Luận án: Vai trò của Laminin α2 trong phát triển Oligodendrocyte và Tủy sống - Jenne Relucio

Stony Brook University The official electronic file of this thesis or dissertation is maintained by the University Libraries on behalf of The Graduate School at Stony Brook University. © ©AAllll R Riigghhttss R Reesseerrvveedd bbyy A Auutthhoorr. The Role of Laminin α2 in Oligodendrocyte Development and CNS Myelination A Dissertation Presented by Jenne Liza Villaranda Relucio to The Graduate School In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Molecular and Cellular Biology (Cellular and Developmental Biology) Stony Brook University May 2011 Stony Brook University The Graduate School Jenne Liza Villaranda Relucio We, the dissertation committee for the above candidate for the Doctor of Philosophy degree, hereby recommend acceptance of this dissertation. – Dissertation Advisor Assistant Professor, Department of Pharmacological Sciences Stony Brook University Joel M.

– Chairperson of Defense Professor, Department of Neurobiology and Behavior Stony Brook University Styliani-Anna E. Professor, Department of Pharmacological Sciences Stony Brook University Gary G. Professor, Department of Neurobiology and Behavior Stony Brook University William J. Professor, Director of Ophthalmic Research Department of Cell Biology SUNY Downstate Medical Center This dissertation is accepted by the Graduate School.

Lawrence Martin Dean of the Graduate School ii Abstract of the Dissertation The Role of Laminin α2 in Oligodendrocyte Development and CNS Myelination by Jenne Liza Villaranda Relucio Doctor of Philosophy in Molecular and Cellular Biology (Cellular and Developmental Biology) Stony Brook University 2011 The myelin-producing oligodendrocytes of the central nervous system are critical to normal brain function and physiology. The molecular mechanisms that influence oligodendrocyte development and CNS myelination, however, remain poorly understood. One potential factor that may regulate these processes is the extracellular matrix (ECM) molecule laminin. Children with mutations in one type of laminin – the alpha 2 subunit (LAMA2) – have defects in the size and shape of the forebrain and cerebellum, along with white matter abnormalities.

However, it is not clear whether laminin α2 modulates oligodendrocytes in vivo, and if so, through which cellular signaling pathways. In this dissertation, I used LAMA2-deficient (dy/dy) and LAMA2-knockout (dy3k/dy3k) mice to investigate the influence of iii laminin α2 on oligodendrogenesis, oligodendrocyte maturation, and CNS myelination. We found that laminin α2 regulates the organization and cellular composition of the postnatal subventricular zone (SVZ), a major gliogenic niche. Loss of LAMA2 resulted in abnormal oligodendrogenesis, such that initially LAMA2-knockout mice had fewer oligodendrocyte progenitor cells (OPCs) compared to their wildtype littermates.

During the active myelination period, LAMA2-deficient brains were also found to have a developmental delay in oligodendrocyte maturation. These defects in OPC genesis and maturation correlated with axonal dysmyelination in various CNS white matter regions. We also identified several signaling abnormalities that may be contributing to these oligodendrocyte defects. LAMA2-deficient brains showed dysregulated Fyn (a Src Family Kinase known to regulate myelination) and elevated levels of C-terminal Src kinase (Csk) and Csk-binding-protein (Cbp), proteins that suppress Fyn activity.

Laminin substrates were furthermore found to modulate Fyn regulation and to promote the transition of cultured oligodendrocyte progenitors to newly-formed oligodendrocytes in a Fyn-dependent manner. These findings indicate that the dysregulation of signaling pathways required for normal oligodendrocyte development may contribute to CNS abnormalities observed in congenital muscular dystrophy type 1A (MDC1A), and identify novel mechanisms by which laminins regulate oligodendrogenesis, oligodendrocyte lineage progression, and CNS myelination in the developing brain. iv For those whose encouragement, understanding, and love helped me make molehills out of mountains and kept me going against insurmountable odds My father Mel My mother Grace My husband Tom v Table of Contents List of Tables. ix List of Figures.

x List of Abbreviations. xix Vita, Publications, and Field of Study. xxi CHAPTER I: GENERAL INTRODUCTION. 1 The extracellular matrix.3 Functional roles for laminins in central nervous system development.6 Laminins and neuroepithelial (NEP) cells.7 Laminins and neurogenesis in the embryonic ventricular zone.9 Laminins and neurogenesis in adult neural stem cell niches.

12 Laminins and oligodendrocytes: from oligodendrogenesis to myelination. 13 Laminin α2 and oligodendrogenesis: insights from the neocortical niche. 14 Oligodendrocyte development in vivo. 15 Laminin α2 and myelination: insights from the peripheral nervous system.

16 A working model: laminin α2 in oligodendrogenesis, development, and CNS myelination. 19 CHAPTER II: LAMININ ALTERS FYN REGULATORY MECHANISMS AND PROMOTES OLIGODENDROCYTE DEVELOPMENT. 28 MATERIALS AND METHODS. 38 Laminin-deficient brains show delayed oligodendrocyte maturation.

