DOCSLIB.ORG

Modeling Neurodevelopment and Cortical Dysfunction in SPG11-Linked Hereditary Spastic Paraplegia Using Human Induced Pluripotent Stem Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Modeling Neurodevelopment and Cortical Dysfunction in SPG11-Linked Hereditary Spastic Paraplegia Using Human Induced Pluripotent Stem Cells Modeling neurodevelopment and cortical dysfunction in SPG11-linked hereditary spastic paraplegia using human induced pluripotent stem cells Untersuchung der Neuronalentwicklung und kortikalen Dysfunktion der SPG11 assoziierten Heriditären Spastische Paraplegie unter Zuhilfenahme von humanen induzierten pluripotenten Stammzellen Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. Vorgelegt von: Himanshu Kumar Mishra aus Bhagalpur, Indien Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 26.02.2016 Vorsitzender des Promotionsorgans: Prof. Dr. Jörn Wilms Gutachter/in: Prof. Dr. Jürgen Winkler PD. Dr. Andreas Gießl “I have no special talents. I am only passionately curious.” ― Albert Einstein Table of Contents Table of Contents 1 List of figures .......................................................................................................................... 01 2 List of tables ............................................................................................................................ 04 3. Declaration .............................................................................................................................. 05 4. Summary/Zusammenfassung ................................................................................................ 06 4.1 Summary ............................................................................................................................. 06 4.2 Zusammenfassung .............................................................................................................. 08 5. Introduction ............................................................................................................................ 10 5.1 Motor neuron diseases ...................................................................................................... 10 5.2 Hereditary spastic paraplegias (HSP) ................................................................................ 13 5.2.1 Genetic heterogeneity and clinical symptoms ....................................................... 15 5.2.2 Neuropathology and emerging molecular mechanisms ........................................ 17 5.2.3 Treatment of HSP .................................................................................................. 19 5.3 SPG11/Spatacsin ............................................................................................................... 20 5.3.1 Clinical symptoms and disease etiology of SPG11 ............................................... 20 5.3.2 Mutation spectrum of SPG11 ................................................................................ 21 5.3.3 Cellular localization and and functions of spatacsin ............................................. 23 5.4 Human induced pluripotent stem cell (iPSC) technology ................................................. 26 5.4.1 Reprogramming strategies to generate iPSCs ...................................................... 26 5.4.2 Modeling neurodegenerative diseases using iPSCs ............................................... 27 5.5 Aims and hypotheses of the thesis ..................................................................................... 28 6. Materials and Methods .......................................................................................................... 31 6.1 SPG11 patients and Control subjects ................................................................................. 31 6.2 Cell culture ........................................................................................................................ 31 6.2.1 Fibroblasts derivation ............................................................................................ 31 6.2.2 Generation of iPSCs .............................................................................................. 33 6.2.3 Neural differentiation paradigm ............................................................................ 33 6.2.4 Karyotyping ........................................................................................................... 35 6.2.5 Neurite length and arborization analysis ............................................................... 35 6.2.6 Electrophysiology .................................................................................................. 36 6.3 Animals.............................................................................................................................. 36 6.3.1 Synaptosomes preparation ..................................................................................... 37 6.4 Molecular Biology ............................................................................................................. 38 6.4.1 Real Time PCR analysis ........................................................................................ 38 6.4.2 Western Blot .......................................................................................................... 39 Table of Contents 6.4.3 Transfections of siRNA and plasmid DNA ........................................................... 40 6.4.4 TOP-flash/FOP-flash Luciferase reporter assay .................................................... 40 6.5 Immunofluorescence staining and analysis ....................................................................... 41 6.5.1 Proliferation analysis ............................................................................................. 