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The Effects of TAR DNA Binding Protein Mutations on RNA Processing Associated with Amyotrophic Lateral Sclerosis

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The Effects of TAR DNA Binding Protein Mutations on RNA Processing Associated with Amyotrophic Lateral Sclerosis The effects of TAR DNA binding protein mutations on RNA processing associated with Amyotrophic Lateral Sclerosis By Afnan Ali Al Sultan Submitted for the degree of Doctor of Philosophy (PhD) Sheffield Institute for Translational Neuroscience University of Sheffield November, 2016 This PhD thesis is dedicated to my dear loving husband, Ahmed Alamer, Without your tremendous support, encouragement and love my dream would not have been possible 1 Abstract Introduction: Amyotrophic Lateral Sclerosis (ALS) is a devastating, chronic progressive neurodegenerative disorder, characterized by the loss of the upper motor neurons in the motor cortex and the lower motor neurons of the brainstem and spinal cord. This leads to muscle weakness, atrophy and paralysis. Death usually occurs 3-5 years from onset. In familial ALS, mutations in TARDBP, encoding the RNA binding protein TDP-43, cause 5% of cases. TDP-43 is mainly localized in the nucleus and has multiple functions, of which the best characterised is regulation of splicing/alternative splicing of hnRNA. In ALS TDP- 43 mislocates to the cytoplasm causing the characteristic protein aggregations. The current work investigates the possible effects of both TARDBP missense mutations and a truncation mutation on RNA processing. This was approached by examining the changes in gene expression in both the cytoplasm and nucleus in fibroblasts derived from familial ALS-TARDBP patients. Hypothesis: The cytoplasmic and nuclear transcriptomic profile from mutant TARDBP fibroblasts will generate different transcriptomic profiles than control fibroblasts and will establish transcripts and pathways dysregulated in the presence of mutations in TARDBP. The objectives were 1) to optimize the separation of nuclear and cytoplasmic RNA from patient and control fibroblasts, 2) to compare the expression profiles of the cytoplasmic and nuclear compartments from control and mutant fibroblasts and 3) to determine the effect of both mutation types on gene expression in fALS. Methodology: Fibroblast cell culture from fALS- TARDBP missense mutation, truncation mutation and controls was performed. In addition, cell fractionation and RNA extraction were performed by two methods, osmotic pressure and Trizol and commercially available kit. Gene expression profiling was achieved using the Human Exon Array 1.0 ST, Human Transcriptome Array 2.0 (HTA) and RNA Sequencing. Findings: The presence of a TARDBP mutation causes change in gene expression in fALS. Cytoplasmic fALS-TARDBP missense mutations were significantly enriched with dysregulated RNA processing genes using both the Human Exon Arrays 1.0 ST and the HTAs while cytoplasmic fALS-TARDBP truncated mutation were enriched with dysregulated angiogenesis using the HTA and dysregulated vesicle mediated transport genes using RNA sequencing. The nuclear fALS-TARDBP missense 2 mutations demonstrated dysregulated RNA splicing using both the Human Exon Arrays 1.0 ST and the HTA while nuclear fALS-TARDBP truncated mutation was mainly enriched with G-protein coupled receptors using the HTAs. Therefore, fALS-TARDBP subtype mutations revealed distinct affected biological processes. Conclusion: The different types of TARDBP mutations assayed here have different effects on gene expression and subsequently on cellular pathways involved in TARDBP-related ALS. 3 Acknowledgement and dedication I would like to thank my supervisors, Dr Paul Heath and Dr Janine Kirby for encouragement, support and guidance throughout my PhD. I would like extend my sincerest thanks to Mrs Catherine Gelsthorpe and Mr Matthew Wyles for providing me with technical assistance in the lab. I would also like to thank my friends in Sheffield for support. My deepest gratitude goes to ALS patients who donated samples for my project. I would also like to thank the University of Dammam for funding my project. I would like to dedicate this PhD thesis to my adorable three years old daughter, Huda. Also to my loving parents Ali and Bahia, my brother Adam and my sister Eman. 4 Table of contents Abstract ............................................................................................................. 2 Acknowledgement and dedication .................................................................. 4 List of tables ................................................................................................................ 12 List of figures .................................................................................................. 16 List of abbreviations ....................................................................................... 