo Duration: 2011-2013

PhD Dissertations

• Author: Ruben Gómez Sánchez • Author: Marta Rodriguez-Carpio Serrano Title: Papel de la proteína PINK1 en la enfermedad de Title: Perfiles genéticos de riesgo a padecer obesidad mór- Parkinson: Su implicación en daño mitocondrial y autofagia bida Date: 5 July 2013 Date: 21 November 2013 Supervisor: José Manuel Fuentes Rodríguez Supervisor: José Manuel Fuentes Rodríguez Group Justo García de Yébenes Prous

Hospital Ramón y Cajal

2 Carretera de Colmenar, km 9,1 28034 Madrid (Spain) Tel.: +34 913 368 821 Program Fax: +34 913 369 016 [email protected] / [email protected].

María Ángeles Mena Conceição Bettencourt Principal Investigator Postdoctoral Fellow Justo García de Yébenes Prous Raquel Ros Neurologist Postdoctoral Fellow Guillermo García Ribas Carolina Ruiz Neurologist PhD student José Luis López Sendón Marian Fernández Neurologist PhD student Juan García Caldentey Andrea Mohedano Neurologist Nurse Patricia Trigo Ana Gómez Neurophychologist Technician María José Casarejos María Paz Muñoz Associate Researcher Technician Juan Perucho María Ángeles Robles Postdoctoral Fellow Administrative assintant

130 Justo García de Yébenes Prous Group 131

Summary

In 2013 our group has maintained the research in neurodegenerative disorders mainly dementias like Alzheimer disease, Parkinson’s disease, Progressive supranuclear palsy, Huntington’s disease, dystonias, ataxias and paraparexias.

Our work centred in:

• Clinical and molecular characterization of neurodegenerative diseases: Identification of new genes and mutations implicated in these diseases. We continued in collaboration with others groups, Spanish, Portuguese, Dutch and English. We participated in the identification of new genes using the whole-exome sequencing in patients with ataxia and PSP diseases. • Clinical studies on neuroprotection compounds in these diseases. This is the case of the clinical trial of SATIVEX drug in Hungtinton patients. • Study in experimental models (transgenic and cell models) of the pathogenic mechanism and the neuroprotective treatments in these diseases. We continued the study of the amyloidogenic role of volatile anesthetics and the role of biomarkers in human cerebrospinal fluid in dementia, the systems of cellular processing of abnormal proteins in models of Parkinson disease and the role of cannabinoids in Huntington’s disease. • In the second half of 2013 we have initiated a new line of work, the development of digital multicentric registries for the investigation of neurological diseases and for the evaluation of movement disorders. We have designed a digital registry for Parkinson´s disease that is going to be implemented in the next few weeks by the Federation of associations of patients with Parkinson’s disease and one method for the quantitative analysis of dyskinesias of the head.

Keywords

Neurodegeneratives diseases, Neurogenetics, Cell cultures, Transgenics mouses, Neuroprotection

Publications

• Lopez-Sendon J, Mena MA, de Yebenes JG. Drug-induced parkinsonism. Expert opinion on drug safety. 2013 Jul;12(4):487-96. • Lopez-Sendon Moreno JL, Garcia-Caldentey J, Regidor I, Alamo MD, Garcia de Yebenes J. A 5-year follow-up of deep brain stimulation in Huntington’s disease. Parkinsonism & Related Disorders. 2013. • Hubers AA, van Duijn E, Roos RA, Craufurd D, Rickards H, Bernhard Landwehrmeyer G et al.; REGISTRY investigators of the European Huntington’s Disease Network. Suicidal ideation in a European Huntington’s disease population. J Affect Disord. 2013;151(1):248-58. • Perucho J, Casarejos MJ, Gomez A, Ruiz C, Fernandez-Estevez MA, Munoz MP, et al. Striatal infusion of glial conditioned medium diminishes huntingtin pathology in r6/1 mice. PloS one. 2013;8(9):e73120. • Metzger S, Walter C, Riess O, Roos RA, Nielsen JE, Craufurd D; REGISTRY Investigators of the European Huntington’s Disease Network, Nguyen HP. The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients. PLoS One. 2013;8(7):e68951. • Llorente-Folch I, Sahun I, Contreras L, Casarejos MJ, Grau JM, Saheki T, et al. AGC1-malate aspartate shuttle activity is critical for dopamine handling in the nigrostriatal pathway. Journal of neurochemistry. 2013;124(3):347-62. • Bettencourt C, Lopez-Sendon J, Garcia-Caldentey J, Rizzu P, Bakker I, Shomroni O, et al. Exome sequencing is a useful diagnostic tool for complicated forms of hereditary spastic paraplegia. Clinical Genetics. 2013. • Squitieri F, Landwehrmeyer B, Reilmann R, Rosser A, de Yebenes JG, Prang A, et al. One-year safety and tolerability profile of pridopidine in patients with Huntington disease. Neurology. 2013;80(12):1086-94. • Casarejos MJ, Perucho J, Gomez A, Munoz MP, Fernandez-Estevez M, Sagredo O, et al. Natural cannabinoids improve dopamine neurotransmission and tau and amyloid pathology in a mouse model of tauopathy. Journal of Alzheimer´s disease. 2013;35(3):525-39. Research Projects

• Code: CB06/05/59 • Code: CAM2011/BMD 2308 Title: Enfermedades Neurodegenerativas Title: Neurofarmacología del sistema endocanabinoide: del Principal Investigator: María Ángeles Mena laboratorio a la clínica CIBERNED’s collaboration: No Principal Investigator: María Ángeles Mena CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: Intramurales Other CIBER’s collaboration: No Funding Agency: CIBERNED Type: CCAA Funding: 120.000 € Funding Agency: CAM Duration: 2013 Funding: 81.000 € Duration: 2013 • Code: 3133K1-3001-WW Title: Ensayo de fase 3, multicéntrico, aleatorizado, en do- • Code: PI2010/06 ble ciego, controlado con placebo y de Groups paralelos, Title: Neuroprotección en enfermedad de Huntington sobre la eficacia y la seguridad del Bapineuzumab (AAB- Principal Investigator: Justo García de Yébenes Prous 001, ELN115727) en sujetos con enfermedad de Alzheimer CIBERNED’s collaboration: Yes de grado leve o moderado que no son CIBERNED’s groups: G 304; G 303; G 305; G 306; G 307 Principal Investigator: Justo García de Yébenes Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: Intramurales CIBERNED’s groups: Funding Agency: CIBERNED Other CIBER’s collaboration: No Funding: 417.600 € Type: Privado Duration: 2010-2013 Funding Agency: Wyeth Farma SA Funding: 30.000 € • Code: MENA MA Duration: 2009-2013 Title: Neuroprotective properties of Sativex® or related phytocannabinoid medicines in Huntington´s disease • Code: 3133K1-3002-WW Principal Investigator: María Ángeles Mena Title: Ensayo de fase 3, multicéntrico, aleatorizado, en do- CIBERNED’s collaboration: No ble ciego, controlado con placebo y de Groups paralelos, CIBERNED’s groups: sobre la eficacia y la seguridad del Bapineuzumab (AAB- Other CIBER’s collaboration: No 001, ELN115727) en sujetos con enfermedad de Alzheimer Type: Privado de grado leve o moderado que no son Funding Agency: GW Pharmaceuticals Ltd Principal Investigator: Justo García de Yébenes Funding: 36.000 € CIBERNED’s collaboration: No Duration: 2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: FIS 2010/172 Type: Privado Title: Papel de la proteolisis en modelos experimentales de Funding Agency: Wyeth Farma SA enfermedades neurodegenerativas. Estrategias neuropro- Funding: 30.000 € tectoras Duration: 2013 Principal Investigator: María Ángeles Mena CIBERNED’s collaboration: No • Code: 3133K1-3000-WW CIBERNED’s groups: Title: Ensayo de fase 3, multicéntrico,aleatorizado, en do- Other CIBER’s collaboration: No ble ciego, controlado con placebo y de Groups paralelos, Type: National sobre eficacia y la seguridad del bapineurzumab(abb-001, Funding Agency: FIS eln11572) en sujetos con enfermedad de Alzheimer en gra- Funding: 192.390 € do leve o moderado que no son portad Duration: 2010-2013 Principal Investigator: Justo García de Yébenes CIBERNED’s collaboration: No • Code: CIBERNED 2013/05 CIBERNED’s groups: Title: Identificación y caracterización molecular de subpo- Other CIBER’s collaboration: No blaciones de receptores cannabinoides en poliglutamino- Type: Privado patías Funding Agency: Wyeth Farma SA Principal Investigator: Manuel Guzmán Pastor Funding: 30.000 € CIBERNED’s collaboration: Yes Duration: 2009-2013 CIBERNED’s groups: G 201; G 303; G 304; G 305 Other CIBER’s collaboration: No

132132 Justo García de Yébenes Prous Group Justo García de Yébenes Prous Group 133

Type: Intramurales Principal Investigator: Manuel Guzmán Pastor Funding Agency: CIBERNED CIBERNED’s collaboration: Yes Funding: 50.000 € CIBERNED’s groups: G 305; G 303; G 304 Duration: 2013-2015 Other CIBER’s collaboration: Yes (CIBERSAM) Type: CCAA • Code: S2010/BMD-2308 Funding Agency: Comunidad de Madrid Title: Neurofarmacología del sistema endocannabinoide: Funding: 177.500 € del laboratorio a la clínica Duration: 2012-2015 Group José Manuel García Verdugo

Laboratorio de Neurobiología Comparada

2 Instituto Cavanilles de Biodiversidad y Biología Evolutiva Universidad de Valencia Catedrático José Beltrán, 2 Program 46980 Paterna (Spain) Tel.: +34 963 543 587 Fax: +34 963 543 670

José Manuel García Verdugo Sara García Gil-Perotín Principal Investigator, Full Professor Postdoctoral Fellow Juan Antonio Barcia Albacar Arantxa Cebrián Silla Associate Researcher, Neurosurgeon PhD student José Miguel Soria López María Durán Moreno Associate Researcher PhD student María Salomé Sirerol Piquer Susana González Granero Postdoctoral Fellow CIBERNED Technician Vicente Herranz Pérez CIBERNED Postdoctoral Fellow

134 José Manuel García Verdugo Group 135

Summary

Our group’s main research line is focused on the study of stem cells, especially those responsible for adult neurogenesis and oligodendrogenesis. And our technical expertise in electron microscopy allowed us to perform interesting correlations with the data obtained by molecular studies. As a consequence, our group has a large number of national and international collaborations.

During 2013 we have worked on various topics ranging from the collection and isolation of stem cells from adipose tissue, to the characterization of neural progenitor markers in primates. Many clinical trials in which stem cell therapy is applied, require protocols for cell isolation and subsequent expansion, ensuring that the cells are free from non-human pathogenic organisms. In this regard, we have described a method for obtaining mesenchymal stem cells from human adipose tissue and its subsequent expansion in xenogeneic-free culture media.

We have also performed a detailed ultrastructural characterization of neurospheres obtained from the ventricles of adult mice, which are often used in transplantation models. These cells exhibit abundant binding complexes and few cytoplasmic organelles. It is worth noting the first description of the presence of annulate lamellae in these cells, as indicative of their high proliferative rate.

Another axis of our work was to carry out a better characterization of oligodendrocytes during postnatal development, especially during aging. We have observed that aging causes a drastic decrease in the neurogenic potential while, in contrast, oligodendrogenesis remains constant. In addition, we wanted to get a deeper insight into the capacity to generate new oligodendrocytes in demyelination models combined with focal irradiation. It was found that the subventricular zone of adult mice responds with activation, attempting to repair the loss of myelin, which somehow indicates some level of resistance to radiation in stem cells. This does not mean that the radiation does not affect stem cells, but that they are able to repair, if not completely at least partially, the injury caused by the loss of myelin. While there is an increase in the production of oligodendrocytes, we observed that irradiation of neurogenic niches, non-combined with demyelinating agents, causes an increase in a number of factors, such as TGF-β, which directly reduces the proliferative capacity of stem cells. Interestingly enough, some of these factors are also increased during aging.

Another line of research is to continue the study of the process of cell migration taking place from the ventricle to the prefrontal cortex in the human brain. We are currently analyzing the organization of these cells forming chains to reach its destination. In addition, we expanded our study to both the medial and lateral ganglionic eminences.

Finally, we found a population of Sox-2 positive cells in the nigrostriatal pathway of primates that could be good candidates to act as an endogenous repair mechanism in Parkinson’s disease.

Keywords

Neurogenesis, Stem cells, Cellular therapy, Subventricular zone, Oligodendrocytes, Human brain

Publications

• Perez-Garnes M, Martinez-Ramos C, Barcia JA, Escobar Ivirico JL, Gomez-Pinedo U, Valles-Lluch A, et al. One-dimensional migration of olfactory ensheathing cells on synthetic materials: experimental and numerical characterization. Cell biochemistry and biophysics. 2013 Jan;65(1):21-36. • Gil-Perotin S, Duran-Moreno M, Cebrian-Silla A, Ramirez M, Garcia-Belda P, Garcia-Verdugo JM. Adult neural stem cells from the subventricular zone: a review of the neurosphere assay. Anatomical record (Hoboken, N.J. 2007). 2013 Sep;296(9):1435-52. • Platero-Luengo A, Gonzalez-Granero S, Duran R, Diaz-Castro B, Piruat JI, Garcia-Verdugo JM, et al. An o2-sensitive glomus cell- stem cell synapse induces carotid body growth in chronic hypoxia. Cell. 2014 Jan 16;156(1-2):291-303. • Frasquet M, Bataller L, Torres-Vega E, Duran-Moreno M, Garcia-Verdugo JM, Sevilla T, et al. Longitudinally extensive transverse myelitis with AQP4 antibodies revealing ovarian teratoma. Journal of neuroimmunology. 2013 Jul 27;263(1-2):145-7. • Martinez-Fernandez de la Camara C, Sequedo MD, Gomez-Pinedo U, Jaijo T, Aller E, Garcia-Tarraga P, et al. Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and inflammation in cone-enriched cultures of porcine retina. Experimental eye research. 2013 Jun;111:122-33. • Garcia-Parra P, Naldaiz-Gastesi N, Maroto M, Padin JF, Goicoechea M, Aiastui A, et al. Murine Muscle Engineered from Dermal Precursors: An In Vitro Model for Skeletal Muscle Generation, Degeneration, and Fatty Infiltration. Tissue engineering. Part C, Methods. 2013 Jun 22;20(1):28-41. • Rosillo JC, Olivera-Bravo S, Casanova G, Garcia-Verdugo JM, Fernandez AS. Olfacto-retinalis pathway in Austrolebias charrua fishes: A neuronal tracer study. Neuroscience. 2013 Sep 3;253C:304-15. • Velazquez-Sanchez C, Garcia-Verdugo JM, Murga J, Canales JJ. The atypical dopamine transport inhibitor, JHW 007, prevents amphetamine-induced sensitization and synaptic reorganization within the nucleus accumbens. Progress in neuro- psychopharmacology & biological psychiatry. 2013 Jul;44:73-80. • Escobedo-Lucea C, Bellver C, Gandia C, Sanz-Garcia A, Esteban FJ, Mirabet V, et al. A xenogeneic-free protocol for isolation and expansion of human adipose stem cells for clinical uses. PloS one. 2013;8(7):e67870. • Ordonez C, Moreno-Murciano P, Hernandez M, Di Caudo C, Carril-Mundinano I, Vazquez N, et al. Sox-2 Positive Neural Progenitors in the Primate Striatum Undergo Dynamic Changes after Dopamine Denervation. PloS one. 2013;8(6):e66377. • Fuster-Matanzo A, Llorens-Martin M, Sirerol-Piquer MS, Garcia-Verdugo JM, Avila J, Hernandez F. Dual effects of increased glycogen synthase kinase-3β activity on adult neurogenesis. Human molecular genetics. 2013 Jan 3;22(7):1300-15. • Capilla-Gonzalez V, Cebrian-Silla A, Guerrero-Cazares H, Garcia-Verdugo JM, Quinones-Hinojosa A. The generation of oligodendroglial cells is preserved in the rostral migratory stream during aging. Frontiers in cellular neuroscience. 2013 Sep 11;7:147. • Fiorelli R, Cebrian-Silla A, Garcia-Verdugo JM, Raineteau O. The adult spinal cord harbors a population of GFAP-positive progenitors with limited self-renewal potential. Glia. 2013 Dec;61(12):2100-13. • Martin ML, Liebisch G, Lehneis S, Schmitz G, Alonso-Sande M, Bestard-Escalas J, et al. Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells. Journal of lipid research. 2013 May;54(5):1457-65. • Voliani V, Gonzalez-Bejar M, Herranz-Perez V, Duran-Moreno M, Signore G, Garcia-Verdugo JM, et al. Orthogonal Functionalisation of Upconverting NaYF4 Nanocrystals. Chemistry (Weinheim an der Bergstrasse, Germany). 2013 Sep 27;19(40):13538-46. • Arana M, Gavira JJ, Pena E, Gonzalez A, Abizanda G, Cilla M, et al. Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction. Biomaterials. 2014 Jan;35(1):143-51. • Pineda JR, Daynac M, Chicheportiche A, Cebrian-Silla A, Sii Felice K, Garcia-Verdugo JM, et al. Vascular-derived TGF-β increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain. EMBO molecular medicine. 2013 Apr;5(4):548-62. • Kodani A, Salome Sirerol-Piquer M, Seol A, Garcia-Verdugo JM, Reiter JF. Kif3a interacts with Dynactin subunit p150 Glued to organize centriole subdistal appendages. The EMBO journal. 2013 Feb 20;32(4):597-607.

Research Projects

• Code: SAF2012-33683 Type: National Title: Análisis del proceso de mielinización en el estriado: Funding Agency: Ministerio de Ciencia e Innovación Efectos del EGF e IGF-1 Funding: 178.000 € Principal Investigator: José Manuel García Verdugo Duration: 2011-2014 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: GVPROMETEO 2009-011 Other CIBER’s collaboration: No Title: Células madre adultas: aplicaciones en terapia rege- Type: National nerativa Funding Agency: Ministerio de Economía y Competitividad Principal Investigator: José Manuel García Verdugo Funding: 105.300 € CIBERNED’s collaboration: No Duration: 2013-2015 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: PRI-PIMNEU-2011-1356 Type: CCAA Title: Biomaterials scaffoldings for brain reconstruction in Funding Agency: Conselleria de Educacion stroke. ERA-NET NEURON Funding: 160.600 € Principal Investigator: Juan Antonio Barcia Albacar Duration: 2009-2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: CB06/05/1131 Other CIBER’s collaboration: No Title: CIBERNED

136136 José Manuel García Verdugo Group José Manuel García Verdugo Group 137

Principal Investigator: José Manuel García Verdugo Funding Agency: Instituto de Salud Carlos III CIBERNED’s collaboration: No Funding: 742.521 € CIBERNED’s groups: Duration: 2007-2014 Other CIBER’s collaboration: No Type: National • Code: RD12/0019/0016 Funding Agency: Instituto de Salud Carlos III Title: Red Terapia Celular: NEUROCEL-ELA. Nodo Valencia Funding: 580.369 € Principal Investigator: José Manuel García Verdugo Duration: 2007-2014 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: 121730 /31 /32 Other CIBER’s collaboration: No Title: Efecto de la migración de células madre mediante Type: National campos magnéticos en la recuperación del infarto cerebral Funding Agency: Instituto de Salud Carlos III Principal Investigator: Juan Sahuquillo Barris Funding: 96.600 € CIBERNED’s collaboration: No Duration: 2013-2016 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: RD12/0019/0008 Type: Privado Title: RETIC de Terapia Celular Funding Agency: Fundació la Marató de TV3 Principal Investigator: Isabel Fariñas (coordinador de red: Funding: José María Moraleda) Duration: 2013 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; • Code: FIS101932 G 607 Title: Elección de la diana óptima en la estimulación cere- Other CIBER’s collaboration: No bral profunda para el tratamiento del transtorno obsesivo- Type: National compulsivo Funding Agency: Fondo de Investigaciones Sanitarias Principal Investigator: Juan Antonio Barcia Albacar Funding: 296.700 € CIBERNED’s collaboration: No Duration: 2013-2016 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: PI2010/07 Type: National Title: Reelina y GSK3 como dianas terapéuticas en la enfer- Funding Agency: Instituto de Salud Carlos III medad de Alzheimer Funding: 202.251 € Principal Investigator: Jesús Ávila de Grado Duration: 2011-2013 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 401; G 403; G 113; G 407; G 408; • Code: AC558 G 410 Title: Identificación y caracterización ultraestructural de Other CIBER’s collaboration: No progenitores neurales en el tercer ventrículo de macacos Type: Intramurales sanos y parkinsonianos Funding Agency: CIBERNED Principal Investigator: Rosario Sánchez Pernaute Funding: 540.000 € CIBERNED’s collaboration: No Duration: 2010-2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: RD12/0019/0011 Type: Privado Title: RETIC de Terapia Celular Funding Agency: La Kutxa Principal Investigator: Xavier Navarro Acebes (coordinador Funding: 185.000 € de red: José María Moraleda) Duration: 2010-2013 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; • Code: RD06/0010/0022 G 607 Title: Red de Terapia Celular. Nodo Valencia Other CIBER’s collaboration: No Principal Investigator: José Manuel García Verdugo Type: National CIBERNED’s collaboration: No Funding Agency: Instituto de Salud Carlos III CIBERNED’s groups: Funding: 180.000 € Other CIBER’s collaboration: No Duration: 2013-2016 Type: National Group José González Castaño

Departamento de Bioquímica e Instituto de Investigaciones Biomédicas “Alberto Sols”

2 UAM-CSIC. Lab B13 Facultad de Medicina UAM Arzobispo Morcillo, s/n Program 28029 Madrid (Spain) Tel.: +34 914 975 427 Fax: +34 915 854 401 [email protected]

José González Castaño Raúl Sánchez Lanzas Principal Investigator PhD student Beatriz Álvarez Castelao Postdoctoral Fellow (until March 2013)

138 José González Castaño Group 139

Summary

A link between aging, stress and protein homeostasis (proteostasis) is critical to the development of brain degenerative diseases. Brains from patients with Parkinson Disease (PD) show eosinophilic intracytoplasmic inclusions in dopaminergic neurons, Lewy bodies. Accordingly PD and other synucleinopathies can be classified as protein conformational diseases characterized by a failure of proteostasis due to aging and cellular stress. Alpha-synuclein (Snca) is the main protein component of Lewy bodies, DJ-1 protein has been implicated in several cell functions and may be considered an oxidative stress sensor. Mutations of Snca and DJ-1 genes are associated with dominant and recessive early onset PD, respectively. The main purpose of our work is to determine the mechanisms controlling proteostasis of Snca and DJ-1. Another critical point is to understand the proteostasis of transcription factors, like Nurr1, that are essential to the development and maintenance of dopaminergic neurons. The pathogenetic channels that resulted in PD development, including those mentioned above, were presented in a recent review. Concerning Nurr1 we have shown that is degraded in the cell nucleus by the ubiquitin proteasome system and determined that its N-terminal region (a.a 1–31) is essential for its degradation in the cell. We have not found any effect of Snca expression on Nurr-1 degradation rate in the cell, in contrast with some results published by other authors. The main effect of Snca on Nurr1 expression is mainly due to an inhibitory effect of Snca on Nurr1 gene transcription as reported by another research group. Nurr1 ∆1–31 has a much longer half-life than the full-length Nurr1 and is as potent as Nurr1 full length either in trans-activation of NGFI-B response elements or trans-repression of inducible NO synthases reporters. Accordingly, Nurr1 ∆1–31, because of longer persistence in the cell, can be a good candidate for gene and cell therapies in the treatment of PD.

Keywords

Proteasome, Ubiquitin, Proteostasis, Synuclein, DJ-1, Nurr1, Degradation, Parkinson, Synucleinopathies

Publications

• Castano JG, Gonzalez C, Obeso JA, Rodriguez M. Molecular pathogenesis and pathophysiology of Parkinson Disease: new targets for new therapies. RSC Drug Discovery Series No. 34. Emerging Drugs and Targets for Parkinson’s Disease. 2013;34:26-57. • Alvarez-Castelao B, Losada F, Ahicart P, Castano JG. The N-terminal region of Nurr1 (a.a 1-31) is essential for its efficient degradation by the ubiquitin proteasome pathway. PloS one. 2013;8(2):e55999. • Alvarez-Castelao B, Goethals M, Vandekerckhove J, Castano JG. Mechanism of cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the RedOx state of the N-terminal methionines. Biochimica et Biophysica Acta-Molecular Cell Research. 2013 Dec 3.

Research Projects

• Code: SAF_2012_34556 CIBERNED’s collaboration: Yes Title: Papel de la proteostasis de alfa-sinucleína y DJ-1 en la CIBERNED’s groups: G 401; G 104; G 111; G 307; G 409; función celular y su relación con la patogénesis de la Enfer- G 412 medad de Parkinson Other CIBER’s collaboration: Yes (CIBERER) Principal Investigator: Jose G. Castaño Type: CCAA CIBERNED’s collaboration: No Funding Agency: Comunidad de Madrid CIBERNED’s groups: Funding: 17.250 € Other CIBER’s collaboration: No Duration: 2012-2015 Type: National Funding Agency: MINECO • Code: PI2011/02 Funding: 120.000 € Title: Inicio y progresión de la Enfermedad de Parkinson Duration: 2013 Vulnerabilidad de la vía nigroestriada, eventos en origen y destino • Code: S2010_BMD Principal Investigator: José Ángel Obeso Inchausti Title: Redes de señalización y vías efectoras en modelos CIBERNED’s collaboration: Yes celulares y animales de enfermedades neurodegenerativas CIBERNED’s groups: G 205; G 201; G 104; G 208; G 203; Principal Investigator: Jose Gonzalez Castaño G 404; G 206 Other CIBER’s collaboration: No Funding: 362.000 € Type: Intramurales Duration: 2011-2013 Funding Agency: CIBERNED

140140 José González Castaño Group Group Manuel Guzmán Pastor

Departamento de Bioquímica y Biología Molecular I

Facultad de Biología, Universidad Complutense de Madrid 2 28040 Madrid (Spain)

Tel.: +34 913 944 668 Program Fax: +34 913 944 672 [email protected]

Manuel Guzmán Pastor María Salazar Roa Principal Investigator, Full Professor Postdoctoral Fellow Ismael Galve-Roperh Javier Díaz Alonso Co-Principal Investigator, Associate Professor PhD student Guillermo Velasco Díez Anna Paola Chiarlone Associate Professor PhD student Cristina Blázquez Ortiz Adán de Salas Quiroga Associate Professor PhD student Luigi Bellocchio Eva Resel Mariné Postdoctoral Fellow Lab manager Zaira Ortega Llorente Elena García Tabeada Postdoctoral Fellow Lab technician

141 Summary

Our research focuses globally on the study of the molecular and cellular mechanisms underlying the control of neural cell physiopathology by the endocannabinoid system. Thus, in 2013 we have kept on studying how this system tunes neural progenitor proliferation, differentiation and survival. Specifically, in the context of our CIBERNED program, we have evaluated the role of the endocannabinoid system in Huntington’s disease. As a matter of fact, Huntington’s disease constitutes, in our opinion, the best currently available model disease to assess the pathophysiological relevance and therapeutic potential of the endocannabinoid system in neurodegenerative diseases. This is due to several reasons: 1) CB1 is the most abundant G protein-coupled receptor in the brain; specifically, it is highly expressed in the basal ganglia at synapses established by neurons containing GABA [especially medium-sized spiny neurons (MSNs), the cells that primarily degenerate in Huntington’s disease] or glutamate (especially corticostriatal projecting neurons, which critically control MSN function) as transmitters, and play a key role in the control of motor behaviour (one of the processes that is most characteristically affected in Huntington’s disease). 2) A remarkable and dorsolaterally-selective down-regulation of the CB1 receptor has been documented in the basal ganglia of Huntington’s disease patients and animal models, and, of interest, this loss of CB1 receptors seems to reflect, at least in part, the neuron-damage pattern characteristic of the disease. 3) This loss of CB1 receptors occurs at early stages of Huntington’s disease and prior to the appearance of overt clinical symptoms, neurodegeneration and bulk changes in other neurochemical parameters. In 2013, our studies on the endocannabinoid system in Huntington´s disease have aimed at defining 1) whether stimulation of the endocannabinoid system in Huntington’s disease models promotes neuroprotection and therefore delays the onset and/or attenuates the progression of the pathology, 2) whether the loss of CB1 cannabinoid receptors observed in MSNs and/or corticostriatal terminals at early stages of the disease is involved in the pathogenesis of Huntington’s disease, and 3) whether the induction of CB2 cannabinoid receptors in reactive microglial cells modulates the excitotoxicity processes associated with Huntington’s disease . We aim at unravelling the molecular and cellular bases as well as the potential clinical relevance of those processes.

Keywords

Endocannabinoid system, Huntington’s disease, Neuroprotection, Neurogenesis, Cell signalling, Experimental therapeutics

Publications

• Fogaça MV, Galve-Roperh I, Guimarães FS, Campos AC. Cannabinoids, Neurogenesis and Antidepressant Drugs: Is there a Link? Current neuropharmacology. 2013 May;11(3):263-75. • Bishay P, Häussler A, Lim HY, Oertel B, Galve-Roperh I, Ferreiros N, et al. Anandamide deficiency and heightened neuropathic pain in aged mice. Neuropharmacology. 2013 Aug;71:204-15. • Salazar M, Lorente M, Garcia-Taboada E, Hernandez-Tiedra S, Davila D, Francis SE, et al. The pseudokinase tribbles homologue-3 plays a crucial role in cannabinoid anticancer action. Biochimica et Biophysica Acta-Molecular Cell Biology Of Lipids. 2013 Oct;1831(10):1573-8. • Campos AC, Ortega Z, Palazuelos J, Fogaça MV, Aguiar DC, Diaz-Alonso J, et al. The anxiolytic effect of cannabidiol on chronically stressed mice depends on hippocampal neurogenesis: involvement of the endocannabinoid system. The international journal of neuropsychopharmacology. 2013 Jul;16(6):1407-19. • Bellocchio L, Soria-Gomez E, Quarta C, Metna-Laurent M, Cardinal P, Binder E, et al. Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB₁ receptor blockade. Proceedings of the National Academy of Sciences of the United States of America. 2013 Mar 19;110(12):4786-91. • Galve-Roperh I, Chiurchiù V, Diaz-Alonso J, Bari M, Guzman M, Maccarrone M. Cannabinoid receptor signaling in progenitor/ stem cell proliferation and differentiation. Progress in lipid research. 2013 Oct;52(4):633-50. • Casarejos MJ, Perucho J, Gomez A, Munoz MP, Fernandez-Estevez M, Sagredo O, et al. Natural cannabinoids improve dopamine neurotransmission and tau and amyloid pathology in a mouse model of tauopathy. Journal of Alzheimer´s disease. 2013;35(3):525-39.

142142 Manuel Guzmán Pastor Group Manuel Guzmán Pastor Group 143

Research Projects

• Code: UCM-FAK/2012/01 CIBERNED’s collaboration: No Title: Alteraciones de la función cannabinoide durante el CIBERNED’s groups: desarrollo cortical como factor de riesgo para la esquizo- Other CIBER’s collaboration: No frenia Type: National Principal Investigator: Ismael Galve-Roperh Funding Agency: MINECO-Plan Nacional CIBERNED’s collaboration: No Funding: 290.000 € CIBERNED’s groups: Duration: 2013-2015 Other CIBER’s collaboration: Yes (CIBERSAM) Type: Privado • Code: UCM-GWP/2011/01 Funding Agency: Fundación Alicia Koplowitz Title: Optimización de terapias antitumorales de cannabi- Funding: 73.000 € noides y temozolomida en modelos animales de glioma Duration: 2012-2013 Principal Investigator: Guillermo Velasco Diez y Manuel Guzmán Pastor • Code: CIBERNED 2013/05 CIBERNED’s collaboration: No Title: Identificación y caracterización molecular de subpo- CIBERNED’s groups: blaciones de receptores cannabinoides en poliglutamino- Other CIBER’s collaboration: No patías Type: Privado Principal Investigator: Manuel Guzmán Pastor Funding Agency: GW Pharmaceuticals CIBERNED’s collaboration: Yes Funding: 150.000 € CIBERNED’s groups: G 201; G 303; G 304; G 305 Duration: 2011-2013 Other CIBER’s collaboration: No Type: Intramurales • Code: PI12/00919 Funding Agency: CIBERNED Title: Papel del receptor CB1 cannabinoide en alteraciones Funding: 50.000 € de la neurogénesis cortical: Implicaciones en excitabibili- Duration: 2013-2015 dad neuronal y función corticoespinal Principal Investigator: Ismael Galve Roperh • Code: PI12/02248 CIBERNED’s collaboration: No Title: Mecanismos de regulación de la autofagia en células CIBERNED’s groups: tumorales: muerte mediada por autofagia en respuesta a Other CIBER’s collaboration: No fármacos antitumorales y participación en el control de la Type: National tumorigénesis Funding Agency: MINECO-ISCIII-FIS Principal Investigator: Guillermo Velasco Diez Funding: 147.015 € CIBERNED’s collaboration: No Duration: 2013-2015 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: PI2010/06 Type: National Title: Neuroprotección en enfermedad de Huntington Funding Agency: MINECO-ISCIII-FIS Principal Investigator: Justo García de Yébenes Prous Funding: 191.500 € CIBERNED’s collaboration: Si Duration: 2013-2015 CIBERNED’s groups: G 304; G 303; G 305; G 306; G 307 Other CIBER’s collaboration: No • Code: S2010/BMD-2308 Type: Intramurales Title: Neurofarmacología del sistema endocannabinoide: Funding Agency: CIBERNED del laboratorio a la clínica Funding: 417.600 € Principal Investigator: Manuel Guzmán Pastor Duration: 2010-2013 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 305; G 303; G 304 • Code: S2011/BMD-2336 Other CIBER’s collaboration: Yes (CIBERSAM) Title: Estudio de la biología de células madre neurales para Type: CCAA su empleo en terapia celular en enfermedades neurodege- Funding Agency: Comunidad de Madrid nerativas Funding: 177.500 € Principal Investigator: Rosario Moratalla Duration: 2012-2015 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 204; G 108; G 305 • Code: SAF2012-35759 Other CIBER’s collaboration: No Title: Neuroprotección por el receptor CB1 cannabinoide Type: CCAA en la enfermedad de Huntington: relevancia de las vías cor- Funding Agency: Comunidad Autónoma de Madrid ticoestriatales directa e indirecta Funding: 598.000 € Principal Investigator: Manuel Guzmán Pastor Duration: 2012-2015 PhD Dissertations

• Author: Clara Andradas Arias Title: Papel del receptor huérfano GPR55 en la fisiopatolo- gía del cáncer: implicación en la proliferación e invasión de células tumorales Date: 12 July 2013 Supervisor: Manuel Guzmán Pastor

144144 Manuel Guzmán Pastor Group Group Teresa Iglesias Vacas

Instituto de Investigaciones Biomédicas “Alberto Sols”

(CSIC-UAM) y CIBERNED (ISCIII) 2 Departamento de Fisiopatología Endocrina y del Sistema Nervioso

Arturo Duperier, nº 4 Program 28029 Madrid (Spain) Tel.: +34 915 854 487 Fax: +34 915 854 401 [email protected]

Teresa Iglesias Vacas Andrea Gamir Morralla Principal Investigator, CSIC Tenured Scientist PhD student Celia López Menéndez Julia Pose Utrilla Postdoctoral Fellow Master’s student Lucía García Guerra Daniel García Carrera Postdoctoral Fellow Student Ana María del Puerto del Pino Yolanda Bachiller de la Cruz Postdoctoral Fellow Technician Sara Ramírez Rubio Rosa Maritato PhD student Research visitor

145 Summary

For the last years, our research group has been focussed on the study of PKD (Protein Kinase D) and its substrate Kidins220 (Kinase D interacting substrate of 220 kDa, also known as ankyrin repeat-rich membrane spanning or ARMS), in order to define their role in neuronal physiopathology and their participation in the molecular mechanisms involved in neurodegenerative processes.

During year 2013 we have found an increase in Kidins220 levels in brain necropsies from patients at different stages of Alzheimer´s disease (AD) progression, where it accumulates with tau (López-Menéndez, Gamir-Morralla et al., 2013). Using these human samples of AD, transgenic mice and cellular models of neurodegeneration we have identified some of the molecular mechanisms involved in Kidins220 accumulation. Among these mechanisms, imbalances between phosphorylation/ dephosphorylation systems clearly associated with AD (particularly GSK3 and PP1 phosphatase activities) play a crucial role by modulating the ability of calpain to proteolyse Kidins220. Finally, we propose a model integrating all our results and explaining how some effects of excitotoxicity may drastically differ in acute versus chronic brain damage, and the implications for therapeutic intervention.

Following this line of research, we have continued studying several molecular mechanisms that modulate Kidins220 stability and the effects produced by variations of Kidins220 protein levels on neuronal survival and death. We have designed a neuroprotective strategy based on the use of peptides with possible therapeutic applications for excitotoxicity, a type of neuronal death elicited by acute or chronic exposure of N-methyl-d-aspartate receptors (NMDARs) to high concentrations of glutamate that takes place in AD and other neuronal degeneration-associated disorders.

On the other hand, regarding PKD, we have found that this kinase associates with, phosphorylates and activates neuronal nitric oxide synthases (nNOS). This activation leads to the production of nitric oxide, a molecule known to have important implications in neuronal death and neurodegenerative diseases.

Finally, at the end of the year we started an internal collaborative project formed by five research groups to study the role of GSK-3 β in the alterations of the cortical circuits that occur in Alzheimer´s Disease.

Keywords

Kidins220/ARMS, Protein Kinase D (PKD), Alzheimer´s Disease, GSK3, Neuroprotection, Neurotoxicity

Publications

• Tapia M, del Puerto A, Pallas-Bazarra N, Puime A, Sanchez-Ponce D, Fronzaroli-Molinieres L, et al. GSK3 and β-catenin determines functional expression of sodium channels at the axon initial segment. Cellular and molecular life sciences. 2013;70(1):105-20. • Del Puerto A, Wandosell F, Garrido JJ. Neuronal and glial purinergic receptors functions in neuron development and brain disease. Frontiers in cellular neuroscience. 2013;7:197.

Research Projects

• Code: SAF2011-26233 Funding: 137.140 € Title: Participation of PKD and its substrate, Kidins220, in Duration: 2012-2014 the molecular pathways of neuronal survival and neurode- generation • Code: S2010_BMD Principal Investigator: Teresa Iglesias Vacas Title: Redes de señalización y vías efectoras en modelos CIBERNED’s collaboration: No celulares y animales de enfermedades neurodegenerativas CIBERNED’s groups: Principal Investigator: José González Castaño Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: National CIBERNED’s groups: G 401; G 104; G 111; G 307; G 409; Funding Agency: MINECO G 412

146146 Teresa Iglesias Vacas Group Teresa Iglesias Vacas Group 147

Other CIBER’s collaboration: Yes (CIBERER) Principal Investigator: Teresa Iglesias Vacas Type: CCAA CIBERNED’s collaboration: Yes Funding Agency: Comunidad de Madrid CIBERNED’s groups: G 401; G 403; G 111; G 504; G 307 Funding: 17.250 € Other CIBER’s collaboration: No Duration: 2012-2015 Type: Intramurales Funding Agency: CIBERNED • Code: 2013/07A Funding: 158.000 € Title: Papel de GSK-3 β en las alteraciones de los circuitos Duration: 2013-2015 corticales que ocurren en la enfermedad de Alzheimer Group Jaime Kulisevsky Bojarski

Director de la Unidad de Trastornos de Movimiento

2 Servicio de Neurología Hospital de Sant Pau Sant Antoni Maria Claret, 167 Program 08025 Barcelona (Spain) Tel.: +34 935 537 613 Fax: +34 935 537 872 [email protected]

Jaime Kulisevsky Bojarski Roser Ribosa Principal Investigator PhD student Berta Pascual Sedano Fabián Arenas Researcher Researcher Alexandre Gironell Carrero María-Victoria Sosti Researcher Researcher Javier Pagonabarraga Saúl Martínez Horta Researcher PhD student Carmen García Sánchez Ramón Fernández de Bobadilla Researcher PhD student Antonia Campolongo Perillo Carle Roig Arnall Researcher (Nursing) Researcher

148 Jaime Kulisevsky Bojarski Group 149

Publications

• Martinez-Horta S, Pagonabarraga J, Fernandez de Bobadilla R, Garcia-Sanchez C, Kulisevsky J. Apathy in Parkinson’s disease: more than just executive dysfunction. Journal of the International Neuropsychological Society : JINS. 2013 May. 19 (5): 571-82. • Jaime Kulisevsky,Saül Martinez-Horta,Javier Pagonabarraga. Cognitive assessment in Parkinson’s disease. 2013. 53-64. • Jaime Kulisevsky,Javier Pagonabarraga,Carolina Villa-Bonomo. Drug treatment and behavior. 2013. 211-229. • Kulisevsky J, Luquin MR, Arbelo JM, Burguera JA, Carrillo F, Castro A et al. [Advanced Parkinson’s disease: clinical characteristics and treatment (part 1)]. Neurologia (Barcelona, Spain). 2013 Oct. 28 (8): 503-21. • Kulisevsky J, Luquin MR, Arbelo JM, Burguera JA, Carrillo F, Castro A et al. Advanced Parkinson’s disease: clinical characteristics and treatment. Part II. Neurologia (Barcelona, Spain). 2013 Nov-Dec. 28 (9): 558-83. • Kulisevsky J, Fernandez de Bobadilla R, Pagonabarraga J, Martinez-Horta S, Campolongo A, Garcia-Sanchez C et al. Measuring functional impact of cognitive impairment: Validation of the Parkinson’s Disease Cognitive Functional Rating Scale. Parkinsonism & related disorders. 2013 Jun 14. • Pagonabarraga J, Soriano-Mas C, Llebaria G, Lopez-Solà M, Pujol J, Kulisevsky J. Neural correlates of minor hallucinations in non-demented patients with Parkinson’s disease. Parkinsonism & related disorders. 2013 Dec 10. • Volkmann J, Albanese A, Antonini A, Chaudhuri KR, Clarke CE, de Bie RM et al. Selecting deep brain stimulation or infusion therapies in advanced Parkinson’s disease: an evidence-based review. Journal of neurology. 2013 Jan 5. • Pagonabarraga J, Corcuera-Solano I, Vives-Gilabert Y, Llebaria G, Garcia-Sanchez C, Pascual-Sedano B et al. Pattern of regional cortical thinning associated with cognitive deterioration in Parkinson’s disease. PloS one. 2013. 8 (1): e54980. • Berardelli A, Wenning GK, Antonini A, Berg D, Bloem BR, Bonifati V et al. EFNS/MDS-ES recommendations for the diagnosis of Parkinson’s disease. European journal of neurology : the official journal of the European Federation of Neurological Societies. 2013 Jan. 20 (1): 16-34. • Fernandez de Bobadilla R, Pagonabarraga J, Martinez-Horta S, Pascual-Sedano B, Campolongo A, Kulisevsky J. Parkinson’s disease-cognitive rating scale: psychometrics for mild cognitive impairment. Movement disorders : official journal of the Movement Disorder Society. 2013 Sep. 28 (10): 1376-83. • Serrano-Munuera C, Corral-Juan M, Stevanin G, San Nicolas H, Roig C, Corral J et al. New subtype of spinocerebellar ataxia with altered vertical eye movements mapping to chromosome 1p32. JAMA neurology. 2013 Jun 1. 70 (6): 764-71. • Tolosa E, Hernandez B, Linazasoro G, Lopez-Lozano JJ, Mir P, Marey J et al. Efficacy of levodopa/carbidopa/entacapone versus levodopa/carbidopa in patients with early Parkinson’s disease experiencing mild wearing-off: a randomised, double-blind trial. Journal of neural transmission. 2013 Nov 20.

Research Projects • Code: 2011 FI-DGR • Code: PI10/01498 Title: Contracte AGAUR2010 FI-DGR 2010 Title: Definición y estudio longitudinal del deterioro cog- Principal Investigator: Jaime Kulisevsky nitivo leve en la enfermedad de Parkinson: definición de CIBERNED’s collaboration: No criterios clínicos operativos y progresion a demencia CIBERNED’s groups: Principal Investigator: Jaime Kulisevsky Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: CCAA CIBERNED’s groups: Funding Agency: AGAUR Other CIBER’s collaboration: No Funding: 61.834 € Type: National Duration: 2011-2014 Funding Agency: FIS Funding: 88.330 € • Code: MIA 2013-1 Duration: 2011 2013 Title: Contrato MIA 2013 Principal Investigator: Jaime Kulisevsky • Code: CIBERNED 2012 CIBERNED’s collaboration: No Title: Desarrollo de modelos experimentales en animales CIBERNED’s groups: en la Enfermedad de Parkinson Other CIBER’s collaboration: No Principal Investigator: Jaime Kulisevsky Type: Privado CIBERNED’s collaboration: No Funding Agency: Fundación Privada Hospital de la Santa CIBERNED’s groups: Creu i Sant Pau Other CIBER’s collaboration: No Funding: 45.223 € Type: Intramurales Duration: 2013-2014 Funding Agency: CIBERNED Funding: 49.000 € CIBERNED’s collaboration: No Duration: 2012-2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: PI12/03005 Type: National Title: Desarrollo, validación y estudio de la sensibilidad al Funding Agency: CENIT cambio de una versión alternativa de la Parkinson’s Disease Funding: 375.000 € - Cognitive Rating Scale Duration: 2011-2014 Principal Investigator: Jaime Kulisevsky CIBERNED’s collaboration: No • Code: EHDN - EUROPEAN HUNTINGTON DISEASE NETWORK CIBERNED’s groups: Title: Huntington’s disease network - REGISTRY Other CIBER’s collaboration: No Principal Investigator: Jaime Kulisevsky Type: National CIBERNED’s collaboration: No Funding Agency: MINECO CIBERNED’s groups: Funding: 28.435 € Other CIBER’s collaboration: No Duration: 2013-2015 Type: International Funding Agency: EHDN • Code: 112610 Funding: 133.875 € Title: Early implementation of music thrapy in rehabilita- Duration: 2011-2013 tion of Aphasic patients after acute adquired brain injury /Efectivity of music therapy in the rehabilitation of acute • Code: PI2011/03 aphasic patients Title: Generación de neuronas dopaminérgicas a partir de Principal Investigator: Jaime Kulisevsky células somáticas de pacientes parkinsonianos con fallo CIBERNED’s collaboration: No cognitivo CIBERNED’s groups: Principal Investigator: Rosario Moratalla Villalba Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: Privado CIBERNED’s groups: G 204; G 106; G 202; G 110; G 108; Funding Agency: Fundació La Marató de TV3 Other CIBER’s collaboration: No Funding: 84.073 € Type: Intramurales Duration: 2012-2015 Funding Agency: CIBERNED Funding: 300.000 € • Code: AC638 Duration: 2011-2013 Title: HENUFOOD Principal Investigator: Jaime Kulisevsky

PhD Dissertations

• Author: María Victoria Sosti Title: Caracterización molecular y determinación del perfil cognitivo en modelos de parkinsonismo en rata inducido por 6-OHDA Date: 29 November 2013 Supervisor: Jaime Kulisevsky Bojarski

150150 Jaime Kulisevsky Bojarski Group Group José Luis Labandeira García

Center for Research in Molecular Medicine and Chronic Diseases (CIMUS)

Universidad de Santiago de Compostela 2 Avenida Barcelona, 22 15782 Santiago de Compostela (Spain) Tel.: +34 881 812 223 / 881 815 471 Program Fax: +34 881 812 378 [email protected]

José L. Labandeira García Juan A. Parga Martín Principal Investigator, Full Professor Postdoctoral Fellow María J. Guerra Seijas Pablo Garrido Gil Full Professor CIBERNED Postdoctoral Fellow Ramón Soto Otero Ana Borrajo López Full Professor PhD student Estefanía Méndez Álvarez A. Domínguez Meijide Full Professor PhD student Jannette Rodríguez Pallares Rita Valenzuela Limiñana Associate Professor Ciberned PhD student Ana M. Muñoz Patiño Iria Novoa Pérez Assistan Professor CIBERNED Specialist Technician Ana I. Rodríguez Pérez Pilar Aldrey García Assistant Professor Specialist Technician Carmen Díaz Ruiz J.A. Trillo Franco Assistant Professor Specialist Technician Begoña Villar Cheda Assistan Professor

151 Summary

We have studied the role of the basal ganglia renin-angiotensin system (RAS) in dopamine neuron vulnerability and progression of the dopaminergic degeneration, and the possible manipulation of RAS activity as neuroprotective strategy. Over the last few years, a considerable number of studies have shown that local /tissue RAS play a major role in aging and aging-related degenerative processes in several tissues including brain tissue. Important functional interactions between RAS and dopamine and dopamine receptors have been observed in several peripheral tissues, particularly in the cardiovascular and renal systems, in which alteration of this interaction plays a major role in the development of degenerative processes. We have shown an important functional interaction between the local RAS and the nigrostriatal system, as well as a major role of the nigral RAS in progression of dopaminergic degeneration. Over the last year, we have studied the RAS in the nigra and its possible role in neurodegeneration and neuroinflammation, and the intracellular pathways involved in the effects of the local RAS on the dopaminergic vulnerability. In the nigra, we have shown interactions between RAS and iron homeostasis, estrogen levels or Rho-Kinasa activity, and their possible interest for neuroprotective strategies. We have investigated L-DOPA-induced dyskinesias in animal models of parkinsonism, as well as potential treatments. In collaboration with other research groups, we have investigated on the development of new potent a selective MAO-B inhibitors as possible neuroprotective drugs for Parkinson´s disease. In collaboration with other research group (member of CIBERNED, Sevilla), we have studied the neuroprotective and rescue effects of carotid body grafts on animal models of parkinsonism. We have participated in several international studies on cell therapy in treatment of neurodegenerative diseases.

