Factors regulating the function and assembly of the sarcoglycan complex in brain A thesis submitted for the Degree of Doctor of Philosophy at Cardiff University School of Medicine Francesca Carlisle 2016 Supervised by Professor Derek Blake and Professor Anthony Isles Thesis summary Myoclonus dystonia (MD) is a neurogenic movement disorder that can be caused by mutations in the SGCE gene encoding ε-sarcoglycan. ε-sarcoglycan belongs to the sarcoglycan family of cell surface-localised, single-pass transmembrane proteins originally identified in muscle where they form a heterotetrameric subcomplex of the dystrophin- associated glycoprotein complex (DGC). Mutations in the SGCA, SGCB, SGCG and SGCD genes encoding α-, β-, γ- and δ-sarcoglycan cause limb-girdle muscular dystrophy (LGMD). There is no phenotypic overlap between MD and LGMD. LGMD-associated sarcoglycan mutations impair trafficking of the entire sarcoglycan complex to the cell surface and destabilise the DGC in muscle, while MD-associated mutations typically result in loss of ε- sarcoglycan from the cell surface. This suggests cell surface ε-sarcoglycan but not other sarcoglycans is required for normal brain function. To gain insight into ε-sarcoglycan’s function(s) in the brain, immunoaffinity purification was used to identify ε-sarcoglycan- interacting proteins. Ubiquitous and brain-specific ε-sarcoglycan isoforms co-purified with three other sarcoglycans including ζ-sarcoglycan (encoded by SGCZ) from the brain. Incorporation of an LGMD-associated β-sarcoglycan mutant into the brain sarcoglycan complex impaired the formation of the βδ-sarcoglycan core but failed to abrogate the association and trafficking of ε- and ζ-sarcoglycan in heterologous cells. Both ε-sarcoglycan isoforms also co-purified with β-dystroglycan, indicating inclusion in DGC-like complexes. Additionally, the brain-specific ε-sarcoglycan isoform co-purified with the perineuronal net component tenascin-R, potentially suggesting a unique function for this isoform in modulating synapses. In common with SGCE, transcripts from the genes encoding α-, β-, δ-, γ- and ζ-sarcoglycans were found to undergo extensive alternative splicing, in some cases producing novel isoforms that affected assembly and trafficking of the sarcoglycan complex. In summary, data presented herein show that alternatively spliced sarcoglycan isoforms are part of the DGC in brain. These data contribute to our understanding of MD pathophysiology and the role of the sarcoglycan protein family. [i] Declarations This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed: Date: 20.09.2016 STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD. Signed: Date: 20.09.2016 STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed: Date: 20.09.2016 STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed Date: 20.09.2016 STATEMENT 4 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed: No bar on access required. Date: 20.09.2016 [ii] Acknowledgements Firstly, I would like to thank my supervisors Professor Derek Blake and Professor Anthony Isles for giving me the opportunity to work on this project. It has been a fascinating and challenging experience. I am immensely grateful to all those who have helped me out in the lab with training, advice, cheer, commiserations and desperate begging for just a tiny bit of this one reagent. In particular, I would like to thank Adrian for his endless advice, support, and willingness to share his ridiculously impressive knowledge of biochemistry. Other stars of the last three years include Alis, Matt, Taniesha, Anna, the other Anna, Lesley, Alex, Magda and Kira. Their help got me through many a trying day. Eloise, Jenny and Sarah – thank you from the bottom of my heart for the coffee dates, bake off hysteria, science jokes and everything else. You helped me keep it together when things got dark, and celebrated with me when things went well. Thank you also to my many friends at Ceroc South Wales, for the silliness and fun we had at each class. Without your good cheer and friendship, I would probably be irredeemably insane by now. To my fellow Aslam House princesses, Alison, Hannah, Stacy and Laura: we made it!!! Last but definitely not least, to my family: Mum, Dad, Emily, Charlie, Grandma, Sally, and my innumerable extended family – I cannot properly express how grateful I am to you for the support, encouragement