38 Correlation between delayed oligodendrocyte differentiation and myelination abnormalities in dy/dy brains. 40 Delayed differentiation of oligodendrocyte progenitors in dy/dy brains. 42 The Src Family Kinase Fyn is dysregulated in dy/dy brains. 44 dy/dy brains contain elevated levels of Csk and Cbp, negative regulators of Fyn.

45 Laminin promotes oligodendrocyte progenitor cell differentiation. 47 Dysregulation of p27 occurs in the dy/dy cerebellum. 48 Oligodendrocyte survival is unchanged in adult laminin-deficient mice. 51 CHAPTER III: LAMININ REGULATES POSTNATAL OLIGODENDROGENESIS.

90 MATERIALS AND METHODS. 101 Laminin expression patterns in the postnatal cerebral cortex. 101 SVZ cell numbers and organization are abnormal in postnatal LAMA2KO mice. 103 Ectopic Sox2(+) intermediate progenitors in the LAMA2KO SVZ.

105 Increased oligodendrocyte progenitor cell death in the laminin α2-null SVZ. 108 Abnormal numbers of oligodendrocyte progenitors in the LAMA2KO corpus callosum. 109 Oligodendrocyte maturation is abnormal in laminin α2 knockout brains. 113 Myelination deficits in laminin α2-knockout brains.

117 Laminin α2, radial glial cells, and intermediate progenitors. 118 Laminin α2-mediated survival of OPCs in the SVZ. 119 Laminin α2 and oligodendrocyte development in white matter tracts. 121 vii CHAPTER IV: CONCLUSIONS AND FUTURE CONSIDERATIONS.

138 Basement membrane laminins versus non-BL laminins: delineating functional contributions through in vitro models. 138 Laminins and cerebellar development. 140 Laminin, astrocytes, and the blood brain barrier. 147 viii List of Tables Table 1.

CC1(+)cells per mm2. PDGFRα(+)cells per mm2. TUNEL(+)cells per mm2. 86 ix List of Figures Figure I-1.

Laminin heterotrimers are assembled from three different laminin subunits, and feature globular, coiled-coil, and rod-like domains in their general structure. Oligodendrocyte formation, maturation, and myelination in vivo can be identified through the expression of stage-specific markers. A working model: roles for laminin α2 in oligodendrogenesis, development, and CNS myelination. Delayed oligodendrocyte maturation in dy/dy brains.

Myelin abnormalities in dy/dy cerebella. Oligodendrocyte progenitor differentiation and myelination are altered in the optic nerves of dy/dy mice. Myelination abnormalities in laminin-deficient dy/dy brains show regional variation. Oligodendrocyte progenitors accumulate in dy/dy brains.

Fyn is dysregulated in dy/dy brains. Laminin promotes the differentiation of oligodendrocyte progenitors using a Fyn-dependent mechanism. Laminin regulates p27 protein levels. Oligodendrocyte death in dy/dy adult brains.

Laminin and oligodendrocyte progenitor development. dy/dy brains show selective loss of laminin α2. Regional and temporal variation in residual dy/dy laminin α2 expression. Laminin expression patterns in the postnatal cerebral cortex.

Abnormal progenitor cell organization and numbers in the postnatal SVZ of laminin α2-knockout mice. Altered Sox2+ progenitor cell positioning in the laminin α2-knockout SVZ. Oligodendrocyte progenitor death in the LAMA2KO subventricular zone. LAMA2KO mice have less OPCs in the corpus callosum initially but accumulate excess progenitors during development.

