Sex divergence and cell specificity of age-related hippocampal DNA modifications

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Abstract Epigenetic processes in the central nervous system may play a mechanistic role in susceptibility to and progression of cognitive decline and age-related neurodegenerative diseases, such as Alzheimer's. DNA modifications, principally cytosine base methylation and hydroxymethylation (mC and hmC respectively), are fundamental regulators of DNA accessibility and gene regulation/expression with differential effects on gene expression depending on the modification (mC/hmC), context CG/non-CG (also known as CH), and genomic location. A barrier to progress in understanding the role of epigenetic mechanisms in brain aging and DNA modifications in particular, has been the lack of quantitatively accurate, genome-wide data in specific cell types. Without the knowledge of the specific genomic locations of altered modifications with aging it is impossible to design well-rationalized, mechanistic studies that unravel the functional effects of epigenetic reconfiguration. Therefore, the critical next step for the field is to generate this genome-wide data of mC and hmC in CG and CH contexts in specific cell types from both sexes across the lifespan. To address this critical issue we have developed cell-type specific, tamoxifen-inducible Cre, transgenic NuTRAP models to allow isolation of nucleic acids (DNA & RNA) from microglia, astrocytes, and neurons. In Aim 1, cell type-specific hippocampal changes in mC/hmC with aging will be examined by whole genome oxidative bisulfite sequencing (WGoxBS) in microglia, astrocytes, and neurons. Paired epigenomic and transcriptomic data from the same animals will be used to: 1) assess aging with `epigenetic clocks' in a cell-type specific fashion, 2) determine the role of altered modification patterns in age-related changes in gene expression, 3) determine enrichment of differential modifications in regulatory regions of the genome, and 4) identify genomic loci for epigenome editing. In prior studies we have determined that age-related DNA modification changes can be prevented by caloric restriction. In Aim 2 we will test whether heterochronic parabiosis can reverse age-related changes once extant in the same Cre-inducible, NuTRAP models. Using a unique database of all publicly available, annotated human methylation data we will validate aspects of our findings in humans. In Aim 3 we will examine the targeting mechanisms that direct changes in mC/hmC to specific genomic regions. These studies will allow the determination of critical genomic regions with altered DNA modification patterns that can be manipulated in future interventional studies. The ultimate goal of the research is to develop clinical interventions that target the epigenome to maintain brain function with aging and prevent age-related neurodegeneration.