A Profiling of DNA Modifications in Neurodegenerative and Neuropsychiatric Disorders Restricted; Files Only
Kuehner, Janise (Spring 2023)
Published
Abstract
Epigenetics refers to heritable changes in gene expression without altering the DNA sequence through mechanisms such as DNA and histone modifications and non-coding RNAs. Mounting evidence implicates critical roles for DNA modifications, specifically 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), in regulating brain development and disease. 5mC is one of the best characterized epigenetic marks and is regarded as a highly stable mark in differentiated cells typically at CpG dinucleotides. On the other hand, 5hmC has emerged as a key DNA modification in the nervous system due to its significant enrichment in the brain and its ability to regulate neuronal-specific gene expression during neural progenitor cell differentiation. Both nervous system development and function can be affected by epigenetic spatiotemporal regulation of gene expression. In the mammalian central nervous system, epigenetic dysregulation is associated with neurodegenerative diseases such as Alzheimer Disease and neuropsychiatric diseases such as major depressive disorder (MDD).
Towards this end, this thesis investigates the roles of 5mC and 5hmC in neurodegenerative and neuropsychiatric diseases. First, using a forebrain organoid model system, we investigated the dynamic changes of 5hmC during mammalian brain development and in Alzheimer’s Disease (AD). 5hmC and transcriptome profiles encompassing several developmental time points of healthy forebrain organoids and organoids derived from several familial AD patients were developed. We observed that stage-specific differentially hydroxymethylated regions (DhMRs) display an acquisition or depletion of 5hmC modifications across development stages. Importantly, our AD organoids corroborate cellular and molecular phenotypes previously observed in human AD brains. These data suggest a highly coordinated molecular system that may be dysregulated in these early developing AD organoids. In the second half of this thesis, we explored the contribution of 5mC and 5hmC underlying the individual differences in stress susceptibility and resilience. Genome-wide 5mC, 5hmC and transcriptome profiles from animals that underwent various durations of social defeat were generated. We characterized the epigenetic impact of chronic stress in young and mature animals to determine if DNA modifications were responsible for increased stress susceptibility with age. Moreover, we observed that 5mC and 5hmC work in parallel in young animals, while in mature animals they function in distinct biological process. Acute stress responses may epigenetically “prime” the animals to either increase or decrease their predisposition to depression susceptibility. Re-exposure studies reveal that the enduring effects of social defeat affect differential biological process between susceptible and resilient animals. Stress-induced 5mC and 5hmC fluctuations across the acute-chronic-longitudinal time course demonstrate that the negative outcomes of chronic stress do not discriminate between susceptible and resilient animals. Finally, 5mC appears to be responsible for acute stress response, whereas 5hmC may function as a persistent and stable modification in response to stress. This study broadened the scope of previous research and offers a comprehensive analysis of the role of DNA modifications in stress-induced depression.
Table of Contents
Chapter 1: Introduction to Dissertation 1
1.1. DNA Methylation 2
1.1.1. Functional Roles of DNA Methylation 2
1.1.2. DNA Methylation in the Brain 5
1.1.3. DNA Methyltransferases 7
1.1.4. DNA Methyltransferases in the CNS 9
1.1.5. Methyl-Binding Proteins 10
1.2. DNA Demethylation 12
1.2.1. Mechanism of DNA Demethylation 12
1.2.2. TET enzymes 12
1.2.3. TET Enzymes in the CNS 13
1.2.4. Roles of 5hmC, 5fC, and 5caC in the CNS 14
1.3. Histone Modifications 15
1.3.2. Polycomb Group Proteins 16
1.3.3. Trithorax Group Proteins 17
1.3.4. PcG and TrxG Proteins in CNS 17
1.3.5. Histone Acetylation 18
1.4. Chromatin Remodeling 19
1.4.2. BAF Chromatin Remodelers 20
1.5. Regulatory RNA 21
1.5.2. microRNAs 22
1.5.3. Long non-coding RNAs 23
1.6. Alzheimer’s Disease 25
1.6.1. The Roles of DNA Methylation and Demethylation in AD 26
1.6.2. The Roles of Polycomb and Trithorax Proteins in AD 28
1.7. Stress and Depression 29
1.7.1. Histone Modifications in Stress and Depression 29
1.7.2. DNA Modifications in Stress and Depression 30
1.7.3. Disruption of DNA Methylation from Environmental Stressors 33
Chapter 2: 5-hydroxymethylcytosine is dynamically regulated during forebrain organoid development and aberrantly altered in Alzheimer’s disease 37
2.1. Summary 38
2.2. Introduction 38
2.3. Results 40
2.3.1. Genome-wide profiling of 5hmC in forebrain organoids during early brain development 40
2.3.2. Dynamics of 5hmC regulation during forebrain organoid development 41
2.3.3. Alzheimer’s disease forebrain organoids recapitulate hallmark AD pathologies 43
2.3.4. 5hmC is globally altered in Alzheimer’s disease organoids 44
2.4. Discussion 46
2.4.1. 5hmC Acquisition and Depletion in Coding and Non-coding Regions During Neurodevelopment 46
2.4.2. Forebrain Organoid Model of Alzheimer’s Disease and the Impact of 5hmC Global Alterations 47
2.5. Figures and Figure Legends 49
2.6. Materials and Methods 62
Chapter 3: Social Defeat Stress Induces Genome-Wide 5mC and 5hmC Alterations in the Mouse Brain 70
3.1. Summary 71
3.2. Introduction 71
3.3. Results 73
3.3.1. Chronic social defeat stress induces social avoidance in young and mature adult mice 73
3.3.2. Global characterization of susceptible and resilient DMRs and DhMRs in young and mature adult mice 74
3.3.3. Comparison Between Shared DMRs and DhMRs in Young and Mature Adult Mice 76
3.3.4. Acute social defeat induced epigenetic alterations that prime chronic stress response 80
3.3.5. Longitudinal social defeat suggests that epigenetic memory may protect against chronic stress 84
3.3.6. 5mC and 5hmC dynamics across the ASDS-CSDS-LSDS time course 86
3.4. Discussion 90
3.5. Figures and Legends 95
3.6. Materials and Methods 121
Chapter 4: Epigenetic Machinery: Tet2 KO and Stereotaxic Experiments 128
4.1. Introduction 129
4.2. Preliminary Results and Discussion 129
4.3. Figures and Figure Legends 131
4.4. Material and Methods 133
Chapter 5: The Third DNA Modification: 6mA and its Writer Alkbh1 135
5.1. Introduction 136
5.2. Preliminary Results and Discussion 136
5.3. Figures and Figure Legends 137
5.4. Materials and Methods: 138
Chapter 6: Conclusions and Future Directions 139
6.1. General Conclusions on Alzheimer’s Disease 140
6.1.1. Alzheimer’s Disease Background 141
6.1.2. A Discussion on Alzheimer’s Disease Model Systems 141
6.1.3. The Incorporation of Inclusive Practices in AD Studies 144
6.1.4. Future Experiments for Alzheimer’s Disease Organoids 145
6.1.5. Alzheimer’s Disease Conclusions 148
6.2. General Conclusions for Chronic Stress 148
6.2.1. Validations and Functional Experiments 149
6.2.2. Additional Stress Responding Brain Regions 151
6.2.3. Cell Type Specific Epigenetic Profiling Using Immunopanning 152
6.2.4. Investigating Stress Response in Females and Alternative Stress Approaches 153
6.2.5. Stress Conclusions 155
6.3. Overall Conclusions 155
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File download under embargo until 22 May 2024 | 2023-04-05 12:35:26 -0400 | File download under embargo until 22 May 2024 |
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