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Implications of Alpha-Dystroglycan Glycosylation Implications of alpha-dystroglycan glycosylation in the proper assembly of the dystroglycan associated protein complex and the polarized distribution of the inwardly rectifying potassium channel, Kir4.1, and the water permeable channel, AQP4, in perivascular glia by Jennifer Mary Elizabeth Rurak B.Sc. Honours Physiology, University of British Columbia, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA March 2007 © Jennifer Mary Elizabeth Rurak, 2007 ABSTRACT The dystroglycan protein complex spans the plasma membrane and provides a link between the cytoskeleton and the extracellular matrix (ECM). Defective O-linked glycosylation of a-dystroglycan (a-DG) within the complex severs this link leading to a subset of muscular dystrophies known as the dystroglycanopathies. These are characterized by muscle weakness and degeneration as well as by brain and ocular defects of unknown etiology. In brain and retina, a-DG and ECM molecules are enriched at astrocytic domains abutting blood vessels where they may be involved in localizing the inwardly rectifying potassium channel, Kir4.1, and aquaporin channel, AQP4, to astrocytic endfeet. To investigate the role of ECM ligand-binding to glycosylated sites on a-DG in the polarized distribution of these channels in vivo, we used the Largemyd mouse, an accepted animal model for dystroglycanopathies. We found that Kir4.1 and AQP4 are lost from astrocytic endfeet in brain while significant labeling for these channels is still detected at analogous cell domains in retina. Furthermore, while both a- and b\- syntrophins are lost from perivascular astrocytes in brain of the Largemyd mouse, labeling for pi-syntrophin is still evident at parallel domains in retina. These findings show that while ligand-binding to glycosylated sites on a-DG in concert with a- and pVsyntrophins is crucial for the polarized distribution of Kir4.1 and AQP4 to functional domains in brain, distinct mechanisms may contribute to their localization in retina. ii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ACKNOWLEDGEMENTS viii DEDICATION ix CO-AUTHORSHIP STATEMENT x 1 INTRODUCTION 1 1.1 Muscular Dystrophy 1 1.2 The Dystroglycan-Associated Protein Complex 3 1.3 Dystrophin 5 1.4 Utrophin 7 1.5 The Syntrophins 9 1.6 Dystrobrevin 11 1.7 The Sarcoglycan-Sarcospan Complex 12 1.8 The Dystroglycans 13 1.9 O-linked glycosylation of a-dystroglycan 15 1.10 Alpha-Dystroglycanopathy Related Muscular Dystrophies 19 1.11 The Dystroglycan Associated Protein Complex in the Central Nervous System 26 1.12 Inwardly Rectifying Potassium Channels (Kir) 31 1.13 Kir4.1 in brain and retina 32 1.14 Aquaporins 36 1.15 Aquaporin 4 in brain and retina 37 1.16 The Largemyd mouse 39 1.17 Project Outline and Hypotheses..'. 42 1.18 Works Cited 44 2 FIRST MANUSCRIPT CHAPTER 58 2.1 Alpha-dystroglycan hypoglycosylation disrupts the polarized distribution of potassium ion and water permeable channels in perivascular astrocytes of the Largemyd mouse 58 2.2 Materials and Methods 62 2.2.1 Tissues 62 2.2.2 Antibodies 64 2.2.3 Immunofluorescence 65 2.2.4 Immunoblotting 65 2.2.5 RNA extraction and RT-PCR 66 2.3 Results 67 2.3.1 Abnormalities in the localization of basement membrane proteins in the Largemyd brain 67 2.3.2 Loss of perivascular localization of AQP4 and Kir4.1 in the Largemyd brain 71 2.3.3 Loss of perivascular localization of a- and p,-syntrophins in the Largemyd brain 76 2.3.4 Expression of Large 1 and Large2 mRNAs in retina and impact of Large 1 mutation on Kir4.1 and AQP4 localization in the Largemyd retina 78 myd 2.3.5 Loss of perivascular localization of a-syntrophin but not prsyntrophin in the Large retina 85 2.4 Discussion 87 2.4.1 AQP4 and Kir4.1 redistribution in brain 87 2.4.2 AQP4 and Kir4.1 distribution in retina 90 2.5 Acknowledgements 92 2.6 Works Cited 93 3 DISCUSSION 98 3.1 Implication of the DAP complex in ion channel localization 98 3.2 Stabilization of AQP4 and Kir4.1 via a-syntrophin 103 3.3 Impact of ct-dystroglycan glycosylation on AQP4 and Kir4.1 expression 105 3.4 Possible physiological implications of AQP4 and Kir4.1 mislocalization 107 3.5 Disruptions in the cortical surface basal lamina 109 3.6 Expression of Large transcripts and glycosylation of a-dystroglycan in retina 111 3.7 Spatial differences in the DAP complex-induced anchoring of AQP4 and Kir4.1 112 3.8 Disruption of the inner limiting membrane and aberrant retinal lamination 117 3.9 Conclusions 117 3.10 Future Directions 118 3.10.1 Involvement of a-DG glycosylation in the laminin-induced clustering of Kir4.1 and AQP4 118 3.10.2 Effect of the Largemyd mutation on other retinal DAP complex proteins 119 3.10.3 Expression of integrins in the retina 120 3.10.4 Pathological implications of AQP4 and Kir4.1 mislocalization in Largemyd brain 121 3.11 Works Cited 123 4 APPENDICES 131 4.1 Appendix