The Role of DNA Damage-Induced Cellular Senescence in the Pathophysiology of Mtbi
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The Role of DNA Damage-Induced Cellular Senescence in the Pathophysiology of mTBI by Nicole Schwab A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Nicole Schwab 2019 The Role of DNA Damage-Induced Cellular Senescence in the Pathophysiology of mTBI Nicole Schwab Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2019 Abstract The pathophysiological mechanism whereby mTBI leads to chronic symptoms and neuropathology is unknown, although it is known that cellular senescence underlies several neurological disorders. I hypothesize that mTBI causes DNA damage-induced cellular senescence, leading to the acute and chronic neurological dysfunction reported in individuals who have suffered from mTBI. Using a cohort of 38 donated brains with mTBI history, I have found extensive DNA damage with immunohistochemistry (γH2AX, a marker of double-stranded DNA breaks) not seen in control brains. Furthermore, gene expression analysis using NanoString revealed activation of DNA damage response pathways, insufficient expression of DNA repair proteins, and upregulation of pro-inflammatory and cellular senescence pathways. This study suggests that DNA damage-induced cellular senescence may be the underlying pathophysiological mechanism of acute and chronic brain dysfunction after mTBI, and represents potential therapeutic targets for treatment of mTBI-induced brain dysfunction. ii Acknowledgements First, I would like to thank my supervisor, Dr. Lili-Naz Hazrati, for having me as her graduate student and providing me with invaluable guidance and consistent support throughout my MSc research. I would like to thank Yvonne Zhong for helping us perform NanoString experiments and guiding me with statistical analysis as well as Paula Marrano for her patience and support in teaching me various immunohistochemistry techniques throughout this project. In addition, I thank our collaborator, Dr. Anne Wheeler, and her lab members for giving me mice to use for preliminary work. Lastly, I would like to thank my advisory committee, Dr. JoAnne McLaurin and Dr. Peter Wells, for their consistent support and constructive feedback, and for committing their time to the success of my research project. iii Table of Contents Abstract ii Acknowledgements iii Table of Contents iv List of Tables and Figures vi List of Abbreviations vii List of Appendices viii 1. INTRODUCTION 1 1.1 Background 1 1.1.1 Mild Traumatic Brain Injury 1 1.1.2 The association between mTBI and neurodegenerative disease 3 1.1.3 From “Punch Drunk” to Chronic Traumatic Encephalopathy 4 1.1.4 Oxidative Stress, DNA damage, and the DNA Damage Response 9 1.1.5 Cellular Senescence and the Senescence-Associated Secretory Phenotype (SASP) 11 1.2 Rationale and Hypothesis 13 1.3 Specific Aims 14 1.4 Scientific Impact 14 2. MATERIALS AND METHODS 16 2.1 Cases and sample acquisition 16 2.2 Immunohistochemistry for Neuropathology and DNA damage 16 2.2.1 Image Analysis 17 2.3 NanoString Gene Expression Assay 17 2.3.1 nSolver Analysis 18 2.4 Mouse CCI Model 18 3. RESULTS 20 iv 3.1 Cohort demographics and clinical presentation 20 3.2 Neuropathological assessment 22 3.3 Immunohistochemistry 23 3.3.1 mTBI brains accumulate extensive DNA damage marked by γH2AX 23 3.4 Nanostring 34 3.4.1 DDR signalling 34 3.4.2 DNA repair genes 36 3.4.3 Regulators of cellular senescence 38 3.4.4 SASP factors 40 3.5 Clinicopathological correlation 42 3.6 DNA damage and cellular senescence are evident in a CCI mouse model 43 4. DISCUSSION 46 4.1 Evidence of DNA damage in human concussed brains 46 4.2 Impaired DNA repair capacity in human concussed brains 48 4.4 Clinicopathological correlation in human concussed brains 51 4.5 Evidence of DNA damage and cellular senescence in a CCI mouse model 53 4.6 Cellular senescence as the pathophysiological mechanism of mTBI-induced brain dysfunction 54 5. FUTURE DIRECTIONS 57 6. CONCLUSION 60 7. REFERENCES 61 APPENDICES 79 v List of Tables and Figures Figure 1: Cellular senescence and the SASP Table 1: Cohort demographics Figure 2: Proportion of neuropathological diagnoses Figure 3: γH2AX control case Figure 4: γH2AX stage 1 staining Figure 5: γH2AX stage 2 staining Figure 6: γH2AX stage 3 staining Figure 7: γH2AX quantification results Figure 8: GFAP staining in a case versus control Figure 9: H3K27Me3 staining in a case versus control Figure 10: Lamin B1 staining in a case versus control Figure 11: Histogram of DDR gene expression in human cohort Figure 12: Boxplot of DNA repair gene expression in human cohort Figure 13: Histogram of DNA repair gene expression in human cohort Figure 14: Boxplot of senescence gene expression in human cohort Figure 15: Boxplot of SASP gene expression in human cohort Table 2: Clinical presentation in senescent and non-senescent cases Figure 16: γH2AX and Lamin B1 staining in CCI mice versus sham Figure 17: Histogram of DDR and senescence gene expression in CCI mice Figure 18: Histogram of SASP gene expression in CCI mice Figure 