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Gene Expression Profiling and Functional Studies of Astrocytes in SOD1-Related Amyotrophic Lateral Sclerosis

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Gene Expression Profiling and Functional Studies of Astrocytes in SOD1-Related Amyotrophic Lateral Sclerosis Gene expression profiling and functional studies of astrocytes in SOD1-related amyotrophic lateral sclerosis David Baker Department of Neuroscience (SITraN) Supervisors: Dr Janine Kirby, Dr Daniel Blackburn and Prof. Dame Pamela Shaw PhD Thesis Submitted March 2015 Abstract Amyotrophic Lateral Sclerosis (ALS) is the most common adult onset motor neuron (MN) disorder, characterised by muscle wasting due to MN death. Astrocytes play an important role in disease progression in the SOD1G93A transgenic mouse model and patients of ALS. Although astrocytes display a selective toxicity to MN, the toxic factor(s) have not been identified. We hypothesise that differential gene expression in SOD1-ALS astrocytes will reveal targets for therapeutic intervention. Microarray analysis was performed upon Laser Capture Microdissected astrocytes isolated from spinal cord of symptomatic (90 day) and late-stage (120 day) SOD1G93A mice and non-transgenic (NTg) littermates, and from post- mortem human SOD1-ALS and control spinal cord. Functional studies were performed using enzymatic activity assays and immunohistochemistry upon spinal cord and in vitro validation studies were performed using murine neonatal cultures of astrocytes, microglia and embryonic MNs and “i-astrocytes” directly converted from human ALS fibroblasts. In murine astrocytes annotation clustering analysis showed increased expression of lysosomal transcripts which were validated by qPCR . Using functional experiments, we have found significantly higher activity of the lysosomal enzyme β-hexosamindase in the spinal cord of SOD1G93A mice. Immune response and phagocytic pathways are also enriched within both datasets, and phagocytosis assays using fluorescently labelled NSC34 cell debris show that SOD1G93A astrocytes engulf significantly higher amounts of neuronal debris compared to NTg controls, highlighting an increased reactivity of astrocytes at symptom onset. Human SOD1- astrocytes show down-regulation of transcripts involved in tight junction formation such as ZO-2, Claudin-5 and Occludin, and we hypothesise that ALS-astrocytes contribute to the breakdown in blood-brain-barrier (BBB) integrity seen in ALS. qPCR confirmed differential expression of BBB-influencing genes such as claudin-5, junctional adhesion molecule 2 and transforming growth factor beta-2. Model BBBs made with i-astrocytes from human patients co-cultured with endothelia show significantly lower transendothelial electrical resistance and dextran-permeability values pointing to an astrocyte role in increased BBB permeability during disease. These studies show the breadth of behaviours displayed by astrocytes during ALS disease progression and will provide an important guide for future therapeutic intervention. i Statement of contribution All of the work in this thesis is my own apart from where stated. ii Acknowledgments First of all, I would like to thank my supervisors (who were numerous!): Dr Laura Ferraiuolo, Dr Daniel Blackburn, Dr Janine Kirby and Professor Pamela Shaw. Your help, along with many of the researchers and PIs present in SITraN, has been greatly appreciated, never less so than in the writing of this thesis. I would also like to thank Dr Brian Kaspar of Ohio State University for allowing me to work in his lab for 3 months of my PhD. It was a once in a lifetime experience and I am extremely grateful to the entire Kaspar lab for their continuing help and friendship. I thank my parents and sister, who have always supported me in whatever I do, even if they do not understand what I am doing! It is no coincidence that I have managed to achieve a PhD after being brought up in such a loving and supportive environment. Finally, and most importantly, I would like to thank my fiancée Nicole, who has been a crutch to me whenever I have needed, and has accepted my travel to America and endless hours spent in the lab and office writing my thesis. Thank you for keeping me grounded. I only have two words left to say: never again. iii Contents Abstract ....................................................................................................................................... i Statement of contribution ......................................................................................................... ii Acknowledgments..................................................................................................................... iii List of Figures ............................................................................................................................. x List of Tables ........................................................................................................................... xiii Abbreviations ........................................................................................................................... xv CHAPTER 1.................................................................................................................................. 1 1. Introduction ....................................................................................................................... 2 1.1 Motor neuron disease ................................................................................................. 2 1.2 Amyotrophic lateral sclerosis ...................................................................................... 2 1.3 Genetic Subtypes of ALS .............................................................................................. 4 1.3.1 Superoxide dismutase 1 ....................................................................................... 7 1.4 Pathology of ALS .......................................................................................................... 9 1.4.1 ALS is a dying back axonopathy with impairment of axonal transport ............... 9 1.4.2 Inclusions ........................................................................................................... 10 1.4.3 Excitotoxicity ...................................................................................................... 11 1.4.4 Oxidative stress and reactive oxygen species (ROS) .......................................... 12 1.4.5 Mitochondrial damage ....................................................................................... 13 1.4.6 Inflammation and immune response ................................................................ 14 1.4.7 RNA processing .................................................................................................. 15 1.5 Non-cell autonomy in ALS ......................................................................................... 16 1.5.1 Astrocytes .......................................................................................................... 18 1.5.2 Astrocytes in ALS ................................................................................................ 22 1.5.3 Microglia ............................................................................................................ 29 1.5.4 Microglia in ALS .................................................................................................. 31 iv 1.5.5 Oligodendrocytes ............................................................................................... 35 1.5.6 The involvement of other cell types in ALS ....................................................... 36 1.6 Gene expression profiling identifies dysregulated pathways in ALS ........................ 38 1.6.1 Microarray technology ....................................................................................... 38 1.6.2 Chip manufacture .............................................................................................. 39 1.6.3 Competing technologies .................................................................................... 40 1.6.4 Application to ALS .............................................................................................. 40 1.7 Summary of the current situation ............................................................................. 43 1.8 Hypothesis and aims ................................................................................................. 45 1.8.1 Microarray of astrocytes from the SOD1G93A mouse model .............................. 45 1.8.2 Microarray of astrocytes from SOD1-ALS human subjects ............................... 46 CHAPTER 2................................................................................................................................ 47 2 Materials and methods .................................................................................................... 48 2.1.1 SOD1G93A transgenic mouse model .................................................................... 48 2.1.2 Cases for human microarray study .................................................................... 48 2.1.3 Chemicals and reagents ..................................................................................... 48 2.2 Methods .................................................................................................................... 50 2.2.1 Gene expression profiling of murine SOD1G93A astrocytes ................................ 50 2.2.2 Quantitative PCR (qPCR) validation ................................................................... 54 2.2.3 Functional studies of murine astrocytes............................................................ 57 2.2.4 Gene expression profiling of human astrocytes ...............................................
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