Keywords

Neurodegeneration, Neuroprotection, Neuroinflammation, Parkinson, Aging, Angiotensin, Cell therapy

Publications

• Pisani L, Catto M, Nicolotti O, Grossi G, Di Braccio M, Soto-Otero R, et al. Fine molecular tuning at position 4 of 2H-chromen- 2-one derivatives in the search of potent and selective monoamine oxidase B inhibitors. Eur J Med Chem. 2013;70:723-39. • Reinhardt P, Glatza M, Hemmer K, Tsytsyura Y, Thiel CS, Höing S, et al. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One. 2013;8(3):e59252. • Bezard E, Munoz A, Tronci E, Pioli EY, Li Q, Porras G, et al. Anti-dyskinetic effect of anpirtoline in animal models of L-DOPA- induced dyskinesia. Neurosci Res. 2013;77(4):242-6. • Rodriguez-Perez AI ,Dominguez-Meijide A, Lanciego JL, Guerra MJ, Labandeira-Garcia JL. Inhibition of Rho kinase mediates the neuroprotective effects of estrogen in the MPTP model of Parkinson’s disease. Neurobiol Dis. 2013;58:209-19. • Dominguez-Meijide A, Villar-Cheda B, Garrido-Gil P, Sierrra-Paredes G, Guerra MJ, Labandeira-Garcia JL. Effect of chronic treatment with angiotensin type 1 receptor antagonists on striatal dopamine levels in normal rats and in a rat model of Parkinson’s disease treated with l-DOPA. Neuropharmacology. 2013 Aug 20. • Labandeira-Garcia JL, Rodriguez-Pallares J, Dominguez-Meijide A, Valenzuela R, Villar-Cheda B, Rodriguez-Perez AI. Dopamine- Angiotensin interactions in the basal ganglia and their relevance for Parkinson’s disease. Mov Disord. 2013;28(10):1337-42. • Garrido-Gil P, Rodriguez-Pallares J, Dominguez-Meijide A, Guerra MJ, Labandeira-Garcia JL. Brain angiotensin regulates iron homeostasis in dopaminergic neurons and microglial cells. Exp Neurol. 2013;250:384-96. • Borrajo A, Rodriguez-Perez AI, Diaz-Ruiz C, Guerra MJ, Labandeira-Garcia JL. Microglial TNF-α mediates enhancement of dopaminergic degeneration by brain angiotensin. Glia. 4 Nov 2013. • Pisani L, Barletta M, Soto-Otero R, Nicolotti O, Mendez-Alvarez E, Catto M, et al. Discovery, biological evaluation, and structure- activity and -selectivity relationships of 6’-substituted (E)-2-(benzofuran-3(2H)-ylidene)-N-methylacetamides, a novel class of potent and selective monoamine oxidase inhibitors. J Med Chem. 2013;56(6):2651-64. • Antonchick AP, Lopez-Tosco S, Parga J, Sievers S, Schürmann M, Preut H, et al. Highly enantioselective catalytic synthesis of neurite growth-promoting secoyohimbanes. Chem Biol. 2013;20(4):500-9. • Dakas PY, Parga JA, Höing S, Schöler HR, Sterneckert J, Kumar K, et al. Discovery of neuritogenic compound classes inspired by natural products. Angew Chem Int Ed Engl. 2013;52(36):9576-81. • Munoz-Manchado AB, Villadiego J, Suarez-Luna N, Bermejo-Navas A, Garrido-Gil P, Labandeira-Garcia JL, et al. Neuroprotective and reparative effects of carotid body grafts in a chronic MPTP model of Parkinson’s disease. Neurobiology Aging. 2013;34(3):902-15.

152152 José Luis Labandeira García Group José Luis Labandeira García Group 153

Research Projects

• Code: 2011/XA033 Type: National Title: Financiación de Groups de referencia competitiva del Funding Agency: Fondo de Investigaciones Sanitarias sistema Universitario gallego Funding: 296.700 € Principal Investigator: José Luis Labandeira García Duration: 2013-2016 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI2011/02 Other CIBER’s collaboration: No Title: Inicio y progresión de la Enfermedad de Parkinson. Type: CCAA Vulnerabilidad de la vía nigroestriada, eventos en origen y Funding Agency: Conselleria de Educacion, Xunta de Galicia destino Funding: 168.000 € Principal Investigator: José Ángel Obeso Inchausti Duration: 2011-2013 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 205; G 201; G 104; G 208; G 203; • Code: XUGA2010/PX195 G 404; G 206 Title: Sistema renina-angiotensina cerebral como diana te- Other CIBER’s collaboration: No rapeutica para desarrollo de terapia neuroprotectora para Type: Intramurales enfermedad de Parkinson Funding Agency: CIBERNED Principal Investigator: José Luis Labandeira García Funding: 362.000 € CIBERNED’s collaboration: No Duration: 2011-2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: RD12/0019/0011 Type: CCAA Title: RETIC de Terapia Celular Funding Agency: Xunta de Galicia Principal Investigator:Xavier Navarro Acebes (coordinador Funding: 73.025 € de red: Jose Maria Moraleda) Duration: 2010-2013 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; • Code: RD12/0019/0008 G 607 Title: RETIC de Terapia Celular Other CIBER’s collaboration: No Principal Investigator: Isabel Fariñas (coordinador de red: Type: National José María Moraleda) Funding Agency: Instituto de Salud Carlos III CIBERNED’s collaboration: Yes Funding: 180.000 € CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; Duration: 2013-2016 G 607 Other CIBER’s collaboration: No Group José Luis Lanciego Pérez

Área de Neurociencias, CIMA

2 Avenida Pío XII, 55 31008 Pamplona (Spain) Tel.: +34 948 194 700 Program Fax: +34 948 194 715 [email protected]

José Luis Lanciego Salvador Sierra San Nicolás Principal Investigator CIBERNED Postdoctoral Fellow, Neurologist Alberto José Rico Martín José Diego Pignataro López CIBERNED Postdoctoral Fellow PhD student Iria González Dopeso-Reyes Elvira Roda Recalde Postdoctoral Fellow Lab Technician Natasha Luquin Barrado Postdoctoral Fellow

154 José Luis Lanciego Pérez Group 155

Summary

Our research group is mainly engaged in the study of the neurobiology of Parkinson disease, with a multiple focus on a number of different topics related to the pathophysiology of this disease, as follows:

Analysis of brain circuits underlying the pathophysiology of Parkinson disease. By using relevant animal models of the disease, we study the ongoing changes in basal ganglia circuits following dopaminergic neurodegeneration.

Study of G-protein-coupled receptors (GPCRs) in Parkinson disease. Our main interest is to address the presence of different GPCRs in basal ganglia nuclei. Among others, we focus on endocannabinoid receptors 1 and 2 (CB1 and CB2), orphan receptor GPR55, adenosine type 2A receptors (A2A) and dopamine receptors types 1 and 2 (D1 and D2). All studies are carried out in control and parkinsonian animals. More specifically, we are very much interested in elucidating which GPCRs are making heteromers, with the ultimate goal of addressing whether these GPCR heteromers may be considered as feasible pharmacological targets.

Cell and gene therapies in Parkinson disease. Together with a number of European collaborators, we have recently started a research project focused on the use of direct reprogramming techniques converting skin fibroblast onto dopaminergic neurons that will further be used for autologous transplantation in relevant animal models of parkinsonism. Moreover, a number of gene therapy-based approaches are currently under implementation to further manipulate basal ganglia circuits of interest.

Keywords

Basal ganglia, Parkinson’s disease, GPCR receptors, Gene therapy, Primate

Publications

• Bonaventura J, Rico AJ, Moreno E, Sierra S, Sanchez M, Luquin N, et al. l-DOPA-treatment in primates disrupts the expression of A2A adenosine-CB1 cannabinoid-D2 dopamine receptor heteromers in the caudate nucleus. Neuropharmacology. 2013 Nov 11;79C:90-100. • Pinna A, Bonaventura J, Farre D, Sanchez M, Simola N, Mallol J, et al. L-DOPA disrupts adenosine A2A-cannabinoid CB1- dopamine D2 receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: Biochemical and behavioral studies. Exp Neurol. 2013. • Gil-Farina I, Di Scala M, Vanrell L, Olagüe C, Vales A, High KA, et al. IL12-mediated liver inflammation reduces the formation of AAV transcriptionally active forms but has no effect over preexisting AAV transgene expression. PloS one. 2013;8(7):e67748. • Paneda A, Lopez-Franco E, Kaeppel C, Unzu C, Gil-Royo AG, D’Avola D, et al. Safety and Liver Transduction Efficacy of rAAV5-cohPBGD in Nonhuman Primates: A Potential Therapy for Acute Intermittent Porphyria. Human gene therapy. 2013 Dec;24(12):1007-17. • Garcia-Barroso C, Ricobaraza A, Pascual-Lucas M, Unceta N, Rico AJ, Goicolea MA, et al. Tadalafil crosses the blood-brain barrier and reverses cognitive dysfunction in a mouse model of AD. Neuropharmacology. 2013 Jan;64:114-23.

Research Projects

• Code: BFU2012-37907 Funding: 127.050 € Title: Coexpresión de receptores de adenosina 2A y de en- Duration: 2013-2015 docannabinoides 1 y 2 en los nucleos de salida de los gan- glios basales en el modelo de enfermedad de Parkinson en • Code: NARSAD monos Title: Immunotoxin-mediated selective removal of specific Principal Investigator: José Luis Lanciego basal ganglia pathways in the monkey model of Parkinson’s CIBERNED’s collaboration: No disease CIBERNED’s groups: Principal Investigator: José Luis Lanciego Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: National CIBERNED’s groups: Funding Agency: Ministerio de Economía y Competitividad Other CIBER’s collaboration: No Type: International Duration: 2013-2015 Funding Agency: Brain & Behavior Researcg Foundation Funding: 100.000 € • Code: PI2011/02 Duration: 2013-2014 Title: Inicio y progresión de la Enfermedad de Parkinson. Vulnerabilidad de la vía nigroestriada, eventos en origen y • Code: COEN-PATHFINDER destino Title: In vivo neuronal cell reprogramming for a new regen- Principal Investigator: José Ángel Obeso Inchausti erative approach in Parkinson’s disease CIBERNED’s collaboration: Yes Principal Investigator:José Luis Lanciego (CIBERNED, Spain) CIBERNED’s groups: G 205; G 201; G 104; G 208; G 203; CIBERNED’s collaboration: No G 404; G 206 CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Intramurales Type: International Funding Agency: CIBERNED Funding Agency: ISCIII-CIBERNED Funding: 362.000 € Funding: 150.000 € Duration: 2011-2013

156156 José Luis Lanciego Pérez Group Group José López Barneo

Instituto de Biomedicina de Sevilla

Hospital Universitario Virgen del Rocío / Universidad de Sevilla 2 (Spain)

Tel.: +34 955 923000 Program Fax: +34 955 923101 [email protected]

José López Barneo Lin Gao Principal Investigator, Full Professor Researcher Aida Platero Luengo Luis María Escudero PhD student Researcher Alberto Pascual Bravo María del Carmen Fernadez-Aguera Rodríguez Researcher PhD student Candela Caballero Erazo Nela Suárez Luna Rio Hortega Post MIR Technician Carmen Calero Acuña Pablo Mir Rivera Rio Hortega Post MIR Médico David Macías Gutiérrez Patricia González Rodríguez Postdoctoral Fellow Postdoctoral Fellow Francisco Javier Villadiego Luque Patricia Ortega Sáenz Postdoctoral Fellow Associate Professor Gloria Paula García Flores Pilar Gómez Garre Technician Researcher Helia Sarmiento Sot Rosana March Díaz Technician Postdoctoral Fellow Ignacio Arias Mayenco Ricardo Pardal Redondo Technician Researcher Ivet López López Victoria Bonilla Henao Technician Specialist Technician José Antonio Rodríguez Gómez Xavier Dánglemont Tassiony Researcher Postdoctoral Fellow Juan José Toledo Aral Researcher

157 Summary

• Pathogenesis of Parkinson’s disease (PD): This year we have made progress in the characterization of a genetically modified animal model of mitochondrial complex I, which will be used to study early changes of neurodegenerative diseases such as PD. Dr. Pablo Mir continues with research on genetic alterations associated with PD and movement disorders diseases and the analysis of changes in cortical function that occur in patients using transcranial magnetic stimulation. Dr. Luis María Escudero has developed a diagnostic method based on the computational analysis of muscle biopsies, which will be useful in the diagnosis of patients with neuromuscular diseases. This has established collaborations with different clinical groups of Hospital Virgen del Rocío, besides generating a patent and several publications.

• Cell therapy of PD: In collaboration with the company Axontherapix has been made transplanted neurospheres CB (carotid body) in animal models Parkinsonian (MPTP generated), and preliminary results indicate that both produce neuroprotection as neurorestauración. In parallel, they still perform human transplants CB in Parkinsonian mice to study trophic action exerted on nigrostriatal neurons by glial cell line-derived neurotrophic factor (GDNF). This year has published a physiological description of human CB. It is the first time that were studied the characteristics of cell and molecular physiology of that providing new insights for the systematic of the pathologies associated with this organ in the human.

• Effect of hypoxia on neurogenic niches in the central and peripheral nervous system: Regarding the characterization of factors that regulate neurogenesis in the neurogenic niche of the CB, we have reported that neuronal cells, (CB type I cells) are responsible for detecting hypoxic stimulus and comunicating with type II cells. We have described that one of the substances involved in the communication between neuronal cells and progenitors of CB is endothelin-1 (ET-1). ET-1 is released by the type I cells in response to the hypoxic stimulus, and is capable of selectively activating the proliferation of the progenitors, thus triggering organ growth. As part of this research is developing a study on the effects of intermittent hypoxia at the level of the neurogenic niche, we have characterized and developed a rodent animal model of intermittent hypoxia. This model is used to analyze different techniques by the existence of changes in the neurogenic niche both centrally (subventricular zone, SVZ) and peripheral (CB).

Keywords

Hypoxia, Ion channels, Carotid body, Movement disorders, Parkinson’s disease

Publications

• Mir P, Rodrigo L, Mercader A, Buendia P, Mateu E, Milan-Sanchez M, et al. False positive rate of an arrayCGH platform for single-cell preimplantation genetic screening and subsequent clinical application on day-3. Journal of Assisted Reproduction and Genetics. 2013;30(1):143-9. • Urena J, Fernandez-Tenorio M, Porras-Gonzalez C, Gonzalez-Rodriguez P, Castellano A, Lopez-Barneo J. A new metabotropic role for L-type Ca(2+) channels in vascular smooth muscle contraction. Current Vascular Pharmacology. 2013;11(4):490-6. • Saez A, Acha B, Montero-Sanchez A, Rivas E, Escudero LM, Serrano C. Neuromuscular disease classification system. Journal of Biomedical Optics. 2013;18(6):066017. • Levitsky KL, Toledo-Aral JJ, Lopez-Barneo J, Villadiego J. Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections. Scientific reports. 2013;14(3): 2937. • Gao L, Hidalgo-Figueroa M, Escudero LM, Diaz-Martin J, Lopez-Barneo J, Pascual A. Age-mediated transcriptomic changes in adult mouse substantia nigra. PLoS ONE. 2013;8(4):e62456. • Sanchez-Gutierrez D, Saez A, Pascual A, Escudero LM. Topological progression in proliferating epithelia is driven by a unique variation in polygon distribution. PLoS ONE. 2013;8(11):e79227. • Munoz-Manchado AB, Villadiego J, Suarez-Luna N, Bermejo-Navas A, Garrido-Gil P, Labandeira-Garcia JL, et al. Neuroprotective

158158 José López Barneo Group José López Barneo Group 159

and reparative effects of carotid body grafts in a chronic MPTP model of Parkinson’s disease. Neurobiology Aging. 2013.;34(3):902-15. • Bishop T, Talbot NP, Turner PJ, Nicholls LG, Pascual A, Hodson EJ, et al. Carotid body hyperplasia and enhanced ventilatory responses to hypoxia in mice with heterozygous deficiency of PHD2. The Journal of Physiology. 2013;591(14):3565-77. • Ortega-Saenz P, Pardal R, Levitsky K, Villadiego J, Munoz-Manchado AB, Duran R, et al. Cellular properties and chemosensory responses of the human carotid body. The Journal of Physiology. 2013;15(591):6157-73. • Munoz-Bravo JL, Hidalgo-Figueroa M, Pascual A, Lopez-Barneo J, Leal-Cerro A, Cano DA. GDNF is required for neural colonization of the pancreas. Development. 2013;140(17):3669-79. • Benitez-Rivero S, Marin-Oyaga VA, Garcia-Solis D, Huertas-Fernandez I, Garcia-Gomez FJ, Jesus S, et al. Clinical features and 123I-FP-CIT SPECT imaging in vascular parkinsonism and Parkinson’s disease. Journal of Neurology, Neurosurgery, and Psychiatry. 2013;84(2):122-9. • Palomar FJ, Suarez A, Franco E, Carrillo F, Gil-Neciga E, Mir P. Abnormal sensorimotor plasticity in CADASIL correlates with neuropsychological impairment. Journal of neurology, neurosurgery, and psychiatry. 2013;84(3):329-36. • Garcia-Gomez FJ, Garcia-Solis D, Luis-Simon FJ, Marin-Oyaga VA, Carrillo F, Mir P, et al. Elaboration of the SPM template for the standardization of SPECT images with 123I-Ioflupane. Revista Espanola de Medicina Nuclear e Imagen Molecular. 2013;32(6):350-6. • Saez A, Rivas E, Montero-Sanchez A, Paradas C, Acha B, Pascual A, et al. Quantifiable diagnosis of muscular dystrophies and neurogenic atrophies through network analysis. BMC medicine. 2013 Mar 20;11:77. • Platero-Luengo A, Gonzalez-Granero S, Duran R, Diaz-Castro B, Piruat JI, Garcia-Verdugo JM, et al. An o2-sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia. Cell. 2013.

Research Projects • Code: Axontherapix pea. FP7-HEALTH-2011 Title: Axontherapix Funding: 100.534 € Principal Investigator: José López Barneo Duration: 2011-2016 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: BFU2011-25734 Other CIBER’s collaboration: No Title: Estudio de enfermedades neurológicas mediante el Type: Privado uso de drosophila y análisis de imagen computerizado Funding Agency: Axontherapix SL Principal Investigator: Luis Mª Escudero Cuadrado Funding: 251.217 € CIBERNED’s collaboration: No Duration: 2011-2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: SAF2012-33816 Type: National Title: Caracterización de los mecanismos intrínsecos impli- Funding Agency: Ministerio de Ciencia e Innovación - FEDER cados en el mantenimiento de las neuronas da de la snpc. Funding: 91.960 € papel de gdnf y de genes modulados por el envejecimiento Duration: 2013 Principal Investigator: Alberto Pascual Bravo CIBERNED’s collaboration: No • Code: PIE 2011 Excelencia (P11-CTS-7685) CIBERNED’s groups: Title: Estudio mediante estimulación magnética transcra- Other CIBER’s collaboration: No neal del papel del circuito cerebelo-talamo-cortical en el Type: National desarrollo de discinesias en la enfermedad de Parkinson y Funding Agency: Ministerio de Ciencia e Innovación - FEDER su modulación por factores genéticos Funding: 187.200 € Principal Investigator: Pablo Mir Rivera Duration: 2013-2015 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: 278367 Other CIBER’s collaboration: No Title: EMTICS (European Multicentre Tics in Children Stu- Type: CCAA dies) Funding Agency: Consejería de Economía, Innovación y Principal Investigator: Pablo Mir Rivera Ciencia. Junta Andalucía CIBERNED’s collaboration: No Funding: 178.400 € CIBERNED’s groups: Duration: 2013 Other CIBER’s collaboration: No Type: International • Code: PAI 2010 (CTS 517) Funding Agency: Séptimo Program Marco. Comisión Euro- Title: Fisiología molecular Principal Investigator: Juan José Toledo Aral L-dopa en la enfermedad de Parkinson. Estudio mediante CIBERNED’s collaboration: No estimulación trascraneal no invasiva y de los factores gené- CIBERNED’s groups: ticos implicados Other CIBER’s collaboration: No Principal Investigator: Pablo Mir Rivera Type: CCAA CIBERNED’s collaboration: No Funding Agency: Consejería de Innovación, Ciencia y Em- CIBERNED’s groups: presa. Junta Andalucía Other CIBER’s collaboration: No Funding: 3.733 € Type: National Duration: 2013 Funding Agency: Instituto de Salud Carlos III Funding: 142.780 € • Code: AECC 12 Duration: 2011-2013 Title: Fisiopatología de células madre cancerosas en neuro- blastoma pediátrico • Code: Instituto de Salud Carlos III Principal Investigator: Ricardo Pardal Title: Search and selection of Parkinson´s disease candidate CIBERNED’s collaboration: No and validation by human genetic analysis CIBERNED’s groups: Principal Investigator: Luis María Escudero Cuadrado Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: Privado CIBERNED’s groups: Funding Agency: Fundación AECC Other CIBER’s collaboration: No Funding: 15.000 € Type: National Duration: 2013 Funding Agency: Instituto de Salud Carlos III Funding: 121.500 € • Code: PIE 2009 Excelencia (P09-CTS-4589) Duration: 2011-2014 Title: Mecanismos moleculares de formación de tumores en modelos genéticos animales con defectos mitocondria- • Code: Fundación Marcelino Botín (Nuevo convenio) les Title: Sensibilidad al Oxígeno y Neurodegeneración Principal Investigator: José Ignacio Piruat Palomo Principal Investigator: José López Barneo CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: CCAA Type: Privado Funding Agency: Consejería de Innovación, Ciencia y Em- Funding Agency: Fundación Marcelino Botín presa. Junta Andalucía Funding: 650.000 € Funding: 238.518 € Duration: 2013-2016 Duration: 2010-2014 • Code: SAF2012/39343 • Code: ERC Title: Sensibilidad al oxígeno y neurodegeneración Title: Physiology of the adult carotid body stem cell niche Principal Investigator: José López Barneo Principal Investigator: Ricardo Pardal Redondo CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: International Funding Agency: Ministerio de Economía y Competividad Funding Agency: Comisión de la Comunidad Europea Funding: 400.000 € Funding: 1.476.000 € Duration: 2013-2015 Duration: 2010-2015 • Code: Red Terapia Celular (RD06/0010/0007) • Code: Fundación Alicia Koplowitz 2012 Title: Terapia Celular Title: Plasticidad neuronal en el síndrome de Tourette Principal Investigator: Juan José Toledo Aral Principal Investigator: Pablo Mir Rivera CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: Privado Funding Agency: Instituto de Salud Carlos III. Ministerio Funding Agency: Fundación Alicia Koplowitz Sanidad y Consumo Funding: 50.000 € Funding: 538.852 € Duration: 2013 Duration: 2006-2013

• Code: PI10/01674 • Code: Red Terapia Celular Exped. RD12/0019/0033 Title: Plasticidad neuronal en las discinesias inducidas por Title: Terapia Celular

160160 José López Barneo Group José López Barneo Group 161

Principal Investigator: Juan José Toledo Aral nidad y Consumo CIBERNED’s collaboration: No Funding: 109.505 € CIBERNED’s groups: Duration: 2013-2015 Other CIBER’s collaboration: No Type: National • Code: RD12/0019/0008 Funding Agency: Instituto de Salud Carlos III. Ministerio Title: RETIC de Terapia Celular Sanidad y Consumo Principal Investigator: Isabel Fariñas (coordinador de red: Funding: 74.175 € Jose María Moraleda) Duration: 2013 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; • Code: PIE 2008 Excelencia (P08-CTS-4121) G 607 Title: Terapia celular en la enfermedad de Parkinson Other CIBER’s collaboration: No Principal Investigator: Juan José Toledo Aral Type: National CIBERNED’s collaboration: No Funding Agency: Fondo de Investigaciones Sanitarias CIBERNED’s groups: Funding: 296.700 € Other CIBER’s collaboration: No Duration: 2013-2016 Type: CCAA Funding Agency: Consejería de Innovación, Ciencia y Em- • Code: PI2010/05 presa. Junta Andalucía Title: Generación de un modelo neuronal dopaminérgico a Funding: 309.924 € partir de células madre pluripotentes inducidas de pacien- Duration: 2009-2013 tes con enfermedad de Parkinson asociada a mutaciones en el gen LRRK2 • Code: PAI 2010 (CTS 630) Principal Investigator: Eduardo Tolosa Sarró Title: Trastornos Del Movimiento CIBERNED’s collaboration: Yes Principal Investigator: Pablo Mir Rivera CIBERNED’s groups: G 207; G 301; G 105; G 109 CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: Intramurales Other CIBER’s collaboration: No Funding Agency: CIBERNED Type: CCAA Funding: 262.500 € Funding Agency: Consejería de Innovación, Ciencia y Em- Duration: 2010-2013 presa. Junta Andalucía Funding: 2.279 € • Code: RD12/0019/0011 Duration: 2013 Title: RETIC de Terapia Celular Principal Investigator:Xavier Navarro Acebes (coordinador • Code: FIS 12 (PI12/02574) de red: Jose María Moraleda) Title: Uso de cuerpo carotídeo en terapia celular en la en- CIBERNED’s collaboration: Yes fermedad de Parkinson CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; Principal Investigator: Juan José Toledo Aral G 607 CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: Instituto de Salud Carlos III Type: National Funding: 180.000 € Funding Agency: Instituto de Salud Carlos III Ministerio Sa- Duration: 2013-2016 Group José Javier Lucas Lozano

Centro de Biología Molecular “Severo Ochoa” CSIC/UAM

2 Nicolás Cabrera, 1 28049 Madrid (Spain) Tel.: +34 911 964 552 / 911 964 582 (Lab) Program Fax: +34 911 964 420 [email protected]

José Javier Lucas Lozan Alberto Parras Rodríguez Principal Investigator PhD student Raquel Gómez Sintes Ivó Hernández Hernández CIBERNED Postdoctoral Fellow Student Jorge Rubén Cabrera Heredia Miriam Lucas Santamaría CIBERNED Postdoctoral Fellow Technician Jesús Torres Peraza María Santos Galindo Postdoctoral Fellow Technician Marta Fernández Nogales Alicia Tomico García PhD student CIBERNED Technician

162 José Javier Lucas Lozano Group 163

Summary

Huntington disease (HD), being inherited monogenic and dominant, is of particular interest to know the keys of neurodegeneration in general. Our work with the inducible animal model of HD showed that symptoms are reversible (Cell. 2000;101:57-66) even in advanced stages when loss of neurons has taken place (J Neurosci. 2005;25:9773-81). More recently we explored the possible inhibition of ubiquitin proteasome system (UPS) (J Neurosci. 2003;23:11653-61; J Neurosci. 2004;24:9361-71; Trends Neurosci. 2004;27:66-9; and J Neurochem. 2006;98:1585-96). By using UPS activity reporter mice we saw that the UPS is not inhibited at steady state in HD mice (PNAS. 2009;106:3986-91). However, in the past year we have seen that, in vivo, mutant huntingtin induces an inhibition of the UPS but this is temporary as it recovers as the neurons build inclusion bodies accumulating mutant huntingtin (J Neurosci. 2010;30:3675-88).

Another issue we explored is the possible alteration of GSK-3 in HD (Cell Death Differ. 2010;7:324-35) and the possible neuroprotective effect of GSK-3 inhibitors. Regarding the latter, we found that besides the reported antiapoptotic effect, inhibition of GSK-3 also can be proapoptotic in certain paradigms and cell types (including striatal neurons) and that the underlying mechanism involves NFAT and FAS (J Clin Invest. 2010;120:2432-45; PLoS One. 2013;8:e70952).We are now combining mice EH model with mice with alterations of GSK-3 activity to clarify the possible therapeutic potential of drug modulators of GSK-3 activity.

We have also reported accumulation of another synaptic protein, Alpha-synuclein, in huntingtin inclusions and explored through the crossing of mice model of EH and knock-out mice lacking Alpha- synuclein its contribution to the pathogenesis of EH (Corrochano et al., Autophagy. 2012;8:431-2; Tomás-Zapico et al., Hum Mol Genet. 2012;25:495-510) and explored the role of synphilin on synuclein clearance (Casadei et al., Hum Mol Genet. 2014;23:767-81).

Finally, given that we have detected a change in the execution of the stress of endoplasmic reticulum (ER-stress) in HD mice and in the postmortem brains of patients (Fernandez-Fernandez et al., Neurobiol Dis. 2011;41:23-32), we decided to explore the possible contribution to neurodegeneration of the different mediators of the ER-stress in an animal model of neurodegenracion with a paradigmatic induction of the ER-stress response. Namely, in the neurodegeneration secondary to epileptic seizures. This work revealed that, contrary to what is thought, adult neurons do express the ATF5 transcription factor (Torres-Peraza et al., 2013) and that the induction of both ATF5 and CHOP exert a neuroprotective role (Engel et al., 2013 and Torres-Peraza et al., 2013).

Keywords

Huntington’s disease, Proteasome, Transgenic mice, GSK-3, Synuclein, ER-stress, ATF5

Publications

• Gomez-Sintes R, Lucas JJ. Neuronal apoptosis and motor deficits in mice with genetic inhibition of GSK-3 are Fas-dependent. PLoS One. 2013;8(8):e70952. • Casadei N, Pöhler AM, Tomas-Zapico C, Torres-Peraza J, Schwedhelm I, Witz A, et al. Overexpression of synphilin-1 promotes clearance of soluble and misfolded alpha-synuclein without restoring the motor phenotype in aged A30P transgenic mice. Hum Mol Genet. 2013;23(3):767-81. • Engel T, Sanz-Rodgriguez A, Jimenez-Mateos EM, Concannon CG, Jimenez-Pacheco A, Moran C, et al. CHOP regulates the p53- MDM2 axis and is required for neuronal survival after seizures. Brain. 2013;136(2):577-92. • Torres-Peraza JF, Engel T, Martin-Ibanez R, Sanz-Rodriguez A, Fernandez-Fernandez MR, Esgleas M, et al. Protective neuronal induction of ATF5 in endoplasmic reticulum stress induced by status epilepticus. Brain. 2013 Apr;136(Pt 4):1161-76. Research Projects

• Code: SAF2012-34177 Funding: 200.000 € Title: Convergencia de alteraciones génicas, transcripciona- Duration: 2010-2013 les y traduccionales en la enfermedad de Huntington Principal Investigator: José Javier Lucas Lozano • Code: HRA_POR/2011/41 CIBERNED’s collaboration: No Title: The proteasome as a treatment target for preventing CIBERNED’s groups: seizure induced neuronal death and epilepsy Other CIBER’s collaboration: No Principal Investigator: David C. Henshall Type: National CIBERNED’s collaboration: No Funding Agency: MINECO, Plan Nacional de I+D+I CIBERNED’s groups: Funding: 330.000 € Other CIBER’s collaboration: No Duration: 2013-2015 Type: International Funding Agency: Irish Health Research Board • Code: AC563 Funding: 20.000 € Title: Dendria. Análisis del potencial terapéutico de nuevos Duration: 2011-2014 compuestos en modelos animales de la enfermedad de Huntington • Code: PI2010/06 Principal Investigator: José Javier Lucas Lozano Title: Neuroprotección en enfermedad de Huntington CIBERNED’s collaboration: No Principal Investigator: Justo García de Yébenes Prous CIBERNED’s groups: CIBERNED’s collaboration: Yes Other CIBER’s collaboration: No CIBERNED’s groups: G 304; G 303; G 305; G 306; G 307 Type: National Other CIBER’s collaboration: No Funding Agency: CDTI, SubProgram de apoyo a consor- Type: Intramurales cios estratégicos nacionales de investigación técnica (CE- Funding Agency: CIBERNED NIT)-2010 Funding: 417.600 € Duration: 2010-2013

164164 José Javier Lucas Lozano Group Group Rosario Moratalla Villalba

Instituto Cajal (CSIC)

Avenida Doctor Arce, 37 2 28002 Madrid (Spain) Tel.: +34 915 854 705 Fax: +34 915 854 754 Program [email protected]

Rosario Moratalla Villalba Sara Ares Santos Principal Investigator, CSIC Professor PhD student Noelia Granado Martínez Irene Ruiz de Diego Postdoctoral Fellow PhD student Luz María Suárez González Isabel Espadas Villanueva Postdoctoral Fellow PhD student Patricia García Sanz José María Caramés Postdoctoral Fellow PhD student Ramiro José González Aparicio María Emilia Rubio Rubio Postdoctoral Fellow (until September 2013) Lab Technician José Rubén García Marco de Mesa Cáceres Postdoctoral Fellow Lab Technician (until June 2013) Óscar Solís Castrejón PhD student

165 Summary

During the year 2013, we have shown that Parkinson’s disease causes a decrease in dendritic spines that equally affects the two major types of striatal neurons, which facilitate the movement and that stop. Chronic treatment with levodopa restores the number of dendritic spines exclusively in neurons that slow motion. However, at the same time, levodopa increases the activity of neurons that facilitate the movement due to the sensitization of dopamine D1 receptor. These changes only occur when the fully denervated striatum, with less than 10% of dopaminergic fibers and may be the morphological and functional mechanisms underlying dyskinesias (Suarez et al., 2014). This work shows for the first time a differential effect of levodopa on the two main populations of striatal neurons, which could be a breakthrough in the study of antiparkinsonian therapy.

On another line of research we showed that methamphetamine, an illicit drug of abuse consumed by 14-50 million users worldwide, can cause irreversible degeneration of dopaminergic neurons in the substantia nigra. As the degeneration of these neurons is the main feature of Parkinson’s disease, our results provide a plausible explanation to why methamphetamine abusers have higher risk to develop Parkinson’s disease in the future. Our data is significant in that it addresses a long-standing question in the field: whether methamphetamine, besides causing degeneration of dopamine terminals, also kills dopamine cell bodies in the substantia nigra. Our study is the first one to provide conclusive direct evidence that methamphetamine kills dopamine neurons in the substantia nigra using silver staining that mark degeneration. We found that methamphetamine induced significant loss of striatal dopaminergic fibers that persisted even two months after its administration. In addition methamphetamine induces significant degeneration of 15-25%, of dopamine neurons in the SN. This neurotoxicity had functional consequences, as a drastic and temporary decrease in motor activity was evident until some of the striatal fiber recover. These data provide direct evidence that methamphetamine administration to mice causes destruction of striatal neurons and dopaminergic fibers, accompanied by a temporal decrease in movement, along with significant irreversible degeneration of cell bodies in the SN. This research helps to understand why methamphetamine use has been related to an increased risk in developing Parkinson’s disease and may indicate that methamphetamine is a risk factor for Parkinson’s disease (Ares-Santos et al., 2014).

Keywords

Parkinson’s disease, Dyskinesia, Neurotoxicity, Dopamine cell death, Addiction and role of the dopaminergic system in learning and memory processes

Publications

• Ares-Santos S, Granado N, Moratalla R. Neurobiology of Methamphetamine. Biological Research on Addiction: Comprehensive Addictive Behaviours and Disorders. Elsevier Inc., San Diego: Academic Press. First Edition, 2013; pp 579-91. • Moratalla R, Ares-Santos S, Granado N. Neurotoxicity of Methamphetamine. Handbook of neurotoxicity. 2013. • Granado N, Ares-Santos S, Moratalla R. D1 but not D4 Dopamine Receptors are Critical for MDMA-Induced Neurotoxicity in Mice. Neurotox Res. 2013 Nov 21. • Granado N, Ares-Santos S, Moratalla R. Methamphetamine and Parkinson’s disease. Parkinson’s disease. 2013. 2013:308052. • Lopez-Jimenez A, Walter NA, Gine E, Santos A, Echeverry-Alzate V, Bühler KM, et al. A spontaneous deletion of α-synuclein is associated with an increase in CB1 mRNA transcript and receptor expression in the hippocampus and amygdala: effects on alcohol consumption. Synapse (New York, NY). 2013 Jun;67(6):280-9. • Shen HY, Canas PM, Garcia-Sanz P, Lan JQ, Boison D, Moratalla R, et al. Adenosine A2A Receptors in Striatal Glutamatergic Terminals and GABAergic Neurons Oppositely Modulate Psychostimulant Action and DARPP-32 Phosphorylation. PloS one. 2013;8(11):e80902. • Peraile I, Granado N, Torres E, Gutierrez-Lopez MD, Moratalla R, Colado MI, et al. Cocaine potentiates MDMA-induced oxidative stress but not dopaminergic neurotoxicity in mice: implications for the pathogenesis of free radical-induced neurodegenerative disorders. Psychopharmacologia. 2013 Nov;230(1):125-35. • Gonzalez-Aparicio R, Moratalla R. Oleoylethanolamide reduces L-DOPA-induced dyskinesia via TRPV1 receptor in a mouse model of Parkinson´s disease. Neurobiol Dis. 2013 Oct 17. • Urrutia A, Granado N, Gutierrez-Lopez MD, Moratalla R, O’Shea E, Colado MI. The JNK inhibitor, SP600125, potentiates the glial response and cell death induced by methamphetamine in the mouse striatum. Int J Neuropsychopharmacol. 2013 Oct 8.

166166 Rosario Moratalla Villalba Group Rosario Moratalla Villalba Group 167

• Ares-Santos S, Granado N, Moratalla R. The role of dopamine receptors in the neurotoxicity of methamphetamine. Journal of internal medicine. 2013 May;273(5):437-53. • Ares-Santos S, Granado N, Espadas I, Martinez-Murillo R, Moratalla R. Methamphetamine Causes Degeneration of Dopamine Cell Bodies and Terminals of the Nigrostriatal Pathway Evidenced by Silver Staining. Neuropsychopharmacology. 2013 Oct 30. • Suarez LM, Solis O, Carames JM, Taravini IR, Solis JM, Murer MG, et al. L-DOPA Treatment Selectively Restores Spine Density in Dopamine Receptor D2-Expressing Projection Neurons in Dyskinetic Mice. Biological psychiatry. 2013 Jun 12.

Research Projects

• Code: Cajal Blue Brain Project CIBERNED’s groups: G 204; G 108; G 305 Title: Cajal Blue Brain Project. International Blue Brain Pro- Other CIBER’s collaboration: No yect Type: CCAA Principal Investigator: Javier de Felipe Funding Agency: Comunidad Autónoma de Madrid CIBERNED’s collaboration: Yes Funding: 598.000 € CIBERNED’s groups: G 204; G 403 Duration: 2012-2015 Other CIBER’s collaboration: No Type: International • Code: PI2011/03 Funding Agency: Ecole Polytechnique Federale de Lausan- Title: Generación de neuronas dopaminérgicas a partir de ne (Suiza), IBM, Universidad Politécnica de Madrid, Consejo células somáticas de pacientes parkinsonianos con fallo Superior de Investigaciones Científicas, España cognitivo Funding: 0 € Principal Investigator: Rosario Moratalla Villalba Duration: 2009-2019 CIBERNED’s collaboration: Yes CIBERNED’s groups: G 204; G 106; G 202; G 110; G 108 • Code: PNSD Other CIBER’s collaboration: No Title: Caracterización de la función de DREAM en la neu- Type: Intramurales rotoxicidad de la metanfetamina y otros psicoestimulantes Funding Agency: CIBERNED Principal Investigator: Rosario Moratalla Villalba Funding: 300.000 € CIBERNED’s collaboration: No Duration: 2011-2013 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: BFU2010-20664 Type: International Title: Papel de DREAM en las disquinesias y en la pérdida de Funding Agency: Ministerio Sanidad Servicios Sociales e neuronas dopaminérgicas en la enfermedad de Parquinson Igualdad. Plan Nacional Sobre Drogas Principal Investigator: Rosario Moratalla Villalba Funding: 120.800 € CIBERNED’s collaboration: No Duration: 2013-2015 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: S2011/BMD-2336 Type: National Title: Estudio de la biología de células madre neurales para Funding Agency: Ministerio de Ciencia e Innovación su empleo en terapia celular en enfermedades neurodege- Funding: 315.000 € nerativas Duration: 2010-2013 Principal Investigator: Rosario Moratalla Villalba CIBERNED’s collaboration: Yes Group José Ramón Naranjo Orovio

CSIC Centro Nacional de Biotecnología

2 Mol. Cell. Biol. Darwin, 3 28049 Madrid (Spain) Program Tel.: +34 915 854 682 [email protected]

Jose Ramón Naranjo Elena Higuera Principal Investigator BMyC–UAM Master’s student Britt Mellström M. Paz González CIBERNED Senior Researcher CSIC Assistant Researcher Juan Casado Xose Manuel Dopazo JAE-DOC Postdoctoral Fellow CIBERNED Assistant Researcher Javier Morón Oset BExAv-UAM Student

168 José Ramón Naranjo Orovio Group 169

Summary

We investigate the nuclear components of activity- and Ca2+-dependent transcriptional responses in neurons to i) understand the molecular determinants of downstream events that are responsible for plastic changes in synaptic functionality during neurodegeneration, ii) to develop tools to expose early markers of the neurodegenerative process and, iii) to design small molecules to intervene in physiological and pathological output processes including learning and memory, motor coordination and neurodegeneration.

Early compensatory changes in transcriptional programs and altered neuronal calcium homeostasis are common features of many neurodegenerative pathologies including Alzheimer’s (AD), Down syndrome (DS) and Huntington’s disease (HD). DREAM (DRE Antagonist Modulator), a Ca2+-dependent transcriptional repressor, also known as calsenilin because its interaction with presenilins, has an important role in neurodegenerative diseases (NDD) through the control of Ca2+ homeostasis. Importantly, changes in DREAM levels are found in several NDD mouse models, including AD, DS and HD. Genetic experiments show that this could be part of a neuroprotective mechanism. In addition, we have found that DREAM is important in the development of L-DOPA-induced dyskinesias.

We foresee the Ca2+-dependent transcriptional repressor DREAM as an active/central component of several different nucleo- protein complexes that mediate specifically in the various transcriptional cascades triggered by membrane depolarization in neurons that are essential in neuronal plasticity and synaptic dysfunction. Work in progress analyzes the role of DREAM in the regulation of transcription in cell and animal models of NDDs to better understand early changes in the transcriptome and epigenome and to explore new venues for therapeutic intervention oriented to boost early endogenous neuroprotective mechanisms.

Keywords

Calciopatia, DREAM, Huntington, Alzheimer, Dyskinesias

Publications

• Baczyk D, Kibschull M, Mellstrom B, Levytska K, Rivas M, Drewlo S, et al. DREAM mediated regulation of GCM1 in the human placental trophoblast. PloS one. 2013;8(1):e51837. • Giacomello M, Oliveros JC, Naranjo JR, Carafoli E. Neuronal Ca(2+) dyshomeostasis in Huntington disease. Prion. 2013 Jan- Feb;7(1):76-84. • Mellström B, Sahun I, Ruiz-Nuno A, Murtra P, Gomez-Villafuertes R, Savignac M, et al. DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways. Mol Cell Biol. 23 December 2013. • Kreiner G, Bierhoff H, Armentano M, Rodriguez-Parkitna J, Sowodniok K, Naranjo JR, et al. A neuroprotective phase precedes striatal degeneration upon nucleolar stress. Cell death and differentiation. 2013 Nov;20(11):1455-64.

Research Projects • Code: S2010_BMD • Code: PI2010/06 Title: Redes de señalización y vías efectoras en modelos Title: Neuroprotección en enfermedad de Huntington celulares y animales de enfermedades neurodegenerativas Principal Investigator: Justo García de Yébenes Prous Principal Investigator: José González Castaño CIBERNED’s collaboration: Yes CIBERNED’s collaboration: Si CIBERNED’s groups: G 304; G 303; G 305; G 306; G 307 CIBERNED’s groups: G 401; G 104; G 111; G 307; G 409; Other CIBER’s collaboration: No G 412 Type: Intramurales Other CIBER’s collaboration: Yes (CIBERER) Funding Agency: CIBERNED Type: CCAA Funding: 417.600 € Funding Agency: Comunidad de Madrid Duration: 2010-2013 Funding: 17.250 € Duration: 2012-2015 • Code: 2013/07A Title: Papel de GSK-3 β en las alteraciones de los circuitos corticales que ocurren en la enfermedad de Alzheimer Type: Intramurales Principal Investigator: Teresa Iglesias Vacas Funding Agency: CIBERNED CIBERNED’s collaboration: Yes Funding: 158.000 € CIBERNED’s groups: G 401; G 403; G 111; G 504; G 307 Duration: 2013-2015 Other CIBER’s collaboration: No

170170 José Ramón Naranjo Orovio Group Group José Ángel Obeso Inchausti

Centre for Applied Medical Research (CIMA)

Avenida Pío XII, 55 2 31008 Pamplona (Spain) Tel.: +34 948 194 700 ext. 2029 Fax: +34 948 194 715 Program [email protected]

José A. Obeso Inchausti Francisco Molinet Dronda Principal Investigator PhD student Hirokazu Iwamuro Anna Quiroga Varela Research staff PhD student Ledia Fernández Hernández David García García Postdoctoral Researcher staff PhD student Inés Trigo Damas Rafael Redondo García Postdoctoral Fellow PhD student Francisco Javier Blesa de los Mozos Carmen Gasca Salas Postdoctoral Fellow PhD student Elena Iglesias López Lab Technician

171 Summary

The Movement Disorders Laboratory is directed by Dr. Obeso and one of the priorities of the group is to study the origin and evolution of Parkinson’s disease (PD). This group has currently different lines of research, but they are all focused on the therapeutic challenges of PD and the need to find treatments that go beyond the traditional replenishment of dopamine characteristic of this disease. In this line, one of the main objectives is to identify potential factors responsible for the selective vulnerability of the Substantia nigra pars compacta to address strategies to stop or slow the progression of Parkinson’s disease.

• Experimental Studies

Compensatory mechanisms Using progressive models of PD in the primate (MPTP) and rat (6-OHDA) we are aiming to increase the understanding of the functional states of the basal ganglia that characterize each stage of the neurodegenerative process with particular attention to the pre-symptomatic stages and the changes that underlie the initial loss of dopamine system keeping this disease silent for years. These studies range from PET imaging techniques (18F-DOPA and 11C-DTBZ) that are close to being finished to electrophysiological, molecular and biochemical studies that are underway with the goal to describe the changes observed during the different stages of the nigrostriatal lesion.

Selective vulnerability in Parkinson’s disease The objective of these studies is to define the anatomical and functional features of the selective vulnerability of dopaminergic neurons of the substantia nigra pars compacta. Using tracers injected in different striatal regions of healthy monkeys we are aiming to define the topography and degree of arborization of the nigro-striatal projections and analyze their vulnerability during the neurodegenerative process. Simultaneously, in intact rats and monkeys, we are using electrophysiology techniques to assess the activity patterns of dopaminergic neurons that are differentiated by their degree of vulnerability to the neurotoxins used traditionally in these animal models. Furthermore, two animal models of Parkinson’s disease are used: a rat model with slow bilateral lesion induced by 6-OHDA and monkeys after MPTP administration (both chronic and progressive models). In both models it is possible to differentiate between symptomatic vs pre-symptomatic stages in order to recognize early changes associated with the neurodegenerative process after nigrostriatal injury.

• Studies in patients with Parkinson’s disease

Oscillatory Activity and Subthalamic nucleus We are recording field potentials in the subthalamic nucleus of PD patients that have previously undergone deep brain stimulation surgery (DBS) with the goal to define the patterns of oscillatory activity associated with different disease states such as dyskinesias and/or impulsivity. We are also interested in functional studies related to cognitive functions such as inhibition of an initiated task (stop-go task) or the selection of a particular action among several options.

Cognitive Impairment in Parkinson’s disease. Functional Neuroimaging Studies We have completed the studies to define the clinical characteristics and risk factors associated with cognitive impairment in PD. The longitudinal and cross-sectional study was completed using a neuropsychological battery of specific tests for parkinsonian patients with mild cognitive impairment and dementia, together with studies by serial PET (18F-FDG) and magnetic resonance imaging (MRI voxel-based morphometry), that allowed us to define the possible anatomical and functional bases of cognitive impairment. Additionally, we have completed the analysis of images based on Z-maps to compare the metabolic abnormalities observed by PET (18F-FDG) (hypometabolism) with structural anomalies objectified by morphometry MRI (atrophy). This will allow us to identify and locate the relationship between the two processes of atrophy (as irreversible brain abnormality) and hypometabolism (as potentially salvageable brain abnormality with pharmacological interventions). Furthermore, complete biochemical characterization by spectroscopic MR univoxel was analyzed and we described a possible metabolite involved in the process of hypometabolism and atrophy of the cerebral cortex in cognitive stages associated with Parkinson’s disease.