19: Overview of DNA damage-induced senescence pathway in mTBI vi List of Abbreviations Note: A complete list of gene names is included in Appendix 1 8-OhDG: 8-oxo-2’-deoxyguanosine AD: Alzheimer’s disease ALS: amyotrophic lateral sclerosis Aβ: amyloid beta BER: base-excision repair CCI: controlled cortical impact CTE: chronic traumatic encephalopathy DDR: DNA damage response DSB: double-stranded break FTD: fronto-temporal dementia GFAP: glial fibrillary acidic protein MCI: mild cognitive impairment mTBI: mild traumatic brain injury NFT: neurofibrillary tangle PD: Parkinson’s disease p-tau: hyperphosphorylated tau protein ROS: reactive oxygen species SAHF: senescence-associated heterochromatin foci SASP: senescence-associated secretory phenotype SSB: single-stranded break TBI: traumatic brain injury TDP-43: TAR DNA-binding protein 43 vii List of Appendices Appendix 1: List of genes included in NanoString custom panel Appendix 2: NanoString mouse neuroinflammation panel viii 1. INTRODUCTION 1.1 Background 1.1.1 Mild Traumatic Brain Injury Traumatic brain injury (TBI) is a leading cause of death and disability worldwide, affecting an estimated 10 million individuals each year (1). In particular mild TBI (mTBI), which includes concussions and sub-concussive blows to the head (2), affects the largest proportion of these individuals. It is difficult to determine the exact incidence of mTBI, as many individuals do not seek medical attention (3), and as a result it is estimated that the true prevalence of mTBI is substantially higher than officially reported (4). It is important to note that some sub-populations experience mTBI at a higher incidence than others, for example professional athletes (5), survivors of domestic abuse (6), and military personnel (7). Individuals in these groups may also underreport their injuries due to various barriers, such as fear of repercussions from a violent partner, not wanting to sit out of play, lack of education on mTBI, or not having the resources to report. It is clear from population statistics that mTBI is extremely common and can be considered a substantial public health issue. For decades it was thought that concussions have little to no consequences to brain health, however it is now clear that the exact opposite is true. mTBI, especially when experienced repetitively (8), is linked to several long-term symptoms which are broad in nature, involving mood, behavior, and cognition (9). In some individuals, mTBI results in acute symptoms such as headache, nausea, fatigue, and confusion (10, 11) which may resolve anywhere from one week to a few months (12). However, an estimated 20% of individuals who experience repetitive mTBIs go on to be diagnosed with post-concussive syndrome (13), defined as the absence of resolution of symptoms 3 months post-injury (14). The long term sequelae of repetitive mTBIs can therefore include symptoms of post-concussive syndrome such as mood disorders (15) (particularly anxiety and/or depression), sleep disturbances (16), cognitive deficits (including memory and attention 1 problems) (17), and an increased risk of being diagnosed with dementia or a neurodegenerative disease later in life (18). The reported prevalence rate of depression following mTBI varies between studies and ranges from 12% (19) to 44% (20), but it has been shown that depression at one month post-injury significantly predicts progression to post-concussive syndrome up to one year post-injury (21). This same study found that over 20% of mTBI patients reported headaches, sleep disturbances, irritability, forgetfulness, and poor concentration one year post-injury (21). In fact, one of the most reported symptoms in individuals with post-concussive syndrome is fatigue and an inability to keep up with their work (22). It is important to note that the prevalence of post- concussive symptoms vary between studies depending on methodology, study population, and timing of assessment. In particular, there is a tendency for mTBI patients to overestimate their pre-injury mood and productivity levels such that they perceive their pre-injury functioning as above average, known as the “good old days” bias (23). In addition, mTBI patients tend to over- report sleep disturbances with subjective measurements, such as self-reporting, when compared to objective measurements (24). Despite possible discrepancies in the reported prevalence of specific symptoms, it is clear that some individuals who experience mTBI recover fairly quickly and some continue to experience debilitating symptoms chronically. Currently, it is unclear what differentiates individuals who recover quickly, and those which go on to experience post- concussive syndrome and/or permanent disability. As mTBI affects nearly all populations, with some sub-populations at higher risk than others, it is considered a significant public health issue which requires further clarification. Currently, the molecular cause of the acute and chronic effects of mTBI remain unknown and no standard clinical tools for diagnosis nor prognosis have been implemented.