Delayed oligodendrocyte maturation in laminin α2-knockout brains. Myelin deficits in the laminin α2-knockout corpus callosum. Preliminary analysis on the cerebellum of laminin α2 knockout mice. 145 xi List of Abbreviations AP apical progenitor APC adenomatous polyposis coli AraC Arabinose C BBB blood-brain barrier BL basal lamina BME beta-mercaptoethanol BP basal progenitor bs brain stem BSA bovine serum albumin Calb calbindin CB cerebellum Cbp C-terminal Src kinase binding protein CD140a PDGFRα, platelet-derived growth factor receptor alpha polypeptide CNPase 2',3'-cyclic nucleotide 3' phosphodiesterase CNS central nervous system xii CO2 carbon dioxide Csk C-terminal Src kinase CTX cerebral cortex CY3 cyanine3 DAPI 4',6-diamidino-2-phenylindole DGC dystrophin-associated glycoprotein complex DMEM Dulbecco's modified Eagle's medium DMSO dimethyl sulfoxide DRG dorsal root ganglia DS donkey serum dy/dy dystrophia muscularis; laminin α2–deficient mouse dy3k/dy3k laminin alpha2 chain-null mutant mouse ECM extracellular matrix EGF epidermal growth factor EGL external granule layer of the cerebellum EM electron microscopy FAK focal adhesion kinase xiii FITC fluorescein isothiocyanate GC granule cells GFAP glial fibrillary acidic protein IgG immunoglobulin G IGL internal granule layer of the cerebellum ILK integrin-linked kinase INM interkinetic nuclear migration iPC intermediate progenitor cell kDa kilodalton L4 laminin internal domain LAMA2 laminin alpha 2 subunit LAMA2KO laminin alpha 2 subunit-knockout mouse LE laminin tandem EGF repeats LG laminin globular domain Lm laminin LmA2 laminin alpha 2 subunit LmC1 laminin gamma 1 subunit xiv LmC3 laminin gamma 3 subunit LN laminin N-terminus domain LSB Laemmli solubilizing buffer Lu/B-CAM Lutheran blood group glycoprotein/basal cell adhesion molecule MAPK mitogen activated kinase-like protein MBP myelin basic protein MDC1A congenital muscular dystrophy type 1A MEB Muscle-Eye-Brain disease MRI magnetic resonance imaging mRNA messenger ribonucleic acid NA numerical apperture NEP neuroepithelium NF neurofilament NG2 nerve/glial antigen 2 NIH National Institute of Health NS not significant NSC neural stem cell xv OCT Optimum Cutting Temperature Olig2 oligodendrocyte lineage transcription factor 2 OPC oligodendrocyte progenitor cell p27 cyclin-dependent kinase inhibitor 1B; Kip1 p57 cyclin-dependent kinase inhibitor 1C; Kip2 PBS phosphate buffered saline PBS-T phosphate buffered saline with Triton PCNA proliferating cell nuclear antigen PDGF platelet-derived growth factor PDGFRα platelet-derived growth factor receptor alpha polypeptide PDL poly-D-lysine PFA paraformaldehyde PI3K phosphoinositide-3-kinase PNS peripheral nervous system POMT1 protein-O-mannosyltransferase 1 POMGnT1 protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase PP2 (4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine QKI quaking homolog, KH domain RNA binding protein xvi RMS rostral migratory stream RNA ribonucleic acid R-Ras related Ras viral oncogene homolog SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM standard error of the mean SFK Src family kinase SGZ subgranular zone Shh Sonic hedgehog Sox2 sex determining region Y-box 2 str striatum SVZ subventricular zone TBS-T Tris buffered saline with Triton TUNEL terminal deoxynucleotidyl transferase 2´-deoxyuridine, 5´-triphosphate nick end labeling VS ventricular surface VZ ventricular zone WM white matter WT wildtype xvii WWS Walker-Warburg Syndrome xviii Acknowledgements The explorer Christopher Columbus has been credited in saying “You can never cross the ocean unless you have the courage to lose sight of the shore.” Almost six years after taking the biggest gamble in my life and leaving one island for another, I remain grateful to the many people who made me the person I am today, and continue to inspire me to become the best version of myself.

I begin by acknowledging my advisor, Dr. Holly Colognato for her mentorship throughout my PhD studies. Joining her lab is arguably one of the best decisions I made in graduate school. I thank Holly for all the trust, encouragement, and guidance that she has given me as a student, a colleague, and a friend.

I would also like to thank the members of my Dissertation Committee, Drs. Joel Levine, Gary Matthews, Stella Tsirka, and Bill Brunken, for all their advice, support, and enormous amounts of patience as they guided me from proposal to defense. All of you have made me a better student of science. I am deeply grateful to the past and present students and members (both de facto and honorary) of the Colognato lab: Dr.

Jason Galvin, Dr. Iva Tzvetanova and Christos Nardis, Christopher Eyermann, Freyja McClenahan, Cindy Leiton, Amy DeMarco, Soyoung Rhee, Michael Menezes, Emory Kuo, Wei Ao, Brian Cho, and Dan Park. You all made graduate school a wonderful experience, and you are truly friends that I will never forget. I also thank our many research collaborators and colleagues who helped us with the laminin α2 knockout and dy/dy projects: Drs.

Emily Chen and Toni Koller, Sara Nik, Dr. Kyungmin Ji, Sue Van Horn, and Laurie Levine from Stony Brook University; Dr. Shinichi xix Takeda from Osaka University; Dr. Bruce Patton and Caitlin Letts from the Oregon Health Sciences University; Drs.Jon and Sabine Lindquist from the Otto-von-Guericke University in Germany; and Drs.

Karine Loulier and Taryk Haydar from the Center for Neuroscience Research. I thank the ever-important (yet often under-appreciated) members of the administrative staff: Carol Juliano and Beverly Piazza of the MCB Graduate Program; and Lynda Perdomo- Ayala, Janice Kito, June Moriarty, Beverly Campbell, and Melissa Daley of the Department of Pharmacology. You kept our lab and my stay at Stony Brook running smoothly.