A-Supplemental Data 131 iv LIST OF TABLES Table 1.1 Summary of sugar-peptide bonds in eukaryotes 16 Table 1.2 Summary of a-DG Hypoglycosylation Related Muscular Dystrophies 24 Table 2.1 Summary of Experimental Animals: Sex, Genotype and Age 63 v LIST OF FIGURES Figure 1.1 Schematic Representation of Molecular Interaction Within the DAP Complex Figure 1.2 O-Mannosyl Glycan Synthesis of a-Dystroglycan Figure 1.3 Schematic of Perivascular Astrocytic Domains Figure 1.4 Schematic of Mammalian Retinal Lamination Figure 1.5 Schematic model of spatial pottasium buffering Figure 2.1 Disruption of the glia limitans and accumulation of agrin and perlecan puncta in the Largemyd brain Figure 2.2 AQP4 and Kir4.1 fail to localize to perivascular astrocyte endfeet in the Largemyd brain Figure 2.3 Total Kir4.1 and AQP4 expression levels are unchanged in the Largemyd brain Figure 2.4 a- and pVsyntrophins fail to localize to perivascular astrocyte endfeet in the Largemyd brain Figure 2.5 Expression profile of Large 1 and Large2 Figure 2.6 a-dystroglycan is hypoglycosylated in the Largemyd retina Figure 2.7 Disruption of the lamination and the inner limiting membrane in the Largemyd retina Figure 2.8 Perivascular AQP4 and Kir4.1 are maintained in the Largemyd retina Figure 2.9 Pi- but not a-syntrophin is still expressed at perivascular astrocytes endfeet in the Largemyd retina Figure 4.1 AQP4 and Kir4.1 fail to localize to the glia limitans in the Largemyd brain. 1 Figure 4.2 Radial Miiller cells appear discontinuous and tortuous in the Largemyd retina. 1 LIST OF ABBREVIATIONS AAV - Adeno Associated Virus LGMD - Limb Girdle MD AchR - Acetylcholine Receptor mAb - Monoclonal Antibody AQP - Aquaporin Man - Mannose Ara - Arabinose MAST - Microtubule Associated Ser/ Asn - Asparagine Thr Kinase CMD - Congenital Muscular Dystrophy MD - Muscular Dystrophy CNS - Central Nervous System MDC - Congenital MD CSF - Cerebrospinal Fluid Mdx - Dystrophin Deficient Mouse DAP - Dystroglycan Associated Protein MEB - Muscle Eye Brain Disease DB -Dystrobrevin Nav- Voltage Gated Sodium Channel DG - Dystroglycan (a or P) NeuAc - Neuraminic Acid DMD -Duchenne Muscular Dystrophy NMJ - Neuromuscular Junction Dol-P-X - Dolichol-Phosphate- nNOS - Neuronal Nitric Oxide Synthase Oligosaccarides OPL - Outer Plexiform Layer Dp - Dystrophin PBS - Phosphate Buffered Saline ECM - Extracellular Matrix PH - Plekstrin Homology Domain EHS - Engelbreth-Holm-Swarm POMGnTl - Protein O-mannose pi,2- ENU - N-ethylnitrosourea N-acetylglucosaminyltransferase ER - Endoplasmic Reticulum POMT - protein O-mannosyltransferase FCMD - Fukuyama CMD (1 or 2) FKRP - Fukutin Related Protein RMP - Resting Membrane Potential Fuc - Fucose SAST - Syntrophin Associated Ser/ Fugu - Takifugu rubripes (puffer fish) Thr Kinase GABA - Gamma aminobutyric Acid Ser - Serine Gal - Galactose SG - Sarcoglycan GalNAc - N-acetyl- galactosamine Sia - Sialic Acid Glc - Glucose SNV sequence - GlcNAc - N-acetylglucosamine SSC - Sarcoglycan Sarcospan Complex HSPG - Heparan Sulfate Proteoglycan SSV sequence- Hyl - Hydroxylysine SU - Syntrophin Unique Domain Hyp - Hydroxyproline Syn - Syntrophin IPL - Inner Plexiform Layer Thr - Threonine + [K ]0 - Extracellular potassium Tyr - Tyrosine concentration w/v - weight per volume Kir - Inwardly Rectifying Potassium W WS - Walker Warburg Syndrome Channel Xyl - Xylose vii ACKNOWLEDGEMENTS This thesis, from its nascence through to its completion, has been shaped and molded by several instrumental and talented researchers. Many, many thanks to: Dr. Hakima Moukhles, my primary investigator, whose insight and patience gave rise to this project, whose support helped it puzzle together into a linear idea and whose valuable criticism gave rise to its completion. My motley crew of lab mates: Daniel (R2) Tham, Geoffroy (where are you from again?) Noel, Leona Lui and Natalie Tarn. You laughed when I laughed and you laughed when I cried. Dr. S. Carbonetto and Nadia Melan, University of Montreal, whose generous donation of Largemyd brain and eye tissue allowed this project to be realized. Dr. S. Froehner, University of Washington, for generously providing antibodies directed against syntrophins. vin DEDICATION For my mother and father Scientists through and through and for Braedon, the orchard of my eye. CO-AUTHORSHIP STATEMENT All components of this manuscript were produced by its primary author, Jennifer Rurak, with the following exceptions: The immunofluoresence experiments and imaging in Figures 2.1 and 2.2 were carried out by Leona Lui. The RT-PCR data in Figure 2.5 were generated by Bharat Joshi. All experiments were carried out under the supervision and guidance of Dr. Hakima Moukhles. x 1 INTRODUCTION 1.1 Muscular Dystrophy The term muscular dystrophy embodies a myriad of heterogeneous, inheritable diseases characterized by progressive muscle weakness and deterioration.
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