41 6.5.2 Cell death analysis ................................................................................................. 43 6.5.3 Acetyl tubulin staining ........................................................................................... 43 6.6 Flow cytometry-PI analysis ............................................................................................... 44 6.7 Pharmacological rescue of NPCs proliferation ................................................................. 44 6.8 Synaptic vesicle transport experiments ............................................................................. 45 6.9 Microscopy ........................................................................................................................ 45 6.9.1 Fluorescence and confocal microscopy ................................................................. 45 6.9.2 Electron microscopy .............................................................................................. 46 6.10 Statistics ............................................................................................................................ 46 7. Results ...................................................................................................................................... 47 7.1 Generation of human models for SPG11 and expression analysis of SPG11 ............ 47 7.1.1 Generation of human iPSCs from SPG11 patients and controls ........................... 47 7.1.2 Differentiation and characterisation of neuronal cultures derived from iPSCs ..... 51 7.1.3 Spatacsin is present in human and mouse cortical projection neurons ................. 52 7.1.4 Spatacsin is expressed in human embryonic and adult mouse neurons................. 54 7.1.5 Spatacsin is expressed throughout mouse brain development .............................. 55 7.1.6 Spatacsin is ubiquitously distributed in axons and dendrites of cortical neurons . 57 7.1.7 Spatacsin is localised in neurites, growth-cones and synapses of neurons ........... 59 7.2 Modeling neurodevelopmental phenotypes of SPG11 using iPSC derived NPCs .... 61 7.2.1 Impaired generation of cortical neural rosettes in SPG11-iPSCs .......................... 61 7.2.2 Transcriptome analysis of control and SPG11-NPCs ............................................ 63 7.2.3 Reduced proliferation and neurogenesis in SPG11-NPCs .................................... 71 7.2.4 Altered cell cycle distribution and stage-specific anomalies in SPG11-NPCs ..... 72 7.2.5 Checkpoint genes are downregulated in SPG11-NPCs ......................................... 75 7.2.6 SPG11-NPCs are less prevalent in cytokinesis and undergo cell death ................ 75 7.2.7 Impaired GSK3ß/ß-Catenin signaling in SPG11-NPCs ........................................ 78 7.2.8 Loss of spatacsin compromises proliferation of neuronal cell line ....................... 80 7.2.9 GSK3 inhibitors rescue proliferation and neurogenesis defects in SPG11-NPCs . 82 7.2.10 Premature differentiation of SPG11-NPCs is ameliorated by GSK3 inhibitor ..... 84 7.2.11 Impairment of autophagy related pathways in SPG11-NPCs ............................... 86 7.3 Modeling neurodegenerative phenotypes of SPG11 using iPSC derived neurons .... 88 7.3.1 Dysfunction of spatacsin in SPG11-dNeurons leads to aberrant gene expression 88 7.3.2 Neurite outgrowth and complexity are compromised in SPG11-dNeurons .......... 90 7.3.3 Disruption of spatacsin destabilizes microtubules in SPG11-dNeurons ............... 93 Table of Contents 7.3.4 Spatacsin dysfunction impairs axonal transport in SPG11-dNeurons ................... 95 7.3.5 Spatacsin dysfunction disturbs Na+/K+ current density in SPG11-dNeurons ....... 97 8. Discussion ................................................................................................................................ 99 8.1 Spatacsin expression in human and mouse cortical neurons
Load more

Recommended publications
  • The Role of Semaphorins and Their Receptors in Vascular Development and Cancer
    EXPERIMENTAL CELL RESEARCH ] ( ]]]]) ]]]– ]]] Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/yexcr Review Article The role of semaphorins and their receptors in vascular development and cancer Chenghua Gua,n, Enrico Giraudob,nn aDepartment of Neurobiology, Harvard Medical School, 220 Longwood Ave, Boston, MA 02115, USA bInstitute for Cancer Research at Candiolo (IRC@C), and Department of Science and Drug Technology, University of Torino, Str. Prov. 142 Km.3,95 10060 Candiolo, Turin, Italy article information abstract Article Chronology: Semaphorins (Semas) are a large family of traditional axon guidance molecules. Through Received 1 February 2013 interactions with their receptors, Plexins and Neuropilins, Semas play critical roles in a Accepted 6 February 2013 continuously growing list of diverse biological systems. In this review, we focus on their function in regulating vascular development. In addition, over the past few years a number of Keywords: findings have shown the crucial role that Semas and their receptors play in the regulation of Semaphorin cancer progression and tumor angiogenesis. In particular, Semas control tumor progression by Plexin directly influencing the behavior of cancer cells or, indirectly, by modulating angiogenesis and Neuropilin the function of other cell types in the tumor microenvironment (i.e., inflammatory cells and Angiogenesis fibroblasts). Some Semas can activate or inhibit tumor progression and angiogenesis, while Cancer others may have the opposite effect depending on specific post-translational modifications. Here Tumor we will also discuss the diverse biological effects of Semas and their receptor complexes on Development cancer progression as well as their impact on the tumor microenvironment. & Vasculature 2013 Elsevier Inc.
