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Defining the Role of the Golgi Apparatus in Juvenile NCL (Batten Disease)

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Defining the Role of the Golgi Apparatus in Juvenile NCL (Batten Disease) Defining the role of the Golgi apparatus in juvenile NCL (Batten disease) Davide Marotta MRC Laboratory for Molecular Cell Biology University College London Dr. Sara E. Mole A thesis submitted for the degree of Doctor of Philosophy in the University College London October 2014 1 Declaration I, Davide Marotta, 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. 2 Abstract The neuronal ceroid lipofuscinoses (NCLs) are a group of severe neurodegenerative lysosomal storage disorders characterised by accumulation of autofluorescent ceroid lipopigments in most cells. NCLs are caused by mutations in at least fourteen recessively inherited human genes. The NCL genes encode both soluble and transmembrane proteins localised to the endoplasmic reticulum, Golgi apparatus or endosomal/lysosomal organelles. Mutations in the CLN3 gene result in juvenile neuronal ceroid lipofuscinoses (JNCL, Batten disease). JNCL represents the worldwide most common form of NCL. Currently more than 40 mutations have been characterised in the CLN3 gene. However, the most common mutation causes a 1-kb deletion. CLN3 encode a multi-pass type III transmembrane protein, which is conserved in single-celled eukaryotes such as the fission yeast Schizosaccharomyces pombe, suggesting a fundamental role for this protein in eukaryotic cells. CLN3 has been functionally linked to many different cellular processes, including lysosomal homeostasis, autophagy, lipid synthesis or modification, cytoskeleton organisation and trafficking. Despite these endeavours, the function of CLN3 remains unclear. The main goal of this project was to investigate the role of the Golgi apparatus in the pathogenesis of juvenile CLN3 disease. The role of CLN3 at the Golgi apparatus was studied in mammalian cells and in fission yeast model. The morphology of the Golgi complex was studied in fibroblast cell lines from patients and in HeLa cells depleted for CLN3 using RNAi. The observed changes in morphology were accompanied by manganese dyshomeostasis within the Golgi complex, ER stress and apoptosis. The morphology of the Golgi complex was studied in S. pombe using electron microscopy in 3 order to confirm the changes observed in mammalian cells. Finally, drugs shown to ameliorate aspects of the yeast model of CLN3 disease were tested for their efficacy in mammalian cells as an early step in therapeutic development. In this study I have shown that both morphology and size of the Golgi apparatus result to be affected by the loss/depletion of CLN3. Moreover, the changes in Golgi complex morphology and size are accompanied by manganese dyshomeostasis within the Golgi complex with activation of ER stress and activation of the proapoptotic protein caspase 2. Together, these data suggest that the loss/depletion of CLN3 activates secretory stress pathways and cell death. A dysfunctional Golgi apparatus may be the key to uncover the role of CLN3 and find new targets for therapeutic development. 4 Table of Contents DECLARATION ............................................................................................................. 2 ABSTRACT ..................................................................................................................... 3 TABLE OF CONTENTS ................................................................................................ 5 TEXT ............................................................................................................................... 5 FIGURES ....................................................................................................................... 10 TABLES ........................................................................................................................ 12 ABBREVIATIONS ....................................................................................................... 14 1. INTRODUCTION ..................................................................................................... 16 1.1 OVERVIEW ............................................................................................................. 17 1.2 LYSOSOMAL STOREAGE DISORDERS ....................................................................... 18 1.3 NEURONAL CEROID LIPOFUSCINOSES ...................................................................... 19 1.4 JUVENILE NCL (JNCL) .......................................................................................... 23 1.4.1 CLN3 ............................................................................................................. 24 1.4.2 The CLN3 orthologue in fission and budding yeast ..................................... 30 1.4.3 Phenotypes in btn1Δ cells ............................................................................. 32 1.5 GOLGI APPARATUS ................................................................................................. 35 1.5.1 Morphology of the Golgi complex ................................................................ 36 1.5.2 The Golgi complex in yeast ........................................................................... 42 1.5.3 ER-Golgi intermediate compartment (ERGIC) ............................................ 43 1.5.4 cis-Golgi network (CGN) .............................................................................. 44 1.5.5 trans-Golgi network (TGN) .......................................................................... 45 1.5.6 transport machinery at Golgi apparatus ...................................................... 46 1.5.6.1 Coat protein I (COPI) .......................................................................... 46 1.5.6.2 Coat protein II (COPII) ....................................................................... 47 1.5.7 Golgi complex in neurodegenerative disorders ............................................ 49 1.5.8 Golgi complex and manganese homeostasis ................................................ 53 1.5.9 Role of manganese in neurodegenerative disorders ..................................... 56 1.6 ENDOPLASMIC RETICULUM (ER) STRESS ................................................................ 61 1.6.1 The UPR in fission and budding yeast .......................................................... 62 1.6.2 The UPR in mammals ................................................................................... 63 5 1.6.3 ER stress and UPR in neurodegenerative disorders .................................... 67 1.7 PROJECT AIMS ........................................................................................................ 69 2. MATERIALS AND METHODS ............................................................................. 70 2.1 MATERIALS ............................................................................................................ 71 2.1.1 List of reagents ............................................................................................. 71 2.1.2 Mammalian cell culture ................................................................................ 74 2.1.2.1 Cell type/lines used .............................................................................. 74 2.1.2.2 Fibroblast cell lines used ..................................................................... 74 2.1.2.3 Cell culture media ................................................................................ 75 2.1.3 siRNA, plasmids and qPCR primers ............................................................. 76 2.1.3.1Primers, siRNA and plasmid used ........................................................ 76 2.1.4 qPCR reaction and FastStart PCR ............................................................... 77 2.1.5 Protein extraction and SDS-PAGE ............................................................... 78 2.1.5.1List of Antibodies used .......................................................................... 78 2.1.5.2 Mammalian cells lysis buffer ............................................................... 79 2.1.5.3 Laemmli buffer ..................................................................................... 79 2.1.5.4 Protein gel buffers ............................................................................... 80 2.1.5.5 Protein gels .......................................................................................... 81 2.1.6 S. pombe strains and maintenance ............................................................... 82 2.1.6.1 S. pombe strains used .......................................................................... 82 2.1.6.2 Growth media ...................................................................................... 83 2.2 METHODS ............................................................................................................... 88 2.2.1 Cell culture and maintenance ....................................................................... 89 2.2.2 siRNA Transfection of HeLa cells using Metafectene®Pro .......................... 89 2.2.3 Quantitative PCR (qPCR) ............................................................................ 91 2.2.3.1 RNA extraction from HeLa .................................................................. 91 2.2.3.2 RNA reverse transcription ................................................................... 91 2.2.3.3 Endpoint PCR using FastStart polymerase ......................................... 92 2.2.3.4 qPCR calibration curve ......................................................................
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