The Role of LRRK2 in Parkinson's Disease
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Universidade de Lisboa Faculdade de Medicina de Lisboa The role of LRRK2 in Parkinson’s disease: from function to dysfunction Patrícia I. da Silva Guerreiro Doutoramento em Ciências Biomédicas Neurociências Dezembro de 2015 Universidade de Lisboa Faculdade de Medicina de Lisboa The role of LRRK2 in Parkinson’s disease: from function to dysfunction Patrícia I. da Silva Guerreiro Tese orientada por: Prof. Doutor Tiago Fleming Outeiro Doutoramento em Ciências Biomédicas Neurociências Todas as afirmações efectuadas no presente documento são da exclusiva responsabilidade do seu autor, não cabendo qualquer responsabilidade à Faculdade de Medicina de Lisboa pelos conteúdos nele apresentados. A impressão desta tese foi aprovada pelo Conselho Científico da Faculdade de Medicina de Lisboa em reunião de 26 de Maio de 2015. Table of contents Acknowledgements …………………………………………………………… VI Abstract …………………………………………………………………………. VII Resumo …………………………………………………………………………. X List of abbreviations ………………………………………………………….. XIII Chapter 1. General Introduction 1. The neurodegenerative disorder of Parkinson’s disease ………………. 2 1.1 Clinical symptoms and pathogenesis of PD …………………………. 2 1.2 Etiology of PD …………………………………………………………… 4 1.2.1 Environmental factors ………………………………………………. 5 1.2.2 Genetic factors ………………………………………………………. 5 1.3. α-synuclein and PD ……………………………………………………. 7 1.4 Association of Tau with PD ……………………………………………. 9 1.5 Leucine-Rich Repeat Kinase 2: a key player in PD …….................. 10 1.5.1 Functional structure of LRRK2 protein …………………………… 11 1.5.2 LRRK2 mutations and PD …………………………...................... 12 1.5.3 Neuropathology of LRRK2 mutations in PD …………………….. 14 1.5.4 Interplay between GTPase and Kinase domains ……………….. 15 1.5.3 LRRK2 interacting proteins and putative functions …………….. 16 1.6 Cellular Quality Control Systems ……………………………………… 18 1.6.1 The ubiquitin-proteasome system (UPS) ………………………... 18 1.6.2 Autophagy ………………………………………………………....... 20 1.7 References ………………………………………………...................... 22 Chapter 2. Aims of the project ……………………………………………… 28 Chapter 3. LRRK2 interactions with α-synuclein in Parkinson’s disease brains and in cell models 3.1 Introduction and main goals …………………………………………… 30 3.2 Materials and Methods …………………………………………………. 31 3.3 Results …………………………………………………………………… 36 3.3.1 LRRK2 co-immunoprecipitates with α-synuclein ……………….. 36 3.3.2 LRRK2 co-localizes with α-synuclein in PD brain and cell model ………………………………………………………………… 37 3.3.3 Knocking down LRRK2 expression reduces α-synuclein aggregation …………………………………………………………... 39 3.3.4 Increasing levels of LRRK2 correlates with α-synuclein in PD 40 3.4 Discussion ……………………………………………………………….. 42 3.5 References ………………………………………………………………. 46 Chapter 4. LRRK2 promotes Tau accumulation, aggregation and release 4.1 Introduction and main goals …………………………………………… 49 4.2 Materials and Methods …………………………………………………. 50 4.3 Results …………………………………………………………………… 54 4.3.1 Tau levels are decreased in LRRK2 knock-out mice …………… 54 4.3.2 LRRK2 physically interacts with Tau ……………………………… 55 4.3.3 Increased levels of Tau depend on LRRK2 expression but not on its kinase activity …………………………………………………. 56 4.3.4 LRRK2 promotes the accumulation of high-molecular weight Tau species…………………………………………………………… 58 4.3.5 LRRK2 impairs proteasomal protein degradation independently of its kinase activity………………………………………………… 60 4.3.6 LRRK2 impairs the proteasomal degradation of Tau but does not interfere with the autophagy pathway ………………………… 62 4.3.7 LRRK2 promotes the cellular release of Tau …………………….. 62 4.4 Discussion ……………………………………………………………….. 64 4.5 References ………………………………………………………………. 68 Chapter 5. LRRK2 interactors and their biological significance 5.1 Introduction and main goals …………………………………………… 71 5.2 Materials and Methods ………………………………………………… 71 5.3 Results …………………………………………………………………… 75 5.3.1 Network of protein interactions…………………………………….. 75 5.3.2 Gene Ontology Analysis ……………………………………………. 75 5.3.3 The effect of LRRK2 expression on the mechanical properties of the cell……………………………………………………………… 81 5.3.4 Different LRRK2 distribution patterns result in different cell stiffness ……………………………………………………………….. 81 5.4 Discussion ……………………………………………………………….. 84 5.5 References ………………………………………………………………. 87 Chapter 6. General discussion and conclusions 6.1 The implications of interaction of LRRK2 with α-synuclein and Tau 90 6.2 LRRK2-interacting protein network and its particular role on cytoskeleton dynamics ………………………………………………….. 93 6.3 Conclusions ……………………………………………………………... 94 6.4 References ………………………………………………………………. 96 Chapter 7. Appendix ………………………………………………………….. 