172172 José Ángel Obeso Inchausti Group José Ángel Obeso Inchausti Group 173

Keywords

Parkinson’s disease, Substantia Nigra pars compacta, Basal Ganglia, Dopamine, Vulnerability, Compensatory mechanisms, Cognitive impairment

Publications

• Pifl C, Hornykiewicz O, Blesa J, Adanez R, Cavada C, Obeso JA. Reduced noradrenaline, but not dopamine and serotonin in motor thalamus of the MPTP primate: relation to severity of parkinsonism. Journal of neurochemistry. 2013 Jun;125(5):657-62. • Rodriguez-Oroz MC, Obeso JA. Commentary. Movement disorders. 2013 Sep;28(10):1349-50. • Bezard E, Olanow CW, Obeso JA. Levodopa-induced dyskinesias in the absence of nigrostriatal degeneration. Movement disorders. 2013 Jul;28(8):1023-4. • Obeso JA, Guridi J, Nambu A, Crossman AR. Motor manifestations and basal ganglia output activity: the paradox continues. Movement disorders. 2013 Apr;28(4):416-8. • Olanow CW, Obeso JA, Stocchi F. The vatican meeting on neuroprotection for Parkinson’s disease. Movement disorders. 2013 Jan;28(1):1-2. • Quiroga-Varela A, Walters JR, Brazhnik E, Marin C, Obeso JA. What basal ganglia changes underlie the parkinsonian state? The significance of neuronal oscillatory activity. Neurobiology of disease. 2013 Oct;58:242-8. • Berg D, Lang AE, Postuma RB, Maetzler W, Deuschl G, Gasser T, et al. Changing the research criteria for the diagnosis of Parkinson’s disease: obstacles and opportunities. Lancet neurology. 2013 May;12(5):514-24. • Schneider SA, Obeso JA. Movement disorders: improved understanding of early disease. Lancet neurology. 2013 Jan;12(1):10-2. • Recasens A, Dehay B, Bove J, Carballo-Carbajal I, Dovero S, Perez A, et al. Lewy body extracts from parkinson’s disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. Annals of neurology. 2013. • Castano JG, Gonzalez C, Obeso JA, Rodriguez, M. Molecular pathogenesis and pathophysiology of Parkinson Disease: new targets for new therapies. RSC Drug Discovery Series No. 34. Emerging Drugs and Targets for Parkinson’s Disease. 2013;34:26-57. • Ballaz S, Morales I, Rodriguez M, Obeso JA. Ascorbate prevents cell death from prolonged exposure to glutamate in an in vitro model of human dopaminergic neurons. Journal of neuroscience research. 2013 Dec;91(12):1609-17. • Morales I, Sabate M, Rodriguez M. Striatal glutamate induces retrograde excitotoxicity and neuronal degeneration of intralaminar thalamic nuclei: their potential relevance for Parkinson’s disease. The European journal of neuroscience. 2013 Jul;38(1):2172-82. • Marin C, Bonastre M, Mengod G, Cortes R, Rodriguez-Oroz MC, Obeso JA. Subthalamic 6-OHDA-induced lesion attenuates levodopa-induced dyskinesias in the rat model of Parkinson’s disease. Experimental neurology. 2013 Dec;250:304-12. • Marin C, Bonastre M, Mengod G, Cortes R, Giralt A, Obeso JA, et al. Early L-dopa, but not pramipexole, restores basal ganglia activity in partially 6-OHDA-lesioned rats. Neurobiology of disease. 2013 Dec 24. • Alvarez-Erviti L, Seow Y, Schapira AH, Rodriguez-Oroz MC, Obeso JA, Cooper JM. Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson’s disease. Cell death & disease. 2013 Mar 14;4:e545. • Obeso I, Wilkinson L, Rodriguez-Oroz MC, Obeso JA, Jahanshahi M. Bilateral stimulation of the subthalamic nucleus has differential effects on reactive and proactive inhibition and conflict-induced slowing in Parkinson’s disease. Experimental brain research. Experimentelle Hirnforschung. Experimentation cerebrale. 2013 May;226(3):451-62. • Alegre M, Lopez-Azcarate J, Obeso I, Wilkinson L, Rodriguez-Oroz MC, Valencia M, et al. The subthalamic nucleus is involved in successful inhibition in the stop-signal task: A local field potential study in Parkinson’s disease. Experimental neurology. 2013 Jan:239:1-12. • Ortega-Cubero S, Clavero P, Irurzun C, Gonzalez-Redondo R, Guridi J, Obeso JA, et al. Effect of deep brain stimulation of the subthalamic nucleus on non-motor fluctuations in Parkinson’s disease: two-years’ follow-up. Parkinsonism & related disorders. 2013 May;19(5):543-7. • Gago B, Marin C, Rodriguez-Oroz MC, Obeso JA. l-dopa-induced dyskinesias in unilateral 6-hydroxydopamine-lesioned rats are not modified by excitotoxic lesion of the entopeduncular nucleus and substantia nigra pars reticulata. Synapse (New York, NY). 2013 Jul;67(7): 407-14. Research Projects • Code: PI2011/02 • Code: MJFF Staff Initiated 2013 Title: Inicio y progresión de la Enfermedad de Parkinson. Title: Prion-like dissemination of synuclein pathology: a Vulnerabilidad de la vía nigroestriada, eventos en origen y non-human primate study destino Principal Investigator:Erwan Bezard (Universite de Bordeaux, Principal Investigator: José Ángel Obeso Inchausti France) CIBERNED’s collaboration: Yes CIBERNED’s collaboration: Yes CIBERNED’s groups: G 205; G 201; G 104; G 208; G 203; CIBERNED’s groups: G 505; G 205; G 109 G 404; G 206 Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: International Type: Intramurales Funding Agency: Michael J. Fox Foundation Funding Agency: CIBERNED Funding: 33.000 € Funding: 362.000 € Duration: 2013-2016 Duration: 2011-2013

174174 José Ángel Obeso Inchausti Group Group Ana María Pérez Castillo

Instituto de Investigaciones Biomédicas (CSIC-UAM)

Dpto. Modelos Experimentales de Enfermedades Humanas 2 Arturo Duperier, 4 28029 Madrid (Spain) Tel.: +34 915 854 436 Program Fax: +34 915 854 401 [email protected]

Ana María Pérez Castillo Elena Hernández Encinas Principal Investigator, CSIC Professor Postdoctoral Fellow Ángel Santos Montes Marina Sanz San Cristóbal Full Professor CIBERNED Technician Elena Giné Sandra Alonso Gil Postdoctoral Fellow Technician José A. Morales García David Martínez Pablo CIBERNED Postdoctoral Fellow Student Diana Aguilar Morante Virginia Alcázar Postdoctoral Fellow (until April 2013) Student, Lab Technician

175 Summary

During the last years our group have demonstrated that inhibitors of the glycogen synthase kinase 3 (GSK-39 and phosphodiesterase 7 (PDE7) are potent neuroprotective and anti-inflammatory agents in different models of Parkinson’s disease (PD). During this year we have studied more in deep these effects and we have demonstrated that administration of SC001, an inhibitor of GSK-3, causes a significant preservation of dopaminergic cells in the 6-OHDA-lesioned model of PD and that these effects were observed not only when administered the same of the toxin but also when the administration occurred 10 days later. This ability to preserve dopaminergic cells in animals with already-established lesions is particularly significant for further clinical intervention.

We have also demonstrated that lentivirus-mediated PDE7B deprivation conferred marked in vitro and in vivo neuroprotection against 6-OHDA and LPS toxicity in dopaminergic neurons, and preserved motor function involving the dopamine system in mouse. Our results substantiate previous data and provide a validation of PDE7B enzyme as a valuable new target for therapeutic development in the treatment of PD.

Lastly, we have finalized the objectives proposed in the cooperative project: “Generation of dopaminergic neurons from somatic cells obtained from Parkinson Disease patients with cognitive failure” coordinated by Dr. Rosario Moratalla (collaborators: Drs. Vicario, Perez-Castillo, Ceña and Kulisevsky). Parkinson disease is usually diagnosed when about 50% of the dopaminergic neurons of the substantia nigra pars compacta (SNpc) have degenerated and the others are already affected by the disease. Thus, it is conceivable that all therapeutic strategies, aimed at neuroprotection, start too late. Therefore there is an urgent medical need to discover new pharmacological targets and novel drugs with disease-modifying properties. In this regard, modulation of endogenous adult neurogenesis toward a dopaminergic phenotype may also provide a new strategy to target Parkinson’s disease by partially ameliorating the dopaminergic cell loss that takes place in this disorder. We have demonstrated that pioglitazone, a PPARɣ ligand, is able to induce endogenous neuroregenerative processes toward a dopaminergic phenotype.

Keywords

GSK-3, Neuroprotection, Neuroinflammation, Neurogenesis, Parkinson, PDE7, PPARγ

Publications

• Morales-García JA, Susín C, Alonso-Gil S, Pérez DI, Palomo V, Pérez C, et al. Glycogen Synthase Kinase-3 Inhibitors as Potent Therapeutic Agents for the Treatment of Parkinson Disease. ACS Chemical Neuroscience. 2013;4(2):350-60. • Gine E, Echeverry-Alzate V, Lopez-Moreno JA, Lopez-Jimenez A, Torres-Romero D, Perez-Castillo A, et al. Developmentally- induced hypothyroidism alters the expression of Egr-1 and Arc genes and the sensitivity to cannabinoid agonists in the hippocampus. Possible implications for memory and learning. Mol Cell Endocrinol. 2013;365(1):119-28.

Research Projects • Code: SAF2010-16365 • Code: FU0159 Title: Assessment of novel PPARγ activators and GSK-3β Title: Identificación y caracterización de nuevas dianas te- and PDE7 inhibitors as possible therapeutic agents for the rapéuticas en el glioblastoma mutiforme treatment of Parkinson Disease and other neurodegenera- Principal Investigator: Ana María Pérez del Castillo tive disorders CIBERNED’s collaboration: No Principal Investigator: Ana María Pérez del Castillo CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: Privado Other CIBER’s collaboration: No Funding Agency: Fundación Mutua Madrileña Type: National Funding: 12.000 € Funding Agency: MICINN Duration: 2012-2013 Funding: 266.200 € Duration: 2011-2013 • Code: PI2011/03 Title: Generación de neuronas dopaminérgicas a partir de

176176 Ana María Pérez Castillo Group Ana María Pérez Castillo Group 177

células somáticas de pacientes parkinsonianos con fallo Type: Intramurales cognitivo Funding Agency: CIBERNED Principal Investigator: Rosario Moratalla Villalba Funding: 300.000 € CIBERNED’s collaboration: Yes Duration: 2011-2013 CIBERNED’s groups: G 204; G 106; G 202; G 110; G 108 Other CIBER’s collaboration: No Group Jordi Pérez Tur

Unitat de Genètica Molecular

2 Institut de Biomedicina de València-CSIC Jaume Roig, 11 46010 València (Spain) Program Tel.: +34 963 391 755 Fax: +34 963 393 774 [email protected]

Jordi Pérez-Tur Irene Nebot Ruiz Principal Investigator, CSIC FPU Researcher Fernando Cardona Serrate Advanced Technical

178 Jordi Pérez Tur Group 179

Summary

This year, our group has deepened, through collaboration with other groups CIBERNED in the genetic basis of neurodegenerative diseases. Basically we worked on defining the impact of variations in risk conferred by CALHM1 against Alzheimer’s disease and how that variation has an important functional effect in the regulation of intracellular calcium. Moreover we have also participated in characterizing an abnormal expansion in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia patients in the c9orf72 locus. Finally, we identified a mutation in a family suffering from a rare disease, benign hereditary chorea, which has led to the first description in a Spanish family of this type of defect.

As for our work in progress, throughout 2013 we continued the study of the genetic factors involved in the onset of dementia in Parkinson’s disease, in characterizing variations or alterations in DNA related to the appearance of ALS and in the definition of the mutation that causes a familial form of Progressive Supranuclear Palsy .

Keywords

Alzheimer, Parkinson, Neurodegeneration, Neurogenetics, ALS

Publications

• Sempere AP,Aparicio S,Mola S,Perez-Tur J. Benign hereditary chorea: clinical features and long-term follow-up in a Spanish family. Parkinsonism Relat Disord. 2013;19(3):394-6. • Rubio-Moscardo F, Seto-Salvia N, Pera M, Bosch-Morato M, Plata C, Belbin O, et al. Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset Alzheimer’s disease patients alter calcium homeostasis. PloS one. 2013;8(9):e74203.

Research Projects • Code: PI2011/01 Title: El factor de transcripción Nrf2 como nueva diana ter- apéutica para la enfermedad de Parkinson Principal Investigator: Antonio Cuadrado Pastor CIBERNED’s collaboration: Yes CIBERNED’s groups: G 101; G102; G103; G 505; G 209 Other CIBER’s collaboration: No Type: Intramurales Funding Agency: CIBERNED Funding: 390.000 € Duration: 2011-2013 Group Manuel Rodríguez Díaz

Departamento de Fisiología, Facultad de Medicina

2 Universidad de La Laguna La Laguna (Spain) Program Tel.: +34 922 319 361 Fax: +34 922 319 397 [email protected]

Manuel Rodríguez Díaz Domingo Alfonso Oramas Principal Investigator, Full Professor Associate Researcher Tomás González-Hernández Ana Manzanedo López Researcher, Full Professor Associate Researcher Magdalena Sabaté Bel Ángel Fuentes Rodríguez Researcher, Associate Professor Technician Ignacio de la Cruz Muros Javier Castro Hernández CIBERNED Researcher Technician Belén González González Josmar Salas-Hernández CIBERNED Researcher Technician Ingrid Morales Pérez Sergio González Martín-Fernández CIBERNED Researcher Technician Catalina Llanos Mendoza PhD student

180 Manuel Rodríguez Díaz Group 181

Summary

We are studying the regulation of the extracellular extrasynaptic glutamate in the striatum, and its possible role as a retrograde excitotoxic in animal models of Parkinson’s disease. The decrease of striatal dopamine increases this pool of glutamate, inhibiting the glutamine synthetase and promoting the massive accumulation of glutamate in astrocytes. These changes are accompanied by a retrograde degeneration of the thalamo-striatal glutamatergic neurons (particularly of those coming from the intralaminar thalamic nuclei). This suggests that retrograde excitotoxicity is a mechanism for the progression of dopamine cell degeneration to other cell groups which, as is the case of intralaminar thalamic neurons, are normally affected in Parkinson’s disease. This process is accompanied by changes in microglial cells of the striatum and thalamus and in oligodendrocytes linked to the thalamo-striatal neurons. The dopaminergic nigro-striatal neurons are not vulnerable to the retrograde excitotoxicity of glutamate (performed on axons and synaptic terminals), but they could be degenerated by the action of this amino acid on their cell somata. “In vitro” studies with SH-SY5Y, which were differentiated to human dopaminergic neurons, showed excitotoxicity of glutamate on the cell somata of these cells. This toxic action was mediated by free radicals, and was prevented by the extracellular administration of ascorbate and increased with the blockade of cell uptake of ascorbate.

The membrane transporter involved in the dopamine uptake by dopaminergic neurons is a relevant factor for vulnerability of mesencephalic dopaminergic neurons in Parkinson’s disease. We have studied the possible role of D3 dopamine receptors on the dopamine transporter activity in mice and cell cultures. D3 agonists decrease dopamine uptake, changing the integration of the transporter in the cytoplasmatic membrane of the dopaminergic synaptic terminals, and the phosforylation and ubiquitinization of the dopamine transporter.

The Mu oscillation in the motor cortex is studied during the planning and execution of different visual-manual motor tasks, and during the simulation of these movements with motor imagery. There is a phase-related activation of Mu oscillation at the beginning of the planning of motor tasks, a fact observed during the planning of both real and simulated movements. Available data support the hypothesis that the Mu wave synchronizes the activity of cortical neurons involved in visual-manual tasks, inhibiting other neurons whose activity could interfere with the ongoing tasks.

By using different magnetic resonance methods (partial correlation, independent components analyses and other methods using the BOLD signal), we have studied the functional and effective connectivity of basal ganglia. Most centers of the direct and indirect pathways showed a functional interactivity compatible with the closed-loop cortico-subcortical circuits of the classical model of basal ganglia. In addition, functional interactions between basal ganglia with no direct structural connections were identified. Functional connectivity of basal ganglia changed when subjects performed complex movements with their hands, showing that these methods are suitable to detect the effect of movements on basal ganglia interactivity. We are paying particular attention to studying the functional connectivity of the intralaminar nuclei of the thalamus with other basal ganglia centers.

Keywords

Glutamatergic Excitotoxicity, Dopamine transporter, Dopaminergic vulnerability, Structural and functional neuroimaging

Publications

• Brito-Armas JM, Baekelandt V, Castro-Hernandez JR, Gonzalez-Hernandez T, Rodriguez M, Castro R. Melatonin prevents dopaminergic cell loss induced by lentiviral vectors expressing A30P mutant alpha-synuclein. Histology and histopathology. 2013 Aug;28(8):999-1006. • Nieto A, Correia R, de Nobrega E, Monton F, Barroso J. Cognition in late-onset Friedreich ataxia. Cerebellum (London, England). 2013 Aug;12(4):504-12. • Llanos C, Rodriguez M, Rodriguez-Sabate C, Morales I, Sabate M. Mu-rhythm changes during the planning of motor and motor imagery actions. Neuropsychologia. 2013 May:51(6):1019-26. • Ballaz S, Morales I, Rodriguez M, Obeso JA. Ascorbate prevents cell death from prolonged exposure to glutamate in an in vitro model of human dopaminergic neurons. Journal of neuroscience research. 2013 Dec;91(12):1609-17. • Morales I, Sabate M, Rodriguez M. Striatal glutamate induces retrograde excitotoxicity and neuronal degeneration of intralaminar thalamic nuclei: their potential relevance for Parkinson’s disease. The European journal of neuroscience. 2013 Jul;38(1):2172-82. • Morales I, Yanos C, Rodriguez-Sabate C, Sanchez A, Rodriguez M. Striatal glutamate degenerates thalamo-striatal neurons. Journal of Neuropathology and Experimental Neurology. 2013;72:286-97. • Castano JG, Gonzalez C, Obeso, JA, Rodriguez M. Molecular pathogenesis and pathophysiology of Parkinson Disease: new targets for new therapies. RSC Drug Discovery Series No. 34. Emerging Drugs and Targets for Parkinson’s Disease. 2013;34:26-57.

Research Projects • Code: 59/2011 Other CIBER’s collaboration: No Title: Cambios cognitivos en el envejecimiento normal: un Type: National estudio de seguimiento Funding Agency: Proyecto I+D+I de la Dirección General de Principal Investigator: Antonieta Nieto Barco Investigación, del Ministerio de Ciencia y Tecnología CIBERNED’s collaboration: No Funding: 108.900 € CIBERNED’s groups: Duration: 2009-2013 Other CIBER’s collaboration: No Type: National • Code: BFU2010-21130 Funding Agency: Ministerio de Sanidad, Seguros Sociales e Title: Regulación de los transportadores de dopamina y Igualdad; Research Projects científica, desarrollo e innova- vulnerabilidad de las neuronas dopaminérgicas mesence- ción tecnológica fálicas Funding: 8.000 € Principal Investigator: Tomás González Hernández Duration: 2012-2014 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PSI2011-24665 Other CIBER’s collaboration: No Title: Cerebelo y Cognición: Deterioro neuropsicológico en Type: National la Ataxia de Friedreich y su relación con la degeneración Funding Agency: Ministerio de Ciencia e Innovación cerebelosa en imagenes de RM Funding: 166.000 € Principal Investigator: Antonieta Nieto Barco Duration: 2010-2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: PI2011/02 Other CIBER’s collaboration: No Title: Inicio y progresión de la Enfermedad de Parkinson. Type: National Vulnerabilidad de la vía nigroestriada, eventos en origen y Funding Agency: Ministerio de Ciencia e Innovación. Re- destino search Projects Fundamental no orientada Principal Investigator: José Ángel Obeso Inchausti Funding: 12.000 € CIBERNED’s collaboration: Yes Duration: 2012-2014 CIBERNED’s groups: G 205; G 201; G 104; G 208; G 203; G 404; G 206 • Code: SAF2008-03746 Other CIBER’s collaboration: No Title: La glía como vector de excitotoxicidad glutamatérgica Type: Intramurales en la enfermedad de Parkinson Funding Agency: CIBERNED Principal Investigator: Manuel Rodríguez Díaz Funding: 362.000 € CIBERNED’s collaboration: No Duration: 2011-2013 CIBERNED’s groups:

182182 Manuel Rodríguez Díaz Group Group Amelia Sánchez Capelo

Servicio Neurobiología - Investigación

Hospital Ramón y Cajal - IRYCIS 2 Carretera Colmenar Viejo, Km 9 28034 Madrid (Spain) Tel.: +34 913 368 000 ext 7806 Program Fax: +34 913 369 016 [email protected]

Amelia Sánchez Capelo Silvia Tapia González Principal Investigator, Neuroscience Area Coordinator CIBERNED Laboratory Technician

183 Publications

• Tapia-Gonzalez S, Munoz MD, Cuartero MI, and Sanchez-Capelo A. Smad3 is required for the survival of proliferative intermediate progenitor cells in the dentate gyrus of adult mice. Cell Communication and Signaling. 2013;11(93):15.

Research Projects

• Code: PI12/00258 Title: Nuevo desarrollo diagnóstico para la enfermedad de Parkinson: mutaciones de TGFbeta/Smad Principal Investigator: Amelia Sánchez-Capelo CIBERNED’s collaboration: No CIBERNED’s groups: Other CIBER’s collaboration: No Type: National Funding Agency: FIS-ISCIII Funding: 74.415 € Duration: 2013-2015

184184 Amelia Sánchez Capelo Group Group Eduard Tolosa Sarró

Hospital Clínic de Barcelona / Universidad de Barcelona

Villarroel, 171 2 08036 Barcelona (Spain) Tel.: +34 932 275 785 Fax: +34 932 275 783 Program [email protected]

Eduard Tolosa Sarró Yaroslau Compta Hinrjy Principal Investigator, Full Professor Specialist María José Martí Doménech Rubén Fernández-Santiago Consultant IDIBAPS Researcher Francesc Valldeoriola i Serra Mónica Serradell Eroles Consultant FCRB PhD student Esteban Muñoz Martínez Carme Junqué Plaja Consultant Full Professor Joan Santamaría Cano Claustre Pont Senior Consultant Michael J Fox Foundation PhD student Josep Valls-Solé Dolores Vilas Senior Consultant Michael J Fox Foundation PhD student Alejandro Iranzo de Riquer Judith Navarro Consultant Emili Letang PhD student Carles Gaig Mercè Bonastre García Specialist IDIBAPS Lab Technician Mario Ezquerra Trabalón Manuel Fernández Sánchez ISCIII Researcher FCRB Lab Technician

185 Summary

We have studied by OMICS technologies dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) generated from patients with idiopathic Parkinson’s disease-associated LRRK2 gene mutations. In these cells we have found epigenetic and transcriptomic changes that precede neurodegenerative phenotypes (neurite shortening and slowing of autophagy) (under review in Nature Neuroscience).

Following up previous research in the preclinical phase of Parkinson’s, during 2013 we have shown that a series of 44 patients with idiopathic REM sleep conduct disorder, followed for a mean of 10 years, most convert to a synucleinopathy. Post-mortem studies in several of these patients have confirmed that most suffered from a synucleinopathy, and we have also documented that a hyposmia is very common among these patients but does not worsen over time, so it is not a good marker of progression pre-motor disease

In another study on epicardial autonomic system involvement in subjects without parkinsonism, we identified pathological synuclein aggregates in 6 percent of cases. This study is a pioneer in detecting preclinical cardiac synucleinopathy.

In the research line of biomarkers of cognitive impairment in Parkinson’s disease and biomarkers of parkinsonism and its neuropathological correlates, we have continued to work on cerebrospinal fluid and neuroimaging (MRI and PET) markers, with projects “Predictive value and diagnosis of dementia Parkinson’s disease pathology markers Beta-amyloid and Tau in cerebrospinal fluid and positron emission tomography” and “PET-FDDNP and in vivo diagnosis of unclassifiable parkinsonism in tauopathy”. A group member (Dr. Yaroslau Compta Hirnyj) defended his doctoral thesis (May 5, 2013) entitled “Cerebrospinal fluid biomarkers in Parkinson’s disease dementia: clinico-pathological rationale and clinical correlates”. Several publications on this subject have been generated during the year, including 3 original articles, a review and an editorial.

Structural and functional cranial magnetic resonance imaging studies in subjects with early Parkinson with variable cognitive impairment and functional connectivity analysis, have allowed us to detect progressive changes in functional networks involved in memory. We have also identified hippocampal subfields involved in cognitive deficits of subjects with Parkinson’s and their relationship in the presence of hallucinations.

During the year 2013 we have developed a research project on apathy in Huntington’s disease; as well as two projects in ataxias, the first of them a study with optical coherence tomography in patients with SCA3 whose results have been published in the journal Eye (London), and the second a videocomputerized analysis of ocular motility and oculovestibular reflexes in patients with various hereditary ataxias, whose results have been submitted for publication (under review).

The group has participated in international collaborative studies of multiple system atrophy (MSA) that has allowed us to cha- racterize the cognition of this “rare” disease and its natural history. It has also participated in the development of new diatonic criteria for parkinsonism associated with tau protein deposits (cortico-basal degeneration).

Keywords

Neuroimaging and cognition, Biological, Diagnostic pre-motor, Non-motor symptoms, Motor complications, Genetic variants, Sleep disturbances, Experimental therapy

Publications

• Cortes M, Elder J, Rykman A, Murray L, Avedissian M, Stampa A, et al. Improved motor performance in chronic spinal cord injury following upper-limb robotic training. NeuroRehabilitation. 2013;33(1):57-65. • Sahuquillo J, Rădoi A, Benejam B, Junque C, Fernandez-Espejo D, Poca MA. Brain activation during speech perception in a patient with a massive left hemisphere infarction. Brain injury: [BI]. 2013;27(12):1470-4. • Luis L, Costa J, Vaz Garcia F, Valls-Sole J, Brandt T, Schneider E. Spontaneous plugging of the horizontal semicircular canal with reversible canal dysfunction and recovery of vestibular evoked myogenic potentials. Otology & neurotology. 2013 Jun;34(4):743-7. • Marques-Iturria I, Pueyo R, Garolera M, Segura B, Junque C, Garcia-Garcia I, et al. Frontal cortical thinning and subcortical volume reductions in early adulthood obesity. Psychiatry research. 2013 Nov 30;214(2):109-15.

186186 Eduard Tolosa Sarró Group Eduard Tolosa Sarró Group 187

• Pujol N, Penades R, Rametti G, Catalan R, Vidal-Pineiro D, Palacios E, et al. Inferior frontal and insular cortical thinning is related to dysfunctional brain activation/deactivation during working memory task in schizophrenic patients. Psychiatry research. 2013 Nov 30;214(2):94-101. • Castellote JM, Van den Berg ME, Valls-Sole J. The StartReact effect on self-initiated movements. Journal of biomedicine and biotechnology. 2013:2013:471792. • Luca G, Haba-Rubio J, Dauvilliers Y, Lammers GJ, Overeem S, Donjacour CE, et al. Clinical, polysomnographic and genome- wide association analyses of narcolepsy with cataplexy: a European Narcolepsy Network study. Journal of sleep research. 2013 Oct;22 (5):482-95. • Siri C, Duerr S, Canesi M, Delazer M, Esselink R, Bloem BR, et al. A cross-sectional multicenter study of cognitive and behavioural features in multiple system atrophy patients of the parkinsonian and cerebellar type. Journal of neural transmission. 2013 Apr;120(4):613-8. • LeWitt PA, Boroojerdi B, Surmann E, Poewe W; SP716 Study Group, SP715 Study Group. Rotigotine transdermal system for long-term treatment of patients with advanced Parkinson’s disease: results of two open-label extension studies, CLEOPATRA- PD and PREFER. Journal of neural transmission. 2013 Jul;120(7):1069-81. • Kumru H, Portell E, Marti M, Albu S, Tormos JM, Vidal J, et al. Mechanical and thermal hyperalgesia in patients with poliomyelitis. Clinical neurophysiology. 2013 Jul;124(7):1431-8. • Navarro-Otano J, Gaig C, Muxi A, Lomena F, Compta Y, Buongiorno MT, et al. 123I-MIBG cardiac uptake, smell identification and 123I-FP-CIT SPECT in the differential diagnosis between vascular parkinsonism and Parkinson’s disease. Parkinsonism & related disorders. 2013 Nov 5. • Navarro-Otano J, Gelpi E, Mestres CA, Quintana E, Rauek S, Ribalta T, et al. Alpha-synuclein aggregates in epicardial fat tissue in living subjects without parkinsonism. Parkinsonism & related disorders. 2013 Jan;19(1):27-31; discussion 27. • Compta Y, Pereira JB, Rios J, Ibarretxe-Bilbao N, Junque C, Bargallo N, et al. Combined dementia-risk biomarkers in Parkinson’s disease: a prospective longitudinal study. Parkinsonism & related disorders. 2013 Aug;19(8): 717-24. • Iranzo A, Serradell M, Vilaseca I, Valldeoriola F, Salamero M, Molina C, et al. Longitudinal assessment of olfactory function in idiopathic REM sleep behavior disorder. Parkinsonism & related disorders. 2013 Jun;19(6):600-4. • Müller T, van Laar T, Cornblath DR, Odin P, Klostermann F, Grandas FJ, et al. Peripheral neuropathy in Parkinson’s disease: levodopa exposure and implications for duodenal delivery. Parkinsonism & related disorders. 2013 May;19(5): 501-7; discussion 501. • Segura B, Baggio HC, Solana E, Palacios EM, Vendrell P, Bargallo N, et al. Neuroanatomical correlates of olfactory loss in normal aged subjects. Behavioural brain research. 2013 Jun 1;246:148-53. • Compta Y, Parkkinen L, Kempster P, Selikhova M, Lashley T, Holton JL, et al. The Significance of α-Synuclein, Amyloid-β and Tau Pathologies in Parkinson’s Disease Progression and Related Dementia. Neuro-degenerative diseases. 2013 Sep 11. • Peraita-Adrados R, Lammers GJ, De Andres C, Santamaria J, Vicario JL, Tafti M. A patient with narcolepsy with cataplexy and multiple sclerosis: two different diseases that may share pathophysiologic mechanisms? Sleep medicine. 2013 Jul;14(7):695-6. • Boeve BF, Silber MH, Ferman TJ, Lin SC, Benarroch EE, Schmeichel AM, et al. Clinicopathologic correlations in 172 cases of rapid eye movement sleep behavior disorder with or without a coexisting neurologic disorder. Sleep medicine. 2013 Aug;14(8):754-62. • Schenck CH, Montplaisir JY, Frauscher B, Hogl B, Gagnon JF, Postuma R, et al. Rapid eye movement sleep behavior disorder: devising controlled active treatment studies for symptomatic and neuroprotective therapy--a consensus statement from the International Rapid Eye Movement Sleep Behavior Disorder Study Group. Sleep medicine. 2013 Aug;14(8):795-806. • Padilla N, Junque C, Figueras F, Sanz-Cortes M, Bargallo N, Arranz A, et al. Differential vulnerability of gray matter and white matter to intrauterine growth restriction in preterm infants at 12 months corrected age. Brain Res. 2013 Dec 17. • Valls-Sole J. Facial nerve palsy and hemifacial spasm. Handbook of clinical neurology / edited by P.J. Vinken and G.W. Bruyn. 2013;115:367-80. • Grau-Rivera O, Gelpi E, Rey MJ, Valldeoriola F, Tolosa E, Compta Y, et al. Prominent psychiatric symptoms in patients with Parkinson’s disease and concomitant argyrophilic grain disease. Journal of neurology. 2013 Dec;260(12):3002-9. • Valldeoriola F, Puig-Junoy J, Puig-Peiro R; Workgroup of the SCOPE study. Cost analysis of the treatments for patients with advanced Parkinson’s disease: SCOPE study. Journal of medical economics. 2013;16(2):191-201. • Eggington S, Valldeoriola F, Chaudhuri KR, Ashkan K, Annoni E, Deuschl G. The cost-effectiveness of deep brain stimulation in combination with best medical therapy, versus best medical therapy alone, in advanced Parkinson’s disease. J Neurol. 2013 Oct 25. • Arino H, Iranzo A, Gaig C, Santamaria J. Sexsomnia: parasomnia associated with sexual behaviour during sleep. Neurologia (Barcelona, Spain). 2013 May 7. • Fernandez-Del-Olmo M, Bello O, Lopez-Alonso V, Marquez G, Sanchez JA, Morenilla L, et al. The effects of startle and non- startle auditory stimuli on wrist flexion movement in Parkinson’s disease. Neuroscience letters. 2013 Aug 26;548:56-60. • Pueyo R, Ariza M, Narberhaus A, Ballester-Plane J, Laporta-Hoyos O, Junque C, et al. Does verbal and gestural expression ability predict comprehension ability in cerebral palsy? Perceptual and motor skills. 2013 Apr;116(2):512-27. • Tolosa E, Hernandez B, Linazasoro G, Lopez-Lozano JJ, Mir P, Marey J, et al. Efficacy of levodopa/carbidopa/entacapone versus levodopa/carbidopa in patients with early Parkinson’s disease experiencing mild wearing-off: a randomised, double-blind trial. Journal of neural transmission. 2013 Nov 20. • Iranzo A. Parkinson disease and sleep: sleep-wake changes in the premotor stage of Parkinson disease; impaired olfaction and other prodromal features. Current neurology and neuroscience reports. 2013 Sep;13(9):373. • Gil-Navarro S, Lomena F, Cot A, Llado A, Montagut N, Castellvi M, et al. Decreased striatal dopamine transporter uptake in the non-fluent/agrammatic variant of primary progressive aphasia. European journal of neurology. 2013 Nov;20(11):1459-e126. • Ferreira JJ, Katzenschlager R, Bloem BR, Bonuccelli U, Burn D, Deuschl G, et al. Summary of the recommendations of the EFNS/ MDS-ES review on therapeutic management of Parkinson’s disease. European journal of neurology. 2013 Jan;20(1):5-15. • Kumru H, Benito J, Murillo N, Valls-Sole J, Valles M, Lopez-Blazquez R, et al. Effects of high-frequency repetitive transcranial magnetic stimulation on motor and gait improvement in incomplete spinal cord injury patients. Neurorehabilitation and neural repair. 2013 Jun;27(5):421-9. • Palacios EM, Sala-Llonch R, Junque C, Roig T, Tormos JM, Bargallo N, et al. White matter/gray matter contrast changes in chronic and diffuse traumatic brain injury. Journal of neurotrauma. 2013 Dec 1;30(23):1991-4. • Serranova T, Sieger T, Dušek P, Růžička F, Urgošik D, Růžička E, et al. Sex, food and threat: startling changes after subthalamic stimulation in Parkinson’s disease. Brain stimulation. 2013 Sep;6(5):740-5. • Respondek G, Roeber S, Kretzschmar H, Troakes C, Al-Sarraj S, Gelpi E, et al. Accuracy of the National Institute for Neurological Disorders and Stroke/Society for Progressive Supranuclear Palsy and neuroprotection and natural history in Parkinson plus syndromes criteria for the diagnosis of progressive supranuclear palsy. Movement disorders. 2013 Apr;28(4):504-9. • Stocchi F, Rascol O, Destee A, Hattori N, Hauser RA, Lang AE, et al. AFQ056 in Parkinson patients with levodopa-induced dyskinesia: 13-week, randomized, dose-finding study. Movement disorders. 2013 Nov;28(13):1838-46. • Navarro-Otano J, Valls-Sole J, Guaita M, Santamaria J, Cardellach F, Munoz E. Chewing-induced segmental myoclonus in a patient with Leigh syndrome. Movement disorders. 2013 Oct;28(12):1756-7. • Compta Y, Revesz T, Lees AJ. The more cortical amyloid-β, the more postural instability in Parkinson’s disease: more grist to the mill for a link between walking, falling, and remembering? Movement disorders. 2013 Mar;28(3):263-4. • Zarei M, Ibarretxe-Bilbao N, Compta Y, Hough M, Junque C, Bargallo N, et al. Cortical thinning is associated with disease stages and dementia in Parkinson’s disease. Journal of neurology, neurosurgery, and psychiatry. 2013 Aug;84(8):875-81. • Quadri M, Fang M, Picillo M, Olgiati S, Breedveld GJ, Graafland J, et al. Mutation in the SYNJ1 gene associated with autosomal recessive, early-onset Parkinsonism. Human mutation. 2013 Sep;34(9):1208-15. • Pereira JB, Valls-Pedret C, Ros E, Palacios E, Falcon C, Bargallo N, et al. Regional vulnerability of hippocampal subfields to aging measured by structural and diffusion MRI. Hippocampus. 2013 Dec 12;23(8):720-8. • Medici C, Barraza G, Castillo CD, Morales M, Schestatsky P, Casanova-Mollà J, et al. Disturbed sensory perception of changes in thermoalgesic stimuli in patients with small fiber neuropathies. Pain. 2013 Oct;154(10):2100-7. • Ferreira D, Molina Y, Machado A, Westman E, Wahlund LO, Nieto A, et al. Cognitive decline is mediated by gray matter changes during middle age. Neurobiology of aging. 2013 Oct 29. • Palacios EM, Sala-Llonch R, Junque C, Roig T, Tormos JM, Bargallo N, et al. Resting-state functional magnetic resonance imaging activity and connectivity and cognitive outcome in traumatic brain injury. JAMA neurology. 2013 Jul;70(7):845-51. • Armstrong MJ, Litvan I, Lang AE, Bak TH, Bhatia KP, Borroni B, et al. Criteria for the diagnosis of corticobasal degeneration. Neurology. 2013 Jan 29;80(5):496-503. • Faraco J, Lin L, Kornum BR, Kenny EE, Trynka G, Einen M, et al. ImmunoChip study implicates antigen presentation to T cells in narcolepsy. PLoS genetics. 2013:9(2):e1003270. • Penades R, Pujol N, Catalan R, Massana G, Rametti G, Garcia-Rizo C, et al. Brain effects of cognitive remediation therapy in schizophrenia: a structural and functional neuroimaging study. Biological psychiatry. 2013 May 15;73(10):1015-23. • Marti MJ, Tolosa E. Parkinson disease: New guidelines for diagnosis of Parkinson disease. Nature reviews. Neurology. 2013 Apr;9(4):190-1. • Iranzo A, Tolosa E, Gelpi E, Molinuevo JL, Valldeoriola F, Serradell M, et al. Neurodegenerative disease status and post-mortem pathology in idiopathic rapid-eye-movement sleep behaviour disorder: an observational cohort study. Lancet neurology. 2013 May;12(5):443-53. • Wenning GK, Geser F, Krismer F, Seppi K, Duerr S, Boesch S, et al. The natural history of multiple system atrophy: a prospective

188188 Eduard Tolosa Sarró Group Eduard Tolosa Sarró Group 189

European cohort study. Lancet neurology. 2013 Mar;12(3):264-74. • Volkmann J, Albanese A, Antonini A, Chaudhuri KR, Clarke CE, de Bie RM, et al. Selecting deep brain stimulation or infusion therapies in advanced Parkinson’s disease: an evidence-based review. Journal of neurology. 2013 Jan 5. • Colom-Cadena M, Gelpi E, Charif S, Belbin O, Blesa R, Marti MJ, et al. Confluence of α-synuclein, tau, and β-amyloid pathologies in dementia with Lewy bodies. Journal of neuropathology and experimental neurology. 2013 Dec;72(12):1203-12. • Colom-Cadena M, Gelpi E, Marti MJ, Charif S, Dols-Icardo O, Blesa R, et al. MAPT H1 haplotype is associated with enhanced α-synuclein deposition in dementia with Lewy bodies. Neurobiology of aging. 2013 Mar;34(3):936-42. • Ortega-Cubero S, Lorenzo-Betancor O, Lorenzo E, Alonso E, Coria F, Pastor MA, et al. Fused in Sarcoma (FUS) gene mutations are not a frequent cause of essential tremor in Europeans. Neurobiology of aging. 2013 Oct;34(10):2441.e9-2441.e11. • Kumru H, Soler D, Vidal J, Navarro X, Tormos JM, Pascual-Leone A, et al. The effects of transcranial direct current stimulation with visual illusion in neuropathic pain due to spinal cord injury: An evoked potentials and quantitative thermal testing study. European journal of pain (London, England). 2013 Jan;17(1):55-66. • Boeckstyns ME, Sørensen AI, Vineta JF, Rosen B, Navarro X, Archibald SJ, et al. Collagen conduit versus microsurgical neurorrhaphy: 2-year follow-up of a prospective, blinded clinical and electrophysiological multicenter randomized, controlled trial. The Journal of hand surgery. 2013 Dec;38(12):2405-11.

Research Projects

• Code: PI041833 • Code: PI041639 (1) Title: Endofenotipos clínicos según el perfil de biomarcado- Title: Gait and motors symptoms in healthy asymptomat- res de beta-amiloide y TAU como posibles predictores de ic relatives of Patients with PD carriers of mutations in the demencia en la enfermedad de Parkinson LRRK2 gene Principal Investigator: Yaroslau Compta Principal Investigator: Anat Mirelaman CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Privado Type: International Funding Agency: Federación Española de Parkinson Funding Agency: Michael J. Fox Foundation for Parkinson’s Funding: 30.000 € Disease Duration: 2013 Funding: 28.125 € Duration: 2012-2013 • Code: PI040438 Title: European Project on Mendelian Forms of Parkinson’s • Code: PI2010/05 Disease (MEFOPA) Title: Generación de un modelo neuronal dopaminérgico a Principal Investigator: Eduardo Tolosa partir de células madre pluripotentes inducidas de pacien- CIBERNED’s collaboration: No tes con enfermedad de Parkinson asociada a mutaciones CIBERNED’s groups: en el gen LRRK2 Other CIBER’s collaboration: No Principal Investigator: Eduardo Tolosa Sarró Type: International CIBERNED’s collaboration: Yes Funding Agency: CP-FP. (Europe Community) CIBERNED’s groups: G 207; G 301; G 105; G 109 Funding: 56.000 € Other CIBER’s collaboration: No Duration: 2010-2013 Type: Intramurales Funding Agency: CIBERNED • Code: CP06/00126 Funding: 262.500 € Title: Factores genéticos y ambientales modificadores de la Duration: 2010-2013 expresividad de las mutaciones del gen LRRK2 en la enfer- medad de Parkinson • Code: 2009SGR1019 Principal Investigator: Eduardo Tolosa Sarró Title: Grup de Recerca de malalties neurològiques CIBERNED’s collaboration: No Principal Investigator: Eduardo Tolosa Sarró CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: Fondo de Investigaciones Sanitarias de la Type: CCAA Seguridad Social (FISS) Funding Agency: Agència de Gestio d’Ajuts Universitaris i Funding: 18.050 € de Recerca (AGAUR) - Generalitat de Catalunya Duration: 2007-2013 Funding: 72.800 € Duration: 2009-2013 Duration: 2013-2018

• Code: PI041639 • Code: PI2013_08-1 Title: ASymptomatic G2019S/ R1441G LRRK2 mutation car- Title: La dinámica mitocondrial y mitofagia como dianas te- riers and Assessment of factors influencing LRRK2-related rapéuticas en las Enfermedades de Parkinson y Huntington PD expression study Principal Investigator: Eduardo Soriano Principal Investigator: Eduardo Tolosa Sarró CIBERNED’s collaboration: Yes CIBERNED’s collaboration: No CIBERNED’s groups: G 301; G 207; G 410; G 109; G 408 CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Intramurales Type: International Funding Agency: CIBERNED Funding Agency: The Michael J. Fox foundation for Funding: 300.000 € Parkinson’s Disease Duration: 2013-2015 Funding: 109.000 € Duration: 2011-2013 • Code: MJF Title: Expansion of the ASAP study (ASymptomatic LRRK2 • Code: 00000 mutation carriers and Assessment of factors influencing Title: The Parkinson’s Progression Markers Initiative (PPMI) LRRK2-related PD expression study) Principal Investigator: Eduardo Tolosa Sarró Principal Investigator: José Félix Martí Massó CIBERNED’s collaboration: No CIBERNED’s collaboration: Yes CIBERNED’s groups: CIBERNED’s groups: G 609; G 207 Other CIBER’s collaboration: No Other CIBER’s collaboration: Yes Type: Intramurales Type: International Funding Agency: The Michael J. Fox Foundation for Funding Agency: Michael J Fox Parkinson’s Research Funding: 180.000 € Funding: 183.0210 € Duration: 2011-2013

190190 Eduard Tolosa Sarró Group Group Carlos Vicario Abejón

CSIC

Avenida del Doctor Arce, 37 2 28002 Madrid (Spain) Tel.: +34 915 854 721 Fax: +34 915 854 754 Program [email protected]

Carlos Vicario Abejón Çagla Defterali Principal Investigator, CSIC Senior Scientist PhD student Jaime Pignatelli Garrigós Eva Rodríguez Traver Postdoctoral Fellow (until March 2013) PhD student Eva Díaz Guerra Mª José Román Postdoctoral Fellow Lab Technician Vanesa Nieto Estévez PhD student, Postdoctoral Fellow (from May 2013)

191 Summary

We have obtained colonies with the morphological and molecular features of genuine induced pluripotent stem cells (iPSCs), transducing the OKSM factors (OCT3/4, KLF4, SOX2, C-MYC). This technique was employed to reprogramme dermal fibroblasts from Parkinson´s disease (PD) patients carrying mutations in the glucocerebrosidase1 gene (GBA1) associated with an increase risk of dementia with Lewy bodies. Cells isolated from two patients and one control subject, and transduced with non-integrative Sendai viral vectors, adopted the typical iPSC morphology and they expressed the pluripotency markers Nanog, TRA-1-60 and SSEA-4 (Rodríguez-Traver et al., Abstract Foro CIBERNED 2013). Moreover, these cells differentiate into dopaminergic neurons.

Our studies have shown that enhancing the levels of the transcription factor Tbr1 in neural stem cells (NSCs) induces the expression of genes associated with early neurogenic events and neuronal differentiation (Dscaml1, Tubb3), while the expression of genes associated to astrocyte generation and differentiation (Gfap, S100beta) was dampened (Díaz-Guerra et al.). We are now using chormatin immunoprecipitation (ChIP) to search for direct Tbr1 binding sites in the potential target genes.

Our data indicate that insulin-like growth factor-I (IGF-I) is a key extracellular factor that regulates the correct neuronal positioning and maturation in the hippocampal (HP) dentate gyrus (Nieto-Estévez et al.). We are now exploring the role of IGF-I and the IGF-IR during adult HP neurogenesis in a conditional Igf-I KO that we have generated using Nestin-Cre and loxP technology, as well as in a mouse line carrying the Igf-IR flanked by loxP sequences that can similarly be deleted using retroviral vectors expressing Cre recombinase.

We have found that the core of the adult mouse olfactory bulb contains endogenous dividing NSCs and progenitors that generate granule and periglomerular neurons, and a very small number of glutamatergic interneurons (Defterali et al.).

Keywords

Parkinson´s disease, Induced pluripotent stem cells (iPSCs), Dopaminergic neurons, Neural stem cells, Transcription factors, Growth factors

Publications

• Diaz-Guerra E, Pignatelli J, Nieto-Estevez V, Vicario-Abejon C. Transcriptional regulation of olfactory bulb neurogenesis. The Anatomical Record. 2013:296(9):1364-82. • Pignatelli J, Alonso-Padilla J, Rodriguez D. Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein. Veterinary research. 2013 Dec 23;44:126. • Nieto-Estevez V, Pignatelli J, Arauzo-Bravo MJ, Hurtado-Chong A, Vicario-Abejon C. A global transcriptome analysis reveals molecular hallmarks of neural stem cell death, survival, and differentiation in response to partial FGF-2 and EGF deprivation. PLOS ONE. 2013;8(1):e53594(1-19). • Rinaldi A, Defterali C, Mialot A, Garden DL, Beraneck M, Nolan MF. HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition. The Journal of Physiology - London (Wiley). 2013 Nov 15;591(Pt 22):5691-709. • Manterola L, Hernando-Rodriguez M, Ruiz A, Apraiz A, Arrizabalaga O, Vellon L, et al. 1-42 β-Amyloid peptide requires PDK1/ nPKC/Rac 1 pathway to induce neuronal death. Translational psychiatry. 2013 Jan 22;3:e219.

