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Glucocerebrosidase Mutations and Parkinson's Disease

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Glucocerebrosidase Mutations and Parkinson's Disease 1 Glucocerebrosidase mutations and Parkinson's disease Dr Alisdair McNeill MRCP (UK) Thesis submitted in partial fulfilment of requirements for the award of the degree of Doctor of Philosphy by University College London January 2013 2 I, Alisdair McNeill confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed: Date: 01/03/2013 3 Acknowledgements Thank you to all the study participants who kindly agreed to participate in the clinical study andundergo skin biopsy. This work would not have been possible without their forebearance and generosity of spirit. I gratefully acknowledge the help of the Gaucher disease clinical team (Dr Hughes, Prof Mehta, Prof Cox, Dr Deegan and all the specialist nurses) in recruiting study participants. I would like to extend my thanks to Dr Matthew Gegg for providing me with outstanding teaching on how to perform the laboratory techniques utilised in this thesis. I acknowledge the supervision of Professor A Schapira and Professor T Warner. Last, but not least, thanks to my wife Ruth for supporting me. 4 Index Chapter I. General introduction, p. 10. Chapter II. Clinical study of prodromal markers of parkinson's disease in Gaucher disease families, p. 62. Chapter III. Study of endoplasmic reticulum associated degradation of mutant glucocerebrosidase in fibroblasts from Gaucher disease patients and Parkison's disease patients with heterozygous glucocerebrosidase mutations, p. 137. Chapter IV. Study of mitochondrial metabolism and markers of oxidative stress in fibroblasts from patients with Gauchers disease and Parkinson's disease with heterozygous glucocerebrosidase mutations, p. 196. Chapter V. General Discussion and conclusions, p. 235. 5 Index of tables and figures Table 1.Environmental risk factors for Parkinson's disease. p. 14. Table 2. Summary of lysosomal storage disorders. p. 30. Table 3. Demographic details of Gaucher disease patients. p. 82. Table 4. Demographic details of carriers. p. 86. Table 5. Montreal cognitive assessment scores. p. 94. Table 6. Correlations between prodromal markers of Parkison's disease. p. 95. Table 7. Demographic details of Parkinson's disease patients. p. 100. Table 8. Cognitive assessment scores in Parkinson's disease patients. p. 102. Table 9. Summary of clinical cases for DaTscan. p. 107. Table 10. Summary of asymmetry index scores. p. 108. Table 11. Summary of monoclonal antibodies used. p. 155. Table 12. Polymerase chain reaction primers used. p. 157. Table 13. Summary of fibroblast lines generated. p. 160. Figure 1. Summary of sphingolipidoses. p. 36. Figure 2. Summary of mucopolysaccharidoses. p. 37. Figure 3. Summary of olfactory function scores. p. 91. Figure 4. Box plot of Unified Parkinson's disease rating scale scores. p. 92. Figure 5. Box plot of Montreal cognitive assessment scores. p. 93. Figure 6. Graph of retinal ganglion cell complex thickness. p. 98. Figure 7. Kaplan-Meier analysis of age related risk of Parkinson's disease. p. 103. Figure 8. Box plots of right:left asymmetry index by genotype. p. 109. Figure 9. Striatal asymmetry index by genotype. p. 110. Figure 10. Representative DaTSCAN images from genetic Parkinson's disease. p. 111. Figure 11. Western blotting of GBA expression in fibroblasts. p. 162. Figure 12. Evidence of endoplasmic reticulum retention of GBA. p. 164. Figure 13. Study of autophagy markers. p. 168. Figure 14. Effect of ambroxol on GCase protein levels. p. 171. Figure 15. Effect of ambroxol on lysosomal markers. p. 173. Figure 16. Effect of ambroxol on alpha-synuclein overexpressing cells. p. 174. Figure 17. Electron transport chain. p. 199. Figure 18. Seahorse analysis profile. p. 211. 6 Figure 19. Analysis of mitochodrial metabolism. p. 218. Figure 20. Analysis of oxidative stress markers. p. 220. Figure 21. Postulated mechanisms by which GBA mutations cause Parkinson's disease. p. 235. 7 List of abbreviations used ADP: adenosine diphosphate ATP: adenosine triphosphate ATF: activating transcription factor ATP: adenosine triphosphate CHOP: CCAAT-enhancer-binding protein homologous protein CI: confidence interval DAPI: 4',6-diamidino-2-phenylindole DHE: dihydroethidium DMSO: dimethyl sulfoxide DNA: deoxyribonucleic acid dH2O: distilled water Endo H: endoglycosidase H ER: endoplasmic reticulum ERAD: endoplasmic reticulum associated degradation ERT: enzyme replacement therapy FCCP: Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone FCS: Fetal calf serum GA: giberallic acid GBA: glucocerebrosidase gene GCC: ganglion cell complex thickness Gcase: glucosylceramidase enzyme GD: Gaucher disease GM: monosialodihexosylganglioside GRP78: Glucose regulatory