    [Show full text]
  • Plexin A4 (PLXNA4) (NM 181775) Human Recombinant Protein – TP761981 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TP761981 Plexin A4 (PLXNA4) (NM_181775) Human Recombinant Protein Product data: Product Type: Recombinant Proteins Description: Purified recombinant protein of Human plexin A4 (PLXNA4), transcript variant 2,full length, with N-terminal GST and C-terminal His tag, expressed in E. coli, 50ug Species: Human Expression Host: E. coli Tag: N-GST and C-His Predicted MW: 86 kDa Concentration: >50 ug/mL as determined by microplate BCA method Purity: > 80% as determined by SDS-PAGE and Coomassie blue staining Buffer: 25mM Tris, pH8.0, 150 mM NaCl, 10% glycerol,1% Sarkosyl. Storage: Store at -80°C. Stability: Stable for 12 months from the date of receipt of the product under proper storage and handling conditions. Avoid repeated freeze-thaw cycles. RefSeq: NP_861440 Locus ID: 91584 UniProt ID: Q9HCM2 RefSeq Size: 2020 Cytogenetics: 7q32.3 RefSeq ORF: 1566 Synonyms: FAYV2820; PLEXA4; PLXNA4A; PLXNA4B; PRO34003 Summary: Coreceptor for SEMA3A. Necessary for signaling by class 3 semaphorins and subsequent remodeling of the cytoskeleton. Plays a role in axon guidance in the developing nervous system. Class 3 semaphorins bind to a complex composed of a neuropilin and a plexin. The plexin modulates the affinity of the complex for specific semaphorins, and its cytoplasmic domain is required for the activation of down-stream signaling events in the cytoplasm (By similarity).[UniProtKB/Swiss-Prot Function] Protein Families: Druggable Genome This product is to be used for laboratory only.
    [Show full text]
  • Expression of a Mirna Targeting Mutated SOD1 in Astrocytes Induces Motoneuron Plasticity and Improves Neuromuscular Function in ALS Mice
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.08.425706; this version posted January 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Expression of a miRNA targeting mutated SOD1 in astrocytes induces motoneuron plasticity and improves neuromuscular function in ALS mice Rochat C.1, Bernard-Marissal N.1,6, Pradervand S.3, Perrin F.E.4, Raoul C.5, Aebischer P.1, Schneider B.L.1,2* 1 Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland 2 Bertarelli Platform for Gene Therapy, Ecole Polytechnique Fédérale de Lausanne (EPFL), Geneva, Switzerland 3 Genomic Technologies Facility, University of Lausanne, Lausanne, Switzerland. 4 INSERM U1198, University of Montpellier, EPHE, Place Eugène Bataillon CC105, F-34095, Montpellier, France 5 The Neuroscience Institute of Montpellier, Inserm UMR1051, Univ Montpellier, Saint Eloi Hospital, Montpellier, France 6 Aix Marseille Univ, INSERM, MMG, Marseille, France *Correspondence should be addressed to B.L.S. ([email protected]) Short title: miRNA gene therapy targeting astrocytes in ALS Study was performed in Lausanne, Switzerland Current address of corresponding author: Bernard Schneider EPFL SV PTECH PTBTG Ch. des Mines 9 CH-1202 Genève Switzerland Email: [email protected] Tel: +41 21 693 95 05 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.08.425706; this version posted January 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • The Expression of Key Guidance Genes at a Forebrain Axon Turning Point Is Maintained by Distinct Fgfr Isoforms but a Common Downstream Signal Transduction Mechanism