Research Projects

• Code: BFU2010-19630/BFI Funding Agency: Dirección GeneraI de Investigación, MI- Title: Neurogénesis en el cerebro embrionario y adulto: CINN mecanismos reguladores y estrategias para la neurorrepa- Funding: 95.590 € ración Duration: 2011-2013 Principal Investigator: Carlos Vicario Abejón CIBERNED’s collaboration: No • Code: 201320E054 CIBERNED’s groups: Title: Regulación de la neurogénesis por factores extracelu- Other CIBER’s collaboration: No lares y de transcripción Type: National Principal Investigator: Carlos Vicario Abejón

192192 Carlos Vicario Abejón Group Carlos Vicario Abejón Group 193

CIBERNED’s collaboration: No Funding Agency: CIBERNED CIBERNED’s groups: Funding: 300.000 € Other CIBER’s collaboration: No Duration: 2011-2013 Type: National Funding Agency: CSIC • Code: S2011/BMD-2336 Funding: 16.000 € Title: Estudio de la biología de células madre neurales para Duration: 2013 su empleo en terapia celular en enfermedades neurodege- nerativas • Code: PI2011/03 Principal Investigator: Rosario Moratalla Title: Generación de neuronas dopaminérgicas a partir de CIBERNED’s collaboration: Yes células somáticas de pacientes parkinsonianos con fallo CIBERNED’s groups: G 204; G 108; G 305 cognitivo Other CIBER’s collaboration: No Principal Investigator: Rosario Moratalla Villalba Type: CCAA CIBERNED’s collaboration: Yes Funding Agency: Comunidad Autónoma de Madrid CIBERNED’s groups: G 204; G 106; G 202; G 110; G 108 Funding: 598.000 € Other CIBER’s collaboration: No Duration: 2012-2015 Type: Intramurales Group Miquel Vila Bover

Neurodegenerative Diseases Research Group

2 Vall d’Hebron Research Institute Mediterranean Building, First Floor, Lab. 102 Passeig Vall d’Hebron, 119-129 Program 08035 Barcelona (Spain) Tel.: +34 934 894 543 / 3885 Fax: +34 934 894 015 [email protected]

Miquel Vila Bover Ariadna Recasens Ibabe Principal Investigator, ICREA Research Professor PhD student Celine Perier Sandra Franco Iborra Ramón y Cajal Researcher PhD student Jordi Bové Badell Annabelle Parent Miguel Servet Researcher Technician Marta Martínez Vicente Thais Cuadros Arasa Miguel Servet Researcher CIBERNED Technician Oriol de Fàbregues-Boixar Nebot Beatriz Rodríguez Galván Collaborator Technician Iria Carballo Carbajal María José Pérez García CIBERNED Postdoctoral Fellow Technician

194 Miquel Vila Bover Group 195

Summary

In 2013, we have shown that α-synuclein species contained in nigral Lewy bodies (LB) from postmortem brains of patients with Parkinson’s disease (PD) have the capacity to trigger a PD-like pathological process when directly injected into the brain of mice and monkeys, including intracellular and pre-synaptic accumulations of pathological α-synuclein in different interconnected brain areas and slowly progressive axon-initiated dopaminergic nigrostriatal neurodegeneration. These pathogenic effects (i) required both human α-synuclein present in LB extracts and host expression of α-synuclein, and (ii) were not observed in animals inoculated with non-LB fractions containing soluble α-synuclein derived from the same nigral PD tissue. In addition, we assessed the potential pathogenic effects of mitochondrial DNA (mtDNA) deletions, which are present in the brains of PD patients, on the dopaminergic nigrostriatal system by using mutant mice possessing a proofreading-deficient form of mtDNA polymerase gamma (POLGD257A). These animals accumulate mtDNA deletions to a similar extent as PD patients. However, despite such high levels of mtDNA deletions, POLGD257A mice do not exhibit dopaminergic neurodegeneration but are resistant to parkinsonian mitochondrial neurotoxins, due to neuroprotective compensatory mechanisms triggered in these neurons at the level of the mitochondrial. Finally, we have shown that in vitro and in vivo PD models exhibit a disruption of OPA1 oligomeric complexes that normally keep mitochondrial cristae junctions tight, resulting in major mitochondrial structural abnormalities These changes are not accompanied by mitochondrial fission but a mobilization of cytochrome c from cristae to intermembrane space, thereby lowering the threshold for activation of mitochondria-dependent apoptosis by cell death agonists in compromised neurons. All these pathogenic changes, including mitochondrial structural remodeling and dopaminergic neurodegeneration are abrogated by OPA1 overexpression in experimental PD models, both in vitro and in vivo.

Keywords

Parkinson’s disease, Lewy bodies, α-synuclein, Mitochondria

Publications

• Dehay B, Martinez-Vicente M, Caldwell GA, Caldwell KA, Yue Z, Cookson MR, et al. Lysosomal impairment in Parkinson’s disease. Movement Disorders. 2013;28(6):725-32. • Martinez-Vicente M, Vila M. Alpha-synuclein and protein degradation pathways in Parkinson’s disease: a pathological feed- back loop. Experimental neurology. 2013 Sep:247:308-13. • Cuadros T, Trilla E, Vilà MR, de Torres I, Vilardell J, Messaoud NB, et al. Hepatitis A virus cellular receptor 1/kidney injury molecule-1 is a susceptibility gene for clear cell renal cell carcinoma and hepatitis A virus cellular receptor/kidney injury molecule-1 ectodomain shedding a predictive biomarker of tumour. Eur J Cancer. 2013 May;49(8):2034-47. • Ramonet D, Perier C, Recasens A, Dehay B, Bove J, Costa V, et al. Optic atrophy 1 mediates mitochondria remodeling and dopaminergic neurodegeneration linked to complex I deficiency. Cell death and differentiation. 2013 Jan;20(1):77-85. • Perier C, Bender A, Garcia-Arumi E, Melià MJ, Bove J, Laub C, et al. Accumulation of mitochondrial DNA deletions within dopaminergic neurons triggers neuroprotective mechanisms. Brain. 2013;136:2369-78. • Recasens A, Dehay B, Bove J, Carballo-Carbajal I, Dovero S, Perez A, et al. Lewy body extracts from parkinson’s disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. Annals of neurology. 2013. • Rue L, Lopez-Soop G, Gelpi E, Martinez-Vicente M, Alberch J, Perez-Navarro E. Brain region- and age-dependent dysregulation of p62 and NBR1 in a mouse model of Huntington’s disease. Neurobiology of Disease. 2013;52:219-28.

Research Projects

• Code: SAF2009-08374 Funding Agency: MICINN Title: Alteracion de la autofagia en la enfermedad de Funding: 93.000 € Huntington Duration: 2010-2013 Principal Investigator: Marta Martínez-Vicente CIBERNED’s collaboration: No • Code: CP09/00184 CIBERNED’s groups: Title: Alteración de la autofagia en la enfermedad de Other CIBER’s collaboration: No Huntington Type: National Principal Investigator: Marta Martínez-Vicente CIBERNED’s collaboration: No disease via cell-fusion-mediated reprogramming CIBERNED’s groups: Principal Investigator: Maria Pia Cosma (CRG, Barcelona) Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: National CIBERNED’s groups: Funding Agency: Fondo de Investigación Sanitaria (FIS) - Other CIBER’s collaboration: No Instituto de Salud Carlos III Type: Privado Funding: 35.000 € Funding Agency: Marató TV3 Duration: 2010-2013 Funding: 138.750 € Duration: 2013-2015 • Code: CP11/00229 Title: Estrategias neuroprotectoras y biomarcadores en la • Code: DENDRIA enfermedad de Parkinson basados en la disfunción lisoso- Title: Soluciones innovadoras para acelerar la identificación mal y desarrollo de nuevos medicamentos para el tratamiento Principal Investigator: Jordi Bové Badell de patologías del sistema nervioso CIBERNED’s collaboration: No Principal Investigator: Miquel Vila Bover CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: FIS-ISCIII Type: National Funding: 60.000 € Funding Agency: Program de consorcios estratégicos nacio- Duration: 2012-2014 nales en investigación técnica (CENIT) Funding: 177.881 € • Code: PI10/00849 Duration: 2010-2013 Title: Initiation, progression and extension of Parkinson’s disease: role of neuromelanin, α-synuclein and Lewy bodies • Code: PI2010/05 Principal Investigator: Miquel Vila Bover Title: Generación de un modelo neuronal dopaminérgico a CIBERNED’s collaboration: No partir de células madre pluripotentes inducidas de pacien- CIBERNED’s groups: tes con enfermedad de Parkinson asociada a mutaciones Other CIBER’s collaboration: No en el gen LRRK2 Type: National Principal Investigator: Eduardo Tolosa Sarró Funding Agency: Fondo de Investigación Sanitaria, Instituto CIBERNED’s collaboration: Yes de Salud Carlos III CIBERNED’s groups: G 207; G 301; G 105; G 109 Funding: 198.000 € Other CIBER’s collaboration: No Duration: 2011-2013 Type: Intramurales Funding Agency: CIBERNED • Code: MJFF Staff Initiated 2013 Funding: 262.500 € Title: Prion-like dissemination of synuclein pathology: a Duration: 2010-2013 non-human primate study Principal Investigator:Erwan Bezard (Universite de Bordeaux, • Code: PI2013_08-1 France) Title: La dinámica mitocondrial y mitofagia como dianas te- CIBERNED’s collaboration: Yes rapéuticas en las Enfermedades de Parkinson y Huntington CIBERNED’s groups: G 505; G 205; G 109 Principal Investigator: Eduardo Soriano Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: International CIBERNED’s groups: G 301; G 207; G 410; G 109; G 408 Funding Agency: Michael J. Fox Foundation Other CIBER’s collaboration: No Funding: 33.000 € Type: Intramurales Duration: 2013-2016 Funding Agency: CIBERNED Funding: 300.000 € • Code: Marato TV3 (120531) Duration: 2013-2015 Title: Regenerating dopaminergic neurons in Parkinson’s

196196 Miquel Vila Bover Group Program 3

Neuromuscular Diseases

Research Groups

Berciano Blanco, José Ángel______201 Muñoz Cánoves, Pura______219 Fernández Chacón, Rafael______207 Navarro Acebes, Xavier______222 Illa Sendra, Isabel______210 Vilchez Padilla, Juan Jesús______226 López de Munain Arregui, Adolfo____ 214

Program 3

Neuromuscular diseases (NMDs) form a heterogeneous group of pathologies affecting the spinal cord and its tracts, nerve roots as well as motor and sensitive peripheral nerves, the neuromuscular junction and muscles. Diagnosis involves using sophisticated methods covering: neurophysiological studies, muscle or nerve biopsy with the use of immunohistochemical techniques and some others, metabolic analysis and tests, RMN studies, quantitative muscle assessment (QMA) and, in many cases, genetic studies.

Just a few epidemiological studies are available in Spain, from which it can be inferred that the prevalence of chronic neurodegenerative and/or hereditary pathology would account for around 60,000 patients in our country.

The members of the neuromuscular pathology program are committed to investigate into specific aspects including:

• Clinical characterization of neuromuscular pathologies and correlation between the clinical phenotype and their genotype (or proteomic characterization). Development of animal models based on the genotypically identified dystrophies. • Research on the physiopathology of neuromuscular diseases. • Development of new therapeutic strategies.

The program comprises 8 groups, four of them are clinical, one basic neuropathology group and three basic research ones.

199 Groups

Principal Investigator Institution Berciano Blanco, José Ángel Marqués de Valdecilla Hospital, Santander

Fernández Chacón, Rafael University of Seville, Institute of Biomedicine of de Seville (IBiS) Illa Sendra, Isabel Santa Creu i Sant Pau Hospital, Barcelona López de Munain Arregui, Adolfo Biodonostia Research Institute, San Sebastián Muñoz Cánoves, Pura Pompeu Fabra University, Barcelona Navarro Acebes, Xavier Autonomous University of Barcelona Vilchez Padilla, Juan Jesús La Fe University Hospital, Valencia

Program 2 is coordinated by Dr. Isabel Illa (Santa Creu i Sant Pau Hospital, Barcelona).

200200 Program 3 Group José Ángel Berciano Blanco

Servicio de Neurología

Hospital Universitario Marqués de Valdecilla 3 Universidad de Cantabria

39008 Santander (Spain) Program [email protected] / [email protected]

José Ángel Berciano Blanco Ana Palanca* Principal Investigator, Full Professor of Neurology FPI MICINN PhD student Onofre Combarros Ana L. Pelayo-Negro Co-Principal Investigator, Full Professor of Neurology Neurologist specialist, Lopez Albo Fellow IDIVAL María Teresa Berciano* Saray Pereda* Full Professor of Cell Biology MICINN Lab Technician Iñigo Casafont* José M. Polo UC Assistant Professor Associate Professor of Neurology Marta de la Fuente Ana Pozueta CIBERNED part-time Administrative Assistant Psychologist, FIS/IFIMAV Fellow Elena Gallardo Javier Riancho-Zarrabeitia Area Specialist Physician (ASP), Assistant Professor of Neurology MIR Radiology Eloy Rodríguez-Rodríguez Antonio García Neurology ASP Clinical Neurophysiology ASP María Ruiz Soto* Isabel González-Aramburu MICINN Technician Neurologist specialist, IFIMAV postMIR Fellow Pascual Sánchez-Juan Andrés González-Mandly Neurology ASP Radiology ASP Coro Sánchez-Quintana Andrea González Suaréz CIBERNED Lab Technician Neurologist specialist, IDIVAL Researcher María J. Sedano Jon Infante Neurology ASP ASP, Assistant Professor of Neurology María Sierra Miguel A. Lafarga* Neurology Specialist, López Albo Fellow IDIVAL Full Professor of Cell Biology José L. Vázquez-Higuera José Ignacio Mateo Neurology ASP Neurology ASP * Basic group members

201 Summary

As noted in previous Annual Reports, the research activity of our group has a clinical component (led by Profs. José Berciano and Onofre Combarros), and other basic (led by Profs. Miguel Lafarga and María Teresa Berciano). The latter is set in the Department of Anatomy and Cell Biology, Faculty of Medicine, University of Cantabria.

On the clinical side, our research activity has focused on the following aspects:

• Clinical-molecular correlations in ataxias and hereditary neuropathies. Our group consists of two EU Concerted projects: EUROSCA, RISCA and AFM/CMT1A. • Analysis of lower limb amyotrophy in CMT by magnetic resonance imaging. • Clinical-molecular correlations in familial Parkinson’s disease. • Risk gene polymorphisms in sporadic Alzheimer’s disease. • Prionopathies

On the basic research side, it has focused on the following points:

• The Cajal body and the nuclear fibrillar centers of the nucleolus as markers of neuronal activity. • Role of the SUMO-1 conjugation pathway in neuronal transcription regulation. • Dynamics of the nuclear aggregation of the protein binding to the tail of poly(A) RNAs in neurons and muscle fibers in oculopharyngeal muscular dystrophy.

Peripheral neuropathies We have continued our studies on the mechanisms of axonal damage in Guillain-Barré syndrome (GBS), proposing that its classification takes into consideration histopathologic findings described in post mortem studies with a large sample of proximal and distal nerve trunks, as those carried out by our group. We have clarified the GBS complex nosology with persistent inexcitability of nerve trunks by MRI examination of the lower limb muscles, showing that this neurophysiological finding does not necessarily associates with axonal degeneration and the corresponding muscular denervation. We have reported the first case of HNPP (neuropatía hereditaria con hipersensiilidad a la presión) with fulminant failure of nerve conduction in lumbosacral roots during an intervention of lumbar fusion, which calls for caution when indicating such surgical procedure in patients with HNPP.

Our group has continued to be integrated in the European Project AFM/CMT (French Association of Myology/Charcot-Marie- Tooth 1A) coordinated by Prof. Michael Sereda (University of Goettingen, Germany), and has entered into the European Project IGOS (International Guillain-Barré Outcome Study), coordinated by Prof. Bart Jacobs (University of Rotterdam, The Netherlands). In 2013, the longitudinal study of clinical and neurophysiological and MRI of legs in CMT1A was completed, showing that in the observation period (2 years) there was a minimal clinical progression.

Hereditary ataxias We have continued the European study Natural History of SCAs within EUROSCA Project, following patients from the RISCA Project (E-Rare JTC 2007/Institute of Health Carlos III, PI071323). Within this project we have contributed to the study of the preclinical stage of the most common forms of SCAs (SCA1, 2, 3 and 6), showing that mild motor impairment and changes in neuroimaging in mutation carriers SCA2 and SCA1 can be detected already at this preclinical stage. We investigated the echogenicity of the substantia nigra in a cohort of patients with Friedreich’s ataxia, finding no significant differences from healthy controls. We have reviewed the peripheral nervous system pathology in hereditary and sporadic ataxias, showing that the sensory neuropathy in Friedreich’s ataxia, a central manifestation of the disease, is due to a mechanism of dying back.

Parkinson’s disease We have carried out a study of clinical and neuroimaging biomarkers in the premotor stage of Parkinson’s disease caused by the G2019S mutation of the LRRK2 gene, finding that 85% of asymptomatic carriers of the mutation show substantia nigra hyperechogenicity and 43% changes in DaTSCAN. We have identified gene polymorphisms influencing uric acid serum levels modify the risk of Parkinson’s disease. We have participated in an international collaborative study aimed at identifying the frequency of the LRRK2 variant S1761R in Spain. Currently, there is an ongoing biomarkers project using multimodal MR imaging

202202 José Ángel Berciano Blanco Group José Ángel Berciano Blanco Group 203

in carriers of this mutation and controls in collaboration with Prof. Olivier Rascol (University of Toulouse, France). We have found that progranulin serum levels are reduced in patients with Parkinson’s disease when compared with the control population.

Alzheimer’s disease Within IGAP the international consortium, a mega-meta-analysis in 74,000 individuals has allowed the identification of 11 new risk genes for Alzheimer’s disease (AD). Within the European consortium EADI, we have found a rare monogenic form of AD associated with the FRMD4A gene. Within the international Epistasis Project we have also found a risk interaction for AD between the glutathione S-transferase gene and the HHEX/IDE/KIF11 gene cluster. In a study in Santander, Barcelona and Pamplona, the analysis of the combined effect of 8 genes associated with AD predicted rapid progression of MCI to AD. Looking at the serum level and progranulin (GRN) mutations in common neurodegenerative diseases, GRN low serum levels were associated with risk of Parkinson but not AD, and GRN mutations were very rare.

Prionopathies Genetic epidemiology of prion diseases. During 2013 we completed a genome-wide association (GWAS) analysis that included 1,543 samples from patients with sporadic Creutzfeldt-Jakob disease, from 7 European countries and Australia, and 4,203 controls. The findings have been replicated in an independent population and the manuscript is ready for submission. Biomarkers of rapidly progressive dementias. We continue our collaboration with Prof. Inga Zerr (University of Goettingen), within the European project JPND DEMTEST. As a result, we currently have an article in press proposing new clinical diagnostic criteria for Fatal Familial Insomnia.

Cell Neurobiology of the Nucleus Studies have focused on the cellular and molecular basis of peripheral neuropathy induced by proteasome inhibition with Bortezomib, a chemotherapy used in multiple myeloma and other solid tumors. In a rat model, we demonstrated that inhibition of proteasome leads to consistent alterations in nuclear retention of mRNAs in nuclear inclusions, nuclear eccentricity, overexpression of nuclear lamins, induction of DNA damage and chromatolysis. Although these alterations lead to severe dysfunction of primary sensory neurons, to counteract the proteotoxic stress induced by proteasome inhibition and to maintain neuronal survival, a reactive nucleolar and Cajal body response occurs. Such a response includes activation of the nucleolar transcription to produce rRNAs, and and increase of Cajal bodies in order to facilitate the nuclear processing of pre-mRNAs and pre-rRNAs.

Keywords

Ataxia, SCA, Charcot-Marie-Tooth disease, Parkinson’s disease, Alzheimer’s disease, Creutzfeldt-Jakob disease, Cajal nuclear bodies, Oculopharyngeal dystrophy

Publications

• Baltanás FC, Berciano MT, Valero J, Gómez C, Díaz D, Alonso JR, et al. Differential glial activation during the degeneration of Purkinje cells and mitral cells in the PCD mutant mice. Glia. 2013 Feb;61(2):254-72. • Berciano J. Recent advances in clinical neurogenetics. J Neurol. 2013 Oct;260(10):2451-7. • Berciano J. Reply to “Motor selectivity: important role in the diagnosis of acute motor axonal neuropathy”. Clin Neurophysiol. 2013 Aug;124(8):1702-3. • Berciano J, de Lucas EM, Combarros O. Thumb, forefinger, and lip numbness: a distinctive thalamic lacunar syndrome. Neurol Sci. 2013 Feb;34(2):253-4. • Berciano J, Gallardo E. Proximal lower-limb weakness in charcot-marie-tooth disease. JAMA Neurol. 2013 Dec 1;70(12):1587. • Berciano J, García A, Berciano MT, Lafarga M. Criteria for Guillain-Barré syndrome: additional insights from clinico-pathological studies. Clin Neurophysiol. 2013 Apr;124(4):819-21. • Berciano J, García A, Infante J. Peripheral nerve involvement in hereditary cerebellar and multisystem degenerative disorders. Handb Clin Neurol. 2013;115:907-32. • Berciano J, Martínez-Agüeros JA, Gallardo E, Martínez-Martínez MÁ, Infante J, García A, et al. Hereditary neuropathy with liability to pressure palsy: fulminant radicular dysfunction during anterolateral lumbar interbody fusion. J Neurol. 2013 Sep;260(9):2411-3. • Bishop MT, Sanchez-Juan P, Knight RS. Splice site SNPs of phospholipase PLCXD3 are significantly associated with variant and sporadic Creutzfeldt-Jakob disease. BMC Med Genet. 2013 Sep 12;14:91. • Breithaupt M, Romero C, Kallenberg K, Begue C, Sanchez-Juan P, Eigenbrod S, et al. Magnetic resonance imaging in E200K and V210I mutations of the prion protein gene. Alzheimer Dis Assoc Disord. 2013 Jan-Mar;27(1):87-90. • Bullock JM, Medway C, Cortina-Borja M, Turton JC, Prince JA, Ibrahim-Verbaas CA, et al. Discovery by the Epistasis Project of an epistatic interaction between the GSTM3 gene and the HHEX/IDE/KIF11 locus in the risk of Alzheimer’s disease. Neurobiol Aging. 2013 Apr;34(4):1309.e1-7. • Delgado-Alvarado M, Palacio-Portilla E, Pelayo-Negro AL, Lerena P, Berciano J. From ileostomy to sudden quadriplegia with electrocardiographic abnormalities: a short and unfortunate path. Neurol Sci. 2013 Aug;34(8):1471-3. • Delgado-Alvarado M, Sedano MJ, González-Quintanilla V, de Lucas EM, Polo JM, Berciano J. Progressive multifocal leukoencephalopathy and idiopathic CD4 lymphocytopenia. J Neurol Sci. 2013 Apr 15;327(1-2):75-9. • Delgado-Alvarado M, Riancho J, Riancho-Zarrabeitia L, Sedano MJ, Polo JM, Berciano J. [Unilateral total ophthalmoplegia without visual loss as the presenting form of a pituitary apoplexy]. Rev Clin Esp. 2013 Oct;213(7):e67-70. • Fernández-Viadero C, Rodríguez Rodríguez E, Combarros Pascual O, Crespo Santiago D. Genetics and Alzheimer’s disease: a population at risk. Rev Esp Geriatr Gerontol. 2013 Jan-Feb;48(1):39-44. • González-Aramburu I, Sánchez-Juan P, Jesús S, Gorostidi A, Fernández-Juan E, Carrillo F, et al. Genetic variability related to serum uric acid concentration and risk of Parkinson’s disease. Mov Disord. 2013 Oct;28(12):1737-40. • Jacobi H, Reetz K, du Montcel ST, Bauer P, Mariotti C, Nanetti L, et al. Biological and clinical characteristics of individuals at risk for spinocerebellar ataxia types 1, 2, 3, and 6 in the longitudinal RISCA study: analysis of baseline data. Lancet Neurol. 2013 Jul;12(7):650-8. • Lambert JC, Grenier-Boley B, Harold D, Zelenika D, Chouraki V, Kamatani Y, et al. Genome-wide haplotype association study identifies the FRMD4A gene as a risk locus for Alzheimer’s disease. Mol Psychiatry. 2013 Apr;18(4):461-70. Erratum in: Mol Psychiatry. 2013 Apr;18(4):521. Caffara, P [corrected to Caffarra, P]. • Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Bellenguez C, et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer’s disease. Nat Genet. 2013 Dec;45(12):1452-8. • Lee SE, Tartaglia MC, Yener G, Genç S, Seeley WW, Sanchez-Juan P, et al. Neurodegenerative Disease Phenotypes in Carriers of MAPT p.A152T, A Risk Factor for Frontotemporal Dementia Spectrum Disorders and Alzheimer Disease. Alzheimer Dis Assoc Disord. 2013 Oct-Dec;27(4):302-9. • Mata IF, Alvarez V, Ribacoba R, Infante J, Sierra M, Gómez-Garre P, et al. Novel Lrrk2-p.S1761R mutation is not a common cause of Parkinson’s disease in Spain. Mov Disord. 2013 Feb;28(2):248. • Mateo I, González-Aramburu I, Pozueta A, Vázquez-Higuera JL, Rodríguez-Rodríguez E, Sánchez-Juan P, et al. Reduced serum progranulin level might be associated with Parkinson’s disease risk. Eur J Neurol. 2013;20:1571-3. • Querol L, Rojas-Garcia R, Casasnovas C, Sedano MJ, Muñoz-Blanco JL, Alberti MA, et al. Long-term outcome in chronic inflammatory demyelinating polyneuropathy patients treated with intravenous immunoglobulin: A retrospective study. Muscle Nerve. 2013 Dec;48(6):870-6. • Riancho J. Commentary on A diagnostic and therapeutic challenge involving a case of dysphagia in association with cervical osteophytosis and a dental pain. J Neurosci Rural Pract. 2013 Jul;4(3):336-7. • Riancho J, Agea L, Infante J, Berciano J. [Progressive binocular diplopia]. Med Clin (Barc). 2013 Sep 21;141(6):e11. • Riancho J, Delgado-Alvarado M, Sedano MJ, Polo JM, Berciano J. Herpes simplex encephalitis: clinical presentation, neurological sequelae and new prognostic factors. Ten years of experience. Neurol Sci. 2013 Oct;34(10):1879-81. • Riancho J, Delgado-Alvarado M, Valero C, Echevarría S, Fariñas MC. Clinical spectrum of peripheral facial paralysis in HIV- infected patients according to HIV status. Int J STD AIDS. 2013 Jan;24(1):39-41. • Riancho J, Infante J, Mateo JI, Berciano J, Agea L. Unilateral isolated hypoglossal nerve palsy associated with internal carotid artery dissection. J Neurol Neurosurg Psychiatry. 2013 Jun;84(6):706. • Riancho J, Infante J, Orizaola P, Mateo I, Viadero R, Delgado-Alvarado M, et al. [Reversible cerebral vasoconstriction and acute encephalopathy as a presentation form of Guillain-Barré syndrome]. Rev Clin Esp. 2013 Apr;213(3):e23-6. • Riancho J, Villalobos I. [Ramsay Hunt syndrome]. Med Clin (Barc). 2013 Jan 19;140(2):96. • Rodríguez-Rodríguez E, Sánchez-Juan P, Vázquez-Higuera JL, Mateo I, Pozueta A, Berciano J, et al. Genetic risk score predicting accelerated progression from mild cognitive impairment to Alzheimer’s disease. J Neural Transm. 2013 May;120(5):807-12. • Rodríguez-Rodríguez E, Vázquez-Higuera JL, Sánchez-Juan P, González-Aramburu I, Pozueta A, Mateo I, et al. Screening for progranulin mutations by serum protein dosage in common neurodegenerative disorders. Parkinsonism Relat Disord. 2013 Aug;19(8):768-9. • Romero AM, Renau-Piqueras J, Pilar Marin M, Timoneda J, Berciano MT, Lafarga M, et al. Chronic alcohol alters dendritic spine

204204 José Ángel Berciano Blanco Group José Ángel Berciano Blanco Group 205

development in neurons in primary culture. Neurotox Res. 2013 Nov;24(4):532-48. • Rubio-Moscardo F, Setó-Salvia N, Pera M, Bosch-Morató M, Plata C, Belbin O, et al. Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset Alzheimer’s disease patients alter calcium homeostasis. PLoS One. 2013 Sep 17;8(9):e74203. • Ruiz A, Dols-Icardo O, Bullido MJ, Pastor P, Rodríguez-Rodríguez E, López de Munain A, et al.; dementia genetic Spanish consortium (DEGESCO). Assessing the role of the TREM2 p.R47H variant as a risk factor for Alzheimer’s disease and frontotemporal dementia. Neurobiol Aging. 2014 Feb;35(2):444.e1-4. • Ruiz-Ontañon P, Orgaz JL, Aldaz B, Elosegui-Artola A, Martino J, Berciano MT, et al. Cellular plasticity confers migratory and invasive advantages to a population of glioblastoma-initiating cells that infiltrate peritumoral tissue. Stem Cells. 2013 Jun;31(6):1075-85. • Sánchez-Ferrero E, Coto E, Beetz C, Gámez J, Corao AI, Díaz M, et al.; Genetics of Spastic Paraplegia study group. SPG7 mutational screening in spastic paraplegia patients supports a dominant effect for some mutations and a pathogenic role for p.A510V. Clin Genet. 2013 Mar;83(3):257-62. • Sedano MJ, Canga A, de Pablos C, Polo JM, Berciano J. Muscle MRI in severe Guillain-Barré syndrome with motor nerve inexcitability. J Neurol. 2013 Jun;260(6):1624-30. • Sierra M, Infante J, Berciano J. Substantia nigra echogenicity in Friedreich’s ataxia patients. Cerebellum. 2013 Aug;12(4):437-40. • Sierra M, Sánchez-Juan P, Martínez-Rodríguez MI, González-Aramburu I, García-Gorostiaga I, Quirce MR, et al. Olfaction and imaging biomarkers in premotor LRRK2 G2019S-associated Parkinson disease. Neurology. 2013 Feb 12;80(7):621-6. Este trabajo va acompañado del siguiente Editorial: Berg D, Bandmann O. Biomarkers for PD: How can we approach complexity? Neurology. 2013 Feb 12;80(7):608-9. • Berciano J, Polo JM, J. Infante, Zarranz JJ. Enfermedades degenerativas espinales y espinocerebelosas. Neurología (5ª edición) Barcelona: Elsevier, 2013; pp 523-36.

Research Projects • Code: FI11/00259 taciones no motoras de la enfermedad de Parkinson Title: Enfermedad de Parkinson causada por la mutación Principal Investigator: María Sierra Peña G2019S de LRRK2 en Cantabria: aspectos genéticos y es- CIBERNED’s collaboration: No tudio de biomarcadores en portadores asintomáticos de la CIBERNED’s groups: mutación Other CIBER’s collaboration: No Principal Investigator: María Sierra Peña Type: National CIBERNED’s collaboration: No Funding Agency: Fundación Marqués de Valdecilla - IFIMAV CIBERNED’s groups: Funding: 7.500 € Other CIBER’s collaboration: No Duration: 2011-2013 Type: National Funding Agency: ISCIII • Code: PI12/02288 Funding: 108.000 € Title: Estudio multimodal de biomarcadores de enferme- Duration: 2011-2015 dad de alzheimer en deterioro cognitivo postoperatorio Principal Investigator: Pascual Sánchez-Juan • Code: WLA 04/11 CIBERNED’s collaboration: No Title: Estudio de biomarcadores en la fase promotora en la CIBERNED’s groups: enfermedad de Parkinson genética y su aplicación al diag- Other CIBER’s collaboration: No nóstico de la enfermedad de Parkinson idiopática Type: National Principal Investigator: María Sierra Peña Funding Agency: FIS CIBERNED’s collaboration: No Funding: 36.905 € CIBERNED’s groups: Duration: 2013-2016 Other CIBER’s collaboration: No Type: National • Code: RD09/0076/00076 Funding Agency: Fundación Marqués de Valdecilla - IFIMAV Title: RETIC Biobancos, nodo Hospital “Universitario Mar- Funding: 50.000 € qués de Valdecilla” Duration: 2012-2014 Principal Investigator: Pascual Sánchez Juan CIBERNED’s collaboration: No • Code: API 11/25 CIBERNED’s groups: Title: Estudio de la ecogenicidad de la sustancia negra me- Other CIBER’s collaboration: No diante sonografía transcraneal en individuos portadores de Type: National la mutación G2019S de LRRK2 y su correlación con manifes- Funding Agency: ISCIII Funding: 524.400 € CIBERNED’s collaboration: No Duration: 2010-2014 CIBERNED’s groups: Other CIBER’s collaboration: No • Code: PI11/00228 Type: International Title: Selección de genes candidato para la enfermedad Funding Agency: Association Française contre les Myo- de Parkinson idiopática a través del análisis diferencial del pathies (AFM) transcriptoma en pacientes y portadores asintomáticos de Funding: 34.000 € mutaciones en LRRK2 Duration: 2011-2013 Principal Investigator: Jon Infante Ceberio CIBERNED’s collaboration: No • Code: PI11/03028 CIBERNED’s groups: Title: DEMTEST: Biomarker based diagnosis of rapid pro- Other CIBER’s collaboration: No gressive dementias Type: National Principal Investigator: Pascual Sánchez Funding Agency: ISCIII CIBERNED’s collaboration: Yes Funding: 105.942 € CIBERNED’s groups: G 114; G 601; G 503; G 609 Duration: 2012-2014 Other CIBER’s collaboration: No Type: International • Code: BFU20011-23983 Funding Agency: Instituto de Salud Carlos III - Comunidad Title: Señalización y reparación del daño en el DNA en neu- Económica Europea ronas. Organización estructural, molecular, espacial y tem- Funding: 10.000 € poral de los focos de lesión/reparación del DNA Duration: 2012-2014 Principal Investigator: Miguel Angel Lafarga Coscojuela CIBERNED’s collaboration: No • Code: PI2010/11 CIBERNED’s groups: Title: Consorcio para generar una base de datos común Other CIBER’s collaboration: No cuya finalidad es implementar la investigación clínica y bá- Type: National sica en enfermedades neuromusculares Funding Agency: Dirección General de Investigación Principal Investigator: Isabel Illa Sendra Funding: 139.150 € CIBERNED’s collaboration: Yes Duration: 2012-2014 CIBERNED’s groups: G 603; G 601; G 605 Other CIBER’s collaboration: No • Code: AFM/CMT1A/2010 Type: Intramurales Title: Validation of prognostic and disease biomarkers in Funding Agency: CIBERNED Charcot-Marie-Tooth disease 1A Funding: 429.000 € Principal Investigator: José Berciano Blanco Duration: 2010-2013

PhD Dissertations

• Author: Ana Lara Pelayo Negro Title: Amiotrofia de extremidades inferiores en la enferme- dad de Charcot-Marie-Tooth tipo 1A: estudio longitudinal clínico, neurofisiológico y de resonancia magnética Date: 9 September 2013 Supervisor: José Ángel Berciano Blanco

206206 José Ángel Berciano Blanco Group Group Rafael Fernández Chacón

Instituto de Biomedicina de Sevilla (IBiS)

Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla 3 Departamento de Fisiología Médica y Biofísica de la Universidad de Sevilla CIBERNED Program Avenida Manuel Siurot, s/n 41013 Sevilla (Spain) Tel.: +34 +34 955 923 047 (office) / 955 923 045 (laboratorio) Fax: +34 955 923 101 [email protected]

Rafael Fernández Chacón José Antonio Martínez López Principal Investigator, Associate Professor PhD student Ana Cristina Calvo Laínez Leonardo Gómez Sánchez Postdoctoral Fellow PhD student Fabiola Mavillard Saborido Macarena Cabrera Serrano Postdoctoral Fellow PhD student Carmen Paradas Ángela Lavado Roldán Postdoctoral Fellow PhD student Gloria Cantero Nieto Alejandro Arroyo Saborido Postdoctoral Fellow (until January 31, 2013) Technician José Luis Rozas Espadas María del Carmen Rivero Mena Postdoctoral Fellow Technician Josif Mircheski Jose Luis Muñoz Bravo Postdoctoral Fellow (until January 31, 2013) (from October 2013) Pablo Luis García-Junco Clemente Postdoctoral Fellow

207 Summary

Our group studies the molecular mechanisms that maintain synaptic function and synaptic structure. A major goal in our lab is to elucidate the role of Cysteine String Protein-alpha (CSP-alpha), a molecular co-chaperone located at the synaptic vesicles that prevents synaptic degeneration. In part, the clinical scope of our studies comes from a recent finding of mutations in the human gene coding for CSP-alpha (DNAJC5) causing autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (NCL4), a devastating neurodegenerative disease in young adults. We have used the neuronal specific promoter Thy1 to generate several mouse transgenic lines expressing three different variants of CSP-alpha (wild-type and the mutants L115R and ΔL116). We have confirmed the transgene expression in different regions of the central nervous system. Such an approach is interesting because it bears an important potential to mimick in mouse neurons the autosomic dominant effect of CSP-alpha mutations observed in humans. In addition, our group is interested in the molecular and cellular mechanisms underlying the brain response to neurodegeneration. Within this context, we are interested in studying the consequences of synaptic degeneration that occurs in parvalbumin positive (PV+) GABAergic interneurons at the hippocampus of CSP-alpha KO mice. Interestingly, it has been recently shown that PV+ interneurons maintain the quiescence of hippocampal neural stem cells. Curiously, we have observed that CSP-alpha KO mice suffer from significant alterations in postnatal neurogenesis that we are currently studying in detail. On the other hand, our group is interested in the use of superresolution microscopy to investigate molecular changes occurring at nerve terminals during neurodegeneration. In order to get familiar with superresolution microscopy, a group member (Jose Antonio Martínez López) has carried out a three months visit in 2013 at the laboratory of Prof. Bernardo Sabatini (HHMI, Harvard Medical School, Boston) funded by the European Molecular Biology Organization, EMBO. Furthermore the IP (Dr. Rafael Fernández-Chacón) have also acquired first hand experience in superresolution microscopy at the Neuroimaging Research Cluster (Marine Biological Laboratory MBL, Woods Hole, Massachusetts) funded by the Grass Foundation (Grass Imaging Award).

Within our group, Dr. Carmen Paradas is leading a traslational research line focused on the description of new muscular dys- trophy phenotypes and identification of the responsible genes. In 2013 she has been working as a postdoc (funded by a BAE fellowship from ISCIII) at the laboratory of Dr. Michio Hirano and Dr. Salvatore DiMauro in Columbia University Medical Center. There, she has used whole exome sequencing (WES) to identify gene mutations linked to several myopathies: 1) mutations at the TK2 gene responsible for a type of mitochondrial myopathy, 2) mutations at the GBE1 gene responsible for a new clincal form of adult polyglucosan body disease with a relapsing-remitting phenotype, and 3) alterations in Notch receptor activation associated to limb-girdle muscular dystrophy. In addition, Dr. Carmen Paradas has been involved in the North American Mito- chondrial Disease Consortium (NAMDC), patient registry and biorepository for the mitochondrial patients in USA and Canada.

Keywords

Synapse, GABA, Neurogenesis, Neuronal ceroid lipofuscinosis, Metabolic myopathies, Muscular dystrophies, Human genetics

Publications

• Saez A, Rivas E, Montero-Sanchez A, Paradas C, Acha B, Pascual A, et al. Quantifiable diagnosis of muscular dystrophies and neurogenic atrophies through network analysis. BMC medicine. 2013 Mar 20;11:77. • Nieto-Gonzalez JL, Jensen K. BDNF Depresses Excitability of Parvalbumin-Positive Interneurons through an M-Like Current in Rat Dentate Gyrus. PloS one. 2013;8(6):e67318. • Paradas C, Akman HO, Ionete C, Lau H, Riskind PN, Jones DE, et al. Branching Enzyme Deficiency: Expanding the Clinical Spectrum. JAMA neurology. 2013 Nov 18. • Paradas C, Camano P, Otaegui D, Oz O, Emmanuele V, DiMauro S, et al. Longitudinal clinical follow-up of a large family with the R357P Twinkle mutation. JAMA neurology. 2013 Nov;70(11):1425-8. • Paradas C, Gutierrez Rios P, Rivas E, Carbonell P, Hirano M, Dimauro S. TK2 mutation presenting as indolent myopathy. Neurology. 2013 Jan 29;80(5):504-6. • Bai B, Hales CM, Chen PC, Gozal Y, Dammer EB, Fritz JJ, et al. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease. Proceedings of the National Academy of Sciences of the United States of America. 2013 Oct 8;110(41):16562-7. • Kastl L, Sasse D, Wulf V, Hartmann R, Mircheski J, Ranke C, et al. Multiple Internalization Pathways of Polyelectrolyte Multilayer Capsules into Mammalian Cells. ACS nano. 2013 Jul 16;7(8):6605-18.

208208 Rafael Fernández Chacón Group Rafael Fernández Chacón Group 209

Research Projects

• Code: 10/02410 Other CIBER’s collaboration: No Title: Descripción de un nuevo fenotipo de distrofia mus- Type: National cular de cinturas autosómica recesiva e identificación del Funding Agency: Ministerio de Ciencia e Innovación gen responsable mediante análisis genómico de ultima ge- Funding: 250.000 € neración Duration: 2011-2013 Principal Investigator: Rafael Fernández Chacón CIBERNED’s collaboration: Yes • Code: 2011/0537 CIBERNED’s groups: G 606; G 603; G 504 Title: Translational Research, Experimental Medicine and Other CIBER’s collaboration: No Therapeutics on Charcot-Marie-Tooth Disease. Proyecto Type: National colaborativo Funding Agency: Fondos de Investigación Sanitaria (FIS) Principal Investigator: Francesc Palau Funding: 106.479 € CIBERNED’s collaboration: Yes Duration: 2011-2013 CIBERNED’s groups: G 606; G 605 Other CIBER’s collaboration: Yes (CIBERER) • Code: BFU2010-15713 Type: International Title: Función molecular de Cysteine String Protein-a en si- Funding Agency: International Rare Diseases Research napsis GABAérgicas en neuronas hipocampales de ratón en Consortium (IRDiRC) / Carlos III cultivo, in situ e in vivo y en neuronas humanas en cultivo Funding: 300.000 € Principal Investigator: Rafael Fernández Chacón Duration: 2013 CIBERNED’s collaboration: No CIBERNED’s groups: Group Isabel Illa Sendra

Hospital de la Santa Creu i Sant Pau

3 Neurología 4º Piso. Edificio Nuevo Sant Antoni Maria Claret, 167 Program 08025 Barcelona (Spain) Tel: +34 935 565 986 Fax: +34 935 565 602 [email protected]

Isabel Illa Sendra Aida Alejaldre Monforte Principal Investigator, Full Professor, CIBERNED PhD student Eduardo Gallardo Vigo Alba Ramos Fransi Researcher Institut de Recerca Sant Pau PhD student Ricardo Rojas García Anna Pastoret Calderó Researcher Neurology Institut de Recerca Sant Pau ”Jain Foundation” Jordi Díaz Manera Postdoctoral Fellow Researcher Neurology Josefa Araque Palacios Gisela Nogales Gadea CIBERNED Technician Sara Borrel FIS Postdoctoral Fellow Esther Ortiz Losada Barbara Flix Ordóñez Institut de Recerca Sant Pau Technician Isabel Gemio Foundation PhD student Miquel Navas Madroñal Xavier Suárez Calvet FIS Intrasalud Technician FIS PhD student Sonia Segovia Simón Luis Querol Gutiérrez Nurse, CIBERNED Project manager Post MIR FIS Rio Hortega Noemí de Luna Eugenia Martínez Hernández CIBERNED Researcher FIS PhD student

210 Isabel Illa Sendra Group 211

Summary

• Immune-mediated neuromuscular diseases Immunopathogenesis studies with characterization of autoantibodies to new targets and their clinical correlation. Its use as biomarkers. Role of autoantibodies in the immunopathogenesis of CIDP and MG. Role of innate immunity in Guillain-Barre Syndrome, Myasthenia, Inflammatory myopathies. Analysis of the effect of new immuno-modulating therapies on functional aspects of immune system cells (response to ligands, production of antibodies, etc.) and clinical response.

• Muscular dystrophy, dysferlinopathy and distal myopathies Clinical characterization, new diagnostic tests for dysferlinopathy and distal myopathies, and study of molecular pathways involved in differentiation and molecular and functional interactions of dysferlin.

• Amyotrophic lateral sclerosis (ALS) Epidemiology Genetic studies

• New therapies in neuromuscular diseases Therapy with new biological agents in subgroups of immune mediated NMD. Bone marrow transplantation in dysferlinopathy (animal model). Advanced therapy with mesoangioblasts. Evaluate the effects of treatment with vitamin D in carriers of a DYSF gene mutation.

• Challenges Advance knowledge of the immunological mechanisms involved in the pathogenesis of immune-mediated neuromuscular diseases. Role of Innate immunity. Search for new antigens using new tissue sources. Development of diagnostic tests using the new antigens. Finding new and more specific immune therapies. Analysis of the natural history of dysferlinopathies Implement new diagnostic methods for limb girdle muscular dystrophies (LGMDs) and distal myopathies. Discovery of new genes causing limb girdle muscular dystrophy. Gain knowledge on the pathogenetic mechanisms involved in muscular dystrophy resulting from a dysferlin deficiency. Set up of a clean room to manipulate cells for treatment in humans. Serum and skeletal muscle miRNA profiling of limb girdle muscular dystrophies. Description of skeletal muscle MRI patterns specific for different LGMDs.

Keywords

Immune neuromuscular diseases (CIDP, MMN, MG, IM), Pathogenesis of Muscular dystrophies, Biological and cell therapies for neuromuscular diseases, ALS

Publications

• Vanhoutte EK, Faber CG, Merkies IS; PeriNomS study group. 196th ENMC international workshop: Outcome measures in inflammatory peripheral neuropathies 8-10 February 2013, Naarden, The Netherlands. Neuromuscular disorders. 2013 Nov;23(11):924-33. • Flix B, de la Torre C, Castillo J, Casal C, Illa I, Gallardo E. Dysferlin interacts with calsequestrin-1, myomesin-2 and dynein in human skeletal muscle. The international journal of biochemistry & cell biology. 2013 Aug;45(8):1927-38. • Gurgel-Giannetti J, Nogales-Gadea G, van der Linden H Jr, Bellard TM, Brasileiro Filho G, Giannetti AV, et al. Clinical and molecular characterization of McArdle’s disease in Brazilian patients. Neuromolecular medicine. 2013 Sep;15(3):470-5. • de Morree A, Flix B, Bagaric I, Wang J, van den Boogaard M, Grand Moursel L, et al. Dysferlin regulates cell adhesion in human monocytes. The Journal of biological chemistry. 2013 May 17;288(20):14147-57. • Sarkozy A, Hicks D, Hudson J, Laval SH, Barresi R, Hilton-Jones D, et al. ANO5 gene analysis in a large cohort of patients with anoctaminopathy: confirmation of male prevalence and high occurrence of the common exon 5 gene mutation. Human mutation. 2013 Aug;34(8):1111-8. • Querol L, Illa I. Myasthenia gravis and the neuromuscular junction. Current opinion in neurology. 2013 Oct;26(5):459-65. • Querol L, Rojas-Garcia R, Casasnovas C, Sedano MJ, Munoz-Blanco JL, Alberti MA, et al. Long-term outcome in chronic inflammatory demyelinating polyneuropathy patients treated with intravenous immunoglobulin: A retrospective study. Muscle & nerve. 2013 Dec;48(6):870-6. • Güell MR, Anton A, Rojas-Garcia R, Puy C, Pradas J; en representacion de todo el Group interdisciplinario. Comprehensive Care of Amyotrophic Lateral Sclerosis Patients: A Care Model. Archivos de bronconeumologia. 2013 Dec;49(12):529-533. • Rojas-Garcia R, Gallardo E, Illa I. Paraproteinemic neuropathies. Presse medicale (Paris, France: 1983). 2013 Jun;42(6 Pt 2):e225-34. • Dols-Icardo O, Garcia-Redondo A, Rojas-Garcia R, Sanchez-Valle R, Noguera A, Gomez-Tortosa E, et al. Characterization of the repeat expansion size in C9orf72 in amyotrophic lateral sclerosis and frontotemporal dementia. Human molecular genetics. 2013 Oct 8. • Flix B, Suarez-Calvet X, Diaz-Manera J, Santos-Nogueira E, Mancuso R, Barquinero J, et al. Bone marrow transplantation in dysferlin-deficient mice results in a mild functional improvement. Stem cells and development. 2013 Nov 1;22(21):2885-94. • Berciano J, Gallardo E. Proximal lower-limb weakness in charcot-marie-tooth disease. JAMA neurology. 2013 Dec 1;70(12):1587. • Berciano J, Martinez-Agüeros JA, Gallardo E, Martinez-Martinez MA, Infante J, Garcia A, et al. Hereditary neuropathy with liability to pressure palsy: fulminant radicular dysfunction during anterolateral lumbar interbody fusion. Journal of neurology. 2013 Sep;260(9):2411-3.