19 Chapter 1: Introduction .................................................................................. 20 1.1 Amyotrophic Lateral Sclerosis ......................................................................... 20 1.1.1 Clinical features ........................................................................................... 20 1.1.2 Diagnosis ...................................................................................................... 21 1.1.3 Treatment and management ..................................................................... 21 1.1.4 Pathogenesis ............................................................................................... 22 1.1.4.1 The role of genetic risk factors in ALS ......................................... 23 1.1.4.1.1 Cu-Zn superoxide dismutase1 (SOD1) ................................. 25 1.1.4.1.2 Trans-active response DNA-binding protein (TARDBP) ....... 25 1.1.4.1.3 Fused in sarcoma/ translocated in liposarcoma (FUS/TLS) .. 27 1.1.4.1.4 Chromosome 9 open reading frame 72 (C9ORF72) ............. 27 1.1.4.2 The role of RNA processing in ALS ............................................. 27 1.1.4.2.1 TAR DNA binding protein 34 (TDP-43) ................................. 31 1.1.4.2.2 Fused in sarcoma/ translocated in liposarcoma (FUS/TLS) .. 32 1.1.4.2.3 Chromosome 9 open reading frame 72 (C9ORF72) ............. 32 1.1.5 Gene expression profiling in ALS using human tissues and cells ....... 33 1.1.5.1 Use of human CNS tissue ........................................................... 34 1.1.5.2 Use of human peripheral tissue ................................................... 34 1.1.5.2.1 Venous whole blood .............................................................. 34 1.1.5.2.2 Fibroblasts ............................................................................ 35 1.1.6 Gene expression profiling .......................................................................... 35 1.1.6.1 In Vitro Transcription (IVT) Array GeneChip® ............................. 36 1.1.6.2 Human Exon 1.0 ST Array GeneChip®........................................ 36 1.1.6.3 Human Transcriptome Array GeneChip® .................................... 37 1.1.6.4 Next generation sequencing (NGS) (RNA sequencing) .............. 37 1.1.7 Studying alternative splicing ...................................................................... 41 1.1.8 The rationale behind the project ‘cytoplasmic and nuclear gene expression profiling’ .............................................................................................. 42 1.1.9 Hypothesis .................................................................................................... 43 5 1.1.10 Objectives .................................................................................................. 43 Chapter 2: Materials and methods ................................................................ 44 2.1 Fibroblast cell culture ......................................................................................... 46 2.1.1 Splitting fibroblasts ...................................................................................... 47 2.1.2 Mycoplasma testing .................................................................................... 47 2.2 Cell fractionation and RNA extraction ............................................................. 47 2.2.1 Cell fractionation and RNA extraction using Norgen kit for Cytoplasmic and Nuclear RNA Purification ............................................................................. 47 2.2.2 Cell fractionation by osmotic pressure ..................................................... 48 2.2.2.1 RNA extraction by Trizol method ................................................. 48 2.3 Glycoblue precipitation method ....................................................................... 48 2.4 Nanodrop 1000 spectrophotometer ................................................................ 49 2.5 Agilent Bioanalyser 2100 .................................................................................. 49 2.6 DNase treatment ................................................................................................ 49 2.7 GeneChip® Arrays .............................................................................................. 50 2.7.1 Sample preparation for both Human Exon Arrays 1.0 ST (HEA) and Human Transcriptome Arrays (HTA) RNA samples ........................................ 50 2.7.2 Poly-A RNA controls preparation for both HEA and HTA RNA samples ................................................................................................................................. 53 2.7.3 Synthesis of first strand cDNA, second strand cDNA and cRNA by In Vitro Transcription (IVT) for both HEA and HTA .............................................. 54 2.7.4 cRNA purification for both HEA and HTA ...............................................
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