    New Research Development The Expression of Key Guidance Genes at a Forebrain Axon Turning Point Is Maintained by Distinct Fgfr Isoforms but a Common Downstream Signal Transduction Mechanism Jung-Lynn Jonathan Yang, Gabriel E. Bertolesi, Stephanie Dueck, Carrie L. Hehr, and Sarah McFarlane https://doi.org/10.1523/ENEURO.0086-19.2019 Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada Abstract During development the axons of neurons grow toward and locate their synaptic partners to form functional neural circuits. Axons do so by reading a map of guidance cues expressed by surrounding tissues. Guidance cues are expressed at a precise space and time, but how guidance cue expression is regulated, and in a coordinated manner, is poorly understood. Semaphorins (Semas) and Slits are families of molecular ligands that guide axons. We showed previously that fibroblast growth factor (Fgf) signaling maintains sema3a and slit1 forebrain expres- sion in Xenopus laevis, and these two repellents cooperate to guide retinal ganglion cell (RGC) axons away from the mid-diencephalon and on towards the optic tectum. Here, we investigate whether there are common features of the regulatory pathways that control the expression of these two guidance cues at this single axon guidance decision point. We isolated the sema3a proximal promoter and confirmed its responsiveness to Fgf signaling. Through misexpression of truncated Fgf receptors (Fgfrs), we found that sema3a forebrain expression is dependent on Fgfr2-4 but not Fgfr1. This is in contrast to slit1, whose expression we showed previously depends on Fgfr1 but not Fgfr2-4.
    [Show full text]
  • Kinesin-3 Family Members in Hereditary Spastic Paraplegia
    fncel-13-00419 September 25, 2019 Time: 15:27 # 1 REVIEW published: 26 September 2019 doi: 10.3389/fncel.2019.00419 Going Too Far Is the Same as Falling Short†: Kinesin-3 Family Members in Hereditary Spastic Paraplegia Dominik R. Gabrych1, Victor Z. Lau1, Shinsuke Niwa2 and Michael A. Silverman1,3* 1 Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada, 2 Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan, 3 Centre for Cell Biology, Development, and Disease, Simon Fraser University, Burnaby, BC, Canada Proper intracellular trafficking is essential for neuronal development and function, and Edited by: when any aspect of this process is dysregulated, the resulting “transportopathy” causes Tomas Luis Falzone, neurological disorders. Hereditary spastic paraplegias (HSPs) are a family of such National Scientific and Technical Research Council (CONICET), diseases attributed to over 80 spastic gait genes (SPG), specifically characterized by Argentina lower extremity spasticity and weakness. Multiple genes in the trafficking pathway such Reviewed by: as those relating to microtubule structure and function and organelle biogenesis are Craig Blackstone, National Institute of Neurological representative disease loci. Microtubule motor proteins, or kinesins, are also causal Disorders and Stroke (NINDS), in HSP, specifically mutations in Kinesin-I/KIF5A (SPG10) and two kinesin-3 family United States members; KIF1A (SPG30) and KIF1C (SPG58). KIF1A is a motor enriched in neurons, Thomas Huckaba, Xavier University of Louisiana, and involved in the anterograde transport of a variety of vesicles that contribute to United States pre- and post-synaptic assembly, autophagic processes, and neuron survival. KIF1C is *Correspondence: ubiquitously expressed and, in addition to anterograde cargo transport, also functions in Michael A.