and love. Even when I thought I couldn’t do it, you did. Thank you. [iii] Abbreviations dATP 2’-deoxyadenosine 5’-triphosphate dCTP 2’-deoxycytidine 5’-triphosphate dGTP 2’-deoxyguanosine 5’-triphosphate dNTP 2’-deoxynucleoside 5’-triphosphate dTTP 2’-deoxythymidine 5’-triphosphate ADom autosomal dominant AR autosomal recessive ATP adenosine triphosphate AP affinity purification α-SG alpha sarcoglycan aa amino acid N-terminus amino-terminus APS ammonium persulfate Amp ampicillin bp base pair BLAST basic local alignment search tool β-SG beta sarcoglycan BLAT BLAST-like alignment tool BSA bovine serum albumin CIP calf intestinal alkaline phosphatase CO2 carbon dioxide C-terminus carboxyl-terminus CNS central nervous system cDNA complementary DNA °C degrees Celsius δ-SG delta sarcoglycan DNA deoxyribonucleic acid DMP dimethylpimelimidate dihydrochloride DMSO dimethylsulphoxide DRD dopamine-responsive dystonia DTT dithiothreitol DMD Duchenne muscular dystrophy DMEM Dulbeccos modified Eagle medium DGC dystrophin-associated glycoprotein complex ER endoplasmic reticulum ERAD endoplasmic reticulum-associated degradation ε-SG epsilon sarcoglycan EDTA ethylenediaminetetraacetic acid EGTA ethyleneglycoltetraacetic acid ECACC European Collection of Authenticated Cell Cultures EST expressed sequence tag ECM extracellular matrix FBS foetal bovine serum fMRI functional magnetic resonance imaging γ-SG gamma sarcoglycan GST glutathione-S-transferase g gram [iv] HA human influenza haemagglutinin Tris-HCl HCl-buffered tris(hydroxymethyl)aminomethane h hour HEK human embryonic kidney IAP immunoaffinity purification IgG immunoglobulin G IP immunoprecipitation kb kilobase kDa kilodalton LGMD limb-girdle muscular dystrophy l litre LB Luria-Bertani media MRI magnetic resonance imaging mRNA messenger ribonucleic acid miRNA micro ribonucleic acid µg microgram µl microliter µM micromolar ml millilitre mM millimolar min minute M molar MD myoclonus dystonia TEMED N,N,N’,N’-tetramethylethylenediamine ng nanogram nM nanomolar NCBI National Centre for Biotechnology Information NE nuclear envelope nt nucleotide OD optical density PNN perineuronal net PBS phosphate-buffered saline PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PET positron emission tomography PTC premature termination codon pre-mRNA pre-messenger ribonucleic acid RIPA radioimmunoprecipitation assay RT-PCR reverse transcriptase polymerase chain reaction rpm revolutions per minute RNA ribonucleic acid SG sarcoglycan SGC sarcoglycan complex s second SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM standard error of the mean Tet tetracycline Thx thioredoxin TMS transcranial magnetic stimulation [v] Tris tris(hydroxymethyl)aminomethane TBST tris-buffered saline-Tween-20 U units UTR untranslated region v/v volume to volume ratio w/v weight to volume ratio Zeo zeocin ζ-SG zeta sarcoglycan β-me β-mercaptoethanol [vi] Table of contents Thesis summary .......................................................................................................................... i Declarations ............................................................................................................................... ii Acknowledgements .................................................................................................................. iii Abbreviations ............................................................................................................................ iv Table of contents ...................................................................................................................... vii List of figures and tables ........................................................................................................... xi Chapter 1: General introduction ............................................................................................ 1 1.1. Introduction ..................................................................................................................... 1 1.2. Overview of dystonia ...................................................................................................... 1 1.2.1. Classification of dystonia ........................................................................................ 2 1.2.1.1. Axis I: clinical features ...................................................................................