Research Projects

• Code: U01 NS042685-01A2 CIBERNED’s collaboration: No Title: A Multi-Center, Single-Blind, Randomized Study Com- CIBERNED’s groups: paring Thymectomy to No Thymectomy in Non-Thymoma- Other CIBER’s collaboration: No tous Myasthenia Gravis (MG) Patients Receiving Prednisone Type: International Principal Investigator: Newsom Davis Funding Agency: Jain Foundation CIBERNED’s collaboration: No Funding: 216.000 € CIBERNED’s groups: Duration: 2011-2016 Other CIBER’s collaboration: No Type: International • Code: Dysferlinopathies 2012-13 Funding Agency: NIH. National Institutes of Health. USA Title: Combined cell transplantation as a therapeutic ap- Funding: 38.700 € proach for dysferlinopathies and transversal study to eval- Duration: 2009-2013 uate the frequency of carriers of dysferlinopathy in Cauca- sian population using a monocyte’s test to detect dysferlin • Code: PI 12/02991 expression Title: Búsqueda de biomarcadores de distrofias musculares Principal Investigator: Eduard Gallardo con debilidad de cinturas CIBERNED’s collaboration: No Principal Investigator: Eduard Gallardo CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: International Other CIBER’s collaboration: No Funding Agency: Jain Foundation: Orchestrating a cure for Type: National dysferlinopathies Funding Agency: Instituto de Salud Carlos III Funding: 91.361 € Funding: 86.515 € Duration: 2012-2013 Duration: 2013-2015 • Code: PI2010/11 • Code: Dysferlinopathy 2012 Title: Consorcio para generar una base de datos común Title: Clinical Outcome Study for Dysferlinopathy cuya finalidad es implementar la investigación clínica y bá- Principal Investigator: Isabel Illa Sendra sica en enfermedades neuromusculares

212212 Isabel Illa Sendra Group Isabel Illa Sendra Group 213

Principal Investigator: Isabel Illa Sendra Principal Investigator: Isabel Illa Sendra CIBERNED’s collaboration: Yes CIBERNED’s collaboration: No CIBERNED’s groups: G 603; G 601; G 605 CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Intramurales Type: National Funding Agency: CIBERNED Funding Agency: Generalitat de Catalunya Funding: 429.000 € Funding: 43.680 € Duration: 2010-2013 Duration: 2010-2014

• Code: FIS 09/1964 INTRASALUD • Code: POMPE-2013 Title: Estudios inmunológicos relevantes para el desarrollo Title: Study of the natural history of adult onset POMPE dis- de estrategias terapéuticas en enfermedades neuromuscu- ease. A prospective study of clinical and imaging predictors lares of POMPE disease course Principal Investigator: Isabel Illa Sendra Principal Investigator: Isabel Illa Sendra CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: National Funding Agency: FIS Funding Agency: Institut de Recerca de l’hospital de la San- Funding: 604.395 € ta Creu i Sant Pau-Genzyme Duration: 2010-2013 Funding: 259.688 € Duration: 2013 • Code: IGOS 2012 Title: International GBS Outcome Study (IGOS) • Code: 10/02410 Principal Investigator: Bart C. Jacobs Title: Descripción de un nuevo fenotipo de distrofia mus- CIBERNED’s collaboration: No cular de cinturas autosómica recesiva e identificación del CIBERNED’s groups: gen responsable mediante análisis genómico de última ge- Other CIBER’s collaboration: No neración Type: International Principal Investigator: Rafael Fernández Chacón Funding Agency: Depts. Neurology and Immunology. Eras- CIBERNED’s collaboration: Yes mus MC. CIBERNED’s groups: G 606; G 603; G 504 Funding: 0 € Other CIBER’s collaboration: No Duration: 2012-2013 Type: National Funding Agency: Fondos de Investigacion Sanitaria (FIS) • Code: 2009 SGR1004 Funding: 106.479 € Title: Research on neuromuscular diseases. Grup de recer- Duration: 2011-2013 ca consolidat de la Generalitat

PhD Dissertations

• Author: Jordi Díaz Manera Date: 26 July 2013 Title: Disferlinopatía: nuevos aspectos diagnósticos y tera- Supervisor: Isabel Illa Sendra péuticos Date: 24 May 2013 • Author: Barbara Flix Ordóñez Supervisor: Isabel Illa Sendra Title: Estudios funcionales de la disferlina y aproximaciones terapéuticas en un modelo murino de miopatía por déficit • Author: Luis Antonio Querol de disferlina Title: Nuevas reactividades antigénicas en neuropatías in- Date: 21 March 2013 munomediadas Supervisor: Eduardo Gallardo Vigo Group Adolfo López de Munain Arregui

Instituto de Investigación Biodonostia

3 Paseo Dr. Beguiristain, s/n 20014 San Sebastián (Spain) Leyre Curto Arzac Program Tel.: +34 943 006 295 [email protected]

Adolfo López de Munain Arregui Juan Bautista Espinal Valencia Fermín Moreno Izco Principal Investigator Researcher Researcher Ana Aiastui Pujana Roberto Fernández Torrón Neia Naldaiz Gastezi Researcher Researcher R e s e a r c h e r Garazi Aldanondo Aristizabal Belén Gago Calderón Nahikari Pastoriza Polo PhD student Researcher R e s e a r c h e r Amaia Azpitarte Ondarra Federico García Bragado Juanjo Poza Aldea Researcher Researcher Researcher Myriam Barandiarán Amillano Patricia García Parra Patricia de la Riva Juez Researcher Researcher Researcher Alberto Bergareche Yarza María Goicoechea Bianchi María Cruz Rodríguez Oroz Researcher Researcher Researcher Vanessa Blázquez García Ana Gorostidi Pagola Javier Ruiz Martínez Technician Researcher Researcher Cristina Caballero Martínez Ohiane Jaka Irizar Amets Sáenz Peña Researcher Researcher Researcher M. Pilar Camaño González Jaione Lacalle Prieto Ernesto Sanz Arigita Researcher R e s e a r c h e r Researcher Leire Casas Fraile José Félix Martí Masso Andone Sistiaga Berrondo Researcher Researcher Researcher Leyre Curto Arzac Pablo Martínez-Lage Alvárez Iván Toral Ojeda A d m i n i s t r a ti v e a s s i s t a n t Researcher R e s e a r c h e r Manuel Delgado Alvarado Alba Mateos Ayerdi Miguel Ángel Urtasun Ocariz Researcher PhD student Researcher Ainara Estanga Alustiza Aroa Méndez Zabalo Ainara Vallejo Illarramendi Researcher R e s e a r c h e r Researcher Elisabeth Mondragón Rezola Olaia Zuriarráin Bergara Researcher Researcher Garazi Aldanondo Aristizabal PhD student

214 Adolfo López de Munain Arregui Group 215

Summary

The dementia research line is focused on frontotemporal dementias. In this field, the group participates in the DEGESCO network, which is composed, among others, by CIBERNED members. The group has already published a collaborative work and maintains partnerships with Alfredo Martinez from the University of Bonn, as well as with Rosa Rademackers for the study of frontotemporal dementias caused by mutations in progranulin. The group also maintains collaboration with Prof. Angeles Martin Requero’s group (CSIC), to study the metabolic pathways involved in progranulin deficiency using a lymphoid cellular model.

Within the Parkinson’s disease line, the group led by the IKERBASQUE Research Prof. María Cruz Rodríguez Oroz, works on the identification of early disease markers in individuals carrying mutations in LRRK2, as well as on impulse control disorders using animal models. This research line holds important collaborations, such as the international projects MEFOPA and Michael J Fox Foundation. In addition, the group holds national projects and has established intramural collaborations with the group of Prof. Fuentes (University of Extremadura) for the study of autophagy and with Dr. Sanchez Pernaute (INBIOMED) to study cytoskeletal proteins in cellular disease models.

In the field of neuromuscular diseases, the group studies molecular pathways and therapeutic strategies in relation with LGMD2A muscular dystrophy. In particular we are analyzing the influence of Calpain 3 protein during myogenesis by using myogenic cellular models obtained by iPS differentiation or by transfection of MyoD into skin fibroblasts. The group collaborates with Angel Raya’s group (Center for Genomic Regulation, Barcelona) for the generation of stem cells by reprogramming, with the group of Pura Muñoz for myogenic differentiation of these cells and more recently with Rita Perlingueiro (Minnesota University) also to achieve these aims. In addition, we are collaborating with Francina Munell from Vall d’ Hebron (CIBERER) to study the use of polymeric extracellular matrices in cellular models of Duchenne muscular dystrophy and with Ruben Artero’s group at the University of Valencia to study miRNAs as biomarkers of muscular dystrophies.

In 2013, Adolfo López de Munain, the group leader, organized the 9th International Congress on Myotonic Dystrophy in San Sebastian, and the 2nd Course for Neurologists, held in Burgos. Both events were partially sponsored by CIBERNED. The Summer Course from the Basque Country University about Parkinson’s disease also took place in San Sebastian, organized by Mari Cruz Rodriguez Oroz.

Keywords

LRRK2, Calpaine, Parkinson Disease, Progranuline, Frontotemporal Dementia, Muscular Dystrophies, Ictus, Autophagy

Publications

• Gago B, Fuxe K, Brene S, Diaz-Cabiale Z, Reina-Sanchez MD, Suarez-Boomgaard D, et al. Early modulation by the dopamine D4 receptor of morphine-induced changes in the opioid peptide systems in the rat caudate putamen. Journal of neuroscience research. 2013 Dec;91(12):1533-40. • Tijero B, Gomez-Esteban JC, Lezcano E, Fernandez-Gonzalez C, Somme J, Llorens V, et al. Cardiac sympathetic denervation in symptomatic and asymptomatic carriers of the E46K mutation in the α synuclein gene. Parkinsonism & related disorders. 2013 Jan;19(1):95-100. • Miquel M, Spampinato U, Latxague C, Aviles-Olmos I, Bader B, Bertram K, et al. Short and long term outcome of bilateral pallidal stimulation in chorea-acanthocytosis. PloS one. 2013;8(11):e79241. • Alvarez-Erviti L, Seow Y, Schapira AH, Rodriguez-Oroz MC, Obeso JA, Cooper JM. Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson’s disease. Cell death & disease. 2013 Mar 14;4:e545. • Ruiz-Martinez J, de la Riva P, Rodriguez-Oroz MC, Rezola EM, Bergareche A, Gorostidi A, et al. Prevalence of cancer in Parkinson’s disease related to R1441G and G2019S mutations in LRRK2. Movement disorders. 2013 Dec 19. • Marti-Masso JF, Bergareche A, Makarov V, Ruiz-Martinez J, Gorostidi A, de Munain AL, et al. The ACMSD gene, involved in tryptophan metabolism, is mutated in a family with cortical myoclonus, epilepsy, and parkinsonism. Journal of molecular medicine (Berlin, Germany). 2013 Dec;91(12):1399-406. • Irwin DJ, McMillan CT, Brettschneider J, Libon DJ, Powers J, Rascovsky K, et al. Cognitive decline and reduced survival in C9orf72 expansion frontotemporal degeneration and amyotrophic lateral sclerosis. Journal of neurology, neurosurgery, and psychiatry. 2013 Feb;84(2):163-9. • Blazquez L, Aiastui A, Goicoechea M, Martins de Araujo M, Avril A, Beley C, et al. In vitro correction of a pseudoexon-generating deep intronic mutation in LGMD2A by antisense oligonucleotides and modified small nuclear RNAs. Human mutation. 2013 Oct;34(10):1387-95. • Rodriguez-Blazquez C, Rojo-Abuin JM, Alvarez-Sanchez M, Arakaki T, Bergareche-Yarza A, Chade A, et al. The MDS-UPDRS Part II (motor experiences of daily living) resulted useful for assessment of disability in Parkinson’s disease. Parkinsonism & related disorders. 2013 Oct;19(10):889-93. • Martinez-Martin P, Rodriguez-Blazquez C, Alvarez-Sanchez M, Arakaki T, Bergareche-Yarza A, Chade A, et al. Expanded and independent validation of the Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS). Journal of neurology. 2013 Jan;260(1):228-36. • Martinez-Martin P, Chaudhuri KR, Rojo-Abuin JM, Rodriguez-Blazquez C, Alvarez-Sanchez M, Arakaki T, et al. Assessing the non-motor symptoms of Parkinson’s disease: MDS-UPDRS and NMS Scale. European journal of neurology. 2013 Apr 22. • Toledo JB, Lopez-Azcarate J, Garcia-Garcia D, Guridi J, Valencia M, Artieda J, et al. High beta activity in the subthalamic nucleus and freezing of gait in Parkinson’s disease. Neurobiology of disease. 2013 Dec 17;64C 60-5. • Lopez de Maturana R, Aguila JC, Sousa A, Vazquez N, Del Rio P, Aiastui A, et al. Leucine-rich repeat kinase 2 modulates cyclooxygenase 2 and the inflammatory response in idiopathic and genetic Parkinson’s disease. Neurobiol Aging. 2013 Nov 22. • Alquezar C, Esteras N, de la Encarnacion A, Alzualde A, Moreno F, Lopez de Munain A, et al. PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers. Neurobiol Aging. 2013 Oct 16. • Delgado-Alvarado M, Gomez-Roman J, Sanchez-Salmon E, Rodriguez-Rodriguez E, Polo JM, Garcia-Castano A, et al. Nonanaplastic Pleomorphic Xanthoastrocytoma with Meningeal Dissemination Presenting with Bilateral Visual Loss. Journal of neuroimaging. 2013 May 23. • McMillan CT, Avants B, Irwin DJ, Toledo JB, Wolk DA, Van Deerlin VM, et al. Can MRI screen for CSF biomarkers in neurodegenerative disease?. Neurology. 2013 Jan 8;80 (2):132-8. • Ortega-Cubero S, Clavero P, Irurzun C, Gonzalez-Redondo R, Guridi J, Obeso JA, et al. Effect of deep brain stimulation of the subthalamic nucleus on non-motor fluctuations in Parkinson’s disease: two-years’ follow-up. Parkinsonism & related disorders. 2013 May;19(5):543-7. • Alegre M, Lopez-Azcarate J, Obeso I, Wilkinson L, Rodriguez-Oroz MC, Valencia M, et al. The subthalamic nucleus is involved in successful inhibition in the stop-signal task: A local field potential study in Parkinson’s disease. Experimental neurology. 2013 Jan;239:1-12. • Manzoni C, Mamais A, Dihanich S, McGoldrick P, Devine MJ, Zerle J, et al. Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation. Biochemical and biophysical research communications. 2013 Nov 29;441(4):862-6. • Garcia-Parra P, Maroto M, Cavaliere F, Naldaiz-Gastesi N, Alava JI, Garcia AG, et al. A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells. BMC neuroscience. 2013 Apr 18;14:48. • Riancho-Zarrabeitia L, Delgado-Alvarado M, Riancho J, Oterino A, Sedano MJ, Rueda-Gotor J, et al. Anti-TNF-α therapy in the management of severe neurosarcoidosis: a report of five cases from a single centre and literature review. Clinical and experimental rheumatology. 2013 Dec 9. • Obeso I, Wilkinson L, Rodriguez-Oroz MC, Obeso JA, Jahanshahi M. Bilateral stimulation of the subthalamic nucleus has differential effects on reactive and proactive inhibition and conflict-induced slowing in Parkinson’s disease. Experimental brain research. Experimentelle Hirnforschung. Experimentation cerebrale. 2013 May;226(3):451-62. • Gatto EM, Parisi V, Converso DP, Poderoso JJ, Carreras MC, Marti-Masso JF, et al. The LRRK2 G2019S mutation in a series of Argentinean patients with Parkinson’s disease: clinical and demographic characteristics. Neuroscience letters. 2013 Mar 14;537:1-5. • de la Riva P, Martinez-Zabaleta MT, Arruti M, Diez-Gonzalez N, Mondragon-Rezola E, Gonzalo-Yubero N, et al. [Acute cerebellitis following Epstein-Barr virus infection in two young women]. Revista de neurologia. 2013 Feb 16;56(4):252-3. • Gago B, Marin C, Rodriguez-Oroz MC, Obeso JA. l-dopa-induced dyskinesias in unilateral 6-hydroxydopamine-lesioned rats are not modified by excitotoxic lesion of the entopeduncular nucleus and substantia nigra pars reticulata. Synapse (New York, NY). 2013 Jul;67(7):407-14. • Tercjak A, Bergareche A, Caballero C, Tunon T, Linazasoro G. Lewy Bodies under Atomic Force Microscope. Ultrastruct Pathol. 2013 Oct 17. • Garcia-Parra P, Naldaiz-Gastesi N, Maroto M, Padin JF, Goicoechea M, Aiastui A, et al. Murine Muscle Engineered from Dermal Precursors: An In Vitro Model for Skeletal Muscle Generation, Degeneration, and Fatty Infiltration. Tissue engineering. Part C, Methods. 2013 Jun 22;20(1):28-41.

216216 Adolfo López de Munain Arregui Group Adolfo López de Munain Arregui Group 217

• Ruiz A, Dols-Icardo O, Bullido MJ, Pastor P, Rodriguez-Rodriguez E, Lopez de Munain A, et al. Assessing the role of the TREM2 p.R47H variant as a risk factor for Alzheimer’s disease and frontotemporal dementia. Neurobiol Aging. 2013 Sep 13. • Marin C, Bonastre M, Mengod G, Cortes R, Rodriguez-Oroz MC, Obeso JA. Subthalamic 6-OHDA-induced lesion attenuates levodopa-induced dyskinesias in the rat model of Parkinson’s disease. Experimental neurology. 2013 Dec;250:304-12. • Riancho J, Infante J, Orizaola P, Mateo I, Viadero R, Delgado-Alvarado M, et al. [Reversible cerebral vasoconstriction and acute encephalopathy as a presentation form of Guillain-Barre syndrome]. Revista clinica espanola. 2013 Apr;213(3):e23-6. • Delgado-Alvarado M, Riancho J, Riancho-Zarrabeitia L, Sedano MJ, Polo JM, Berciano J. [Unilateral total ophthalmoplegia without visual loss as the presenting form of a pituitary apoplexy]. Revista clinica espanola. 2013 Oct;213(7):e67-70. • Riancho J, Delgado-Alvarado M, Valero C, Echevarria S, Farinas MC. Clinical spectrum of peripheral facial paralysis in HIV- infected patients according to HIV status. International journal of STD & AIDS. 2013 Jan;24(1):39-41. • Riancho J, Delgado-Alvarado M, Sedano MJ, Polo JM, Berciano J. Herpes simplex encephalitis: clinical presentation, neurological sequelae and new prognostic factors. Ten years of experience. Neurological sciences. 2013 Oct;34(10):1879-81. • Delgado-Alvarado M, Sedano MJ, Gonzalez-Quintanilla V, de Lucas EM, Polo JM, Berciano J. Progressive multifocal leukoencephalopathy and idiopathic CD4 lymphocytopenia. Journal of the neurological sciences. 2013 Apr 15;327(1-2):75-9. • Lee SE, Tartaglia MC, Yener G, Genç S, Seeley WW, Sanchez-Juan P, et al. Neurodegenerative disease phenotypes in carriers of MAPT p.A152T, a risk factor for frontotemporal dementia spectrum disorders and Alzheimer disease. Alzheimer disease and associated disorders. 2013 Oct-Dec;27(4):302-9. • Gonzalez-Aramburu I, Sanchez-Juan P, Jesus S, Gorostidi A, Fernandez-Juan E, Carrillo F, et al. Genetic variability related to serum uric acid concentration and risk of Parkinson’s disease. Movement disorders. 2013 Oct;28(12):1737-40.

Research Projects • Code: PI10/02714 Funding Agency: Instituto de Salud Carlos III Title: Caracterización clínica de los endofenotipos del tem- Funding: 376.915 € blor esencial familiar y esporádico Duration: 2010-2013 Principal Investigator: Adolfo López de Munain CIBERNED’s collaboration: No • Code: SA- 22012/00129 CIBERNED’s groups: Title: Efecto terapéutico de compuestos moduladores del Other CIBER’s collaboration: No receptor de rianodina Type: National Principal Investigator: Ainara Vallejo Funding Agency: Fondos de Investigación Sanitaria FIS CIBERNED’s collaboration: No Funding: 160.500 € CIBERNED’s groups: Duration: 2010-2013 Other CIBER’s collaboration: No Type: CCAA • Code: SAIO12-PE12BN008 Funding Agency: Gobierno Vasco. Departamento de Indus- Title: Desarrollo de modelos celulares avanzados de enfer- tria. Saiotek medades neuromusculares para aplicaciones terapéuticas Funding: 42.852 € Principal Investigator: Adolfo López de Munain Duration: 2013 CIBERNED’s collaboration: No CIBERNED’s groups: • Code: GV2012MC Other CIBER’s collaboration: No Title: Estudio de Biomarcadores en la fase temprana de la Type: CCAA demencia en la enfermedad de Parkinson Funding Agency: Gobierno Vasco, Departamento de Indus- Principal Investigator: Mari Cruz Rodriguez Oroz tria, Turismo. Saiotek CIBERNED’s collaboration: No Funding: 34.016 € CIBERNED’s groups: Duration: 2013 Other CIBER’s collaboration: No Type: CCAA • Code: PS09/00660 Funding Agency: Gobierno Vasco Title: Desarrollo preclínico de una estrategia de terapia Funding: 52.250 € combinada celular y génica, en modelos celulares y anima- Duration: 2013-2015 les para la distrofia muscular de cinturas tipo 2A Principal Investigator: Adolfo López de Munain • Code: PI10/00848 CIBERNED’s collaboration: No Title: Evaluacion preclínica de una estrategia terapéutica en CIBERNED’s groups: la Distrofia de Cinturas LGMD2A mediante silenciamiento Other CIBER’s collaboration: No génico de diversos sistemas celulares Type: National Principal Investigator: Adolfo López de Munain CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: ISCIII Type: National Funding: 254.265 € Funding Agency: Fondos de Investigación Sanitaria FIS Duration: 2013 Funding: 128.000 € Duration: 2010-2013 • Code: MEFOPA Title: MEFOPA: Mendelian forms of Parkinson’s disease • Code: MJF Principal Investigator: Adolfo López de Munain Title: Expansion of the ASAP study (ASymptomatic LRRK2 CIBERNED’s collaboration: No mutation carriers and Assessment of factors influencing CIBERNED’s groups: LRRK2-related PD expression study Other CIBER’s collaboration: No Principal Investigator: José Félix Martí Masso Type: International CIBERNED’s collaboration: Yes Funding Agency: Ayuda Europea de Enfermedades Raras CIBERNED’s groups: G 609; G 207 Funding: 115.000 € Other CIBER’s collaboration: Yes Duration: 2010-2013 Type: International Funding Agency: Michael J Fox • Code: PI11/01499 Funding: 180.000 € Title: Preclinical characterization of ryanodine receptor sta- Duration: 2011-2013 bilizers in patients with muscular dystrophies Principal Investigator: Ainara Vallejo • Code: PI10/02570 CIBERNED’s collaboration: No Title: Factores ambientales y hábitos que influyen en la CIBERNED’s groups: penetrancia y expresividad de la enfermedad de Parkinson Other CIBER’s collaboration: No asociada a mutaciones en LRRK2/dadarina Type: National Principal Investigator: Adolfo López de Munain Funding Agency: ISCIII CIBERNED’s collaboration: No Funding: 83.940 € CIBERNED’s groups: Duration: 2013 Other CIBER’s collaboration: No Type: National • Code: PI11/03028 Funding Agency: Fondos de Investigación Sanitaria FIS Title: DEMTEST: Biomarker based diagnosis of rapid pro- Funding: 25.000 € gressive dementias Duration: 2010-2013 Principal Investigator: Pascual Sánchez CIBERNED’s collaboration: Yes • Code: PI11/02109 CIBERNED’s groups: G 114; G 601; G 503; G 609 Title: Mecanismos fisiopatológicos del trastorno del control Other CIBER’s collaboration: No de impulsos en la enfermedad de Parkinson: estudio en pa- Type: International cientes y desarrollo de un modelo animal Funding Agency: Instituto de Salud Carlos III - Comunidad Principal Investigator: Mª Cruz Rodriguez Oroz Económica Europea CIBERNED’s collaboration: No Funding: 10.000 € CIBERNED’s groups: Duration: 2012-2014

218218 Adolfo López de Munain Arregui Group Group Pura Muñoz Cánoves

Cell Biology Group

ICREA and Pompeu Fabra University (UPF) 3 Department of Experimental and Life Sciences (DCEXS)

(Spain) Program Tel.: +34 933 160 891 Fax: +34 933 160 901 [email protected]

Pura Muñoz Cánoves Laura Ortet Principal Investigator, ICREA Research Professor Postdoctoral Fellow Antonio L. Serrano Sánchez Patrizia Pessina Senior Researcher Postdoctoral Fellow Eusebio Perdiguero Santamaría Marina Raya CIBERNED Senior Researcher Labmanager Laura García Prat Vanessa Ruíz-Bonilla PhD student CIBERNED Postdoctoral Fellow Susana Gutarra Díaz Mónica Zamora Villafaina Technician Postdoctoral Fellow Mercè Jardí Ripoll Jessica Segales Dalmau Technician Postdoctoral Fellow (from January 2013) Yacine Kharraz Diana Sobral Mesquita Postdoctoral Fellow PhD student (from September 2013) Vera Lukesova Begoña Ampudia Carrasco Technician Animal Facility Technician (from September 2013)

219 Summary

Regulation of skeletal myogenesis by p38 MAPK signaling during myogenesis, at the different stages of the in vivo regeneration process: activation, proliferation, differentiation and return to quiescence (self-renewal).

New roles for the p38/MKP-1 balance in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell–dependent tissue repair. This novel regulatory mechanism may have implications in chronic inflammatory degenerative diseases.

Matrix-associated fibrin-driven inflammation coupled to the αMβ2 leukocyte integrin receptor modulates muscle regeneration and/or fibrosis. We found that fibrin accumulates in dystrophic muscles of DMD patients and mdx mice and disruption of fibrin- αMβ2 interactions attenuated dystrophy progression.

Identification of the extracellular uPA/PAI-1 proteolytic balance as an important regulator of miR–21 biogenesis, controlling age-associated muscle fibrosis and dystrophy progression. Fibrosis in aged mdx mice could be reversed by miR-21- or uPA- selective interference.

Regulation of muscle growth by IL-6. Our results have provided novel functional evidence for the critical involvement of the cytokine IL-6 in mediating hypertrophic muscle growth by controlling muscle stem cell (satellite cell) incorporation to growing myofibers.

Geriatric muscle stem cells switch reversible quiescence into senescence, due to depression of p16INK4a. We demonstrated that p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. This may provide a basis for stem cell rejuvenation in sarcopenic muscles.

International collaborations and external funding

• Collaboration within the European Consortium coordinated by Dr. David Sassoon, aimed to Optimize regeneration in skeletal muscle by stimulating stem cell functions: o Dr. David Sassoon (Institut Nationale de la Santé et de la Recherche Medicale, INSERM, France) o Dr. Benedicte Chazaud (Institut Nationale de la Santé et de la Recherche Medicale, INSERM, France) o Dr. Silvia Brunelli (Fondazione Centro san Raffaele del Monte Tabor, HSR).

• Collaboration with Dr. Ana Cuenda (CNB, Madrid, Spain): Deciphering the function of p38 isoforms in muscle regeneration. Role p38γ in muscle stem cell and macrophage homeostasis during muscle regeneration. Association Française contre les Myopathies (AFM). (2012-2014, 2 years). The goals of this AFM grant are complementary to those of the present Project • Collaboration with: o Dr. Michael Rudnicki, (Ottawa Hospital Research Institute, Department Regenerative Medicine Program, Canada) o Dr. Fabien Le Grand (Institut Cochin, Inserm, France) o Dr. Gillian S. Butler-Browne (Institut de Myologie, Inserm, France).

During the preparation of the E-Rare-2 Call for Proposals 2013 for “European Research Projects on Rare Diseases” presented project aimed to study the role of Wnt7 as a factor to combat Duchenne Muscular Dystrophy.

Merits: Our group has participated in a project selected/funded by the E-Rare-2 Call for Proposals 2013 for “European Research Projects on Rare Diseases”.

Keywords

Skeletal muscle, Regeneration, Inflammation, Fibrosis, Muscle stem cells, Sarcopenia, Aging

220220 Pura Muñoz Cánoves Group Pura Muñoz Cánoves Group 221

Publications

• Sandri M, Barberi L, Bijlsma AY, Blaauw B, Dyar KA, Milan G, et al. Signalling pathways regulating muscle mass in ageing skeletal muscle: the role of the IGF1-Akt-mTOR-FoxO pathway. Biogerontology. 2013 Jun;14(3):303-23. • Garcia-Prat L, Sousa-Victor P, Munoz-Canoves P. Functional dysregulation of stem cells during aging: a focus on skeletal muscle stem cells. The FEBS journal. 2013 Sep;280(17):4051-62. • Munoz-Canoves P, Scheele C, Pedersen BK, Serrano AL. Interleukin-6 myokine signaling in skeletal muscle: a double-edged sword? The FEBS journal. 2013 Sep;280(17):4131-48. • Munoz-Canoves P, Michele D. Special issue: myogenesis: introduction. The FEBS journal. 2013 Sep;280(17):3979. • Acuna MJ, Pessina P, Olguin H, Cabrera D, Vio CP, Bader M, et al. Restoration of muscle strength in dystrophic muscle by angiotensin-1-7 through inhibition of TGF-β signalling. Hum Mol Genet. 2014 Mar 1;23(5):1237-49. • Kharraz Y, Guerra J, Mann CJ, Serrano AL, Munoz-Canoves P. Macrophage plasticity and the role of inflammation in skeletal muscle repair. Mediators of inflammation. 2013;2013:491497.

Research Projects

• Code: ENDOSTEM • Code: OPTISTEM Title: ENDOSTEM Optimization of stem cell therapy for dis- Title: OPTISTEM: Optimization of stem cell therapy for dis- ease of epithelia and skeletal muscle through combined ease of epithelia and skeletal muscle through combined basic and applied research basic and applied research Principal Investigator: David Sassoon Principal Investigator: Giulio Cossu CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: International Type: International Funding Agency: UE Funding Agency: European Union (7th FP) Funding: 425.950 € Funding: 410.000 € Duration: 2010-2014 Duration: 2009-2013

• Code: La Marató-TV3 • Code: SAF2012-38547 Title: Novel approaches for degenerative muscular dystro- Title: Regulation and dysregulation of skeletal muscle re- phies therapies generation and growth Principal Investigator: Pura Muñoz Cánoves Principal Investigator: Pura Muñoz Cánoves CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: Privado Type: National Funding Agency: La Marató-TV3 Funding Agency: MINECO Funding: 120.000 € Funding: 250.000 € Duration: 2013-2015 Duration: 2013-2015 Group Xavier Navarro Acebes

Dept. Cell Biology, Physiology and Immunology

3 Institute of Neurosciences Universitat Autónoma de Barcelona Edif. M, Campus UAB Program 08193 Bellaterra (Spain) Tel.: +34 935 811 966 Fax: +34 +34 935812986 [email protected]

Xavier Navarro Acebes Jordi Badia Casahuja Principal Investigator, Full Professor PhD student Jordi Cuadras Mas Marina Coll Miró Researcher, Emeritus Professor PhD student Esther Udina Bonet Isaac Francos Quijorna Researcher, Associate Professor PhD student Rubén López Vales Francisco González Pérez Researcher, Lecturer PhD student Caty Casas Louzao Renzo Mancuso Researcher, Associate Professor Interim PhD student Jordi Bruna Escuer Elena Redondo Castro Assistant Professor PhD student Stefano Cobianchi Eva Santos Nogueira Postdoctoral Fellow PhD student Jaume del Valle Macià Daniel Santos Rojas Postdoctoral Fellow PhD student Joaquim Hernández Martín Abel Torres Espín Postdoctoral Fellow PhD student Mireia Herrando Grabulosa Jessica Jaramillo Rodríguez Postdoctoral Fellow Technician Albert Alé Miranda Marta Morell Orduña PhD student CIBERNED Technician

222 Xavier Navarro Acebes Group 223

Summary

The Group of Neuroplasticity and Regeneration of the UAB is a multidisciplinary research group working on repair, regeneration and functional recovery after peripheral nerve and spinal cord lesions and in neurodegenerative diseases. The research activities of the Group of Neuroplasticity and Regeneration have focused on the study of physiopathological mechanisms of neural lesions, neuropathic pain and neurodegeneration, and on the application of novel therapeutic strategies for regenerating traumatic and degenerative lesions of the nervous system.

The active research lines include:

• Cellular and molecular mechanisms implicated in degeneration of motoneurons in experimental models. Search for biomar- kers. Neuroprotective strategies based on gene and pharmacological therapy. • Cell therapy by transplantation of mesenchimal stem cells and olfactory glia for the repair of spinal cord injuries and moto- neuron degenerative diseases. • Repair of spinal root avulsion injuries by surgical reimplantation and pharmacological neuroprotection. • Etiopathogenic role of lipid mediators and modulators of the neuroinflammatory response in neurodegeneration induced by central nervous system lesions. • Activity-dependent therapies for enhancing axonal regeneration and functional recovery after peripheral nerve lesions. • Physiopathologic mechanisms in neuropathic pain induced by nerve and spinal cord lesions. Study of the relationship bet- ween neuroinflammation and hyperexcitability. • Study of etiopathogenic mechanisms and potential neuroprotective treatments in peripheral neuropathies induced by dia- betes and by antitumoral agents. • Design and evaluation of neural interfaces for the development of neuroprostheses applied to neurorehabilitation. Study of new intraneural electrodes for the selective stimulation and recording of neural activity.

Keywords

Neurodegeneration, Nerve regeneration, Spinal cord injury, Neuropathic pain, Motoneuron diseases, Peripheral neuropathies, Neural interfaces

Publications

• Gonzalez-Perez F, Udina E, Navarro X. Extracellular matrix components in peripheral nerve regeneration. International review of neurobiology. 2013;108:257-75. • del Valle J, Navarro X. Interfaces with the peripheral nerve for the control of neuroprostheses. International review of neurobiology. 2013;109:63-83. • Auer M, Allodi I, Barham M, Udina E, Neiss WF, Navarro X, et al. C3 exoenzyme lacks effects on peripheral axon regeneration in vivo. Journal of the peripheral nervous system: JPNS. 2013 Mar;18(1):30-6. • de Diego C, Puig S, Navarro X. A sensorimotor stimulation program for rehabilitation of chronic stroke patients. Restorative neurology and neuroscience. 2013 Jan 1;31(4):361-71. • Redondo-Castro E, Garcia-Alias G, Navarro X. Plastic changes in lumbar segments after thoracic spinal cord injuries in adult rats: an integrative view of spinal nociceptive dysfunctions. Restorative neurology and neuroscience. 2013 Jan 1;31(4):411-30. • Torres-Espin A, Hernandez J, Navarro X. Gene expression changes in the injured spinal cord following transplantation of mesenchymal stem cells or olfactory ensheathing cells. PloS one. 2013 Oct 11;8(10):e76141. • Redondo-Castro E, Navarro X. Chronic ibuprofen administration reduces neuropathic pain but does not exert neuroprotection after spinal cord injury in adult rats. Exp Neurol. 2013 Nov 15. • Ale A, Bruna J, Morell M, van de Velde H, Monbaliu J, Navarro X, et al. Treatment with anti-TNF alpha protects against the neuropathy induced by the proteasome inhibitor bortezomib in a mouse model. Exp Neurol. 2013. • Herrando-Grabulosa M, Casas C, Aguilera J. The C-terminal domain of tetanus toxin protects motoneurons against acute excitotoxic damage on spinal cord organotypic cultures. Journal of neurochemistry. 2013 Jan;124(1):36-44. • Allodi I, Casals-Diaz L, Santos-Nogueira E, Gonzalez-Perez F, Navarro X, Udina E. FGF-2 low molecular weight selectively promotes neuritogenesis of motor neurons in vitro. Molecular Neurobiology. 2013 Apr;47(2):770-81. • Modol L, Casas C, Navarro X, Llido A, Vallee M, Pallarès M, et al. Neonatal finasteride administration alters hippocampal α4 and δ GABAAR subunits expression and behavioural responses to progesterone in adult rats. The international journal of neuropsychopharmacology. 2013 Sep 9;1-15. • Torres-Espin A, Corona-Quintanilla DL, Fores J, Allodi I, Gonzalez F, Udina E, et al. Neuroprotection and axonal regeneration after lumbar ventral root avulsion by re-implantation and mesenchymal stem cells transplant combined therapy. Neurotherapeutics. 2013 Apr:10(2):354-68. • Boeckstyns ME, Sørensen AI, Vineta JF, Rosen B, Navarro X, Archibald SJ, et al. Collagen conduit versus microsurgical neurorrhaphy: 2-year follow-up of a prospective, blinded clinical and electrophysiological multicenter randomized, controlled trial. The Journal of hand surgery. 2013 Dec;38(12):2405-11. • Kumru H, Soler D, Vidal J, Navarro X, Tormos JM, Pascual-Leone A, et al. The effects of transcranial direct current stimulation with visual illusion in neuropathic pain due to spinal cord injury: An evoked potentials and quantitative thermal testing study. European journal of pain (London, England). 2013 Jan;17(1):55-66. • Casas C, Herrando-Grabulosa M, Manzano R, Mancuso R, Osta R, Navarro X. Early presymptomatic cholinergic dysfunction in a murine model of amyotrophic lateral sclerosis. Brain and behavior. 2013 Mar;3(2):145-58. • Redondo-Castro E, Hernandez J, Mahy N, Navarro X. Phagocytic microglial phenotype induced by glibenclamide improves functional recovery but worsens hyperalgesia after spinal cord injury in adult rats. The European journal of neuroscience. 2013 Dec;38(12):3786-98. • Redondo-Castro E, Torres-Espin A, Garcia-Alias G, Navarro X. Quantitative assessment of locomotion and interlimb coordination in rats after different spinal cord injuries. Journal of neuroscience methods. 2013 Jan 3;213(2):165-78. • Redondo-Castro E, Navarro X. Peripheral nerve alterations after spinal cord injury in the adult rat. Spinal cord. 2013 Aug;51(8):630-3. • Flix B, Suarez-Calvet X, Diaz-Manera J, Santos-Nogueira E, Mancuso R, Barquinero J, et al. Bone marrow transplantation in dysferlin-deficient mice results in a mild functional improvement. Stem cells and development. 2013 Nov 1;22(21):2885-94.

Research Projects

• Code: SAF2010-17851 and hybrid bionics (MERIDIAN) Title: Activación de los Programs de resolución de la infla- Principal Investigator: Xavier Navarro Acebes mación como nueva terapia neuroprotectora para tratar las CIBERNED’s collaboration: No lesiones agudas de la medula espinal CIBERNED’s groups: Principal Investigator: Rubén López Vales Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: International CIBERNED’s groups: Funding Agency: European Commission Other CIBER’s collaboration: No Funding: 310.000 € Type: National Duration: 2012-2015 Funding Agency: Ministerio de Ciencia e Innovación Funding: 108.900 € • Code: PI11/00464 Duration: 2011-2013 Title: Modulación del arco reflejo de estiramiento para me- jorar la recuperación funcional despues de lesiones nervio- • Code: FP7-278612 sas Title: Biohybrid templates for peripheral nerve regenera- Principal Investigator: Esther Udina Bonet tion (BIOHYBRID) CIBERNED’s collaboration: No Principal Investigator: Xavier Navarro Acebes CIBERNED’s groups: CIBERNED’s collaboration: No Other CIBER’s collaboration: No CIBERNED’s groups: Type: National Other CIBER’s collaboration: No Funding Agency: Fondos de Investigación Sanitaria, Institu- Type: International to Salud Carlos III Funding Agency: European Commission Funding: 56.460 € Funding: 360.000 € Duration: 2012-2014 Duration: 2011-2015 • Code: FP7-602547 • Code: FP7-NMP4-SL-2012-280778 Title: Natural sensory feedback for phantom limb pain Title: Micro and nano engineered bidirectional carbon in- modulation and therapy (EPIONE) terfaces for advanced peripheral nervous system prosthetic Principal Investigator: Xavier Navarro Acebes

224224 Xavier Navarro Acebes Group Xavier Navarro Acebes Group 225

CIBERNED’s collaboration: No Type: National CIBERNED’s groups: Funding Agency: Instituto de Salud Carlos III Other CIBER’s collaboration: No Funding: 180.000 € Type: International Duration: 2013-2016 Funding Agency: Project FP7-602547 Funding: 199.840 € • Code: PIRG05-GA-2009-249274 Duration: 2013 Title: Role of lysophosphatidic acid in the pathophysiology of spinal cord injury (ROLPASCI) • Code: FP7-611687 Principal Investigator: Xavier Navarro Acebes Title: Neurocontrolled bidirectional artificial upper limb CIBERNED’s collaboration: No and hand prosthesis (NEBIAS) CIBERNED’s groups: Principal Investigator: Xavier Navarro Acebes Other CIBER’s collaboration: No CIBERNED’s collaboration: No Type: International CIBERNED’s groups: Funding Agency: Marie-Curie International Reintegration Other CIBER’s collaboration: No Grant, European Commission FP7 Type: International Funding: 100.000 € Funding Agency: European Commission Duration: 2010-2014 Funding: 517.424 € Duration: 2013 • Code: TV32011-110310 Title: Therapeutic role of IL-37 in spinal cord injury • Code: TV32011-110430 Principal Investigator: Rubén López Vales Title: Potenciación de los mecanismos endógenos de neu- CIBERNED’s collaboration: No roprotección y reparación del sistema nervioso despues de CIBERNED’s groups: daño agudo Other CIBER’s collaboration: No Principal Investigator: Caty Casas Type: Privado CIBERNED’s collaboration: No Funding Agency: Fundació Marató-TV3 CIBERNED’s groups: Funding: 200.000 € Other CIBER’s collaboration: Yes (CIBERER) Duration: 2012-2014 Type: Privado Funding Agency: Fundació Marató-TV3 • Code: RD12/0019/0008 Funding: 189.000 € Title: RETIC de Terapia Celular Duration: 2012-2014 Principal Investigator: Isabel Fariñas (coordinador de red: Jose María Moraleda) • Code: RD12/0019/0011 CIBERNED’s collaboration: Yes Title: RETIC de Terapia Celular CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; Principal Investigator:Xavier Navarro Acebes (coordinador G 607 de red: Jose María Moraleda) Other CIBER’s collaboration: No CIBERNED’s collaboration: Yes Type: National CIBERNED’s groups: G 301; G 102; G 113; G 208; G 105; Funding Agency: Fondo de Investigaciones Sanitarias G 607 Funding: 296.700 € Other CIBER’s collaboration: No Duration: 2013-2016

PhD Dissertations

• Author: Elena Redondo Castro • Author: Abel Torres Espín Title: Neuropathic pain after thoracic spinal cord injuries. Title: Terapia celular para lesiones que afectan a la médula The importance of plasticity in lumbar segments and glial espinal modulation Date: 27 June 2013 Date: 6 April 2013 Supervisor: Xavier Navarro Acebes Supervisor: Xavier Navarro Acebes Group Juan Jesús Vilchez Padilla

Hospital Universitari i Politecnic La Fe

3 Servicio de Neurología Torre D, planta 5 Bulevar Sur, s/n Program 46026 València (Spain)

Tel.: +34 961 244 000 (centralita) / +34 961 245 543 (secretaria neurología) [email protected]

Juan Jesús Vilchez Padilla Fernando Mayordomo Principal Investigator, Associate Professor, Researcher, Morphology and Neuromuscular Pathology Chief of Neurology department Inmaculada Azorín Villena Teresa Sevilla Mantecón CIBERNED Postdoctoral Fellow Neurologist Pilar Martí Luís Bataller Alberola PhD student Neurologist E. Torres Vega María José Chumillas PhD student Clinical Neurophysiologist Clara Gomis Coloma Nuria Muelas Gómez PhD student MD, Medical Specialist in Neurology Lorena Perpiñá Gómez Rafael Sivera CIBERNED Technician Neurologist Roger Vilchez Medina Juan Francisco Vázquez Costa Lab Technician Post-MIR

226 Juan Jesús Vilchez Padilla Group 227

Summary

In the area of genetic neuropathies, particularly in Charcot Marie Tooth Disease (CMT) neuropathies, in the annuity 2013, it has been established the constitution of clinical Workpage led by Dr. Sevilla within the cooperative project TREAT-CMT who was elected for funding by the European call -ISCIII-IRDYC-2012. Among his achievements is the creation of a unified database which contains a casuistry of CMT over 700 patients in which prominent Spanish centers (La Paz, Bellvitge, Rocío, etc.) has been involved. As an example of this work has been published a series CMT full genetic characterization (Sivera et al., Neurology 2014) which is a reference in this field. Other contributions of interest are clinical and genetic reassessment of hereditary motor and sensory neuropathy type Russe associated with a founder mutation affecting a predominantly Spanish Gypsies (Sevilla et al., Clin Genet 2013), also has published a study original on the vestibular involvement as a primary symptom in CMT4A neuropathy (Sivera et al., J Neurol Neurosurg Psychiat 2014). A milestone in the field of motor neuron disease, also led by Dr. Sevilla, it has been obtaining a fellowship in Post-MIR public call by the ISS La Fe awarded to Juan Francisco Vazquez Costa, on a research oriented ELA project, developed in collaboration with the group of Neurogenetic Dr. Jordi Pérez-Tur, Institute of Biomedicine of Valencia.

In the field of congenital myopathies and myasthenias, led by Dr. Muelas and Dr. Vilchez, the highlights were our participation in the discovery of a mutation in the gene that is responsible Trasnportina III Muscular Dystrophy LGMD1F in collaboration with several Spanish and international groups (Melià MJ et al., 2013). This form of dystrophy is unique to a Spanish family whose clinical following up is mainly performed in our hospital, which has allowed the publication of additional data on the clinical phenotype in collaboration with Italian researchers (Pertele et al., 2013), and also it has put forward the basis for undertaking projects with prestigious groups in order to decipher the pathogenesis and treatment of this myopathy and to deepen transport in the pathogenesis of HIV virus that depends on the Transportina III as well. Another notable collaboration is the study of miRNA dysregulation in a model of both Drosophila and human biopsies of patients with myotonic dystrophy has clarified that data on the pathogenesis of the disease (Fernandez- Costa JM et al., Human Molec Genet 2013).

In the field of neuromuscular diseases and autoimmune encephalopathies led by Dr. Bataller should be noted the confirma- tion of the presence of ACS against aquaporin serum of patients with paraneoplastic myelitis - hyperCKemia as expression of an ovarian teratoma (Frasquet M et al., 2013). We also noticed two major collaborations on the involvement of NMDA recep- tor Abs against both pediatric encephalitis (Amargue T et al., J Pediatrics 2013) and late encephalitis (Titulaer MJ et al., 2013). This field should also be noted obtaining a predoctoral fellowship EAI (Valencian Health Department) for a degree in Biology (E. Torres Vega) under the direction of Dr. Bataller develop a project to decipher the pathogenesis of the thymus in developing neuromyotonia, myasthenia syndromes.

Keywords

Charcot-Marie-Tooth disease, Genetic neuropathy type Russe, CMT4G, Gen HK1, CMT4A, Gen SH3-TCH, Vestibular neuropathy, Muscular dystrophy LGMD1F, Gen Transpotina III, Myotonic dystrophy, CTG expansions, miRNA, Limbic encephalitis, Anti-NMDA receptor Abs, Anti-Aquoporin Abs

Publications

• Aida L, Parkhutik V, Tembl JI, Martin N, Frasquet M, Bataller L. Embolism and impaired washout: a possible explanation of border zone strokes in hypereosinophilic syndrome. Journal of the neurological sciences. 2013 Feb 15;325(1-2):162-4. • McDonald CM, Henricson EK, Abresch RT, Florence JM, Eagle M, Gappmaier E, et al. The 6-minute walk test and other endpoints in Duchenne muscular dystrophy: longitudinal natural history observations over 48 weeks from a multicenter study. Muscle & nerve. 2013 Sep;48(3):343-56. • Peterle E, Fanin M, Semplicini C, Padilla JJ, Nigro V, Angelini C. Clinical phenotype, muscle MRI and muscle pathology of LGMD1F. Journal of neurology. 2013 Aug;260(8):2033-41. • Sivera R, Sevilla T, Vilchez JJ, Martinez-Rubio D, Chumillas MJ, Vazquez JF, et al. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series. Neurology. 2013 Oct 29;81(18):1617-25. • Titulaer MJ, McCracken L, Gabilondo I, Iizuka T, Kawachi I, Bataller L, et al. Late-onset anti-NMDA receptor encephalitis. Neurology. 2013 Sep 17;81(12):1058-63. • Melià MJ, Kubota A, Ortolano S, Vilchez JJ, Gamez J, Tanji K, et al. Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene. Brain. 2013 May;136(Pt 5):1508-17. • Frasquet M, Bataller L, Torres-Vega E, Duran-Moreno M, Garcia-Verdugo JM, Sevilla T, et al. Longitudinally extensive transverse myelitis with AQP4 antibodies revealing ovarian teratoma. Journal of neuroimmunology. 2013 Jul 27;263(1-2):145-7. • Querol L, Rojas-Garcia R, Casasnovas C, Sedano MJ, Munoz-Blanco JL, Alberti MA, et al. Long-term outcome in chronic inflammatory demyelinating polyneuropathy patients treated with intravenous immunoglobulin: A retrospective study. Muscle & nerve. 2013 Dec;48(6):870-6.

