A Profiling of DNA Modifications in Neurodegenerative and Neuropsychiatric Disorders Open Access

Kuehner, Janise (Spring 2023)

Permanent URL: https://etd.library.emory.edu/concern/etds/8336h3277?locale=en


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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