Phipps_whole_thesis.pdf (25.69 MB)
DNA methylation and histone modification dynamics in neurons in aging and Alzheimer's disease
thesisposted on 2023-05-28, 09:29 authored by Andrew PhippsAndrew Phipps
The epigenome dynamically regulates chromatin structure to control cellular function and homeostasis in a highly specific fashion and mediates the complex interaction between the DNA and our environment. Epigenetic mechanisms such as DNA methylation and posttranslational histone modifications contribute to the maintenance of cellular structure, identity, and plasticity in a cell type specific manner. Despite the key role epigenetics has in the regulation of the genome, there have been relatively few studies that have characterised the epigenome in neurons, neither in the healthy aging brain nor in neurodegenerative disease. Alzheimer's disease (AD) is a terminal progressive neurodegenerative disorder. While familial mutations account for approximately 10% of cases, the cause of 90% of AD cases is unknown. In the absence of highly penetrant risk alleles, epigenetics is well poised to contribute to the pathogenesis and progression of AD, but this role is yet to be fully explored. The research in this thesis has characterised DNA methylation in post-mortem human AD brains in a cell type specific manner, and is the first to characterise key histone modifications in neurons across a time-course of healthy aging, and across a time-course in a model of AD. Firstly, global DNA methylation (5mC) and hydroxymethylation (5hmC) levels were assessed in neuronal and glial cell types in the inferior temporal gyrus of human AD cases and age matched controls. Neurofilament (NF)-labelled pyramidal neurons, known to be vulnerable to AD pathology were deficient in extranuclear 5mC in AD cases compared to controls. This work also demonstrated that fewer astrocytes exhibited nuclear 5mC and 5hmC marks in AD cases compared to controls. However, there were no alterations in global levels of 5mC and 5hmC in disease-resistant calretinin-interneurons or microglia in AD. Furthermore, no alteration in the density of 5mC or 5hmC labelled nuclei was detected in near-plaque versus plaque-free regions in late-AD cases. 5mC and 5hmC were present in a high proportion of neurofibrillary tangles, suggesting no loss of DNA methylation marks in tangle bearing neurons. This demonstrated that global epigenetic dysregulation may be occurring in astrocytes and NFpositive pyramidal neurons in AD. Histone modifications aid in the regulation and compaction of DNA into chromatin. The second part of this PhD study was the first to characterise H3K27ac and H3K4me3 markers of active enhancer and promoter elements, using chromatin immunoprecipitation and next generation sequencing (ChIP-seq) in neurons from the forebrain of C57/BL6 mice at 3, 6, 12, and 24 months of age (n=5 per genotype). H3K27ac marking was enriched in young neurons at 3 months of age and old neurons at 24 months of age at a range of genomic regulatory regions including promoters proximal to transcriptional start sites, CpG islands around transcriptional start sites, enhancers distal to transcriptional start sites, and at known cortical super enhancers. H3K4me3 was also enriched at promoters in neurons at 3, 12, and 24 months of age, and at enhancers at 3 months of age. Gene ontology analysis predicted that H3K27ac and H3K4me3 aid in the regulation of neuronal processes including synaptic plasticity, ion channel binding, transporter activity, calcium channel activity and cellular metabolic processes. These data also point towards a partial recapitulation of a juvenile-like epigenetic state in late aging and demonstrate the dynamic nature of the histone landscape in neurons in the aging brain. APP/PS1 AD mice closely recapitulate the pathology present in human early-AD cases, including beta-amyloid plaque deposition, neuritic dystrophy and plaque-associated synapse loss. APP/PS1 mice allow for the examination of the earliest pre-pathology epigenetic changes that occur in AD, as well as measurement across a time course of disease progression. The third part of this PhD thesis used neuronal nuclei from the forebrain of APP/PS1 mice and age matched wild-type control mice (n=5 per genotype) at 3, 6 and 12 months of age that were subject to ChIP-seq using antibodies detecting H3K27ac and H3K4me3. These data identified enrichment of H3K27ac marking at the transcriptional start site and at cortical super enhancers, but depletion from enhancers, in neurons prior to pathology onset in APP/PS1 mice. H3K4me3 marking was also enriched at the transcriptional start site in neurons prior to pathology onset, and in pathology rich cases in APP/PS1 mice. Gene ontology analysis predicted that key neuronal specific pathways were disrupted early in AD. These included pathways involved in synaptic plasticity, membrane depolarisation and protein localisation, as well as pathways involved in cellular maintenance. Both H3K27ac and H3K4me3 marking evolved over time in APP/PS1 neurons. In addition, these data point towards a partial recapitulation of a juvenile like epigenetic state epigenome in pathology rich neurons, with numerous H3K27ac and H3K4me3 marked sites shared between 3 and 12 months of age and the results have also identified several novel genes and pathways yet to be investigated in AD. In summary, the data presented here show the complex nature of the neuronal epigenome and its dynamic response to neuropathology. The neuronal epigenome undergoes dramatic change in early adulthood and undergoes a partial recapitulation of a juvenile-like epigenetic state in late aging. The epigenome undergoes cell-type specific global loss of DNA methylation, occurring in both NF+ pyramidal neurons and astrocytes in AD. Furthermore, the neuronal epigenome is dysregulated prior to pathology onset in AD mice, and major restructuring of the histone landscape occurs with dense AD pathology. Taken together, the data presented within this thesis demonstrate the evolution of the neuronal epigenome in aging, and dysregulation of both DNA methylation and histone modifications in AD.
Rights statementCopyright 2019 the author Portions of chapter 3 appear to be the equivalent of a pre-print version of an article published as: Phipps, A. J., Vickers, J. C., Taberlay, P. C., Woodhouse, A., 2016. Neurofilament-labeled pyramidal neurons and astrocytes are deficient in DNA methylation marks in Alzheimer's disease, Neurobiology of aging, 45, 30-42