Research Projects

• Code: Proyecto de Investigación Terapia en Distrofias Mus- • Code: PI12/00946 culares / Fundación Isabel Gemio Title: Estudio mutacional de una serie de pacientes con Title: Biología, fisiopatología y terapia de las células satélite neuropatía hereditaria motora distal del músculo esquelético Principal Investigator: Mª Teresa Sevilla Mantecón Principal Investigator: Juan José Vilchez Padilla CIBERNED’s collaboration: No CIBERNED’s collaboration: No CIBERNED’s groups: CIBERNED’s groups: Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: Privado Funding Agency: Instituto de Salud Carlos III / FIS Funding Agency: Fundación Isabel Gemio Funding: 110.000 € Funding: 180.000 € Duration: 2013 Duration: 2011-2014 • Code: 2011/0537 • Code: FIS- PI 12-01031 Title: Translational Research, Experimental Medicine and Title: Estudio de anticuerpos contra antígenos de membra- Therapeutics on Charcot-Marie-Tooth Disease. Proyecto na neuronal en el síndrome opsoclonus-mioclonus, la neu- colaborativo Principal Investigator: Fracesc Palau romiotonia y las epilepsias de probable causa autoinmune CIBERNED’s collaboration: Yes Principal Investigator: Luis Bataller CIBERNED’s groups: G 606; G 605 CIBERNED’s collaboration: No Other CIBER’s collaboration: Yes (CIBERER) CIBERNED’s groups: Type: International Other CIBER’s collaboration: No Funding Agency: International Rare Diseases Research Con- Type: National sortium (IRDiRC) / Carlos III Funding Agency: ISCIII-FIS Funding: 300.000 € Funding: 90€ Duration: 2013 Duration: 2013 • Code: PI2010/11 • Code: PI11/01330 Title: Consorcio para generar una base de datos común Title: Estudio de la Hiperckemia Asintomática o Paucisin- cuya finalidad es implementar la investigacion clínica y bá- tomática sica en enfermedades neuromusculares Principal Investigator: Juan José Vilchez Padilla Principal Investigator: Isabel Illa Sendra CIBERNED’s collaboration: No CIBERNED’s collaboration: Yes CIBERNED’s groups: CIBERNED’s groups: G 603; G 601; G 605 Other CIBER’s collaboration: No Other CIBER’s collaboration: No Type: National Type: Intramurales Funding Agency: FIS Funding Agency: CIBERNED Funding: 66.000 € Funding: 429.000 € Duration: 2012-2014 Duration: 2010-2013

228228 Juan Jesús Vilchez Padilla Group Cooperative

2013 Annual Report 231

Cooperative Research

The cooperative research program began in 2010 with the publication of a first call for intramural projects. This program constitutes one of the cornerstones of CIBERNED research activity and aims to stimulate cooperative research between different research groups, joining forces and exploiting synergies and complementary capabilities. The goal is to encourage translational research between clinical and basic research groups, combining both approaches and fostering the sharing of resources and knowledge so that complex issues related to the causes, diagnosis and treatment of neurodegenerative diseases can be addressed. It pretends to identify research projects in collaboration having a highly innovative and translational nature and in which the cooperative effort significantly increase the added value of the research activity. This first call resulted in 6 approved projects:

Nº Title Coordinator

1 Reelin and GSK3 as therapeutic targets in Alzheimer's disease Ávila de Grado, Jesús 2 BESAD-P: Biomarkers of Early Stages of Alzheimer Disease-Prevention Ferrer Abizanda, Isidre Glial activation during the neuroinflammatory process: a potential Vitorica Ferrández, Francisco 3 therapeutic target for Alzheimer's disease Javier García de Yébenes Prous, 4 Neuroprotection in Huntington's disease Justo Generating a dopaminergic neuronal model from induced pluripotent stem 5 cells from patients with Parkinson's disease associated to mutations in the Tolosa Sarró, Eduardo LRRK2 gene Consortium to generate a common database aimed at implementing 6 Illa Sendra, Isabel clinical and basic research on neuromuscular diseases

These six projects have brought together the efforts of 28 CIBERNED research groups and 12 external ones, and has represented an economic assignment of 750,000€ per year. Each proposal has a coordinator and other CIBERNED participating groups, and in some cases external groups (not funded). The proposals, although still subject to the approval of the Steering Committee, are reviewed externally by the National Evaluation and Foresight Agency (ANEP), so that the allocation of funds is transparent. The projects are part of each program research lines, bearing in mind that the participation of groups from other programs is also allowed. The objective pursued is to bring together the best groups working on important issues within research on neurodegeneration. In 2011, enhancement of the existing cooperative program was seen as an essential scientific tool for CIBERNED. Once evaluated through the defined internal process and given the success obtained, the projects from the first call were extended another year with funding equivalent to that previously assigned for the two previous annuities. The second call for CIBERNED cooperative projects was launched during 2011, with the same grounds and features than the first one. In this case the outcome of the evaluation made by ANEP resulted in the approval of 4 new projects: Nº Title Coordinator

The transcription factor Nrf2 as a new therapeutic target for Parkinson's 7 Cuadrado Pastor, Antonio disease Onset and progression of Parkinson's disease. Vulnerability of the 8 Obeso Inchausti, José Ángel nigrostriatal pathway, events at origin and destination Generation of dopaminergic neurons from somatic cells of parkinsonian 9 Moratalla Villalba, Rosario patients with cognitive failure Multicenter study on LCR and neuroimaging biomarkers in the continuum 10 Lleó Bisa, Alberto preclinical- prodromal Alzheimer's disease (SIGNAL Study)

These new projects brought together 21 CIBERNED research groups with a fixed duration of two years. Both calls have generated a cooperative research activities and networking, which has involved 49 of the 58 groups CIBERNED plus external others. During the month of May 2013 the third call for cooperative projects CIBERNED as launched, which was evaluated again by the ANEP and ended up in early September with the award of 5 new collaborative projects, listed below:

Nº Title Coordinator

Emergent properties of the neuron-glia relationship underlying 11 Torres Alemán, Ignacio neurodegeneration and dementia in Alzheimer's disease Mitochondrial dynamics and mitophagy as therapeutic targets in 12 Soriano García, Eduardo Parkinson's and Huntington's diseases Identification and molecular characterization of cannabinoid receptors 13 Guzmán Pastor, Manuel subpopulations in poliglutaminopatías Role of GSK-3β in the cortical circuits alterations occurring in Alzheimer's 14 Iglesias Vacas, Teresa disease Synaptic degeneration and dysregulation of adult neurogenesis in murine 15 Fernández Chacón, Rafael models of neurodegeneration

This third call for projects, endowed with 688,000€ per year for two years, involves 23 CIBERNED research groups and an external partner groups. A brief description of the activity performed by each of the completed projects (calls for 2010 and 2011) and a summary of the objectives for the approved projects under the third call, initiated during the second half of 2013 is presented.

232 2013 Annual Report 2013 Annual Report 233

Project 1 Title: Reelin and GSK3 as therapeutic targets in Alzheimer’s disease The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Ávila de Grado, Jesús CIBERNED, CBMSO CSIC-UAM, Madrid de Felipe Oroquieta, Javier CIBERNED, Center of Biomedical Technology, UPM, Madrid García Verdugo, José Manuel CIBERNED, University of Valencia Sáez Valero, Javier CIBERNED, Miguel Hernández University, Elche Soriano García, Eduardo CIBERNED, IRB, University of Barcelona Trullás Oliva, Ramón CIBERNED, IDIBAPS-CSIC, Barcelona Rábano Gutiérrez del Arroyo, Alberto Associated group, CIEN Foundation, Madrid

In this collaborative project we have studied, inter alia, the role of the Reelin/GSK3 pathway in the pathogenesis of sporadic Alzheimer’s disease (SAD). During the first year the effects of overexpression of GSK3 on axonal transport of mitochondria were analyzed by the Soriano and Avila groups whereas the Avila and García-Verdugo groups analyzed the effect of the GSK3 overexpression in neurogenesis occurring in the dentate gyrus. Regarding objective 1 of the collaborative project, the group of Trullas in collaboration with the group of Soriano have investigated the regulation of AD-associated signaling pathways by NP1 (Pentraxin Neuronal 1). They have studied the effect of the gain and loss of function of NP1. With regards to objectives 2 and 6 of the collaborative project, the García-Verdugo group, in collaboration with the group of Soriano, has assessed the potential involvement of Reelin/GSK3 in the proliferation of neuronal precursors finding that, in the absence of additional validation, Reelin appears to have no effect on the proliferation of these precursors after adding mouse reelin to primary cultures of neurospheres from the subventricular zone (SVZ). Currently, the Sáez Valero group is leading, in collaboration with other CIBERNED groups, the study of the function of brain acetyl cholinesterase in tauopathies. In the second part of the project, they have continued to analyze the two main objectives of the project: a) the regulation of signaling pathways associated with Alzheimer’s disease, mainly studying the involvement of GSK3 protein, reelin, tau, Aß and NP1; b) the regulation of adult neurogenesis by GSK3 or reelin/GSK3 signaling pathways. These two objectives have been carried out both at the level of collaboration between two or more groups or at a single group level. A the collaborative level, the Ávila, Soriano and de Felipe groups have continued studying the involvement of GSK3 in the neurogenesis in the adult dentate gyrus. Finally, the Sáez-Valero group has examined whether there is any relationship between acetylcholinesterase and tau phosphorylation. Also the Sáez-Valero group, in collaboration with some of the other groups, has studied how the beta-amyloid peptide affects reelin signaling or how ApoER2 receptor processing by presenilin 1 modulates the expression of reelin. Moreover, de Felipe’s group described the effect of phosphorylated tau on dendritic spines in neurons from Alzheimer’s patients. It is also noteworthy the Trullas’s group studies indicating that the presence of mitochondrial DNA in the cerebrospinal fluid may be an excellent early biomarker for Alzheimer’s disease. Project 2 Title: BESAD-P: Biomarkers of early stages of Alzheimer disease - prevention The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Ferrer Abizanda, Isidre CIBERNED, IDIBELL, Barcelona Camins Espuny, Antonio CIBERNED, University of Barcelona del Río Fernández, José Antonio CIBERNED, Catalonian Institute of Bioengineering, Barcelona Wandosell Jurado, Francisco CIBERNED, CBMSO CSIC-UAM, Madrid Carro Díaz, Eva CIBERNED, 12 de Octubre Hospital, Madrid Portero Otín, Manuel Associated group, University of Lleida Bullido Gómez-Heras, María Jesús CIBERNED, CBMSO CSIC-UAM, Madrid López de Ceballos Lafarga, María CIBERNED, Cajal Institute CSIC, Madrid Mengod Los Arcos, Guadalupe CIBERNED, IDIBAPS-CSIC , Barcelona Martí Puig, Eulàlia Associated group, CIBERESP, CRG, Barcelona

The project is focused on the study of transcriptome abnormalities at very early stages of AD-related pathology (Braak stage I-II) that may be serve to: 1. Increase understanding of early molecular damage of key pathways. 2. Identify putative early biomarkers. 3. Identify putative targets of therapeutic intervention geared to delay or reduce disease progression. For these purposes human post-mortem brains obtained and stored under optimal conditions have been used for epigenetics, genomics, transcriptomics, lipidomics and metabolomics followed by bioinformatics processing. Several deregulated pathways have been identified at first and early stages of AD, including inflammation, olfactory receptors, purine metabolism, energy metabolism, and protein synthesis. The role of certain virus has also been explored. This has been analyzed in mice models and cell lines in order to validate altered molecular pathways. Already identified putative targets of therapeutic intervention have been tested in experimental models of AD, mainly APP/PS1 mice, by using varied therapeutic approaches under randomized conditions. The following treatment procedures have been assessed: 1. Hormonal treatment assays: effects of estradiol. 2. β-amyloid clearance and trophic factors from choroid plexus. 3. Treatment with CB1 and CB2 cannabinoid receptor agonists; effects of cannabinoids in murine models of AD. 4. Treatment with erythropoietin (Epo) and CEPO. 5. Ketogenic diets and triheptanoin. 6. JNK inhibitors.

234 2013 Annual Report 2013 Annual Report 235

7. Cellular and animal models have been subjected to TAT-like singular peptide libraries to modify β-amyloid production and tau hiperphosphorylation. 8. Anti-inflammatory therapies: PK11195, ligand of the translocator protein 18kDa. All these aspects have been conducted in collaboration with the different partners. All the procedures have been carried out following the guidelines on the use of human brain tissues and biological products for research, and the appropriate utilization of animals for experimental procedures.

Project 3 Title: Glial activation in the neuroinflammatory process: a potential therapeutic target for Alzheimer’s disease The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Vitorica Ferrández, Francisco Javier CIBERNED, University of Seville Gutiérrez Pérez, Antonia CIBERNED, University of Malaga Comella Carnice, Joan Xavier CIBERNED, Vall d'Hebron University Hospital, Barcelona Torres Alemán, Ignacio CIBERNED, Cajal Institute CSIC, Madrid Rodríguez Álvarez, José CIBERNED, Autonomous University of Barcelona

Microglial response during the development of Alzheimer’s disease, and other diseases, is complex. The general idea of assuming a neuroprotective quiescent state and a cytotoxic active state microglia is a very unrealistic simplification regarding the development of pathology. More likely, the microglia responds gradually altering its differentiation state in response to the many variations within the microglia-neuron- glia system. At each stage the microglial response would reflect the integration of multiple environmental stimuli (probably negative). Moreover, the role of the various stages of differentiation, as well as its influence on the rest of the cells in its surroundings, remains unknown. We had proposed in this project the evaluation of the inflammatory response in Alzheimer’s patients and the modification of this response by manipulating murine models. The following can be highlighted as a summary of the most important activities in the coordinated project: 1. Study of the neuroinflammatory process in AD patient samples As a first activity, we have collected a population of 10-12 different controls, Braak II, Braak III-IV and V-VI sample, of which, only patients classified as Braak V-VI had Alzheimer-type dementia. To unify the data, mRNAs, cDNAs and total protein have been extracted and prepared. We have also been extracted in parallel the soluble fractions of the different samples. This biological material has been sent to the rest of the groups. Also in parallel, the Gutierrez group has obtained and processed for immunohistochemistry a similar sample population. We have started to characterize the inflammatory response in the development of Alzheimer’s disease from this biological material. In particular, Gutiérrez and Vitorica groups have analyzed the state of microglial activation whereas Comella’s group is examining the dual role of TNF-alpha together with soluble Abeta oligomers in neuronal death and the Torres-Alemán group is analyzing the astrocyte response stimulated by the soluble extracts. The results of this collaborative project until now have been submitted for publication (Carriba et al., Cell Death and Differentiation; Heneka et al., Lancet Neurol, submitted) or in the process of completion and analysis for publication. This delay has been due to the high heterogeneity in the results that, together with the limited number of samples (12 per group), decreases the statistical significance of the observed changes. Furthermore, we have also faced the lack of information on the medical history of patients, so that we know neither the duration of the disease nor the degree of it, which can therefore influence the heterogeneity of the study population. Moreover, it is also noteworthy that the inflammatory response observed in patients is very different from that found in Alzheimer transgenic models. This finding is perhaps the most relevant and we are currently reassessing our working hypothesis to continue our study. Finally, we have analyzed in collaboration with CNIC’s Proteomics Department (J. Vázquez) the soluble fractions of the different populations. The data are currently being evaluated by the IBIS bioinformatics service 2. Phenotypic characterization of murine models of Alzheimer’s disease and repair of new models with modified neuroinflammatory response Regarding murine models of disease we have analyzed within this collaborative project in collaboration with Dr. Torres-Alemán the role of astrocyte activation through calcineurin activity in the development of Abeta peptide pathology (Fernandez et al., Mol Psychiatry. 2012;17:705-18); the role of autophagy in Abeta accumulation and formation of dystrophic axons (Sanchez-Varo et al., Acta Neuropathol. 2012;123:53-70; Torres et al., Mol Neurodegener. 2012;7:59) and the possible pharmacological modulation (in this case with lithium) of plaques toxicity (Trujillo-Estrada et al., Acta Neurophatol Commun. 2013;12:73), in collaboration with Dr. Gutiérrez. Moreover, the neuroprotective role of death receptor antagonists such as Faim-L on the progression of the disease in these models it is being evaluated in collaboration with Dr. Comella. With respect to the generation of new models, Dr. Comella has generated a model of Faim-L overexpression in glutamatergic neurons. This protein has a neuroprotective action on TNF-alpha. These mice are currently being crossed with an Alzheimer’s model. Finally, to assess the role of IL-4-induced alternative activation (M2) in the progression of the disease, our group has developed a model APPxIL-4KO. This model is currently aging and in the process of being characterized.

Project 4 Title: Neuroprotection in Huntington’s disease The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution García de Yébenes Prous, Justo CIBERNED, Ramón y Cajal University Hospital, Madrid Guzmán Pastor, Manuel CIBERNED, Complutense University of Madrid Lucas Lozano, José Javier CIBERNED, CBMSO CSIC-UAM, Madrid Fernández Ruiz, Javier CIBERNED, Complutense University of Madrid Naranjo Orovio, José Ramón CIBERNED, National Center of Biotechnology CSIC, Madrid

236 2013 Annual Report 2013 Annual Report 237

There is a great interest in developing neuroprotective therapies in Huntington’s disease (HD) and different strategies are currently under evaluation from inhibition of transcription of mutant huntingtin by selective antisense oligonucleotides and transplantation of stem cells to alternative high energy alternative metabolic substrates. There is also information about the involvement of cannabinoids in the pathogenesis of HD and as putative mediators of the therapeutic response.

Original Project Objectives Four platforms for the study of disease progression-modifying drugs in HD will be created: 1. In vitro studies in knock-in cells from striatum with normal (7 glutamines, Q7) and pathological (111 glutamines, Q111) expansions in the amino terminal portion of huntingtin. 2. In vitro studies in animal models (R6/2 mice). The R6/2 mice are transgenic mice with polyglutamine expansions of ≈ 150. These animals show attention problems at 3-4 weeks of postnatal life, and motor disorders from 5-7 weeks. 3. Safety and neuroprotection studies using disease biomarkers in asymptomatic subjects carrying the mutation of huntingtin. These individuals may show abnormalities in certain biomarkers (behavioral, motor, cognitive, neuroimaging and biochemical) for periods as long as 15 years prior to developing diagnostic symptoms of the disease. 4. Disease progression modification studies (neuroprotection, symptomatic effect and safety) by candidate drugs in patients with Huntington’s disease. Compounds to be used are already under investigation in other clinical indications or are useful in the symptomatic treatment of HD. They belong to the following families: 1. Dopaminergic stabilizers. 2. Cannabinoid compounds. 3. DREAM inhibitors. Ramón y Cajal Hospital (PI: JG Yébenes) In the Ramón y Cajal Hospital there have been a number of studies related to the project that can basically fit into three categories: a. Studies in experimental models of the disease in order to determine its pathogenesis and explore new therapeutic avenues. b. Studies in subjects with the mutation but asymptomatics in order to determine early clinical or biological markers that may help begin the possible neuroprotective treatment in early stages of disease. c. Clinical trials of compounds undergoing clinical investigation in subjects with symptoms of the disease.

School of Medicine, Complutense University of Madrid (PI: J Fernández Ruiz) Two types of studies under the CIBERNED intramural project coordinated by Justo García de Yébenes have been conducted: a. Preclinical studies with Sativex or its components in experimental models of Huntington’s disease: We have studied the neuroprotective effects of the combination of Sativex phytocannabinoids in 3NP-lesioned rats, a model of mitochondrial damage and oxidative stress in which it has been described that Sativex is capable of preserve striatal neurons and reduce oxidative damage and calpain activation produced by the toxin antioxidant effects independent of cannabinoid receptor (published study). A similar type of study has been conducted in malonate-lesioned rats, a rather pro-apoptotic that is dependent of glial activation, in which Sativex was also effective in preserving striatal neurons primarily through glial effects mediated by the CB1 and CB2 receptors (published study). We have also studied the combination of Sativex phytocannabinoids in R6/2 mice, but in this case no neuroprotective effects were obtained at two different doses, in contrast to the fact that one of its components (∆9-THC) turned out to be neuroprotective when administered individually (through effects mediated by CB1 receptors) in one of the studies included in this collaborative project led by the group of Manuel Guzmán. This has led to hypothesize that it would be necessary to use a variant of Sativex with increased amount of ∆9-THC and decreased (or none) cannabidiol. These experiments are currently underway and the results will be announced by mid-2014. b. Analysis of markers in the Sativex clinical trial in patients with Huntington’s disease: Our group has contributed to the development of the clinical trial with Sativex by analyzing gene expression of the cannabinoid system (receptors and enzymes) in lymphocytes from patients treated with drug or placebo. The only marker that showed differences was the expression of the CB2 recepto, that showed an increase in patients treated with Sativex compared to what happened during the placebo phase.

Faculty of Sciences, Complutense University of Madrid (PI: M Guzmán) a. Aims 1 and 2 (STHdh cells and R6/2 mice), two compounds (cannabinoids) - Study of the neuroprotective role of the cannabinoid CB1 receptor A bilateral stereotaxic injection of an adenovirus vector encoding the CB1 receptor (or the corresponding empty vector as a control) in the striatum of 4 weeks-old R6/2 mice (and the corresponding wild-type controls) was performed. At 8 weeks of age, the animals were subjected to behavioral testing (RotaRod), neuropathological evaluation (striatal volume by MRI) and analysis of molecular markers (DARPP-32, GAD-67, PSD-95, BDNF). The data obtained in all parameters measured show that the re-expression of CB1 receptor in the R6/2 animals is neuroprotective. - Study of neuronal subpopulations expressing the CB1 cannabinoid receptor We have assessed the expression of CB1 receptor on glutamatergic (corticostriatal) and GABAergic (striatal) terminals in R6/2 (and corresponding wild-type controls) mice by analyzing mRNA (qPCR, in situ hybridization) and protein (immunofluorescence brain slices and synaptosomes) levels. The data support the view that loss of CB1 receptors throughout the progression of the disease is selectively restricted to GABAergic terminals, while receptor expression in glutamatergic terminals remains unchanged. To test the hypothesis that the population of CB1 receptors located on glutamatergic terminals may help buffer the excitotoxicity associated with advanced stages of the disease, we selectively knocked-out CB1 receptor expression in GABAergic and glutamatergic cells. Thus, from the model R6/2, we generated a new model to study the functionality of the CB1 receptor: mouse R6/2:CB1floxed/floxed. This allows for both receptor removal in a spatiotemporally-selective manner by using vectors expressing Cre recombinase. We injected a rAAV-CaMKII-Cre vector in the deep layers of the motor cortex or in the dorsal striatum of these mice to eliminate the expression of CB1 receptor selectively in cortico-striatal projection neurons or MSNs, respectively. RotaRod and expression of DARPP-32 analysis show that the neuroprotection mediated by the CB1

238 2013 Annual Report 2013 Annual Report 239

receptor lies in a restricted population of receptors, specifically those located in glutamatergic terminals, whereas the most widely distributed in GABAergic terminals do not appear to play a relevant neuroprotective role. b. Aim 4 (HD patients), two compounds (cannabinoids compounds, Sativex) Clinical trial with Sativex:

- Analysis of the viability of patient’s skin fibroblasts in response to excitotoxins (H2O2, malonate, quinolinate, kainate, rotenone). - Analysis of miR-34b levels in plasma of patients (collaboration with Laura García-Bermejo, Ramón y Cajal Hospital). - Analysis of endocannabinoid levels in CSF of patients, also compared with patients with aphasia, dementia and hydrocephalus (collaboration with Silvia Ortega, UCM). No difference was found between Sativex and placebo-treated patients. However, anandamide levels were decreased in HD patients (regardless of treatment) compared with those with aphasia or hydrocephalus.

Alcorcón Hospital Foundation (PI: J Romero) We first proceeded to determine the levels of an important neurotrophic factor in Huntington’s disease such as BDNF in samples of cerebrospinal fluid (CSF). Despite using high sensitivity kits (R&D), all samples showed levels below the detection limit, so we could not draw conclusions of any kind. TNFa measurements in CSF suffered the same problem, with all the samples below the detection limit. We then decided to try to determine the BDNF levels in serum samples from patients enrolled in the trial. This time, quantifiable levels of BDNF were indeed detected, although the results grouped by treatment did not show the existence of significant differences.

Project 5 Title: Generating a model of dopaminergic neurons from induced pluripotent stem cells from patients with Parkinson’s disease associated with mutations in the gene LRRK2 The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Tolosa Sarró, Eduardo CIBERNED, Clinic Hospital, Barcelona Alberch Vie, Jordi CIBERNED, University of Barcelona López Barneo, José CIBERNED, Virgen del Rocío Hospital, University of Sevilla Raya Chamorro, Ángel Associated group, Institute Bioengineering, Barcelona Vila Bover, Miquel CIBERNED, Vall d'Hebron University Hospital, Barcelona

Mutations in leucine-rich repeat protein kinase 2 gene (LRKK2) are the most common known genetic cause of familial and sporadic Parkinson’s disease (PD). These cases with mutations in LRRK2 largely mimic the clinical and pathological characteristics of idiopathic cases, including cardinal motor symptoms as well as neurodegeneration of dopaminergic cells in the substantia nigra. However, the pathogenic processes associated with mutations in LRRK2 has remained elusive up to now, so that the generation of valid experimental models that recap PD phenotype, as well as the research on new drugs, could be very helpful to a better understanding of PD. Populations of dopaminergic (DA) neurons (DAn) will be generated from induced multipotent stem cells (iPSCs) derived from isolated fibroblasts from dermal biopsies of subjects carrying mutations in the LRRK2 gene (LRRK2-PD), idiophatic PD patients (ID-PD) and healthy controls. The obtained neurons will be broadly characterized by using different analytical approximations (biochemical, proteomic, and electrophysiological).

Results We have generated iPSC lines from seven patients with ID-PD, four patients with familial LRRK2-PD and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients’ genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type.

Project 6 Title: Consortium to build a common database aimed at implementing clinical and basic research in neuromuscular diseases The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Illa Sendra, Isabel CIBERNED, Santa Creu i Sant Pau Hospital, Barcelona Infante Ceberio, Jon CIBERNED, Marqués de Valdecilla Hospital, Santander López de Munain Arregui, Adolfo CIBERNED, Biodonostia Research Institute Olive Plana, Montserrat CIBERNED, IDIBELL, Barcelona Paradas López, Carmen CIBERNED, Virgen del Rocío Hospital, University of Sevilla Vílchez Padilla, Juan Jesús CIBERNED, La Fe University Hospital, Valencia Andreu Periz, Antonio Luis Associated group, CIBERER, Vall d'Hebron University Hospital Coll Canti, Jaume Associated group, Germans Trias Hospital, Barcelona Casasnovas Pons, Carlos Associated group, Bellvitge Hospital Esteban Pérez, Jesús Associated group, 12 de Octubre Hospital Guerrero Sola, Antonio Associated group, San Carlos Clinical Hospital Marquéz Infante, Celedonio Associated group, Virgen del Rocío University Hospital

240 2013 Annual Report 2013 Annual Report 241

Principal Investigator Institution Muñoz Blanco, José Luis Associated group, Gregorio Marañón University Hospital Navarro Fernández-Balbuena, Carmen Associated group, Vigo University Hospital Complex

Neuromuscular diseases (NMD) are a heterogeneous group of pathologies. They are unusual and have different etiologies, including neurodegenerative mechanisms, hereditary and inmunomediated. NMD are an enormous social and health problem due to the disability cause to patients having progressive and chronic muscular weakness, without curative treatment in the majority of the cases. Our aim was having an operative NMD database with clinical information, complementary test, neuroimaging and biological samples. Its design should allow us not only a better clinical coordination but a significant change in the research area in NMD in Spain. First of all we have agreed with the methodology and the design of the different databases. Four work teams were created, each of them formed by a core of experts in the different areas. Several meetings were performed, to define the aims of each database and to develop their contents. Databases include between 70 and 540 items. Once the consensus was reached by the core of experts, databases were submitted to the rest of the participants asking for the final approval. A web master was contracted and the databases were hosted in a web domain. The definitive name of these databases was NMD-ES (Neuromuscular Database - Spain). During 3 years 6 databases have been designed: Myasthenia Gravis (1000 patients), Hereditary Polineuropathies (931 patients), Motorneuron disease (968 patients), Muscular Dystrophies (1100 patients), Inflammatory Neuropathies (130 patients) and Pompe disease (22 patients). So far, 4161 patients have been introduced. Twenty-four hospitals were participating by the end of the project. Neurologists are responsible of introducing data. All patients must have previously signed a consent form. The identity of the patients is preserved, since all the personal data are encoded. All the neurologist that introduce data are responsible for the information. To ensure the quality of the data a curator checks them periodically, in silico and in person, to verify the information and detect mistakes that have to be mended. The Project has provided us with a large amount of information that will be analyzed for a long time, and has already produced two publications: “Guidelines for molecular diagnosis of Charcot-Marie-Tooth disease”, Neurología 2012, y “Analysis of the C9orf72 gene in patients with amyotrophic lateral sclerosis in Spain and different populations worldwide”, Human Mutation 2013. Moreover, NMD-ES database has been presented in international meetings and allowed us to join the TREAT-NMD and be part of the Global Database Oversight Committee (TGDOC), responsible for planning and recruiting patients for clinical trials. This collaborative project not only has accomplished the objectives proposed but also has managed to be one of the largest NMD existing databases with a design internationally recognized. New funding is actively searched for the sake of continuing this ambitious project. Project 7 Title: The transcription factor Nrf2 as a new therapeutic target for Parkinson’s disease The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Cuadrado Pastor, Antonio CIBERNED, Autonomous University of Madrid Fariñas Gómez, Isabel CIBERNED, University of Valencia Herrero Ezquerro, María Trinidad CIBERNED, University of Murcia Fuentes Rodríguez, José Manuel CIBERNED, University of Extremadura, Cáceres Pérez Tur, Jordi CIBERNED, Institute of Biomedicine CSIC, Valencia

The transcription factor NRF2 (Nuclear factor (erythroid -derived 2)-like 2) is a master regulator of cellular homeostasis that regulates the expression of more than 2% of the human genome. These genes exert functions of defense against oxidative, proteotoxic, and inflammatory stress and adapt cellular metabolism to the needs of the moment. Several evidence exist linking decreased NRF2 transcriptional activity with risk of Parkinson’s disease (PD): 1) NRF2 activity declines with age, the main risk factor for PD; 2) this protein is abnormally found in the cytosol in nigrostriatal dopaminergic neurons from PD patients; 3) there is a genetic association between a haplotype comprising three SNPs in NRF2 promoter, that produce an increase in its basal expression, and reduced incidence of PD. Therefore, in this multidisciplinary project we have studied how NRF2 modulates several aspects characterizing PD pathology including proteinopathy, inflammation and mobilization of neural progenitors. One of the most important features of PD is the accumulation of protein aggregates rich in α-synuclein (α-SYN), which form Lewy bodies. Although the aggregation of α-SYN is a hallmark of sporadic and familial PD, it is unclear how it contributes to the initial events of PD pathogenesis such as oxidative and inflammatory stress. In this project, we have addressed this issue in a new animal model based on stereotactic administration of an adeno-associated virus vector (rAAV) for expression of human α-SYN in the ventral midbrain of mice lacking the transcription factor NRF2 (Nrf2-/-). Two months after inoculation with rAAV-α-SYN, the NRF2-/- mice showed an exacerbated nigrostriatal degeneration compared to wild mice (Nrf+/+), with an increase of dystrophic dendrites, reminiscent of Lewy neurites, which correlated with altered NRF2-dependent gene expression, including several proteasome genes. The loss of dopaminergic neurons is associated with increased neuroinflammation and gliosis which intensified in Nrf2-/- mice. These studies were completed with experiments in cell cultures of microglia, by stimulation with recombinant α-SYN. Microglia from mice Nrf2-/- failed to activate the expression of two anti-inflammatory genes, heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate quinone oxidorreductase-1 (NQO1). This NRF2-mediated lack of adaptive response correlated with a change in the profile of microglial activation, to the increased production of pro-inflammatory markers, IL6, IL1 and iNOS and reduced phagocytic capacity. Postmortem brain tissue samples from patients in early-mid PD progression showed increased expression of HO-1 in astrocytes and microglial cells, suggesting an attempt of the diseased brain to compensate for these distinctive features of PD through the activation of NRF2 pathway. This study demonstrates that α-SYN and NRF2 deficiency cooperate in protein aggregation, neuroinflammation and neuronal death and provides a bifactorial animal model to study early PD.

242 2013 Annual Report 2013 Annual Report 243

Considering the importance of autophagy in cellular proteostasis and NRF2 participation in the regulation of at least two key genes in this process, p62 and NDP52, in this project we compared the impact of the parkinsonian toxins paraquat (PQ) and 1-methyl-4-phenyl pyridine (MPP+) in autophagy in cells from Nrf2-/- and Nrf2+/+ mice as well as mice deficient in the adapter protein KEAP1 (Keap1-/-) which directs NRF2 to its ubiquitination and subsequent degradation by the proteasome. All mouse lines were sensitive to the toxic effects of PQ and MPP+ by a cell death mechanism consistent with oxidative stress-mediated apoptosis, but the mouse line deficient in NRF2 was the most sensitive while KEAP1-deficient mice showed a slight resistance. This greater sensitivity shown by the Nrf2-/- line against oxidative stress caused by PQ and MPP+ is consistent with the loss of expression of antioxidant and cytoprotective genes regulated by NRF2. Interestingly, the Nrf2-/- cells have more basal autophagy that the Keap1-/- and Nrf2+/+ mouse lines. The Keap1-/- line has a lower induction of autophagy but exhibits a stronger degradation compared with Nrf2+/+ and also appears to show some sort of alteration in the lysosomal membrane proton pump ATPase, since the effects of Bafilomycin A1, a highly selective inhibitor of this ATPase are attenuated. Finally, pharmacological activation of NRF2 with sulforaphane induced expression of antioxidant genes in Nrf2+/+ cells and decreased the levels of reactive oxygen species produced by PQ and MPP+. Moreover, sulforaphane activated a degradative autophagic mechanism. These results suggest that the pharmacological intervention on NRF2 may be helpful in the maintenance of cell proteostasis parkinsonian against toxins. Another distinguishing feature of PD is the alteration of neurogenesis. The reduction in the number and in the neurogenic contribution from neural (NSCs) in the subventricular zone cells during PD, suggests that the ability of these self-renewing cells is negatively affected when producing functional progeny. Although the molecular mechanisms altered in PD are not known yet, we do know that the high levels of reactive oxygen species present in PD are associated with stem cells dysfunction. As part of this project, we have investigated how NRF2 regulates the behavior of subependymal NSCs by analyzing the subventricular zone and neurosphere cultures of Nrf2-/- mice and Nrf2+/+. The results suggest complex interactions between oxidative stress and the maintenance of the NSCs in this niche. While complete loss of NRF2 leads to premature depletion of NSCs both in vivo and in vitro, low levels of NRF2 present in heterozygous animals (Nrf2+/-), activated NSC proliferation but decreased self-renewal. These results provide an explanation of how the subventricular zone neurogenic niche during aging, main risk factor for PD, a condition that correlates with progressive loss of NRF2 activity. All the results obtained in this multidisciplinary project, both at the basic mechanisms that operate in the regulation of NRF2 in experimental PD, as well as the correlations observed in postmortem samples from PD patients, have paved the way for clinical studies to determine whether NRF2 is a novel therapeutic target for PD. Project 8 Title: Onset and progression of Parkinson’s disease. Vulnerability of the nigrostriatal pathway, origin and destination events The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Obeso Inchausti, José Ángel CIBERNED,CIMA, University of Navarra, Pamplona Canela Campos, Enric Isidre CIBERNED, University of Barcelona González Castaño, José CIBERNED, Autonomous University of Madrid Labandeira García, José Luis CIBERNED, University of Santiago de Compostela Lanciego Pérez, José Luis CIBERNED,CIMA, University of Navarra, Pamplona Matute Almau, Carlos CIBERNED, University of the Basque Country, Bilbao Rodríguez Díaz, Manuel CIBERNED, University of La Laguna, Tenerife

To determine the synuclein toxicity-associated anatomical-functional basis and molecular factors involved in the selective vulnerability of dopaminergic neurons of the substantia nigra pars compacta (SNc) in Parkinson’s disease. To determine whether the focal onset of motor manifestations and of striatal dopaminergic deficit is associated with compensatory changes in target (striatum) and origin (SNc), which can perpetuate and even accelerate the progressive evolution of the neurodegenerative process.

Objectives 1. Define the pattern of onset and progression of Parkinson’s disease. 2. Recognize the “oil slick” progression pattern and rate of progression. 3. Recognize whether a topographic organization of the nigrostriatal pathway exists, which corresponds to the pattern of dopaminergic denervation. 4. Demonstrate whether the degree of dendritic arborization and synaptic contacts of the nigrostriatal projection is determinant of selective vulnerability. 5. Study the relationship between vulnerability, striatal axonal arborization and energy consumption at the cellular level in nigrostriatal organotypic cultures. Demonstrate harmful effects of synuclein. 6. Focal administration by reverse dialysis of dopamine (DA), glutamate, etc. to study the interaction DA/glutamic and potential excitotoxicity. 7. Identify which G protein-coupled receptors (GPCRs) are involved in the onset and progression of the disease, both at origin and destination.

Results 2011-2014 The project started administratively in October 2011 and today is considered close to its completion. It follows the major developments of the research undertaken and the difficulties and limitations encountered in its development.

244 2013 Annual Report 2013 Annual Report 245

a. Objectives 1 and 2. Pattern of onset and progression of PD. Clinical-neuroimaging correlation (Obeso, Rodríguez-Díaz) We performed a study using PET with 18F-DOPA on the topography of striatal dopaminergic deficit in newly diagnosed PD patients (n=36) with marked clinical asymmetry (unilateral signs). The dopaminergic deficit rostro-caudal pattern has been shown, being described for the first time that the depletion is more intense and early in the putamen region corresponding to the upper extremity (Pavese et al., Brain, submitted for publication). Currently, 18F-DOPA PET studies on 16 patients are being repeated to define the progression pattern. At cortical level it has been defined the progression pattern of hypometabolism (PET-2-FDG) and atrophy (MRI) in cognitively normal PD with mild cognitive impairment and dementia. The concept of degenerative penumbra corresponding to hypometabolic areas that will evolve towards atrophy (Gonzalez-Redondo et al., Brain, in press) has been defined. We have been unable to begin a prospective study in pre-motor PD with PET 14C-dihydrotetrabenazine because, despite the routine availability of radioligand for experimental studies (Blesa et al., 2010, 2012), we have not yet received regulatory approval (after 2 years) by the Medicines Agency, essential and unavoidable requirement for its use in humans. Tractography, functional connectivity, and effective connectivity studies with functional magnetic resonance imaging (fMRI) in 20 controls and 20 PD patients have been performed in La Laguna. Obtained data have allowed us to establish a global model of connectivity in the basal ganglia that as a whole is consistent with the classical model proposed from studies in animals. In addition we evaluated the functional relevance of the thalamus intralaminar nuclei projection to the striatum (publication in preparation). These results in PD patients have helped to define for the first time a specific pattern (somatotopic) of nigro-striatal and cortical dopaminergic deficit, and a new concept of degenerative penumbra, as well as to establish tools to assess alterations of sub-cortical circuits in PD b. Objectives 3 and 4. Neuronal vulnerability in the MPTP monkey model (Lanciego, Obeso) Two retrograde neuronal tracers were injected at different sites within the caudate and putamen in a total of six Macaca fascicularis primates (3 controls and 3 parkinsonian animals). 1. Nigro-striatal topography in the monkey and ventro-lateral SNc vulnerability. SNc neurons are grouped by zones according to nigrostriatal projection destination, although no specific topographical correspondence with the pattern of dopaminergic denervation in the SNc has been found.

2. Striatal axonal arborization and vulnerability of the SNc ventral side. The results indicate that there are no dopaminergic neurons doubly labeled with both tracers, that is, the degree of axonal arborization in destination (striatum) is not extensive enough to consider that a single neuron is simultaneously innervating dopaminergic the caudate and putamen nuclei, nor two distant regions of the putamen. Thus, the original working hypothesis of increased axonal arborization of the SNc ventro-lateral neurons has not been confirmed. Furthermore, the content in calbindin in dopaminergic neurons has been evaluated to obtain a correlation with the potential differential vulnerability of such neurons against MPTP-induced neurodegeneration. In control animals, the highest percentages of colocalization between tyrosine hydroxylase (TH; used as dopaminergic marker) and calbindin (CB) have been observed in the ventral tegmental area (VTA, 58%), followed by neurons in the SNcd dorsal region (34%). As expected, no co-localization at the level of the SNcv ventral region (0%) was observed. The presence of CB was examined in neurons identified according to their projection targets to the caudate, putamen and internal globus pallidus (GPi) in the two control animals receiving a Fluoro-Gold injection in the caudate nucleus and a cholera toxin injection in the putamen, plus two control animals that were injected with cholera toxin in the GPi. As expected, all observed SNc efferent neurons were dopaminergic neurons (TH+), although none of the projection neurons to the caudate/ putamen showed immunoreactivity for calbindin. This feature makes them particularly vulnerable to neurodegeneration. A very interesting and original finding of this study is that the only SNcd neurons exhibiting CB content are those that project through the nigro-extraestriatal pathway to innervate the GPi, possibly implying that these neurons are less vulnerable to parkinsonian neurodegeneration. These data minimize the possible role of a significantly increased axonal arborization as a distinctive feature of the dopaminergic vulnerability, demonstrate the relation between calbindin and vulnerability and very originally show the greater resistance of extra-striatal projection dopaminergic neurons, which could be important in the compensatory mechanisms of dopaminergic deficit and in the action of anti-parkinsonian drugs in PD. These results have been presented at the SENC2013 congress (Oviedo) and will be published in a Special Issue of Frontiers in Neuroanatomy co-edited by Drs. Obeso, Blesa and Lanciego, with focus on the selective vulnerability of dopaminergic neurons, along with other CIBERNED researchers as authors of several intended contributions. c. Objective 5. Organotypic culture and vulnerability cellular factor (González-Castaño, Matute) Different wild type Synuclein (Snca) and A30P, A53T and E46K point mutants were purified and used either directly or after fluorescent labeling with rhodamine to study entry in N2a neuroblastoma cells or cortical neurons. The results indicate that the wild type Snca and point mutants were very inefficiently transported into the cell. Moreover, Snca aggregated for 20 days resulted highly toxic to cells (N2a and primary neurons), but less so when aggregation was undertaken only for 3-5 days. To verify the cellular uptake of α-synuclein and its axonal transport we have used two different murine cell models: the primary cell culture (cortical neurons, astrocytes, oligodendrocytes, microglia) and organotypic culture of the nigrostriatal pathway (s. nigra-striatum-cortex). The human α-synuclein supplied in solution is incorporated into all cell types cultured. In a more complex cell culture system such as the organotypic, after injection into substantia nigra or striatum human α-synuclein is detected only in astrocytes and neurons. In no case anterograde axonal transport of synuclein was observed in this preparation of organotypic cultures nor with microfluidic techniques (AXIS) in primary neuronal cultures. Further experiments are needed to determine whether retrograde transport in organotypic cultures of striatal cortex exists, as well as transport in neuronal primary cultures with microfluidic techniques in the presence of human α-synuclein or Lewy bodies extracts. The results show that aggregated Snca is more toxic than Snca native and it is translocated to both neurons and glial cells. Furthermore, it has been shown that the Snca aggregation process leads to a protein that is not degraded by the proteasome and that inhibition of degradation of aggregated Snca was not due to the actual aggregation, but rather to the oxidation of Met in Snca. An interesting and novel finding is that Snca preferentially incorporates into cortex and striatum, and to a lesser extent in the substantia nigra. In addition, the incorporation of α-synuclein partially co-localizes with the cell death marker (Sytox). These results indicate that in response to oxidative stress a large majority of proteins are degraded by the proteasome, but certain proteins such as Snca exhibit inhibition of the degradation

246 2013 Annual Report 2013 Annual Report 247

justifying their tendency to accumulate and cause cellular dysfunction and death. Axonal transport mechanisms associated with toxicity at the soma level and its trans-synaptic progression requires further study, perhaps using protein aggregates from patients with PD (Recasens et al., Ann Neurol. 2014). d. Objective 6. Dopamine-glutamate striatal interaction and excitotoxicity and progression of Parkinson’s disease (Rodríguez-Díaz, González Castaño) We have studied the interaction dopamine-glutamate in striatum with microdialysis techniques. The reduction of striatal dopamine was accompanied with increased extracellular glutamate, inhibiting the glutamine synthetase and generating a massive and persistent accumulation of glutamate in astrocytes. These effects are followed with retrograde degeneration of glutamatergic neurons innervating the striatum, which is accompanied by astrocytosis and microgliosis in the regions in which the degenerating neurons have their soma. These phenomena have been particularly studied in the intralaminar nuclei of the thalamus, suggesting that degeneration of non- dopaminergic neurons in PD is triggered by the previous reduction dopaminergic and subsequent genesis of retrograde excitotoxic degeneration. These studies have been completed in vitro in human dopaminergic neurons differentiated from SH- SY5Y cells demonstrating that excitotoxicity can also affect dopaminergic neurons when glutamate acts on the neuronal soma with anterograde propagation. This type of excitotoxicity is dependent on the generation of free radicals, being prevented by intracellular ascorbate. These results suggest therapeutic strategies acting on excitotoxic mechanisms to modify the progression of the neurodegenative process in PD. e. Objective 7. Dopamine, Adenosine, Cannabinoids receptor interaction and Renin-Angiotensin system (Canela, Labandeira, Lanciego) We have shown the existence of heteromeric receptors for adenosine 2A (A2A), dopamine type 2 (D2) and cannabinoid type 1 (CB1) in the striatum of PD animal models (rat and primate), characterizing their structural changes as a result of neurodegeneration and the effect of levodopa associated with involuntary movements (e.g. dyskinesias). We have studied the allosteric modulation produced by the binding of a ligand to a receptor (A2A and/or CB1) on the binding of a ligand to the D2 receptor using the technique of radioligand binding, and the proximity ligation assay (PLA) to detect in situ the interaction between two proteins. Thus it was found that heteromeric A2A-D2-CB1 receptors in the brain striatum are abundant in the caudate of control monkeys treated with MPTP. On the other hand, treatment with L-DOPA wiped allosteric modulation between ligands and reduced the expression of heteromers. The most striking result was the disappearance of these heteromers in dyskinetic animals, both rats (Exp Neurol. 2014;253:180-91) and primates (Neuropharmacol. 2014;79:90-100). These results constitute the first evidence of the alteration in the expression of a heteromeric receptor in the striatum associated with the parkinsonian state. A the GPi level, we have documented the disappearance of these heteromers in the dyskinetic state. Similar results have been obtained to document changes in heteromeric A2A-CB1 and A2A- CB2 receptors in the GPi of dyskinetic primates. More recently, it has been shown the presence of heteromeric CB1 and GPR55 (a recently ‘deorphanized’ cannabinoid receptor) in the striatum of rats and primates, as shown in a recently submitted article (Martínez-Pinilla et al., submitted). Several recent studies by the Labandeira group have shown that receptors (GPCRs) of the renin- angiotensin system (RAS) are involved in the progression of neurotoxin-induced dopaminergic degeneration, acting as enhancer of oxidative stress (via activation of the NADPH oxidase complex) and the microglial inflammatory response. In this project we have studied the intracellular pathways involved in these processes. Thus, we have studied the role of PPAR-γ, Rho kinase, ATP- dependent mitochondrial channels or TNF-α in the effects of RAS GPCRs receptors on dopaminergic degeneration. The existence of angiotensin intracellular receptor in the nucleus and mitochondria in dopaminergic neurons has been categorized. We are currently characterizing its function using preparations of mitochondria and isolated nuclei and a broad methodological approach that includes mitochondrial respirometry, electron microscopy, etc. All together, these results show several previously unknown molecular mechanisms at the level of receptor interaction in both the SNc and striatum, which are affected by the dopaminergic deficit. While the precise meaning of these results will require further investigation, it can be argued that they represent a new dimension in the molecular pathophysiology of PD.