    [Show full text]
  • Trka Mediates Retrograde Semaphorin 3A Signaling Through Plexin A4 To
    © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1802-1814 doi:10.1242/jcs.184580 RESEARCH ARTICLE TrkA mediates retrograde semaphorin 3A signaling through plexin A4 to regulate dendritic branching Naoya Yamashita1,2,*, Masayuki Yamane1, Fumikazu Suto3 and Yoshio Goshima1,* ABSTRACT subcellular compartments of neurons – axons, dendrites and cell – Semaphorin 3A (Sema3A), a secretory semaphorin, exerts various body need to communicate with each other. biological actions through a complex between neuropilin-1 and plexin- Semaphorin 3A (Sema3A), one of the first repulsive axon As (PlexAs). Sema3A induces retrograde signaling, which is involved guidance molecules to be recognized (Raper, 2000; Tran et al., in regulating dendritic localization of GluA2 (also known as GRIA2), 2007), plays an important role in proper neuronal network formation an AMPA receptor subunit. Here, we investigated a possible by regulating not only growth cone motility but also dendritic – interaction between retrograde signaling pathways for Sema3A and development and maturation through neuropilin-1 (NRP1) Plexin- nerve growth factor (NGF). Sema3A induces colocalization of PlexA4 A (PlexA) receptor complexes (Campbell et al., 2001; Morita et al., (also known as PLXNA4) signals with those of tropomyosin-related 2006; Polleux et al., 2000; Sasaki et al., 2002; Shelly et al., 2011; kinase A (TrkA, also known as NTRK1) in growth cones, and these Suto et al., 2003; Yamashita et al., 2014). Sema3A accelerates colocalized signals were then observed along the axons. The time- endocytosis during growth cone collapse (Fournier et al., 2000; lapse imaging of PlexA4 and several TrkA mutants showed that the Hida et al., 2012), and also induces bidirectional axonal transport kinase and dynein-binding activity of TrkA were required for Sema3A- (Goshima et al., 1999, 1997; Li et al., 2004; Yamane et al., 2012).
    [Show full text]
  • Mutation Screening of SEMA3A and SEMA7A in Patients with Congenital Hypogonadotropic Hypogonadism
    nature publishing group Clinical Investigation Articles Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism Johanna Känsäkoski1,2, Rainer Fagerholm1,3, Eeva-Maria Laitinen2, Kirsi Vaaralahti1,2, Peter Hackman4, Nelly Pitteloud5, Taneli Raivio1,2 and Johanna Tommiska1,2 BACKGROUND: Congenital hypogonadotropic hypogonad- FGF8, PROK2, PROKR2, CHD7, WDR11, GNRHR, GNRH1, ism (HH), a rare disorder characterized by absent, partial, or KISS1R, KISS1, TAC3, and TACR3 (5,6), have been shown delayed puberty, can be caused by the lack or deficient num- to cause the disorder. In the majority of cases, however, the ber of hypothalamic gonadotropin-releasing hormone (GnRH) molecular genetic cause remains unresolved, implying the neurons. SEMA3A was recently implicated in the etiology of the existence of additional genes underlying the condition (7). disorder, and Sema7A-deficient mice have a reduced number We have described the phenotypic and genotypic features of of GnRH neurons in their brains. Finnish patients with Kallmann syndrome (KS) (8). A molecu- METHODS: SEMA3A and SEMA7A were screened by Sanger lar genetic cause was obtained for ~40% of Finnish KS patients, sequencing in altogether 50 Finnish HH patients (34 with suggesting the presence of mutations in genes not yet impli- Kallmann syndrome (KS; HH with hyposmia/anosmia) and 16 cated to underlie this syndrome. with normosmic HH (nHH)). In 20 patients, mutation(s) had Semaphorins are a class of secreted and membrane proteins already been found in
    [Show full text]
  • Targeting Neuropilin-1 with Nanobodies Reduces Colorectal Carcinoma Development
    cancers Article Targeting Neuropilin-1 with Nanobodies Reduces Colorectal Carcinoma Development 1, 2,3, 1 Yannick De Vlaeminck y , Stefano Bonelli y, Robin Maximilian Awad , Maarten Dewilde 4 , Sabrina Rizzolio 5, Quentin Lecocq 1 , Evangelia Bolli 2,3, Ana Rita Santos 4, Damya Laoui 2,3 , Steve Schoonooghe 2,3, Luca Tamagnone 6,7 , 1 8,9 1, , Cleo Goyvaerts , Massimiliano Mazzone , Karine Breckpot * z and 2,3, , Jo A. Van Ginderachter * z 1 Laboratory for Molecular and Cellular Therapy, Department of Biomedical Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium; [email protected] (Y.D.V.); [email protected] (R.M.A.); [email protected] (Q.L.); [email protected] (C.G.) 2 Laboratory for Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1040 Brussels, Belgium; [email protected] (S.B.); [email protected] (E.B.); [email protected] (D.L.); [email protected] (S.S.) 3 Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, 1040 Brussels, Belgium 4 VIB Discovery Sciences, 3000 Leuven, Belgium; [email protected] (M.D.); [email protected] (A.R.S.) 5 Candiolo Cancer Institute-FPO, IRCCS, 10060 Candiolo, Italy; [email protected] 6 Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, 00100 Rome, Italy; [email protected] 7 Department of Oncology, Fondazione Policlinico Universitario “A. Gemelli”, IRCCS, 00100 Rome, Italy 8 Laboratory of Tumor Inflammation and Angiogenesis, VIB Center for Cancer Biology, 3000 Leuven, Belgium; [email protected] 9 Department of Oncology, Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology, KU Leuven, 3000 Leuven, Belgium * Correspondence: [email protected] (K.B.); [email protected] (J.A.V.G.) These authors contributed equally as co-first author.