protein 78 (also known as BiP) HA: haemagluttinin HDACi: histone deacetylase inhibitor HSP: heat shock protein 8-OHG: 8-hydorxy guanyl IIF: indirect immunofluorescence IQR: interquartile range IRE1: inositol requiring enzyme-1 LAMP1: Lysosomal associated membrane protein-1 LIMP2: Lysosomal integral membrane protein-2 8 4-HNE: 4-hydroxynonenal NMC: non-manifesting carriers PBS: phosphate buffered saline PCR: polymerase chain reaction PD: Parkinson's disease PERK: PKR-like endoplasmic reticulum kinase PSI: proteasome inhibition MCB: monochlorbamine MCI: mild cognitive impairment MMSE: minmental state examination MOCA: Montreal cognitive assessment MPS: mucopolysaccharidoses LBD: Lewy body dementia LRRK2: Leucin rich repeat kinase-2 LSD: lysosomal storage disorder NCL: Neuronal ceroid-lipofuscinoses NMSS: non-motor symptoms scale OCR: oxygen consumption rate Optical coherence tomography: OCT OR: Odds ratio PINK1: PTEN induced putative kinase-1 PD – GBA: Parkinson's disease with heterozygous GBA mutation PD – S: Parkinson's disease without GBA or LRRK2 G2019S mutation RBD: Rapid eye movement sleep behaviour disorder RNA: ribonucleic acid RNFL: retinal nerve fiber layer ROS: reactive oxygen species SNCA: alpha-synuclein SRT: substrate reduction therapy TFEB: transcription factor EB MDS-UPDRS: Movement Disorders Society Unified Parkinson's Disease Rating Scale TMT : timed motor tests UMSARS: unified Multiple Systems Atrophy Rating scale UPR: unfolded protein response UPSIT: University of Pennsylvania Smell Identification Test 9 XBP1: x-box binding protein 1 Abstract Objectives - Gaucher disease (GD) is caused by bi-allelic mutations in the glucocerebrosidase gene (GBA). GD and heterozygous carriage of GBA mutations significantly increase the risk of developing Parkinson's disease (PD). Here we studied GD patients and carriers to identify a cohort of individuals with clinical signs of prodromal PD and generated fibroblast lines from them to study why GBA mutations cause PD. Methods - 83 patients with Type I GD and 41 of their heterozygous carrier relatives were recruited from lysosomal storage disorder clinics at the Royal Free Hospital and Addenbrooke's Hospital Cambridge, along with 30 mutation negative matched controls. They were clinically screened for hyposmia (University of Pennsylvania Smell Identification Test), cognitive impairment (Montreal Cognitive Assessment), autonomic dysfunction, REM sleep behaviour disorder and motor signs of PD. Two hundred and thirty cases of sporadic PD were screened for GBA gene mutations. Fibroblasts were generated from skin biopsies taken from a selection of patients. GBA metabolism (Western blotting for protein levels, enzyme activity, immunofluorescent localisation), mitochodrial metabolism, endoplasmic reticulum and oxidative stress markers were assayed in the cell lines. Results – GD patients and heterozygous carriers had significantly lower olfactory and cognitive function scores than controls. Several GD patients and carriers had motor signs of PD (e.g. rest tremor) while controls did not. Thirteen PD patients with heterozygous GBA mutations were identified. Their clinical phenotype was similar to mutation negative PD cases. GD fibroblast lines (n=5), and lines from heterozygous GBA mutation carriers with (n=4) and without PD (n=2) had reduced GBA enzyme activity due to endoplasmic reticulum retention of GBA protein. This was associated with upregulation of endoplasmic reticulum stress markers and oxidative stress (increased rate of dihydroethidium oxidation). Conclusions – a subset of GD patients and carriers express clinical markers of prodromal PD. Study of fibroblasts from these individuals indicates that endoplasmic reticulum and oxidative stress may contribute to increased PD risk in these individuals. 10 Chapter I. GENERAL INTRODUCTION. 11 General Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease in the United Kingdom (U.K), with an age dependent prevalence of 1/150-1/350 (Lees et al, 2009). PD is characterised by rest tremor, rigidity, bradykinesia and postural instability. The classical motor syndrome of PD becomes apparent once greater than 50% of dopaminergic neurons in the substantia nigra pars compacta have degenerated. Neuronal degeneration in PD is associated with the presence of eosinophilic inclusions in surviving neurons known as Lewy bodies. Lewy bodies are composed of alpha-synuclein (SNCA), a synaptic protein of uncertain function. Before the motor syndrome of PD occurs there is a long prodromal period during which non-motor signs and symptoms of PD predominate (Schapira & Tolosa, 2010). Olfactory dysfunction, autonomic dysfunction, depression and subtle motor slowing are well recognised as clinical markers of the prodromal phase. The aetiology of PD is
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