DNA methylation and nucleosome occupancy landscapes of excitatory neurons in human sporadic Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative condition and is the main cause of dementia affecting the elderly population. The vast majority of AD cases are sporadic cases that occur after 65 years of age. While multiple environmental and genetic risk factors have been identified for sporadic AD, a large proportion of sporadic AD risk remains unexplained. Epigenetics, the study of the processes that modify gene activation or repression, are at the interface of the genome and the environment. Previous research has shown that epigenetic changes may play a role in AD risk and biology. The majority of these studies have been completed using whole brain homogenate, an approach that obscures cell-type specific epigenetic signatures, making it difficult to define changes occurring in individual cell types. This is particularly important when considering the molecular aetiology of AD as excitatory neurons are the most vulnerable to dysfunction, degeneration, and death; yet, only make up an estimated 30% of the cells in the brain. This research project developed and optimised a new fluorescence activated nuclei sorting protocol to purify excitatory neuronal nuclei from human brain tissue. This enabled DNA methylation and nucleosome occupancy patterns to be investigated in vulnerable excitatory neurons in human sporadic AD for the first time.

Nucleosome occupancy and methylome sequencing (NOMe-seq) was performed on excitatory neurons purified from the inferior temporal gyrus (ITG) of low (control), intermediate and high pathology AD cases (based on brain-wide pathology load, n=10 per case type). There were approximately 1000 differentially methylated CpG regions (DMRs) in excitatory neurons from low compared to intermediate pathology cases, ~900 DMRs from intermediate compared to high AD cases and ~2000 DMRs from low compared to high pathology cases. Between 57-67% of these DMRs were hypermethylated in excitatory neurons from cases with higher AD pathology. These AD-associated DMRs were predominantly located in proximity to promoters, exons, and enhancer elements. Gene ontology (GO) analyses of these AD-associated DMRs showed enrichment in neurogenesis, neuronal development, regulation of transcription from RNA polymerase II promoter and transcription factor binding pathways.

To better understand the impact of AD pathology on DNA methylation in excitatory neurons, AD pathological hallmarks, beta-amyloid (Aβ) plaques and total tau pathology, were quantitated in the TTG of this AD human brain cohort. This enabled the identification of DMRs associated with Aβ plaque and total tau pathology measurements. Between 1786-2985 DMRs associated with Aβ plaque measurements (density, load, and size), between 1423-1683 DMRs associated with tau measurements (density, load, and size), and 1718 DMRs associated with neurofibrillary tangle (NFT) density. Hypermethylation of 45-67% of these DMRs occurred with higher Aβ plaque, tau, and NFT pathology. These pathology-associated DMRs were also mainly located in proximity to promoters, exons, and enhancers. DMRs that associated with Aβ plaque density and load were enriched in protein kinase, cell surface receptor, Wnt signalling and neuron differentiation pathways. DMRs that associated with tau density and load were enriched in chromatin and transcription factor binding as well as neuron fate commitment. DMRs that associated with NFT density were enriched in the GTPase regulatory activity, enzyme activator, phospholipid, and chromatin binding pathways. Overall, GO enrichment of these DMRs differed between Aβ plaque and tau pathology, with only neuronal differentiation pathways in common.

Nucleosome depleted regions (NDRs) in excitatory neurons were identified for each AD case type. Low pathology cases had >4000 associated NDRs, followed by high AD cases with ~2500 related NDRs, and intermediate AD cases with ~1250 related NDRs. NDRs were located adjacent to promoters and enhancers in all case types; however, NDRs in low pathology cases also localised in exons whereas NDRs in intermediate and high AD cases overlapped intergenic regions. AD-associated NDRs in low and intermediate cases were enriched for similar pathways related to neuron fate commitment and cell morphogenesis, whereas a single pathway (post-transcriptional regulation of gene expression) was enriched for the AD-associated NDRs in high AD cases.

NDRs in excitatory neurons that associated with Aβ plaque, and total tau pathology measurements in the ITG were also assessed. An increased number of NDRs were associated with higher levels of pathology measurements in the ITG. The greatest number of NDRs were associated with high tau load and density (-2700 NDRs), followed by high NFT density (-2500 NDRs) and then high Aβ plaque load (~2000 NDRs). NDRs that associated with AD pathology located in intergenic regions and in proximity to promoters and enhancers. NDRs associated with high Aβ plaque and tau pathology were enriched in similar biological pathways including transcription regulation from RNA polymerase II promoter, neurogenesis, and neuron differentiation and development.

These results are the first to demonstrate that human sporadic AD progression is associated with alterations in DNA methylation and nucleosome organisation within vulnerable excitatory neurons. The nuclei purification protocol was essential to investigate the epigenetic profile of excitatory neurons. The number of DMRs increased with AD progression, ~55-67% of these DMRs were hypermethylated, suggesting ongoing, atypical, and increasingly divergent spread of gene repression in excitatory neurons across the progression of AD. The number of NDRs also diminished as AD progressed, concomitant with increasing DNA methylation events, and consistent with physical impediment of gene expression patterns in these vulnerable neurons. A pattern of greater DMR gain concomitant with greater NDR loss was observed with increasing Aβ plaque compared to tau pathology, which may indicate that Aβ plaque pathology has greater repressive influence on gene expression in excitatory neurons than tau pathology. Functional enrichment of genes in proximity to DMRs and NDRs that associated with each AD pathological hallmark in excitatory neurons identified common pathways involved in neuron development and fate commitment. Interestingly, DMRs that associated with tau pathology were also enriched in chromatin and transcriptional regulation pathways, whereas DMRs associated with Aβ plaque pathology were enriched in a variety of categories including intracellular signalling pathways and transcriptional regulation. These data reveal a key role of disease-related changes in DNA methylation and nucleosome occupancy that may affect neuronal and transcriptional pathways specifically in vulnerable neurons during the progression of AD.