99 Acknowledgements During my PhD I was exposed to a variety of challenges, developing my passion for working in science and having the opportunity to meet amazing people, who became amazing friends. During this journey, I had the essential support of several people, which allowed me to grow and learn a lot on how to be a scientist and to whom I am extremely greatful. I would like to thank my supervisor Prof. Dr. Tiago Outeiro, for taking me as a graduate student, for his support since the beginning of my project, for always being available to discuss my ideas and for encouraging me to work hard to achieve my goals. Also, for givinging me the opportunity to join the UNCM, at IMM in Lisbon, where I am proud to have been part of this extraordinary working group. Later on, for supporting my move to his group in Goettingen, allowing me to belong to an outstanding community of scientists. I am thankful to all the members of UNCM group, that in one way or another helped me during my PhD. A very friendly thank to my colleagues and good friends: Teresa Pais, Leonor Fleming, Ana Oliveira, Oldriska Chutna, Elisa Basso, Sandra Jacinto, Hugo Miranda and Sandra Tenrreiro, with whom I shared fruitful scientific discussions and for the good moments and unconditional support in the lab. Also a special thank to Federico Herrera and Rita Oliveira for being so patient in helping me with the scientific writing, always with a very constructive criticism. I would also like to thank all the members from the group in Göttingen, for receiving me so well, and having jointly started a new working group. I owe my gratitude to my german colleagues and good friends Ellen Gerhardt, Christiane Fahlbusch, Sonja Reisenauer and Omar Diaz, and to my special collaborator and friend Katrin Eckermann, for all the essential support, for always making my life easier, in and outside the lab, and for making me feel at home in the (sometimes) cold Germany. A very special thank to my buddy Pauline, with whom I shared my time in Germany, for our long and hard hours in the lab, and also all the good moments outside the lab, and for always being with me in overcoming many Germans hills. Lastly, I am extremely thankful to all my family for their unconditional support, in particular to João, for always supporting my life decisions, and to Mariana, for helping me to have a four-hand writing of this thesis. VI Abstract Parkinson’s disease (PD) belongs to the group of neurodegenerative disorders and it is currently considered the most common progressive movement disorder. Neurodegenerative disorders, such as Alzheimer’s, Huntington’s, fronto- temporal dementia and amyotrophic lateral sclerosis, share several dysfunctional molecular pathways and impairments in basic cell mechanisms. Despite intense efforts to understand to decipherthe triggers underlying these disorders, to date, there is no effective cure. This results in a growing number of cases and, consequently, in a complex social and economic problem. Therefore, it is of extreme importance to understand the common biological mechanisms involved in the pathogenesis of this devastating group of diseases, in order to develop effective therapies. The majority of the PD cases are sporadic, however, in the last decades, it has been recognized that rare genetic mutations are patholgical for PD in a number of inherited cases. Futhermore, these mutations can be as well a risk factor for sporadic PD, supporting the idea that familial and sporadic PD can share common pathlogical mechanisms. This study focused on a key player protein in PD, Leucine-rich repeat kinase 2 (LRRK2). Mutations in LRRK2 gene are the most frequent cause of autosomal dominant forms of PD and they are also consider a risck factor for sporadic cases. A central catalytic GTPase and kinase core, flanked by protein interaction domains, composes this large and complex multi-domain protein. The most frequent LRRK2 PD-related mutation occurs at the animoacid 2019, a glycine subtitution for a serine (G2019S), precisely on the kinase domain of the protein resulting in its toxic gain of function. LRRK2 is known to play a role in distinct cellular mechanisms such as vesicular trafficking, microtubule network regulation and mitochondrial morphology. However, the function of LRRK2 in these important mechanisms and their related pathways is not fully understood, which is crucial for developing new therapeutic targets. Here, we investigated LRRK2 function by characterizing/identifying its protein interactors and, in particular, by exploring its relationship with two central proteins in neurodegenerative disorders, α-synuclein and Tau. In PD brain samples, we show that levels of LRRK2 are positively correlated to an increase in α-synuclein phosphorylation and aggregation in affected brain regions, where both proteins co-localize in neurons and Lewy body VII inclusions. In a cell line model, this co-localization