    [Show full text]
  • CRMP2 Mediates Sema3f-Dependent Axon Pruning and Dendritic Spine
    bioRxiv preprint doi: https://doi.org/10.1101/719617; this version posted July 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine remodeling Jakub Ziak1,8, Romana Weissova1,8,¶, Kateřina Jeřábková2,¶, Martina Janikova3, Roy Maimon4, Tomas Petrasek3, Barbora Pukajova1,8, Mengzhe Wang5, Monika S. Brill5,6, Marie Kleisnerova1, Petr Kasparek2, Xunlei Zhou7, Gonzalo Alvarez-Bolado7, Radislav Sedlacek2, Thomas Misgeld5, Ales ‡ Stuchlik3, Eran Perlson4 and Martin Balastik1, 1Dpt. of Molecular Neurobiology, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic 2Dpt. of Transgenic Models of Diseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic 3Dpt. Of Neurophysiology of the Memory, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic 4Dpt. of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel 5Institute of Neuronal Cell Biology, Technical University Munich, Munich, Germany 6German Center for Neurodegenerative Diseases and Munich Cluster for Systems Neurology, Munich, Germany 7Institute of Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany 8Faculty of Science, Charles University, Prague, Czech Republic ¶These authors contributed equally ‡ All correspondence should be addressed to Martin Balastik, Tel.: +420 241 062 822; E-mail: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/719617; this version posted July 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
    [Show full text]
  • Patient Induced Pluripotent Stem Cell Model
    RESEARCH ARTICLE GSK3ß-Dependent Dysregulation of Neurodevelopment in SPG11-Patient Induced Pluripotent Stem Cell Model Himanshu K. Mishra, PhD,1 Iryna Prots, PhD,1 Steven Havlicek, PhD,1 Zacharias Kohl, MD,2 Francesc Perez-Branguli, PhD,1 Tom Boerstler, BSc,1 Lukas Anneser, MSc,1 Georgia Minakaki, MSc,2 Holger Wend,1 Martin Hampl, MSc,3 Marina Leone, PhD,4 Martina Bruckner,€ 5 Jochen Klucken, MD,2 Andre Reis, MD,6 Leah Boyer, PhD,7 Gerhard Schuierer, MD,8 Jurgen€ Behrens, PhD,5 Angelika Lampert, MD,3,9 Felix B. Engel, PhD,4 Fred H. Gage, PhD,7 Jurgen€ Winkler, MD,2 and Beate Winner, MD1 Objective: Mutations in the spastic paraplegia gene 11 (SPG11), encoding spatacsin, cause the most frequent form of autosomal-recessive complex hereditary spastic paraplegia (HSP) and juvenile-onset amyotrophic lateral sclerosis (ALS5). When SPG11 is mutated, patients frequently present with spastic paraparesis, a thin corpus callosum, and cognitive impairment. We previously delineated a neurodegenerative phenotype in neurons of these patients. In the current study, we recapitulated early developmental phenotypes of SPG11 and outlined their cellular and molecular mechanisms in patient-specific induced pluripotent stem cell (iPSC)-derived cortical neural progenitor cells (NPCs). Methods: We generated and characterized iPSC-derived NPCs and neurons from 3 SPG11 patients and 2 age- matched controls. Results: Gene expression profiling of SPG11-NPCs revealed widespread transcriptional alterations in neurodevelop- mental pathways. These include changes in cell-cycle, neurogenesis, cortical development pathways, in addition to autophagic deficits. More important, the GSK3ß-signaling pathway was found to be dysregulated in SPG11-NPCs.