Project 9 Titulo: Generation of dopaminergic neurons from somatic cells in parkinsonian patients with cognitive failure The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Moratalla Villalba, Rosario CIBERNED, Cajal Institute CSIC, Madrid Vicario Abejón, Carlos CIBERNED, Cajal Institute CSIC, Madrid Pérez Castillo, Ana María CIBERNED, IIB CSIC-UAM, Madrid Ceña Callejo, Valentín CIBERNED, University of Castilla-La Mancha, Albacete Kulisevsky Bojarski, Jaime CIBERNED, Santa Creu i Sant Pau Hospital, Barcelona Vela, Lydia Associated group, Alcorcón Hospital, Madrid

The DA neurons (DAn) derived from iPS cells (iPSC, induced pluripotent stem cells) in parkinsonian patients with cognitive deficit may be useful to study molecular, cellular, functional and genetic aspects of PD and to lay the foundations for a cell therapy. Specific aims 1. To obtain iPS cells from age-matched parkinsonian patients and control subjects. We will focus on patients with mutations in the glucocerebrosidase gene (GBA) and microtubule-associated protein tau (MAPT), which, in addition to suffering Parkinson, show cognitive deficits. 2. To differentiate iPS cells to dopaminergic neurons, that will be characterized by their morphological, neurochemical, molecular, genetic, and functional features. To analyze the effect of the drugs pioglitazone and TDZD-8 on proliferation of neural stem cells (MNC) from mouse ventral mesencephalon and its differentiation into dopaminergic neurons.

248 2013 Annual Report 2013 Annual Report 249

3. To establish a cellular model of PD to study the impact of GBA and MAPT mutations in the processes and molecular mechanisms of dopaminergic neuronal death that occurs in PD. 4. Perform cell transplants in early postnatal mouse models and in parkinsonian adults to determine the degree of survival, differentiation, maturation and functionality of neurons derived from patients compared to those derived from controls. To study motor recovery in PD models. To determine the degree of differentiation and migration towards the damaged zone of endogenous neural precursors in injured mice after treatment with pioglitazone and TDZD-8.

Project 10 Title: Multicenter study of CSF biomarkers and neuroimaging in the continuum preclinical-prodromal Alzheimer disease (SIGNAL Study) The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Lleó Bisa, Alberto CIBERNED, Santa Creu i Sant Pau Hospital, Barcelona Bermejo Pareja, Félix CIBERNED, 12 de Octubre University Hospital, Madrid Cantero Lorente, José Luis CIBERNED, Pablo de Olavide University, Sevilla Calero Lara, Miguel Associated group, ISCIII - CIEN Foundation Frank García, Anna CIBERNED, La Paz University Hospital, Madrid Antúnez Almagro, Carmen Associated group, Virgen de la Arrixaca Hospital, Murcia Pastor Muñoz, María Asunción CIBERNED, CIMA, University of Navarra, Pamplona Sánchez-Juan, Pascual CIBERNED, Marqués de Valdecilla Hospital, Santander García Ribas, Guillermo Associated group, Ramón y Cajal Hospital, Madrid Boada Rovira, Mercé Associated group, ACE Foundation, Barcelona Yusta Izquierdo, Antonio Associated group, University Hospital of Guadalajara Martínez-Lage, Pablo Associated group, CITA Foundation, San Sebastián

Overall aim To search for new biomarkers in CSF and its relationship with other established markers in CSF, neuroimaging and genetic markers in early stages (pre-dementia) of AD. In order to do that, a national multicenter cohort aged 50-75 comprised of a group of healthy controls (GDS 1), subjective cognitive impairment (GDS 2) and slight cognitive impairment (MCI; GDS 3) will be recruited.

Specific aims 1. Characterize new biomarkers in CSF in the pre-dementia continuum of AD, in particular amyloidogenic pathway and inflammatory markers and analyze its relationship with already established markers in CSF (Aβ42, tau and p-tau). 2. Analyze the relationship between new markers and genetic (genotype APOE) and MRI neuroimaging markers. 3. Harmonize the methodology used in the application of biomarkers in AD (clinical, neuropsychological assessment protocols, genetic determination, collection and analysis of CSF and structural imaging (MRI)) across the spectrum of pre-dementia AD).

Results In the last year we have completed the recruitment of subjects for the SIGNAL project with a common protocol for clinical evaluation, neuropsychological examination, collection of biological samples (blood and CSF) and structural imaging (MRI 3 Tesla). Measures were agreed prior to recruitment on clinical evaluation and neuropsychological, pre-analytical conditions for collecting blood and CSF and MRI acquisition parameters. The total number of subjects recruited for the study was 132 (controls, SCI and DCL). All subjects have clinical, neuropsychological formal evaluation, CSF and blood sample with a protocol common to all centers. Due to variations in MRI facilities between centers we could only harmonize the neuroimaging protocol in two of the them so that the subjects with common neuroimaging study protocol finally amounted to 88 cases: 48 controls, 25 SCI, and 15 with MCI. The analysis of clinical and neuropsychological data is currently ongoing. Biological samples were sent to the centers responsible for processing (Blood - Marques de Valdecilla Hospital; LCR - Santa Creu i Sant Pau Hospital) whereas MR images were sent to the Pablo Olavide University for processing. At present, the APOE genotyping is being completed in all subjects. We have so far determined the core CSF biomarkers (Aβ42, tau and phosphorylated tau) in all samples and the determination of markers of APP processing (specifically BACE1, soluble forms of Aβ) and inflammatory (YKL-40) is also in the process of completion. On the other hand, analysis of MR structural imaging and its relation with the AD biochemical signature (Aβ42 levels, p-tau and tau in CSF) is being completed as well. Specifically, the different layers of hippocampus and white matter lesions are being measured, and their correlation with CSF biomarkers analyzed. We expect to complete these analyzes in the next two months in order to write a scientific manuscript for publication.

Project 11 Title: Emerging properties of neuron-glia relationship underlying neurodegeneration and dementia in Alzheimer’s disease The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Torres Alemán, Ignacio CIBERNED, Cajal Institute CSIC, Madrid Vicario Abejón, Carlos CIBERNED, Cajal Institute CSIC, Madrid Vitorica Ferrándiz, Francisco Javier CIBERNED, Pablo de Olavide University, Sevilla Comella Carnicé, Joan Xavier Associated group, ISCIII - CIEN Foundation Gutiérrez Pérez, Antonia CIBERNED, La Paz University Hospital, Madrid Moratalla Villaba, Rosario Associated group, CIBERNED, Cajal Institute CSIC, Madrid Boada Rovira, Mercé Associated group, ACE Foundation, Barcelona

250 2013 Annual Report 2013 Annual Report 251

The importance of astroglia as metabolic support of the neuron and protective barrier against oxidative stress, or as key cellular component (“brain macrophages”) of the immune system is beyond doubt. However, the more than likely distortions of bidirectional neuron-glia homeostasis underlying Alzheimer ‘s disease (AD) in particular are not well understood. The sum of these distortions, which ultimately are adaptive responses leads to “emergent properties” (phenotypic/functional abnormalities) of the system that result in the onset and gradual progression of pathology as well as clinical symptoms. In this project we propose to identify the mechanisms underlying the appearance of these emergent properties and their impact on the development of synapto-toxicity and subsequent neuronal death (likely basis of the dementia) using patient samples and animal models. The ultimate goal is to identify potential therapeutic targets to modulate the neuron-glia relationship and avoid/minimize neuronal degeneration and thus the clinical progression of AD. Specifically we pretend to evaluate specific aspects of the above cellular elements from a multidisciplinary and translational perspective. We will analyze separately the emergent properties of microglia, astroglia and neurons (combining studies in animal models and human material) bearing in mind the constant and close functional relationship between them. We propose the binomial Aβ/tau as the guiding thread of the emergent properties of the 3 cell types. Being aware of the enormous attention on the inflammatory process in AD and therefore intense competition, we propose to determine the impact of Aβ/tau in the process of microglial differentiation typical of AD (by in vitro manipulation after its characterization in regions with high impact pathological in patients with AD) as the first adaptive event that generates, together with Aβ/tau, emergent properties in astroglia and neurons through intercellular mediators (we will consider only cytokines and growth factors). We will look in astrocytes at the Aβ/tau-induced appearance (probably related with microglial TNFα) of an interaction between the phosphatase calcineurin (involved in inflammatory processes) and the FOXO transcription factor (involved in responses to oxidative stress) as well as adaptive changes in IGF-I neuroprotective factor signaling. We will also analyze in neurons the interrelationship between TNFα-induced survival mechanisms, neuronal death mechanisms (death receptor and autophagy), and neuroinflammation (Fas-ligand) as well as sinapto-toxicity processes that involve e.g. loss of synaptic plasticity, and IGF-I and BDNF sinapto-trophic processes. Methodologically we will emphasize cell models developed by consortium groups (neurons and astrocytes derived from iPS cells from patients with sporadic AD, “tet-off” and viral transduction systems) and corroborate the findings in models, including obtaining necessary descriptive data, in patient samples.

Project 12 Title: Mitochondrial dynamics and mitophagy as therapeutic targets in Parkinson’s Disease and Huntington The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Soriano García, Eduardo CIBERNED, University of Barcelona Vila Bover, Miquel CIBERNED, Vall d'Hebron University Hospital, Barcelona Trullás Oliva, Ramón CIBERNED, IDIBAPS-CSIC , Barcelona Alberch Vié, Jordi CIBERNED, University of Barcelona Tolosa Sarró, Eduardo CIBERNED, Clinic Hospital, Barcelona Mitochondrial dysfunction is a key process in the pathogenesis of many neurodegenerative diseases, including Parkinson’s disease (PD), and Huntington’s disease (HD). In addition to mitochondrial alterations associated with cellular respiration and ATP production, calcium homeostasis, and oxidative stress, recent studies suggest a second type of mitochondrial alterations that converge on two main processes for neuronal viability and physiology: mitochondrial transport and dynamics, and recycling of mitochondria by mitophagy. While we know some of the genes involved in these processes, the exact mechanisms that regulate mitochondrial dynamics and mitophagy in neurons, as well as their involvement in the pathogenesis of PD and EH - especially in early stages, remain unknown. The objective of this project is to dissect the role of mitochondrial proteins recently identified by the groups from this consortium as regulators of mitochondrial dynamics and mitophagy processes (Armcx/Armc10, Pentraxin1/NP1 and HTT family) and in the pathogenesis of PD and HD. Second, the potential therapeutic utility in treating these diseases will be determined. Three specific objectives are proposed to achieve that: 1. Dissect the molecular mechanisms by which Armcx/Armc10, NP1 and HTT genes regulate mitochondrial dynamics and mitophagy. 2. Characterize the pathological alterations in mitochondrial dynamics and mitophagy during PD and HD (in brains of patients as well as in disease cellular models, including fibroblasts and differentiated neurons from patients iPSCs) and their relationship to the mentioned genes. 3. Investigate whether manipulating the expression of these genes is able to reverse the mitochondrial deficits associated with PD and HD and restore neuronal function/viability both in vitro (fibroblasts and neurons derived from patient - iPSCs ) as well as in in vivo experimental models. The results of this project will help to decipher the role of abnormal mitochondrial dynamics and mitophagy in the pathogenesis of PD and HD and will be used to assess whether Armcx/Armc10, NP1 and HTT genes may represent new therapeutic targets for the treatment of these disorders.

Project 13 Title: Identificación y caracterización molecular de subpoblaciones de receptores cannabinoides en poliglutaminopatías The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Guzmán Pastor, Manuel CIBERNED, Complutense University of Madrid Fernández Ruis, Javier CIBERNED, Complutense University of Madrid McCormick, Peter Joseph CIBERNED, University of Barcelona Mena Gómez, Mª Ángeles CIBERNED, La Paz University Hospital, Madrid

Understanding the process of neuronal survival is essential for the characterization of the etiology and progression of neurodegenerative diseases and, therefore, for the rational design of therapies for treatment. In this context, in recent years the four groups of this consortium have studied how the cannabinoid system modulates these processes, and what may be the therapeutic soundness of cannabinoid receptors

252 2013 Annual Report 2013 Annual Report 253

as neuropharmacological targets. Specifically, the CB1 cannabinoid receptor is the most abundant mammalian brain metabotropic receptor and it is highly expressed in the basal ganglia and cerebellum, where it plays an essential role in controlling the motor activity and coordination, respectively. It has been reported that expression of the CB1 receptor decreases in basal ganglia of patients and animal models of Huntington’s disease (HD), the most studied neurodegenerative disease among all caused by triplet expansion. Although there have been detailed studies on the protective role of cannabinoid receptors in HD models, we still do not know the precise subpopulations of cannabinoid receptors involved in the neuroprotective effects. The existence of subpopulations for the CB1 receptor have recently been suggested by the discovery that this receptor has different efficacy to signal depending on its location on GABAergic and glutamatergic terminals and the possibility to form heteromers with other metabotropic receptors in transfected cells or tissues ex vivo. Therefore, the overall objective of this project is to identify and molecularly characterize subpopulations of cannabinoid receptors (CB1 and CB1- adenosine receptors or CB1-other cannabinoid receptors heteromers) and their potential as new targets to induce neuroprotection in poly-glutaminopathies. These goals can be broken down into the following specific objectives: 1. Studying the differential expression of the CB1 receptor heteromers and its neuroprotective role in GABAergic and glutamatergic terminals. 2. Studying the neuroprotective role of the CB1 receptor and its heteromers in murine models of two poly-glutaminopathies (HD and SCA3) and in animals in which the selective activation of neuronal populations is induced by the pharmacogenetics technique Designer Receptors Exclusively Activated by Designer Drugs (DREADD). 3. Studying possible alterations in the expression of CB1 and its heteromers in postmortem brain tissue from patients with two poly-glutaminopathies (HD and SCA3). These studies, which will combine the complementary expertise and capabilities of the four groups of the consortium will help to clarify the molecular basis and potential clinical relevance of two important issues: (i) whether the alterations of the cannabinoid system are involved in the pathogenesis of polyglutamine disorders, and (ii) whether the stimulation of the cannabinoid signaling promotes neuroprotection and thus delay the onset and/or attenuate the progression of these diseases.

Project 14 Title: Role of GSK-3β in the cortical circuits alterations occurring in Alzheimer’s disease The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Iglesias Vacas, Teresa CIBERNED, IIB CSIC-UAM, Madrid Ávila de Grado, Jesús CIBERNED, CBMSO CSIC-UAM, Madrid de Felipe Oroquieta, Javier CIBERNED, Cajal Institute CSIC, Center of Biomedical Technology, Madrid Naranjo Orovio, José Ramón CIBERNED, National Center of Biotechnology CSIC, Madrid Lleó Bisa, Alberto CIBERNED, Santa Creu i Sant Pau Hospital, Barcelona Alzheimer’s disease (AD) is the most common form of dementia and the risk for it increases with age, appearing 50-70% of cases in people over 65. This disease is currently incurable and is accompanied by progressive neurodegeneration, severe synaptic and neuronal loss, especially in the cerebral cortex. One of the major research topics in AD aims to elucidate the relationship between progressive cognitive impairment and its relation to the molecular, physiological and morphological alterations of cortical circuits that occur in the disease. It is generally considered that altered calcium levels and over-activation of glycogen synthase kinase-3β (GSK-3β) are directly related to the pathogenesis of AD, hence therapies aimed at modulating calcium homeostasis and/or inhibiting GSK-3β, applied in early stages of the disease, could have great potential for treatment. In this project we propose to analyze the changes/alterations associated with GSK-3β hyperactivity in the continuum of AD and its relationship to early alterations in neuronal calcium homeostasis, to identify those changes associated with disease in prodromal or preclinical stages that can help make an early diagnosis of AD. For our studies will be mainly use GSK-3β-overexpressing inducible mouse models as well as brain samples and cerebrospinal fluid (CSF) of patients at different stages of AD. This project tries to i) further our understanding of the molecular basis by which GSK-3β is involved in the etiopathogenesis of AD, ii) identify markers that assist in the early detection of the disease in the future in order to be able to intervene therapeutically before the appearance of greater signs of cognitive impairment.

Project 15 Title: Synaptic degeneration and dysregulation of adult neurogenesis in mouse models of neurodegeneration The groups involved in this project are as follows (coordinator in bold):

Principal Investigator Institution Fernández Chacón, Rafael CIBERNED, Santa Creu i Sant Pau Hospital, Barcelona Lucas Lozano, José Javier CIBERNED, 12 de Octubre University Hospital, Madrid Fariñas Gómez, Isabel CIBERNED, Pablo de Olavide University, Sevilla

It is widely accepted that neurodegenerative processes have, from the early stages of pathology, synaptic- type dysfunction. However, synaptic alterations are more difficult to analyze experimentally that the neuronal vulnerability, which contributes to the lack of information on how they can participate in neurodegenerative processes. In addition, there is recent evidence that synaptic activity can regulate adult neurogenesis processes thus contributing to the progression of certain symptoms in neurodegenerative disorders and to the possible brain palliative response to these processes. The identification, in recent years, of molecules involved in neurotransmitter release and related to various neurodegenerative diseases, has opened the possibility to study the connection between the maintenance of synaptic function and diseases such as Huntington’s disease (HD) or Parkinson’s disease (PD). Therefore, this consortium intends to study the relationship between synaptic activity and neurodegeneration, through a strategy consisting of the use of murine models which have altered three of these molecules: CSP-alpha, alpha-synuclein and

254 2013 Annual Report 2013 Annual Report 255

huntingtin in order to investigate 1) adult neurogenesis processes in the context of synaptic alterations, and 2) endoplasmic reticulum stress as a basic pathogenic mechanism in response to synaptic alterations. This consortium will study the relationship between synaptic activity, regulation of adult neurogenesis and neurodegeneration in several genetically modified mouse models: a model of polyglutamine toxicity (R6/1 mice with mutant huntingtin), a model of presynaptic degeneration (KO mice lacking Cysteine String protein-alpha (CSP-alpha)) and models with genetic modifications of the synaptic protein alpha-synuclein. Previous results suggest an intriguing connection, yet poorly studied, between huntingtin and CSP in neurodegenerative mechanisms. On the other hand, functional interactions between CSP-alpha and alpha- synuclein have been described and both indirectly affect neurotransmitter release by acting on the SNARE complex. Unpublished data indicate impaired hippocampal neurogenesis and susceptibility to epilepsy in CSP-alpha KO mice, in part due to a deficit of GABAergic inhibition. In this project we will investigate if they have effects on neurogenesis by studying neurogenic niches of the subventricular and subgranular zone. In particular, we will characterize the effects on neurogenesis of the degeneration of synaptic terminals that innervate the neurogenic niche and synaptic dysfunction in various models. In parallel, we will study the specific weight of the endoplasmic reticulum stress in neuronal death in our murine models and the ability to therapeutically reverse degeneration by pharmacological modulation of this process

International

2013 Annual Report 259

International Relations

Neurodegenerative diseases (ND) are responsible for mitigating states, largely untreated and are closely linked with age. Among these disorders, dementias are responsible for the greatest burden of disease, with Alzheimer’s disease and related disorders causing impairment of approximately 7 million people in Europe. This figure is expected to double every 20 years, due to the progressive aging of the population. Currently, healthcare and treatment of patients with some form of dementia in Europe, represents a cost around € 130 billion per year, according to estimations of the European Commission Joint Programming on Neurodegenerative Diseases. This highlights that age-associated neurodegenerative diseases constitute one of the main medical and social challenges facing our society. International scientific collaboration increases more and more, not only because of the availability of international funding and the drive of modern communication technologies, but also because science itself has become a truly international collaborative activity. In particular, the scope and scale of the problem of neurodegenerative diseases in today’s society require a global response to confront this great challenge and thus has been recognized by various international institutions such as the European Union (EU), the Organization for Economic Cooperation and Development (OECD), the World Health Organization (WHO), etc.), and the industrialized countries that constitute the G8. The leaders of governments, businesses and academia also recognize the need for a coordinated strategy to address this major global challenge for health systems. There is consensus among all stakeholders on the need to build capacities, infrastructures and R&D resources in the field of neurodegenerative diseases. There is also a pressing need for global participation and a commitment to a significant increase in investment in skills and resources to reduce the duration of these chronic brain pathologies and/or the number of people at risk. This budgetary effort should be accompanied by sound policies and legislative initiatives to encourage public-private partnerships. History has shown that collaboration between academic researchers, government agencies and pharmaceutical and biotechnology companies is an essential ingredient in promoting this type of ambitious initiatives, especially when resources are limited. In this context, in recent years CIBERNED, has given a boost to its relations with international organizations in the area of research in neurodegenerative diseases such as the EU Joint Programme for Research in Neurodegenerative Diseases (JPND) and the Network of Centres of Excellence in Neurodegeneration (CoEN), among other initiatives.

European Union Joint Programming on Neurodegenerative Disease Research (JPND)

The EU Joint Programming for Research on Neurodegenerative Diseases (JPND) is an innovative collaborative research initiative created to address the growing challenges posed by these disorders. The JPND is a pioneering example of joint programming for the promotion of research within the European Union aimed at scientific challenges requiring a response that exceeds the capacity of a single country, based on the alignment of national research programs devoted to these challenges. Its objective is to enhance the impact of research by aligning existing national research programs and the identification of common objectives whose scope would benefit from joint action. The JPND Scientific Advisory Committee has significant participation of two CIBERNED researchers, Drs. Jesús Avila and Jesús de Pedro, as well as Dr. Ángel Cedazo-Mínguez, from the Karolinska Institute in Stockholm and member of the CIBERNED Scientific External Advisory Committee. The Research Strategy designed by JPND provides a framework for future investments and shows that the research effort within the European Union can be leveraged to improve care on prevention, diagnosis and treatment of patients suffering from these diseases. To achieve impact there is a need to encourage novel as well as multidisciplinary approaches, and to strengthen and extend existing capabilities across the full spectrum of basic, clinical, health and social care, and translational research. A number of thematic priorities for future research have been identified: • The origins of neurodegenerative diseases (ND): Further knowledge is needed regarding the causes of specific ND, the factors that determine people’s risk and resilience, and the triggering events leading to illness. • Disease mechanisms and models: A better understanding of the underlying disease mechanisms is required to underpin the development of new diagnostic and therapeutic approaches, as well as to identify appropriate time-windows for intervention. • Healthcare and social care: At present there is inefficient co-ordination between health and social care systems in individual countries, needing an evaluation of the equity of access to, as well as the effectiveness and cost-effectiveness of, pathways to diagnosis, treatment, care and support for ND across Europe. • Disease definitions and diagnosis: Standard clinical assessments fail to capture the presumed complexity of common ND, needing refinement and updating of the current diagnostic criteria. • Treatment and prevention: Progress in the identification of new therapeutic targets and drug development against them will be further strengthened through the promotion of a two-way connection between studies in cell and animal models as well as in patients To ensure progress of scientific priorities described above, will also require the development of a series of complementary activities: • Knowing our research capability, in order to identify both research gaps and those opportunities that can be addressed through improved co-ordination and investment. • Supportive infrastructure and platforms, such as integration and harmonisation of data and equipment, and promote an open-access approach to their use. • Working in partnership with industry, specially from the pharmaceutical, diagnostic and biotechnology sectors. • Working with regulatory organizations, to promote effective translation of research and find patient benefits. • International partnership beyond Europe, recognizing that clinical research and the social impact of diseases ND is a global problem, and therefore may be opportunities to join research efforts that are taking place around the world in this area. • Capacity building, through strengthening of certain areas of research that lack adequate development and the establishment of networks across and between disciplines and researchers • Education and training, based on a good understanding of the disorder, the patient needs characteristic of these conditions, and the available evidence-based options for treatment. • Connection to policy makers, by establishing a framework through which they can detect and define relevant issues, to be considered by national policies and their alignment and engagement with major international initiatives. • Communication and outreach, in order to achieve an effective translation across the different sectors of society. This Research Strategy also provides a framework of opportunities for countries involved in JPND and willing to participate in joint actions, which will be implemented through co-operative activities that realign or link national investments to achieve increased impact, and the provision of new funding. A guiding principle for its delivery will be that the research to be supported is of the highest scientific quality.

260 2013 Annual Report 2013 Annual Report 261

In this regard, during 2011 took place the first call for European research projects JPND. Under the theme “Optimization of biomarkers and harmonization of their use in the clinic”, a total of four transnational projects were awarded for the period 2012-2014. As quoted below, two of the approved projects have participation CIBERNED research groups.. BIOMARKAPD: Biomarkers for Alzheimer’s disease and Parkinson’s disease Coordinator: Bengt Winblad, Institutet Karolinska, Sweden CIBERNED Participation: Groups A. Lleó y J. Sáez-Valero DEMTEST: Biomarker based diagnosis of rapid progressive dementias-optimisation of diagnostic protocols Coordinator: Inga Zerr, University Medical Center Göttingen, Germany CIBERNED Participation: P. Sánchez-Juan (Group J. Berciano) and M. Calero (Group J. de Pedro) During the year 2013 the second JPND call for collaborative research projects was launched, with a focus on two thematic areas of particular interest identified in the aforementioned research strategy was resolved: 1) Risk factors (genetic, epigenetic, environmental) for the development of neurodegenerative diseases such as Alzheimer’s; and 2) Strategies for health and social care for people living with these debilitating diseases. This call for projects was funded with a total of 29 million Euros to be distributed between the two areas. 19 million for the thematic area 1 and 10 million for thematic area 2. A total of five transnational projects were awarded for the period 2013-2015, one of which has participation from CIBERNED: COURAGE-PD: COmprehensive Unbiased Risk factor Assessment for Genetics and Environment in Parkinson‘s Disease Coordinator: Thomas Gasser, University of Tubingen, Germany CIBERNED Participation: Group E. Tolosa It is important to underscore that the Spanish participation in this call has been made possible by a financial effort from CIBERNED, which has entirely funded it from its own budget, in exchange for having to halve the allocation for intramural cooperative projects that have been so effective for the promotion of CIBERNED translational activity and to fulfill the objectives in past years. This shows CIBERNED responsibility and interest in maintaining relationships and international commitments in this area. We hope this is a temporary situation and that in the near future we do not find ourselves in the dilemma of having to choose between national and international calls, both necessary and complementary.

Centers of Excellence in Neurodegeneration (CoEN)

A major obstacle to the advancement of research on neurodegenerative diseases is the relative lack of common standards and mechanisms for validation of potentially relevant results in preclinical studies, and clinical studies based on population. One approach to deal with these challenges on a large scale is through more effective use of large centers and institutes, where there is already the necessary critical mass of resources and expertise. Increased collaboration between national centers of excellence should also provide the opportunity to accelerate progress in understanding the basic mechanisms of disease, and the identification of new therapeutic approaches. To this end, on June 10, 2010, the Canadian Institutes of Health Research (CIHR), the German Centre for Neurodegenerative Diseases (DZNE, Germany) and the Medical Research Council (MRC, UK) launched a funding initiative to establish a collaborative approach to research in neurodegenerative diseases, called “Centers of Excellence in Neurodegeneration (CoEN)”. These founding members were later joined by other European institutions and thus, in December 2011 the CoEN membership application by CIBERNED-CIEN Foundation was approved, recognizing the scientific excellence in both basic and clinical science of the institution which became part of the CoEN Oversight Group. Current CoEN members are: • Canadian Institutes of Health Research (CIHR) • Deutsche Zentrum für Neurodegenerative Erkrankungen (DZNE, Germany) • Medical Research Council (MRC, United Kingdom) • Flanders Institute of Biotechnology (VIB Flanders, Belgium) • Health Research Board (HRB) / Science Foundation Ireland (SFI), Ireland • Ministero della Salute (MDS, Italy) • Centre of Excellence for Brain Research, Slovakia • CIBERNED-CIEN Foundation, Spain The overlapping of the CoEN group members with those of the JPND will ensure that their complementary objectives progress in close cooperation with each other. This is accomplished through a two-step process, involving expert workshops for the analysis of needs, followed by a call for proposals for collaborative teams between PIs within the participating national Centers of Excellence. Since 2012, CIBERNED and CIEN Foundation form part of the Oversight Group to actively participate in the design of CoEN future scientific strategy. Both institutions are represented in the CoEN Oversight Group by Dr. Miguel Medina, CIBERNED Deputy Scientific Director and member of the Scientific Advisory Committee of the CIEN Foundation. The first phase of the CoEN initiative began in late 2010 and has been aimed at the development of common resources and methodological approaches that underpin future studies. Some of the key issues addressed are: the development and validation of cell and animal models of disease; development of new measures to define subgroups of patients for clinical studies; identification of new biomarkers to support translational research; the development and harmonization of the battery of cognitive tests for the diagnosis and monitoring of disease progression; and the establishment of common platforms for improved data analysis and exchange. Phase II of the initiative was launched during the year 2013, with the aim of catalyzing collaborative research between centers with a critical mass of resources and expertise to thus promote a radical change in research on neurodegeneration. To do this, the countries participating in CoENs contributed a total amount of 5,5 million euros (of which Spain has provided 600,000 euros) in this call to establish an innovative and progressive research program to address the major challenges in this field. The call is intended to encourage the community to think outside the pre-established frameworks and stimulate new and creative approaches and solutions to the challenges of research in neurodegeneration. This call of Pathfinder projects intends to combine the strengths of research groups through Centers of Excellence in at least two partner countries to provide a truly collaborative effort and value that will advance our approach to research neurodegeneration. The projects would address issues that are not easily financed through standard grant mechanisms from CoEN partners, and is expected to further

262 2013 Annual Report 2013 Annual Report 263

collaboration between Centers of Excellence, the projects would also serve to provide a platform for future collaboration with industry. A total of eighteen project applications were submitted, eight of which included a CIBERNED research group (as co-ordinator in four of them). The result of the evaluation of these applications by an international panel of experts resulted in the approval of five research projects, three of which inlude Spanish participation (which is an excellent 37.5% success rate for CIBERNED applications) and a total committed funding for two years of 570,000 euros. The three projects involving CIBERNED are described below: - Title: WNT signaling: biomarker and target evaluation in Alzheimer’s disease Participant groups: Antonio Cuadrado (coordinator, CIBERNED), James Woodgett (Canada) and Simon Lovestone (UK) The invariant failure of Biopharmaceutical pipelines to modify Alzheimer’s disease (AD) progression indicates the need for new creative solutions beyond currently active areas of genetics, amyloid-β, and Tau pathology. Very recent work from our groups and others indicate that novel elements of the canonical and noncanonical WNT signaling pathways could be targeted to modify and monitor AD progression. In this project we will use multidisciplinary approaches, including brain oriented drug screening, a novel proprietary mouse model of AD, new genetic and pharmacological tools for modulation of WNT/GSK3 and WNT/JNK pathways, human blood samples from our and the IMI/ EMIF platform to identify novel biomarkers of AD progression and a pilot study on metabolomics, to explore the impact on AD of novel WNT elements including but not limited to clusterin, DKK1 and Nrf2. Our results in this pathfinder phase with the identification of new candidate targets and drugs for clinical studies will pave the way to therapeutic innovations in the long-term programme. Indeed, several Biopharma companies have initiated drug development programs aimed at targeting certain components of the WNT signaling pathway, including ligands and other small molecule regulators of Axin1and GSK-3, and Nrf2 that will be interested in our findings. - Title: In vivo neuronal cell reprogramming for a new regenerative approach in Parkinson’s disease Participant groups: Vania Broccoli (coordinator, Italy), Alexander Dityatev (Germany) and José Luis Lanciego (CIBERNED, Spain) Neurodegenerative diseases cause a significant burden on the elderly population in Europe. Parkinson’s disease (PD) affects 1,2 million people in Europe and with the increasing life expectancy this number will rise, putting more pressure on health care. Treatment of PD is only symptomatic, and therefore, there is an urgent need for more efficient therapies. Degeneration of mesencephalic DA neurons triggers the initial phases of PD, which raises the concept that cell replacement might represent a long-term restorative option for this neuropathology. Indeed, previous studies in PD patients have indicated that cell therapy has the potential to significantly sustain an enduring symptomatic relief. However, these studies suffered for lacking an ideal source of transplantable human DA neurons. We have recently developed a methodology that promotes transdifferentiation of mouse and human fibroblasts into functional induced dopaminergic neurons (iDANs), which display sophisticated neuronal properties including pacemaking firing activity, synaptic integration, and activity-dependent dopamine release. Therefore, iDAN cells offer an unprecedented cellular source with ideal features for cell therapy in PD, since they can be generated from the patients in high amounts. Here, we propose to push forward this technology by elaborating methods of direct in vivo reprogramming to further induce in situ local neuronal transdifferentiation in relevant animal models of PD (mouse and monkey). - Title: microRNA as novel therapeutic targets and disease biomarkers in Alzheimer’s Disease, Frontotemporal dementia and Amyotrophic lateral sclerosis (NEURO-MIR) Participant groups: Jochen Prehn (coordinator, Ireland), Andre Fischer (Germany), Pierre Lau (Flanders, Belgium), José Lucas (CIBERNED, Spain) Alzheimer’s disease (AD), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are distinct neurodegenerative disorders, but share common disease pathways and susceptibility genes. Recent data suggest that deregulation of epigenetic mechanisms including non-coding RNAs contribute to the pathogenesis of these disorders. miRNA regulate up to 30% of all protein-coding genes, with each miRNA controlling multiple, often hundreds of proteins. Due to their unique biological function and in vivo-stability, miRNA is arguably one of the most interesting area for drug and biomarker development. NEURO-MIR partners have identified neuronal miRNAs (miR- 29,-34c,–134) that modulate synaptic activity, network (hyper)excitability, protein processing and degradation, autophagy and apoptosis. However, their complex biology makes effects of miRNA and miRNA manipulation difficult to predict, and their potential as therapeutic targets and biomarkers has not yet been systematically studied. NEURO-MIR aims to explore the full potential of miRNAs as key contributors to disease progression and as therapeutic targets and biomarkers. NEURO-MIR also realises that the complexity of miRNA biology can only be tackled through bioinformatics and computational modelling approaches, and will develop a systems biology platform that will allow for the first time to approach the role of miRNA in neurodegeneration on a true systems level. Moreover, on November 29, 2013, the CoEN Oversight Group brought together leaders of the 15 innovative supported initiatives with representatives of the pharmaceutical industry, including Pfizer, Sanofi, GSK, MSD, Lilly and others. The result of this meeting exceeded expectations. Industry representatives expressed their deep satisfaction of being able to engage in conversation and exchange with academic leaders. They emphasized the fact that innovative collaboration supported by the CoEN initiative research was exactly the type needed to provide new insights and avenues to explore possible solutions to dementia activity, and said CoEN was “an important piece in the overall portfolio of innovation needed to address this critical public health challenge“. Finally, a novelty in 2013 has been the combination of the IX International Symposium on Advances in Alzheimer’s disease, which annually promoted the Queen Sofia Foundation and CIEN Foundation, with the 7th Scientific Forum CIBERNED, that each year brings together more than 500 researchers that constitute CIBERNED. A more detailed description of this event can be found in the following section.

International Congress for Research and Innovation in Neurodegenerative Diseases (CIIIEN)

During 23rd and 24th of September 2013, on the occasion of the World Alzheimer’s Day celebrated on September 21, was held in Madrid the first International Congress for Research and Innovation in Neurodegenerative Diseases (CIIIEN) promoted by the Queen Sofia Foundation in collaboration with the CIEN Foundation. The main objective of CIIIEN is to have a meeting forum where to share progress and relevant information about neurodegenerative disease among the scientific community The CIIIEN arises from the unifying of the two major scientific conferences on neurodegenerative diseases in general, and in particular Alzheimer’s disease, organized in Spain: the IX International Symposium on Advances in Alzheimer’s disease, which annually promoted the Queen Sofia Foundation and CIEN Foundation, and the 7th CIBERNED Scientific Forum, which annually brought together more than 500 researchers that constitute CIBERNED. The unification of the two events is a first step in creating a new

264 2013 Annual Report 2013 Annual Report 265

operating structure in the two main institutions involved in research of neurological and neurodegenerative diseases in Spain: CIEN Foundation and CIBERNED, both under the Ministry of Economy and Competitiveness through the Institute of Health Carlos III. This new structure seeks greater effectiveness and efficiency in research, promoting the interaction of the different research groups. The first edition of CIIIEN was chaired by Her Majesty the Queen and the scientific program consisted of three main blocks: Alzheimer’s disease, Parkinson’s and Huntington’s diseases, and neuromuscular diseases. Among the speakers at the Congress were include some international researchers that are reference in its field of research such as: Kenneth S. Kosik (University of California, Santa Barbara), Lawrence Goldstein (University of California, San Diego), Steven Finkbeiner (University of California, San Francisco), David J. Brooks (Imperial College, London), Davide Pareyson (Neurological Institute C. Besta, Milan) and Yves Agid (Pitié-Salpêtrière Hospital, Paris). Thus, this event establishes on its first edition as a meeting point for the world’s leading experts in neurodegenerative diseases, allowing sharing of knowledge, working methods, new developments and discoveries in a field in which international cooperation between institutions and is becoming increasingly important for obtaining optimal results in research.

European Month of the Brain

The European Commission designated May 2013 as the European Month of the Brain and on this occasion CIBERNED, in collaboration with the Department of Molecular Neurobiology of the Center for Molecular Biology Severo Ochoa, and the Center for Networked Biomedical Research on Mental Health (CIBERSAM), jointly organized a scientific symposium with the participation of George Perry, Professor at the University of Texas and Chairman of CIBERNED Scientific Advisory Committee; Dr. Josep M. Haro, CIBERSAM researcher; and Prof. José A. Esteban, Center for Molecular Biology Severo Ochoa. The title of the lecture by Prof. Perry was “Is Alzheimer’s disease a metabolic disorder?”. So far there is no effective treatment to prevent or cure Alzheimer’s, the most prevalent dementia in both the Spanish society and the world. Researchers are looking for new risk factors to identify strategies for the prevention and treatment of this disease, lately focusing on metabolic syndrome and obesity as having a possible role in the development of this dementia. Dr. Haro’s lecture was titled “The ROAMER project, towards a research agenda, in Coordinated mental health in Europe.” The Roamer project, funded by the European Commission 7th Framework Programme (FP7), aims at developing a research plan for the comprehensive and integrated mental health, oriented to translational research, that helps to reduce the enormous impact of mental disorders in European society, increasing knowledge about the factors that contribute to it, on how to prevent them and how to improve the efficiency and effectiveness of treatments. There are clear indications that research funding for mental health disorder is much lower than the corresponding impact on health. Finally, in his presentation entitled “PIP3 signaling at the synapse and its relevance for Alzheimer’s disease”, Dr. Esteban described the intracellular signaling pathways that regulate the function of synaptic connections in the brain. The continuous remodeling of synapses is critical for the establishment and maturation of neuronal circuits in the brain and also to memory and learning. Indeed, an early pathological event in Alzheimer’s disease is the appearance of subtle alterations in synaptic efficacy in the hippocampus, leading to a marked memory impairment in the earliest clinical stage. Recent studies in this lab have shown that signaling pathways mediated by lipids are altered in synapses in Alzheimer’s disease. This research opens the possibility to manipulate these mechanisms therapeutically in order to reverse the synapses to their physiological state and alleviate cognitive impairment. Bilateral UK-Spain Meeting

On May 8 was held in the Queen Sofia Foundation Alzheimer Center a meeting between representatives and researchers from CIBERNED, CIEN Foundation and Queen Sofia Foundation together with members of the scientific section of the British Foreign Office in which a round table was held on dementia attended by Robin Grimes, head scientific advisor of the British Foreign Ministry, Jesús Ávila, CIBERNED scientific director, and Pablo Martínez, scientific director of the Alzheimer Project Research Unit (UIPA) of the Queen Sofia Foundation. Professor Grimes, presented attendees with the British program to improve the treatment of dementia in the United Kingdom (UK Dementia Challenge) promoted by British Prime Minister David Cameron last March. In a country in which dementia affects about 670,000 people we are living a “historic” moment, said Grimes, because it is “the first time a Premier makes this problem a priority.” Jesús Ávila, explained to members of the British Ministry the nature and implications of the association CIEN Foundation-CIBERNED-Queen Sofia Foundation, that complements the institutional and financial support of the latter with the multidisciplinary study of human subjects in CIEN Foundation and basic and translational research on animal models in CIBERNED. Synergies such as this, he said, can make qualitative progress in neurodegeneration research, and specifically Alzheimer’s, as it is essential to establish “earliest markers and make the diagnosis in the symptomatic phase to succeed.” Finally, Pablo Martínez focused his intervention in the operation and projects associated with the Research Unit he heads, focusing primarily on the Vallecas Project, which for five years is studying volunteers aged between 70 and 85 years, cognitively healthy, to obtain biological, clinical and neuroimaging data in order to try to identify biological markers of disease. In this sense, Martínez wanted to highlight “the need for social collaboration in research in Alzheimer’s to move towards early detection of the disease,” crucial for improving the quality of life and treatment of those affected by it. Attendees included also Matthew Desoutter, director of Department of Economic Growth of the British Embassy; Hakim Yadi, head of Life Sciences sector from UK Trade & Investment; Arturo Coello, secretary of the Queen Sofia Foundation; José Ignacio Fernández, program coordinator of the Directorate General of the Elderly of the Department of Social Affairs of the Region of Madrid; Mª Ángeles Pérez, managing director of CIEN Foundation; Miguel Medina, deputy scientific director of CIBERNED; Laura Fernández, managing director of the Alzheimer Center Queen Sofia Foundation; and CIBERNED researchers José Ramón Naranjo and Javier de Felipe. After the panel discussion, attendees visited the facility and met in detail the operation and structure of the Queen Sofia Foundation Alzheimer Center and the Alzheimer Project Research Unit.

Tau Workshop

It has been over a quarter century since the discovery in the mid-1980s that the paired helical filaments of neurofibrillary tangles were made up of abnormally hyprphosphorylated tau. A decade earlier tau had been first isolated from porcine brain as a heat stable protein essential for microtubule assembly. The following years clearly established tau protein as a microtubule-associated protein (MAP) that under physiological conditions regulates microtubules (MT) assembly, dynamic behaviour, and spatial organization, and has also been shown to regulate the axonal transport of organelles, including mitochondria. Further studies during the following couple of decades revealed the importance of post-translational modifications, mainly phosphorylation but also truncation, in tau function and dysfunction. Prominent tau pathology is present in

266 2013 Annual Report 2013 Annual Report 267

a number of neurodegenerative disorders, predominantly within the neuronal compartment, but also within glial cells. Because of this shared histopathological feature, they are referred collectively as tauopathies, although they constitute a group of etiologically heterogeneous, clinically and neuropathologically overlapping disease entities. Clinicopathological studies have shown that tau pathology is associated with progressive neuronal loss and cognitive decline. In the brains of AD patients, tau pathology propagates following an anatomically defined pattern described by the commonly used neuropathological Braak sequential staging. Mounting evidence strongly suggests that accumulation of abnormal tau might be mediated through spreading of seeds of the protein from cell to cell and point to the involvement of extracellular tau species as the main agent in the interneuronal propagation of neurofibrillary lesions and spreading of tau toxicity throughout different brain regions in these disorders. That would support the concept that pathology initiates in a very small part of the brain many years before becoming symptomatic, spreading progressively to the whole brain within 10-20 years, reminiscent of some features of the prion protein propagation mechanism. Precisely, this prion-like behavior of tau prompted us together with Karen Duff and Ken Kosik to organize a workshop entitled “Is tau a protein-like protein? Implications for physiology and pathology”, held in Madrid on October 28-30, 2013 as part of the Cantoblanco Workshops in Biology (http://www.cbm.uam.es/tau). This workshop brought together world class experts to discuss up-and-coming areas in the tau field that may reveal novel mechanisms and targets for therapeutic intervention, inspiring new insights and approaches in the battle against neurodegenerative diseases. Session topics included biochemistry and processing of tau in physiological and pathological conditions; cell-to-cell tau propagation and in vivo spreading; novel therapeutic approaches including tau immunization. The list of invited speakers for this workshop included Khalid Iqbal, Eckhard Mandelkow, Susanne Wegmann, Akihiko Takashima, Alejandra Alonso, Illana Gozes, Ratan Bhat, Laura Sayas, Ana María Cuervo, Amritpal Mudher, Natura Myeku, George Perry, Garth Hall, Luc Buée, Maria Grazia Spillantini, Emmanuel Planel, Alberto Rábano, Eva-Maria Mandelkow, Jean Pierre Brion, Naruhiko Sahara, George Bloom, Jurgen Götz, Einar Sigurdsson, Norbert Zilka, Rakez Kayed, and Marc Diamond. A compilation of contributions from some of the speakers related to the topic of the symposium served to compose a special issue of the Journal of Alzheimer’s Disease, published in early 2014.

Scientific Productivity and

2013 Annual Report 271

Analysis of CIBERNED Scientific Productivity in 2013

To complete this section we provide a table of bibliometric indicators for 2012 and 2013. A quick analysis allows to observe a strengthened of the improved scientific production in 2013 (in line with the ongoing historical improvement observed in the last years since the creation of the Centre), not only in quantity but also especially in quality (percentage of publications in quartiles 1 and 2, number of publications in high impact journals, average impact factor, international collaborations).

Indicator 2012 2013 % change

(A) Total number of publications1 593 572 -6.91 (B) Number of publications in quartiles (Q) 1 y 2 489 479 -2.04 (C) Number of publications in Q1 y Q2 as percentage of total (A) 82.46 86.78 5.23 (E) Number of publications in 1st decile 153 141 -7.84 (F) Number of publications (1st decile) as percentage of total (A) 25.80 25.54 -1.00 (G) Number of publications in Q1 (incluye 1st decile) 368 330 -10.33 (H) Number of publications in Q1 as percentage of total (A) 62.06 59.78 -3.67 (I) Number of publications in Q1 as percentage of total Q1 and Q2 (B) 75.26 68.89 -8.45 (J) Number of publications in Q2 121 149 23.14 (K) Number of publications in Q2 as percentage of total (A) 20.40 26.99 32.29 (L) Number of publications (Q1 + Q2) led by CIBERNED groups (1st author or corresponding 263 244 -7.22 author) (M) Number of publications in Q1 and Q2 led by CIBERNED groups as percentage of total (B) 53.78 50.94 -5.29 (N) Number of publications (Q1 and Q2) not led by CIBERNED groups (1st author or 226 234 3.54 corresponding author) (O) Number of publications in Q1 and Q2 not led by CIBERNED groups as percentage of total 46.22 48.85 5.70 (B) (P) Number of publications in Q1 and Q2 in which “CIBERNED” appears in the afiliation (as 51.33 59.50 15.92 percentage of total (B)) (Q) Number of publications in Q1 and Q2 led by CIBERNED groups in which “CIBERNED” 78.71 73.36 -6.79 appears in the afiliation (as percentage of total (L)) (R) Number of publications in Q1 and Q2 not led by CIBERNED groups in which “CIBERNED” 74.78 66.24 -11.42 appears in the afiliation (as percentage of total (N)) (S) Number of publications in Q1 and Q2 with international groups 131 165 25.95 (T) Number of publications in Q1 and Q2 with international groups (S) as percentage of total 26.79 34.24 27.80 number of publications (B) (U) Number of publications in Q1 and Q2 with international groups, and led by CIBERNED 53 67 26.42 groups (V) Number of publications in Q1 and Q2 with international groups, and led by CIBERNED 40.46 40.61 0.37 groups as percentage of all international publications (S) (W) Number of publications in Q1 and Q2 with other CIBERs 26 31 19.23 (X) Percentage of publications in Q1 and Q2 with other CIBERs led by CIBERNED groups 65.38 35.48 -45.73 (Y) Number Percentage of published in Q1 y Q2 1 0 -100.00 (Z) Percentage of Percentage of led by CIBERNED groups 60 100 66.67 Indicator 2012 2013 % change (A2) Addition of impact factors of journals (Q1 y Q2) in which articles have been published 2759.625 2741.119 -0.67 (B2) Average of impact factors of journals (Q1 y Q2) in which articles have been published 5.64 5.51 -2.30 (C2) Number of publications in journals with impact factor >15 11 15 36.36 Number of publications in Q1 and Q2 with 2 CIBERNED groups 57 61 7.02 Number of publications in Q1 and Q2 with 3 CIBERNED groups 11 10 -9.09 Number of publications in Q1 and Q2 with 4 CIBERNED groups 2 1 -50.00 Number of publications in Q1 and Q2 with 5 CIBERNED groups 0 1 100.00 Number of publications in Q1 and Q2 with 6 CIBERNED groups 1 1 0.00 Cooperative projects – Program 1 6 8 33.33 Cooperative projects – Program 2 7 9 28.57 Cooperative projects – Program 3 2 3 50.00 Program 1 cooperative projects 112 90 -19.64 Program 2 cooperative projects 138 123 -10.87 Program 3 cooperative projects 58 49 -15.52 Projects with other CIBERs/RETICs/CIEN networks 34 31 -8.82 Basic-type projects 163 144 -11.66 Translational-type projects 145 118 -18.62 Projects with other national groups 148 139 -6.08 Projects with other international groups 92 50 -45.65 Projects with industry 27 24 -11.11

1 Number of publications indexed in ISI (Web of Knowledge).

272 2013 Annual Report 2013 Annual Report 273

Some figures are included below to summarize some of the main bibliometric indicators on CIBERNED’s scientific production during 2013:

Ciberned 2013 Publications

Q1 60 %

Q2 27 %

Others 13 %

CIBERNED 2013 Publications included in the first decile

D1 26 % Others 74 % Journals with 5 or more CIBERNED publications in 2013

PLoS ONE Movement Disorders Parkinsonism & Related Disorders Neurobiology of Aging Journal of Alzheimer’s disease Neurology Neuropharmacology European Journal of Neurology Brain Neurobiology of Disease Journal of Neurology Experimental Neurology Biochimia et Biophysica Acta Journal of Neurology, Neurosurgery & Psychiatry Human Molecular Genetics Glia Cell Death & Disease Lancet Neurology Journal of Neuropathology and Experimental Neurology Journal of Neurochemistry Journal of Neural Transmission JAMA Neurology Current Pharmaceutical Design 0 5 10 15 20 25 30 35 40

Most common subject categories (ISI -WoK-JCR ) in CIBERNED 2013 publications

Clinical Neurology Neurosciences Biochemistry & Molecular Biology Pharmacology & Pharmacy Biology Geriatrics & Gerontology Medicine Multidisciplinary Sciences Cell Biology Chemistry, Medicinal Pathology Psychiatry Genetics & Heredity Chemistry, Multidisciplinary Surgery Immunology Biotechnology & Applied Microbiology

0 20 40 60 80 100 120

274 2013 Annual Report 2013 Annual Report 275

Transversal Programs

Neurological tissue banks

1. Neurological Tissue Bank and Biological Samples of the IDIBEL Institute of Neuropathology, Bellvitge Hospital (Head: Dr. Isidre Ferrer). 2. Tissue Bank for Neurological Research (BTIN) and CIEN Foundation (Head: Dr. Alberto Rábano). 3. Navarra Neurological Tissue Bank (Head: Dr. María Teresa Tuñón). 4. Neurological Tissue Bank (TNB) at the University of Barcelona (Head: Dr. Eduardo Tolosa). 5. Vigo Neurological Tissue Bank (TNB) (Head: Dr. Carmen Navarro).