    [Show full text]
  • Gpr97 Exacerbates AKI by Mediating Sema3a Signaling
    BASIC RESEARCH www.jasn.org Gpr97 Exacerbates AKI by Mediating Sema3A Signaling Wei Fang,1 Ziying Wang,1 Quanxin Li,1 Xiaojie Wang,1 Yan Zhang,1 Yu Sun,1 Wei Tang,2 Chunhong Ma,3 Jinpeng Sun,4 Ningjun Li,5 and Fan Yi1,6 1Departments of Pharmacology, 2Pathogenic Biology, 3Immunology, and 4Biochemistry and Molecular Biology, The Key Laboratory of Infection and Immunity of Shandong Province, School of Basic Medical Science, and 6The State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; and 5Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia ABSTRACT Background G protein-coupled receptors (GPCRs) participate in a variety of physiologic functions, and several GPCRs have critical physiologic and pathophysiologic roles in the regulation of renal function. We investigated the role of Gpr97, a newly identified member of the adhesion GPCR family, in AKI. Methods AKI was induced by ischemia–reperfusion or cisplatin treatment in Gpr97-deficient mice. We assessed renal injury in these models and in patients with acute tubular necrosis by histologic examination, and we conducted microarray analysis and in vitro assays to determine the molecular mechanisms of Gpr97 function. Results Gpr97 was upregulated in the kidneys from mice with AKI and patients with biopsy-proven acute tubular necrosis compared with healthy controls. In AKI models, Gpr97-deficient mice had significantly less renal injury and inflammation than wild-type mice. Gpr97 deficiency also attenuated the AKI-induced expression of semaphorin 3A (Sema3A), a potential early diagnostic biomarker of renal injury. In NRK- 52E cells subjected to oxygen–glucose deprivation, siRNA-mediated knockdown of Gpr97 further in- creased the expression of survivin and phosphorylated STAT3 and reduced toll-like receptor 4 expression.
    [Show full text]
  • Prdc Cdna Insert Product Line
    hPlexin A4 VersaClone cDNA Catalog Number: RDC1214 Specifications: Description This shuttle vector contains the complete ORF for the gene of interest, Gene: hPLXNA4 along with a Kozak consensus sequence for optimal translation initiation. It is inserted NotI to AscI. The gene insert is flanked with Accession: NP_065962 convenient multiple cloning sites which can be used to easily cut and transfer the gene cassette into your desired expression vector. Insert size: 5697bp Preparation and Storage Concentration: 10µg at 0.2μg/μL Formulation cDNA is provided in 10 mM Tris-Cl, pH 8.5 Shipping Ships at ambient temperature Stability 1 year from date of receipt when stored at -20°C to -80°C Storage Use a manual defrost freezer and avoid repeated hPlexin A4 cDNA freeze-thaw cycles. Plasmid PLXNA4 plexin A4 [ Homo sapiens (human) ] HpaI HindIII SspI BamHI Also known as: PLEXA4; PLXNA4A; BmtI NheI NotI PLXNA4B; FAYV2820; PRO34003 EagI BsiWI Summary: AMP EcoNI PLXNA4 is a member of the Plexin family of semaphorin signal transducers. It is found on sensory, COLE1 PshAI autonomic and motor neurons and oligodendrocytes, plus T cells and RDC1214 dendritic cells. PLXNA4 regulates cell 8431 bps Eco47III migration, activation, and axon XhoI guidance via repulsion. It serves as a PspXI SalI receptor for transmembrane XbaI BssHII NsiI semaphorins, Sema6A and 6B, and AscI hPlexin-A4 (1-1894) as a coreceptor with neuropilin 1 for the secreted semaphorin, Sema3A. NruI SphI Alternatively spliced transcripts AccIII encoding different proteins have been described.
    [Show full text]