Other platforms

- DNA Analysis Service (Head: Dr. Jordi Pérez Tur). - Electron Microscopy Service (Head: Dr. José Manuel García Verdugo). - Neuroimaging Service (Head: Dr. José Luis Cantero Llorente). Events and other activities

CIBERNED has organized or participated in a number of events during the year, particularly worth mentioning is the VII Annual Scientific Forum (held in Madrid on September 23-24) and the Meeting with Associations of Relatives of Alzheimer Patients held in conjunction with the Alzheimer’s World Day (September 21), as well as other scientific, outreach and international cooperation events.

VII CIBERNED Scientific Forum

The objective of this annual Forum is to become a meeting point between CIBERNED researchers and collaborators, where they can present, share and disseminate the most relevant research in neurodegenerative diseases over the past year. New in 2013 was the combination of the IX International Symposium Advances in Alzheimer’s disease, which has been annually promoted by the Queen Sofia Foundation and the CIEN Foundation, together with CIBERNED’s VII Scientific Forum, which annually brings together the more than 500 researchers constituting CIBERNED. Thus, during 23 and 24 September 2013, on the occasion of World Alzheimer’s Day that is celebrated on September 21, was held in Madrid the first International Congress for Research and Innovation in Neurodegenerative Diseases (CIIIEN). A more detailed description of this event is available at the International Relations section of this report.

Alzheimer Social Forum

On the occasion of World Alzheimer’s Day on September 21 CIBERNED, in collaboration with CIEN Foundation and Queen Sofia Foundation, met at the Melia Castilla Hotel in Madrid with associations of relatives of Alzheimer’s disease patients and several researchers for the presentation of the documentary “Dialogues on Alzheimer’s” by M. Ángeles Pérez, managing director of the CIEN Foundation and CIBERNED. Afterwards there was a discussion between patients and associations moderated by Dr. Alberto Lleó, a CIBERNED researcher and Clinical Head of the Memory of the Neurology Hospital de la Santa Creu i Sant Pau Barcelona, a summary of which is available at the following link: http://ciberned.es/es/noticias/517-el-alzheimer-un-problema-de-todos.html.

Excellence in Neurodegeneration Seminar Series During 2013 CIBERNED has launched a new initiative in conjunction with the Queen Sofia Foundation and CIEN Foundation: the lecture series “Excellence in Neurodegeneration Seminar Series”, offering seminars by leading international experts in their areas of research on all aspects of cutting-edge research in neurodegenerative diseases (molecular, cellular, genetic, cognitive, clinical, animal models, biomarkers, neuroimaging, etc.) and related areas. Taking advantage of the designation of May as the “European Month of the brain” by the European Commission, the inaugural seminar of this series took place on Thursday, May 23 at the Severo Ochoa

276 2013 Annual Report 2013 Annual Report 277

Molecular Biology Center (CBMSO), CSIC-UAM, under a symposium jointly organized between CIBERNED, the CBMSO Department of Molecular Neurobiology Center, and the Center for Biomedical Research in Mental Health (CIBERSAM), described below. This first seminar was given by Professor George Perry, who is also the chairman of CIBERNED External Scientific Advisory Committee. During his visit Professor Perry was also appointed to the Royal Academy of Exact, Physical and Natural Sciences (RAC) as Foreign Correspondent in a ceremony held at the RAC headquarters in Madrid, where he delivered a lecture entitled “Living with aging”, which gave an overview of his years as a researcher in Alzheimer’s disease, mainly addressing the reason for the recent media attention and knowledge about the disease, that not less than 30 years ago was largely unknown. After his speech, Professor Perry received the diploma certifying him as a member of the scientific corporation. Below are listed the seminars held during 2013 in this series: Speaker: George Perry, University of Texas, San Antonio Title: Is Alzheimer’s Disease A Metabolic Disorder? Speaker: Kenneth Kosik, University of California, Santa Bárbara Title: Bridging genomics and transcriptomics to the study of the familial Alzheimer’s disease kindred in Colombia Speaker: Lawrence Goldstein, University of California, San Diego Title: Probing mechanism of neurodegenerative disease with pluripotent stem cell technology Speaker: Yves Agid, Pitié-Salpêtrière Hospital, Paris Title: Myths in Parkinson’s disease Speaker: Steven Finkbeiner, University of California, San Francisco Title: Insights into Huntington’s disease from studying the lives of single cells Speaker: David Brooks, Imperial College, London Title: Insights into Parkinson’s disease through imaging Speaker: Davide Pareyson, C. Besta Neurological Institute, Milan Title: Charcot-Marie-Tooth disease: an overview

Dementia Genetics Spanish Consortium (DEGESCO) During the last quarter of 2012, sponsored by CIBERNED and in collaboration with Fundació ACE at Barcelona and the University Hospital Marqués de Valdecilla at Santander, a telematic survey was conducted among thirteen Spanish research centers. These centers were selected because of their systematic performing of research in Alzheimer’s disease and other neurodegenerative dementias molecular genetics. Survey results showed, conclusively, the interest of a majority of the centers (89% of respondents) in constitutinh a working group or stable national consortium that would improve the statistical power and hence the accuracy of studies performed by each group individually. Similarly, we found creating such consortium would allow joint project applications and favor the coordinated participation in various international multicenter projects. While the said interest was detected, the survey conducted a small inventory of biological samples available from the emerging collaborative group. The figures are approximately 7,200 cases of Alzheimer’s disease and 4,500 controls (2,500 population controls and 2,000 “hypercontrols” or neurologically evaluated controls). After several rounds of contacts via e-mail between stakeholders, CIBERNED hosted a meeting of this initiative at the Center of Molecular Biology Severo Ochoa (Madrid) on January 17, 2013 with participation of all DEGESCO founders including CIBERNED. After a second meeting held during the 7th CIBERNED Scientific Forum in Madrid in September 23, 2013, the development of a preliminary roadmap was decided, which included launching some nation-wide consortium pilot studies; the estimation of biomaterial available by creating a common register; accept Dr. Adolfo López de Munain proposal to adopt the name of the initiative and its acronym DEGESCO (Dementia Genetics Spanish Consortium); and drafting of a Memorandum of Understanding that will provide the basis to formalize the scientific and long-term relationship between the parties.

Young Investigator Award As every year, CIBERNED awards a prize to a emerging young scientist involved in the basic or clinical research and its part of CIBERNED. Candidates must be under 35 and senior authors of an article published in 2013. New for this year, CIBERNED has decided to establish also a biennial prize to an emerging scientist involved in clinical research. In this case, the Young Clinical Investigator Award of the Year CIBERNED will be awarded to a scientist with a maximum age of 40, who belongs to a CIBERNED research group. Both call remits were announced to all CIBERNED Principal Investigators by email as well as through the Center’s website. After the open period for nominations, a total of five applications to the Young Investigator Award and two to the Young Clinical Investigator Award were received that met the specifications of the call in a timely manner. The jury was made up of members of the Steering Committee, which assessed the quality and impact of scientific research. After completion of the evaluation process of the applications received, CIBERNED Scientific Management agreed to award the said Awards the following candidates, based on the scientific quality and impact of the publication, and its contribution to it: - Young Investigator 2013: Guillermo López-Doménech (E. Soriano group), from the Cell Biology Department, University of Barcelona, for his paper “The Eutherian Armcx genes regulate mitochondrial trafficking in neurons and interact with Miro and Trak2”. López-Doménech et al. Nat Commun. 2012;3,814. - Young Clinical Investigator: Luis A. Querol Gutiérrez (I. Illa group), from the Neurology Department, Santa Creu i Sant Pau Hospital, for his paper “Antibodies to Contactin-1 in Chronic Inflammatory Demyelinating Polyneuropathy”. Querol et al. Ann Neurol. 2013;73(3):370-80.

278 2013 Annual Report 2013 Annual Report 279

- Summary (López-Doménech et al. Nat Commun. 2012;3:814) Brain function requires neuronal activity-dependent energy consumption. Neuronal energy supply is controlled by molecular mechanisms that regulate mitochondrial dynamics, including Kinesin motors and Mitofusins, Miro1-2 and Trak2 proteins. Here we show a new protein family that localizes to the mitochondria and controls mitochondrial dynamics. This family of proteins is encoded by an array of armadillo (Arm) repeat-containing genes located on the X chromosome. The Armcx cluster is unique to Eutherian mammals and evolved from a single ancestor gene (Armc10). We show that these genes are highly expressed in the developing and adult nervous system. Furthermore, we demonstrate that Armcx3 expression levels regulate mitochondrial dynamics and trafficking in neurons, and that Alex3 interacts with the Kinesin/Miro/Trak2 complex in a Ca(2+)-dependent manner. Our data provide evidence of a new Eutherian-specific family of mitochondrial proteins that controls mitochondrial dynamics and indicate that this key process is differentially regulated in the brain of higher vertebrates. - Summary (Querol et al. Ann Neurol. 2013;73(3):370-80) OBJECTIVE: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a frequent autoimmune neuropathy with a heterogeneous clinical spectrum. Clinical and experimental evidence suggests that autoantibodies may be involved in its pathogenesis, but the target antigens are unknown. Axoglial junction proteins have been proposed as candidate antigens. We examined the reactivity of CIDP patients’ sera against neuronal antigens and used immunoprecipitation for antigen unraveling. METHODS: Primary cultures of hippocampal neurons were used to select patients’ sera that showed robust reactivity with the cell surface of neurons. The identity of the antigens was established by immunoprecipitation and mass spectrometry, and subsequently confirmed with cell-based assays, immunohistochemistry with teased rat sciatic nerve, and immunoabsorption experiments. RESULTS: Four of 46 sera from patients with CIDP reacted strongly against hippocampal neurons (8.6%) and paranodal structures on peripheral nerve. Two patients’ sera precipitated contactin-1 (CNTN1), and 1 precipitated both CNTN1 and contactin-associated protein 1 (CASPR1). Reactivity against CNTN1 was confirmed in 2 cases, whereas the third reacted only when CNTN1 and CASPR1 were cotransfected. No other CIDP patient or any of the 104 controls with other neurological diseases tested positive. All 3 patients shared common clinical features, including advanced age, predominantly motor involvement, aggressive symptom onset, early axonal involvement, and poor response to intravenous immunoglobulin. INTERPRETATION: Antibodies against the CNTN1/CASPR1 complex occur in a subset of patients with CIDP who share common clinical features. The finding of this biomarker may help to explain the symptoms of these patients and the heterogeneous response to therapy in CIDP.

Financial

2013 Annual Report 283

CIBERNED, as the rest of the CIBER, were established with a view to generate large translational research centers, multidisciplinary and multi-institutional, where to integrate basic, clinical and epidemiological research in order to develop a single joint research program focused on some diseases that are relevant to the National Health System due to its prevalence or are considered strategic for it owing to their social impact. The genesis of the CIBER was conducted through a strategy of coordination of research exploiting syner- gies between different research groups involved in biomedical research in these areas. It seeks to promo- te quality research in Biomedicine and Health Sciences carried out in the National Health System (NHS) and the System of Science and Technology through the development and enhancement of Networked Research Structures. The aim is to boost and fund, through the Institute of Health Carlos III, CIBER by brin- ging together research groups, linkable to the NHS, helping to substantiate scientifically the NHS policies and programs in the National R&D&I Strategy priority areas. Following provisions in their new statutes, CIBERNED replaced the former 6 subject areas in which initially was organized, which collected various neurodegenerative diseases, with three programs: • Program 1: “Alzheimer’s disease and other degenerative dementias.” • Program 2: “Parkinson’s disease, Huntington’s chorea and other movement disorders.” • Program 3: “Neuromuscular Diseases.” CIBERNED was initially set up with 48 research groups endorsed by different universities, hospitals and the CSIC, each led by a principal investigator or responsible. It was expected that new groups would in- corporate to the center through annual calls, so that it would bring the most internationally respected and recognized Spanish neuroscientists; hence, 14 new groups in 2008 and one more joined CIBERNED in 2009. Beginning 2010 the Center had 63 research groups. After an internal evaluation conducted during April 2010, discontinuation of three groups was proposed and later endorsed by the Governing Council in December 2010. Thus, from January 2011 the number of CIBERNED research groups went from 63 down to 58, which has kept constant through 2012 and 2013. The Scientific Director Office has been set since October 2011 at the Center for Molecular Biology “Se- vero Ochoa” (CSIC) in Madrid. The headquarters, including the General Manager Office is located in the Alzheimer Center of the Queen Sofia Foundation, Valderrebollo street number 5 in Madrid. Funding

The main source of funding for the activities and objectives of the Consortium of the Institute of Health Carlos III, which, as promoter and major funding agency, contributes about 75% of available resources for 2013. The Consortium was created by virtue of the call for of stable cooperative research structures dated March 30, 2006 (BOE April 7, 2006). Said call in its paragraph 13 states that the aid granted to the Centers for Biomedical Networked Research (CIBER) may reach a maximum of 80 percent of the budget of the consortium activities. Several resolutions of the Institute of Health Carlos III were published on February 11th, May16, Sept- ember 2, and December 4, 2013 whereby financial support is granted to the CIBER in the subject area of Neurodegenerative Diseases. These resolutions set out in their respective annexes that the Institute of Health Carlos III will transfer the nominative grants reflected there, totaling four million four hundred thirty-two thousand five hundred and seventy euros (€ 4,432,570.00), distributed over four million two hundred sixty-nine thousand one hundred ninety euros (€ 4,269,190.00) for current expenditure, and one hundred and sixty-three thousand three hundred and eighty euros (€ 163,380.00) for capital expenditure. This financial provision by the Institute of Health Carlos III is subject to the requirements and objectives contained in the Implementation Agreement for Funding and Exploitation of Results. In compliance with the requirements of paragraph 13.1 of the 30 March, 2006 Resolution of the Institute of Health Carlos III, by which it calls for grants intended to finance stable structures for cooperative re- search in the field of Biomedicine and Health Sciences, within the framework of the Ingenio 2010, CONSO- LIDER program, the Consortium institutions will provide the remaining 20% of the budget.

284 2013 Annual Report 2013 Annual Report 285

Basis of presentation of the accounts

True and fair view The financial statements have been prepared from the accounting records of the Institution, having applied the existing legal provisions on accounting matters, and especially, to present fairly the assets, financial situation and results of the Institution. Accounts are displayed in the standard model and following the accounting principles suggested in the General Public Accounting Plan. More specifically, the Adjustment Plan Public Accounting for government agencies whose spending budget is an estimate is applied, as approved by the 28 July, 2011 Resolution of the General Comptroller of the State Administration. These financial statements were prepared by the Managing Director of the Institution and will be submit- ted to approval by the Governing Council

Accounting principles All mandatory accounting principles that have a significant impact on these accounts have been applied. Non-binding accounting principles have not been applied.

Aspects derived from the comparison of the information The figures for 2013 are comparisons with the previous year, without producing any reclassifications or changes from those contained in the annual accounts for the previous year.

Changes in accounting policies and correction of errors There have been no changes in accounting policies during the year, nor adjustments of errors detected in the year incurred in prior years. Balance Sheet Year 2013

ASSETS

FIZED ASSETS 1,058,409.65 TANGIBLE ASSETS 33,453.96 Computer applications 33,453.96 INTANGIBLE ASSETS 749,955.69 Technical facilities and other intangible assets 749,955.69 LONG-TERM RECEIVABLES 275,000.00 CURRENT ASSETS 3,198,040.50 CASH 2,559,102.96 SHORT-TERM INVESTMENTS 602.70 TRADE AND OTHER ACCOUNTS RECEIVABLES 638,334.84 TOTAL ASSETS 4,256,450.15

EQUITY AND LIABILITIES

EQUITY 3,013,561.52 GENERATED EQUITY 2,862,391.76 Results from previous years 2,496,964.27 Results of the year 342,202.07 Surplus 23,225.42 OTHER ASSETS INCREASES PENDING OF ALLOCATION TO RESULTS 151,169.76 LIABILITIES 275,000.00 LONG-TERM LIABILITIES 275,000.00 Other liabilities 20,675.00 CURRENT LIABILITIES 967,888.63 SHORT-TERM LIABILITIES 406,753.91 Other liabilities 406,753.91 Creditors and other accounts payable 561,134.72 Creditors by management operations 442,681.34 Other payables to Public Administration 118,453.38 grand total liabilities 4,256,450.15

286 2013 Annual Report 2013 Annual Report 287

Financial Equity Income Statement 2013

ORDINARY OPERATING REVENUES

TRANSFERS AND GRANTS RECEIVED 5,600,682.57 IN THE YEAR 4,831,441.18 Grants received to finance expenditure for the year 273,871.18 Wire transfers 4,557,570.00 Grant attributions for non-financial assets 769,241.39 NET SALES AND SERVICES 111,664.80 SERVICES PROVIDED 111,664.80 OTHER REGULAR OPERATING INCOME 46,020.64 TOTAL REGULAR OPERATING INCOME 5,758,368.01

ORDINARY OPERATING EXPENSES

PERSONNEL COSTS 3,096,579.97 WAGES, SALARIES AND RELATED 2,329,987.41 SOCIAL SECURITY TAXES 766,592.56 TRANSFERS AND GRANTS AWARDED 116,489.46 SUPPLIES 736,063.39 CONSUMPTION OF GOODS AND OTHER SUPPLIES 736,063.39 OTHER ORDINARY OPERATING EXPENSES 661,841.78 SUPPLIES AND EXTERNAL SERVICES 643,627.29 TAXES 17,725.47 AMORTIZATION OF ASSETS 812,820.86 TOTAL ORDINARY OPERATING EXPENSES 5,423,795.46

REVENUES FROM NON-FINANCIAL OPERATIONS (SAVINGS) 334,572.65 REVENUES 7,629.42 NET REVENUES FOR THE EXERCISE (SAVINGS) 342,202.07 Identification of programs

Programs and other activities, have the following distribution of economic resources for its implementa- tion, the description of each, as well as data and information that is detailed in the scientific section of this report:

PERSONNEL IMPLEMENTATION COSTS TOTAL

Administration, management and coordination 119,806.73 259,690.77 379,497.50 Program 1 "Alzheimer's disease and other degenerative 1,010,355.42 159,184.53 1,169,539.95 dementias" Program 2 "Parkinson's disease, Huntington's chorea 653,610.71 182,911.63 836,522.34 and other movement disorders" Program 3 "Neuromuscular Diseases" 363,229.69 35,252.38 398,482.07

Deputy Scientific Direction / Supporting platforms 92,263.02 10,986.63 103,249.65 Training 16,258.48 16,258.48

Collaborative project 1 (1st call) 304,767.17 202,142.72 506,909.89 Collaborative project 1 (2nd call) 309,779.56 290,775.93 600,555.49 Collaborative project 1 (3rd call) 26,580.68 28,646.06 55,226.74

COMPETITIVE PROJECTS 216,186.99 133,919.79 350,106.78 FPVII 64,484.90 31,321.97 95,806.87 MS08/0088 37,245.68 0 37,245.68 COEN Projects 19,448.95 13,538.66 32,987.61 PI11/03028 24,881.99 24,881.99 Alzheimer's Association 26,247.15 26,247.15 FRS-CIBERNED 47,585.65 82.30 47,667.95 MJFF-JFR 35,887.65 35,887.65 SYLENTIS-CIBERNED 7,757.75 1,960.07 9,717.82 NOSCIRA (Dendria-CENIT) 39,664.06 39,664.06

ASSOCIATED INSTITUTIONS AGREEMENTS 60,688.41 EVENTS 76,080.58 76,080.58 AMORTIZATION OF FIXED ASSETS 812,820.86 3,096,579.97 1,395,849.50 5,365,938.74

288 2013 Annual Report 2013 Annual Report 289

The data in the table above show that CIBERNED allocates € 2,404,544.36 to implementing research pro- grams, which represents 44.81% of the ordinary operating expenses (excluding taxes). The amount allocated in 2013 to cover the costs of the internal call for collaborative projects adds to € 1,162,692.12, representing 21.67% of the overall budget. The sum of these amounts represents the allocation of more than 66% of expenditure directly related to basic, clinical and population-based research. In line with the policy of engagement in maintaining investment in research and strengthening the com- mitment to the value added by our researchers, spending on staff in 2013 accounts for 57% of the ordinary operating costs. While the share of spending from the previous year experienced a significant increase (+/- 12 points), the accounting increase on this item is 222,359.12 euros, basically derived from the application in 2012 of the provisions of the Royal Decree law 20/2012 of July 13 on measures to ensure fiscal stability and promoting competitiveness. Consumption of resources devoted to administration amounts decreases up to 7%, excluding the cost de- rived from the organization and hosting of the scientific and social fora. Depreciation of existing assets represents a 15.2%, a 4-point decrease from 2012.

Management 7 % Program 1 "Alzheimer's disease and other degenerative dementias" 22 % Program 2 "Parkinson's disease, Huntington's chorea and other movement disorders" 16 % Program 3 "Neuromuscular Diseases" 7 %

Deputy Scientific Direction 0 %

Supporting platforms 0 %

Training 2 %

Collaborative project (1st call) 22 %

COMPETITIVE PROJECTS 4 %

OTHER PROJECTS 3 %

AGREEMENTS WITH ASSOCIATED INSTITUTIONS 1 %

EVENTS 1 %

AMORTIZATION OF FIXED ASSETS 15 % Human resources, grouped into the following categories, own and attached staff, as well as the analysis of its distribution are showed at the next table:

Associated Grupo Principal Investigator Institution Hired researchers

101 Cuadrado Pastor, Antonio Autonomous University of Madrid 3 2 102 Fariñas Gómez, Isabel University of Valencia 12 2 103 Fuentes Rodríguez, José Manuel University of Extremadura, Cáceres 9 3 104 González Castaño, José Autonomous University of Madrid 2 0 105 López Barneo, José Virgen del Rocío University Hospital, University 29 5 of Sevilla 106 Ceña Callejo, Valentín University of Castilla-La Mancha, Albacete 5 3 107 Sánchez Capelo, Amelia Ramón y Cajal University Hospital, Madrid 0 1 108 Vicario Abejón, Carlos Cajal Institute CSIC, Madrid 3 0 109 Vila Bover, Miquel Vall d’Hebron University Hospital, Barcelona 8 3 110 Pérez Castillo, Ana María Institute of Biomedical Research CSIC-UAM, Madrid 6 3 111 Iglesias Vacas, Teresa Institute of Biomedical Research CSIC-UAM, Madrid 5 2 113 García Verdugo, José Manuel Cavanilles Institute, University of Valencia 6 2 114 del Río Fernández, José Antonio Catalonian Institute de Bioengineering, Barcelona 0 1 201 Canela Campos, Enric Isidre University of Barcelona 6 3 202 Kulisevsky Bojarski, Jaime Santa Creu i Sant Pau Hospital, Barcelona 7 1 203 Lanciego Pérez, José Luis Center of Applied Medical Research, 7 2 Univ. Navarra, Pamplona 204 Moratalla Villalba, Rosario Cajal Institute CSIC, Madrid 3 2 205 Obeso Inchausti, José Ángel Center of Applied Medical Research, 10 5 Univ. Navarra, Pamplona 206 Rodríguez Díaz, Manuel University of La Laguna, Tenerife 2 2 207 Tolosa Sarró, Eduardo Hospital Clínic of Barcelona 11 2 208 Labandeira García, José Luis University of Santiago de Compostela 12 3 209 Pérez Tur, Jordi Institute of Biomedicine of Valencia, CSIC 5 0 301 Alberch Vie, Jordi University of Barcelona 19 2 303 Fernández Ruiz, Javier Complutense University of Madrid 12 5 304 Mena Gómez, María Ángeles Foundation for Biomedical Research, University 2 4 Hospital Ramón y Cajal 305 Guzmán Pastor, Manuel Complutense University of Madrid 9 2 306 Lucas Lozano, José Javier Center for Molecular Biology “Severo Ochoa” 6 4 CSIC-UAM, Madrid 307 Naranjo Orovio, José Ramón National Center of Biotechnology, CSIC, Madrid 3 2 401 Ávila de Grado, Jesús Center for Molecular Biology “ Severo Ochoa” 8 9 CSIC-UAM, Madrid 402 Camins Espuny, Antonio University of Barcelona 5 1 403 de Felipe Oroquieta, Javier Cajal Institute CSIC, Technical University, Madrid 12 4 404 Matute Almau, Carlos University of the Basque Country, Bilbao 15 2 406 Rodríguez Álvarez, José Autonomous University of Barcelona 7 2

290 2013 Annual Report 2013 Annual Report 291

Associated Grupo Principal Investigator Institution Hired researchers

407 Sáez Valero, Javier Miguel Hernández University, Elche 7 1 408 Soriano García, Eduardo University of Barcelona 14 6 409 Torres Alemán, Ignacio Cajal Institute CSIC, Madrid 4 3 410 Trullás Oliva, Ramón Institute of Biomedical Research IDIBAPS-CSIC, 5 2 Barcelona 411 Vitorica Ferrández, Francisco Javier University of Seville 6 1 412 Wandosell Jurado, Francisco Center for Molecular Biology “Severo Ochoa” 3 3 CSIC-UAM, Madrid 413 Comella Carnice, Joan Xavier Vall d’Hebron University Hospital, Barcelona 11 4 415 Gutiérrez Pérez, Antonia University of Málaga 10 2 502 Bermejo Pareja, Félix 12 de Octubre University Hospital, Madrid 3 3 503 Ferrer Abizanda, Isidre Bellvitge Institute of Biomedical Research, 12 3 Barcelona 504 Lleó Bisa, Alberto Santa Creu i Sant Pau Hospital, Barcelona 12 3 505 Herrero Ezquerro, María Trinidad University of Murcia 18 0 506 López de Ceballos Lafarga, María Cajal Institute CSIC, Madrid 1 1 507 Pastor Muñoz, María Asunción Center of Applied Medical Research, Univ. 8 0 Navarra, Pamplona 508 Mengod de Los Arcos, Guadalupe Institute of Biomedical Research IDIBAPS-CSIC, 6 2 Barcelona 509 de Pedro Cuesta, Jesús Institute of Health Carlos III, Madrid 11 1 510 Bullido Gómez-Heras, María Jesús Autonomous University, Madrid 8 2 511 Cantero Lorente, José Luis Pablo de Olavide University, Sevilla 8 0 601 Berciano Blanco, José Ángel Marqués de Valdecilla Foundation, Santander 19 3 603 Illa Sendra, Isabel Santa Creu i Sant Pau Hospital, Barcelona 12 4 604 Muñoz Cánoves, Pura Pompeu Fabra University, ICREA, Barcelona 12 3 605 Vílchez Padilla, Juan Jesús La Fe University Hospital, Valencia 6 2 606 Fernández Chacón, Rafael University of Seville 10 2 607 Navarro Acebes, Xavier Autonomous University of Barcelona 19 2 609 López de Munain Arregui, Adolfo Biodonostia Research Institute 34 3 Oficina técnica 0 7 Since inception, CIBERNED has considered its human capital as the most important asset. In fact, it comprises a team of internationally renowned, strongly motivated, highly skilled and specialized that bring with its talent innovation and expertise to the scientific research. Only responsible, entrepreneurs and committed researchers can perform pioneering research to generate solutions to current problems. We at CIBERNED work every day to have a competitive team, professionally regarded and identified with our values, strategies and objectives. The process of expansion and internationalization in which we are involved, offers people a solid project for continuous learning and a unique professional experience. Therefore, we keep a thorough selection process in which we reject favoritism selecting professionals with a willingness for permanent development. The long-term success of CIBERNED depends on its ability to attract, motivate and train researchers capable of settling our research on a solid and consistent basis. The know-how, skills and competencies of our staff are critical factors for achieving the objectives of the institution and, through a permanent approach to excellence, to bring our research to society and to serve as a reference at national and international level. Research groups and the Technical Office manage and support research effectively and efficiently with innovative and quality tools and criteria, offering professional service and personal attention oriented to user satisfaction, working and promoting the objective of CIBERNED to promote research, innovation and the transfer of results to the service of society.

Calls for Personnel

CIBERNED has established a systematic procedure approved by the Board of Directors and the Governing Council in order to cover job vacancies. This procedure is in agreement with section 6.2 of ISO 9001:2008. All job announcements are posted on the website of the consortium Institutions and in compliance with the obligations acquired with the various centers, members of CIBERNED Governing Council and managers of the Institutions are informed of the call contents. Looking to expedite and offer CIBERNED Principal Investigators with a quick and easy method of managing calls for personnel, an integrated management system of the offers posted was implemented in 2008 through the intranet. This system has allowed to streamline the selection process and the filling of job vacancies offered, significantly reducing the time between the decision and the hiring of workers. Implementation of this system has facilitated the rapid covering of the needs in filling job vacancies, foreseen or unforeseen, as well as improved and streamlined the organizational functioning by introducing performance and quality criteria in the internal HR operation following the guidelines of the CIBERNED Quality Plan established under ISO 9001:2008, thus ensuring the Investigator that the position will be filled in the desired date. All CIBERNED selection processes are conducted under criteria of ability, merit and advertising. During the year 2013, we have posted a total of 8 calls for employment, with 34 positions available. There have been a total of 716 applicants.

292 2013 Annual Report 2013 Annual Report 293

List of Published calls during 2012-2013

Available Applicants / Call year Calls Applicants positions position 2012 6 31 512 16.52 2013 8 34 716 21.05

All jobs posted are defined with a specific profile, required qualifications, job requirements and roles to be developed space as well as the working hours and location of the workplace. Out of the 34 positions filled-in during 2013, eleven people had a PhD degree, sixteen were graduates, one was a technician and six were advanced technicians.

Category 2012 2013

Doctor (PhD) 11 11 Graduate (BSc, MSc) 15 16 Technician 1 1 Advanced technician 4 6 31 34

CIBERNED welcomes its new workers

The integration of new researchers in CIBERNED is as important as the selection of candidates. All workers beginning their employment at CIBERNED are provided “Welcome Handbook” consisting of a brief information as a guide, that will be useful in providing an overview of our organization as well as basic guidelines to ease their working relationship. Its content includes a brief presentation of CIBERNED, its origin and objectives, job classification, organization of work, leaves and vacations, a summary of Occupational Risk Prevention (ORP) and templates to handle vacations and ORP. CIBERNED ensures the existence of a work environment that does not accept any form of discrimination, promotes equality of conditions and works to ensure equal opportunities. One example is that wages do not differ between men and women but according to the responsibilities and work of each. CIBERNED provides an environment that favors its researchers health and welfare, according to the highest standards of reliability, safety and hygiene. So, any suggestion on potential improvement is welcomed and treated with the utmost care. Jobs posted 2012-2013

20

15

10

5

0 Doctor (Phd) Graduate Technician Advanced Technician

Distribution of research groups personnel during 2013

During 2013 the distribution of personnel by type of contract was 49 permanent and 99 part-time.

CIBERNED personnel by type of contract

Permanent 49 Part-time 99

294 2013 Annual Report 2013 Annual Report 295

The distribution of personnel according to their degree was 51 Doctors (PhD), 51 graduates, 7 technicians, 29 advanced technicians, and 10 administrative assistants.

CIBERNED personnel according to degree

Administrative 10 % Undergraduate 7 % PhD 51 % Graduate 51 % Technician 29 %

CIBERNED workforce during 2013 consisted of 103 women and 45 men.

CIBERNED personnel by gender

Men 45 Women 103 The geographic distribution of personnel of the research teams by regions was as follows:

CIBERNED personnel by region

Andalucía

Cantabria

Cataluña

Castilla-La Mancha

Extremadura

Galicia

Islas Canarias

Madrid

Navarra

País Vasco

Valencia

0 10 20 30 40 50 60

Training program Bearing in mind that the primary objective of health research is to get a deeper insight into our knowledge of the different disease mechanisms and human health problems as well as to develop strategies for prediction, prevention, treatment and rehabilitation, continuing education is necessary to impact on the development of CIBERNED professionals through their ranks, levels and specialties. The training program is aimed at updating knowledge, improving skills and attitudes, provide solutions to the problems of our professionals and increase their competences and maintaining their professional capacities. Completion of the following training activities: during 2013 can be highlighted: • Two two-months training periods in the Department of Chemistry at Texas Christian University in Fort Worth, Texas, USA. • Two months stay at the Institute of Biomedicine, University of Barcelona. • “International Conference on Research and Innovation in Neurodegenerative Diseases (CIIIEN)”. • “First Meeting on Translational Research in Mental and Neurodegenerative Diseases”. • Workshop “Cantoblanco Workshops on Biology: Is tau a prion-like protein? Implications for physiology and pathology”. • Presentation “CB2 receptors as a promising target for disease-modifying develop developing therapies in neurodegenerative disorders.” 7th Conference on Cannabinoids in Medicine. • “Neuroimaging, Neurosciences & Brain Diseases” course. • “II Neurogenetics course.”

296 2013 Annual Report 2013 Annual Report 297

Prevention of Occupational Risks CIBERNED, through the establishment of its preventive policy is committed to develop and implement a system of risk prevention that will be integrated into the whole of its activities and decisions, both in the technical processes as well as in the organization of work and the conditions in which it develops, including all levels of the company. All of this with a double objective: • Ensuring the safety and health of workers, controlling and minimizing workplace hazards. • Compliance with established legislative obligations regarding the prevention of occupational risks. The following prevention activities have been performed during 2013: • Design, planning and organization of the Center’s preventive training program. Initial training with basic knowledge regarding the risks of the workplace and preventive measures to be taken. • Delivery of “Welcome Handbook” for new workers. • Delivery of Information Sheets on risks, preventive measures, first aid and rules of action in case of emergency. • Conducting 78 medical examinations. • Investigation of two workplace accidents without sick leave. No workplace accidents wick results of sick leave has occurred during 2013, keeping CBERNED below sector accident rates.

Quality During 2013 CIBERNED management area has successfully passed the certification monitoring of the Quality Management System according to TÜV Rheinland-certified ISO 9001:2008. Quality objectives are established annually in order to achieve a continuous improvement in purchasing and staffing procedures as well as to obtain higher levels of customer (both external and internal) satisfaction. CIBERNED’s Quality policy seeks to ensure and optimize processes related with: • Orientation towards the external and internal customer. • Leadership. • Staff involvement. • Process-based approach. • Continuous improvement. The Quality Management System is based on processes. To do this, the basic processes of the Centre are continuously analyzed, which is a continuous improvement tool that enables to meet customers’ requirements, applicable laws and regulations, as well as optimize resources. The tools used to carry out the review of the Quality Management System are the following: • Internal and external audit reports. • Evaluation of suppliers. • Complaints, suggestions and information from customers. • Results of customer satisfaction studies. • Evaluation of corrective and preventive actions. • Indicators of processes quality. • Quality objectives. • Internal and external modifications with influence on the Quality System

298 2013 Annual Report 2013 Annual Report 299

Collaboration agreements

Signed cooperation agreements with other entities are detailed in the section on CIBER-specific scientific activities of this report. Results as a consortium. Its economic detail is as follows:

No. Institution Subject Start Date End Date Amount

113/01 Salud 2000 Foundation - Research grant on "Characterization of 1-feb-13 1-feb-14 10,000.00 € CIBERNED the proteasome degradation system and autophagy in a AAV6-a-sinuclein model of Parkinson's disease: involvement of the transcription factor Nrf2" (Isabel Lastres - Dr. Cuadrado) 113/02 CSIC - CIBERNED - Johnson & Confidentiality agreement 15-may-13 5 years - Johnson (patent application 201231807) 113/03 CIBERNED - CSIC - Neuron Confidentiality agreement 23-may-13 10 years - Bio (patent application P201231654) 113/04 CIBERNED - FISEVI Research collaboration agreement 27-may-13 31-dec-13 -12,000.00 € (PI2010/11-1) 113/05 CIBERNED - CSIC - FCIEN Co-ownership agreement on patent 27-may-13 Vigencia EP13382108.2 (Drs. Naranjo - Rábano) patente 113/06 CIBERNED - BIocross Non-disclosure agreement 27-may-13 10 years

113/07 CIEN Foundation and Organization Forum-Symposium CIIIEN 28-may-13 31-dec-13 -75,000.00 € CIBERNED 113/08 ANEP Scientific evaluation of application to the 8-jul-13 31-dec-13 -2,583.76 € CIBERNED 2013 call for projects 113/09 CSIC - CIBERNED - Bio Rad Confidentiality agreement 9-jul-13 3 years Laboratories Inc (patent application 201231807) 113/10 CIBERNED - Parque Association to Parque Científico de Madrid 16-sep-13 31-dec-14 Científico de Madrid and office rent 113/11 CIBERNED - Biogen Idec Research project (Dr. Cuadrado): 1-oct-13 165,000 USD "Evaluating the potential for Nrf2 activation via DMF treatment in modifying Parkinson’s disease progression in the AAV6‐α‐ synuclein mouse model" 113/12 CIBERNED - IRB Agreement for the transfer of 50% 31-oct-13 31-oct-16 45,000.00 € ownership of the microscope Andor (in relation with purchase agreement 107/04)

113/13 GW Pharma Desarrollo de Proyecto de Investigación 1-oct-13 31-mar- 34,000.00 € que se desarrollará en la Universidad 15 Complutense de Madrid 113/14 CIBERNED - FIBIO Hospital Acuerdo copropiedad de patente "Modelo 4-dec-13 Patent life 12 de Octubre animal de déficit cognitivo, procedimiento de obtención y aplicaciones" (Dra. Eva Carro) 113/15 CIBERNED - ISCIII Convenio colaboración Plan Estatal de 1-jan-13 31-dec-13 125,000.00 € Investigación Científica y Técnica de Innovación 2013-2016 Acquisitions of fixed assets

The acquisition of assets made during the year are as follows:

Vat Activated Value Total Equipment Description Invoice Code Invoice Date Tax Base Ip Code % Pro-Rate 31% Invoice

Esteromicroscopio Stemi 2000 5548001833 09/01/2013 5,553.90 0.21 6,358.66 6,720.22 109 + complementos Combi Cíclico clase A++ mod. F13000167 11/01/2013 756.98 0.21 866.67 915.95 606 CP4023 - Liebher Congelador estático vertical F13000167 11/01/2013 1,327.10 0.21 1,519.40 1,605.79 606 mod G5216 Liebher Impresora laser 400 HP 13158 19/03/2013 398.00 0.21 455.67 481.58 110 M451AN Toshiba portege R930-10X A/1064 25/03/2013 1,073.55 0.21 1,229.11 1,299.00 606 Toshiba C855D-164/E1- 60591833 25/03/2013 333.88 0.21 382.26 403.99 306 1200AMD Recond LKB 7800 Knifemaker 428521 10/03/2013 2,564.25 0 2,564.25 2,564.25 403 Mcllwain Tissue Chopper (cortador automático de 24924 20/04/2013 1,838.00 0.21 2,104.33 2,223.98 111 cerebro) T100 THERMAL CYCLER + S01/21309323 26/04/2013 1,800.00 0.21 2,060.82 2,178.00 103 PLATES Sistema HPLC Isocrático + FV13-008160 30/04/2013 31,737.00 0.21 36,335.69 38,401.77 505 complementos Microscopio Primo Star Zeiss FV13-008160 30/04/2013 1,263.00 0.21 1,446.01 1,528.23 505 Ordenador BCB + teclado + 184 13/05/2013 1,765.00 0.21 2,020.75 2,135.65 403 ratón Monitor 184 13/05/2013 - 0 - - 403 Rack ventilado + accesorios 96646 08/05/2013 26,023.58 0.21 29,794.40 31,488.53 401 Compact power and Energy MI2422754 27/05/2013 1,166.04 0.21 1,335.00 1,410.91 205 meter console digital 4" LCD Portátil ASUS UX31 A-C4027H 20130745 05/06/2013 1,318.18 0.21 1,509.18 1,595.00 203 Office Home & Buss 2010 20130746 25/06/2013 280.99 0.21 321.71 340.00 203 32/64 SPA Objetivo LCI Plan-Neofluar 5548002240 27/06/2013 8,591.00 0.21 9,835.84 10,395.11 403 63x1,3 lmm Torre HP 4300P SFF G2020 13F/1301888 14/10/2013 368.00 0.21 421.32 445.28 306 4.0G 39 PC HP lasejet pro 400 M401dn C-2013/2927 13/09/2013 254.01 0.21 290.82 307.35 111 impresora B/W Fluoimager: módulo de 3301409 29/08/2013 4,800.00 0.21 5,495.52 5,808.00 305 software para NewCast Microimager: módulo de software para adquisición y 3301409 29/08/2013 5,200.00 0.21 5,953.48 6,292.00 305 sampleado Imaris bitplane-filament tracer BPAG-102013- 24/10/2013 9,325.08 0.21 10,676.28 11,283.35 403 7.6 252928-1

300 2013 Annual Report 2013 Annual Report 301

Vat Activated Value Total Equipment Description Invoice Code Invoice Date Tax Base Ip Code % Pro-Rate 31% Invoice Servidor NZXST caja ATX H2 negra classic series, disco duro, 2-4T-2013 29/10/2013 1,453.00 0.21 1,663.54 1,758.13 305 ventilador, intel core i7 3770

Mini transblot/power pack hc S01/21322847 25/10/2013 1,834.00 0.21 2,099.75 2,219.14 407 Software update for existing Neurolucida "Workstation" license and 091013-521 09/10/2013 3,350.00 0.21 3,835.42 4,053.50 403 existing Neurolocuda/Stereo Investigador licenses on Microscope-controlling system ordenador de mesa Apple Core i% de intel de cuatro núcleos, SE/16687 12/11/2013 1,139.67 0.21 1,304.81 1,379.00 105 2.7 Ghz, 8 GB de SDRAM DDR, 1600 mhz Ordenador Hp 7500MT 13F/1302116 15/11/2013 1,087.08 0.21 1,244.60 1,315.37 410 I153470 1TB Ordenador Hp 7500MT 13F/1302116 15/11/2013 1,087.08 0.21 1,244.60 1,315.37 410 I153470 1TB Ordenador Hp 7500MT 13F/1302116 15/11/2013 1,087.08 0.21 1,244.60 1,315.37 410 I153470 1TB Ordenador Hp 7500MT 13F/1302116 15/11/2013 1,087.08 0.21 1,244.60 1,315.37 410 I153470 1TB Monitor HP W2072A 20" INCH 13F/1302116 15/11/2013 - 0 - - 410 LED Monitor HP W2072A 20" INCH 13F/1302116 15/11/2013 - 0 - - 410 LED Monitor HP W2072A 20" INCH 13F/1302116 15/11/2013 - 0 - - 410 LED Monitor HP W2072A 20" INCH 13F/1302116 15/11/2013 - 0 - - 410 LED HP Workstation Z220C ZC 3.4 13F/1302116 15/11/2013 1,595.08 0.21 1,826.21 1,930.05 410 Monitor HP W2072A 20" INCH 13F/1302116 15/11/2013 - 0 - - 410 LED Portátil Sony Ultrabook vaio 13F/1302116 15/11/2013 1,595.97 0.21 1,827.23 1,931.12 410 pro 13" i7-4500u 1,8 Ghz Sony I5-4200u/4/128G/IN/ 131028/001/ 28/10/2013 990.91 0.21 1,134.49 1,199.00 305 W8/13/TUB 9785/027 Torre HP P3500 MT I3 13F/1301953 25/10/2013 570.00 0.21 652.59 689.70 1 3240/500GB Monitor HP W2072A 20" INCH 13F/1301953 25/10/2013 - 0 - - 1 LED Torre HP P3500 MT I3 13F/1301953 25/10/2013 570.00 0.21 652.59 689.70 1 3240/500GB Monitor HP W2072A 20" INCH 13F/1301953 25/10/2013 - 0 - - 1 LED Monitor HP W2072A 20" INCH LED + TECLADO LOGITECH 13F/1301953 25/10/2013 151.50 0.21 173.45 183.32 1 MK330 RETAIL Vat Activated Value Total Equipment Description Invoice Code Invoice Date Tax Base Ip Code % Pro-Rate 31% Invoice Equipo black side I5 MN2- A/1485 10/12/2013 694.42 0.21 795.04 840.25 606 windows 7 progr Img. Recd. Chembr. W Remv 97529 14/11/2013 1,068.00 0.21 1,222.75 1,292.28 606 Elec Mag Baño de ácero inoxidable 95 o 200 º C agua 7 litros + tapa 4090150401 23/12/2013 666.00 0.21 762.50 805.86 504 tejadillo WNB

127,764.41 145,905.91 154,056.44

302 2013 Annual Report Principal Investigators Index

Alberch Vie, Jordi programa 2 103 Ávila de Grado, Jesús programa 1 21 Berciano Blanco, José Ángel programa 3 201 Bermejo Pareja, Félix programa 1 25 Bullido Gómez-Heras, Mª Jesús programa 1 28 Camins Espuny, Antonio programa 1 31 Canela Campos, Enric Isidre programa 2 107 Cantero Lorente, José Luis programa 1 35 Ceña Callejo, Valentín programa 2 111 Comella Carnicé, Joan Xavier programa 1 38 Cuadrado Pastor, Antonio programa 2 114 de Felipe Oroquieta, Javier programa 1 41 de Pedro Cuesta, Jesús programa 1 44 del Río Fernández, José Antonio programa 2 117 Fariñas Gómez, Isabel programa 2 120 Fernández Chacón, Rafael programa 3 207 Fernández Ruiz, Javier programa 2 123 Ferrer Abizanda, Isidre programa 1 49 Fuentes Rodríguez, José Manuel programa 2 127 García de Yébenes Prous, Justo programa 2 130 García Verdugo, José Manuel programa 2 134 González Castaño, José programa 2 138 Gutiérrez Pérez, Antonia programa 1 54 Guzmán Pastor, Manuel programa 2 141 Herrero Ezquerro, María Trinidad programa 1 57 Iglesias Vacas, Teresa programa 2 145 Illa Sendra, Isabel programa 3 210 Kulisevsky Bojarski, Jaime programa 2 148 Labandeira García, José Luis programa 2 151 Lanciego Pérez, José Luis programa 2 154 Lleó Bisa, Alberto programa 1 61 López Barneo, José programa 2 157 López de Ceballos Lafarga, María programa 1 65 López de Munain Arregui, Adolfo programa 3 214 Lucas Lozano, José Javier programa 2 162 Matute Almau, Carlos programa 1 67 Mengod de los Arcos, Guadalupe programa 1 70 Moratalla Villalba, Rosario programa 2 165 Muñoz Cánoves, Pura programa 3 219 Naranjo Orovio, José Ramón programa 2 168 Navarro Acebes, Xavier programa 3 222 Obeso Inchausti, José Ángel programa 2 171 Pastor Muñoz, María Asunción programa 1 73 Pérez Castillo, Ana María programa 2 175 Pérez Tur, Jordi programa 2 178 Rodríguez Álvarez, José programa 1 77 Rodríguez Díaz, Manuel programa 2 180 Sáez Valero, Javier programa 1 80 Sánchez Capelo, Amelia programa 2 183 Soriano García, Eduardo programa 1 85 Tolosa Sarró, Eduardo programa 2 185 Torres Alemán, Ignacio programa 1 87 Trullás Oliva, Ramón programa 1 89 Vicario Abejón, Carlos programa 2 191 Vila Bover, Miquel programa 2 194 Vilchez Padilla, Juan Jesús programa 3 226 Vitorica Ferrández, Francisco Javier programa 1 92 Wandosell Jurado, Francisco programa 1 95

C/ Valderrebollo, 5 28031 Madrid GOBIERNO MINISTERIO 913 852 200 DE ESPAÑA DE ECONOMÍA www.ciberned.es Y COMPETITIVIDAD Instituto de Salud

Informe Anual 2013 Informe Carlos III