ABSTRACT Here we propose a Supplement to the research proposed in the parent grant Combinatorial regulation of the enhancer codes in senescence to perform a logical extension of our initial Specific Aims regarding cellular aging/senescence to achieve initial insights into the distinction between these events and distinct pathological causal features in each CNS cell type that may represent the underlying mechanisms underlying sporadic Alzheimer’s Disease (AD). The Specific Aims of the initial grant employed and developed biostatistical tools relevant to cellular aging and replicative senescence, including an examination of underlying epigenomic alterations and enhancer activation codes ultimately leading to cellular senescence approaches and proposed application of single cells approaches. Appling these specific aims/approaches is particularly suitable for enhancing our understanding of the potential initial causal events that eventuate in clinical sporadic AD, an aging- associated disease affecting both men and, to a greater extent, women. In concert with the original Aim of understanding the molecular basis for enhancer-mediated programs of cellular aging and senescence, in this Supplement, we propose to extend our original Specific Aims to uncover the enhancer program underlying the aging events in each CNS cell type, to permit examination of the central question whether the altered enhancer and transcriptome changes in AD in each cell type represent a trajectory distinct from the normal aging-related alterations in these cell types. We hypothesize that, while each cell type will, of course, exhibit specific features of enhancer activation characteristic of aging and even cellular senescence, as we have uncovered in the parent grant, AD represents a distinct trajectory for these cell types. This Supplement is licensed by our ability by our development of the technology to perform simultaneous quantitation of single nucleus (sn) RNA-seq and snATAC-seq using archival samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD. We have carefully piloted this approach to ensure that we are technically able to obtain high quality data and that all of the proposed informatic pipelines and our ability to successfully aggregate such massive data sets is fully established. The Supplement would license our ability to scale the analysis to obtain data sets capable of generating statistically significant results and, therefore, informative conclusions, which can be ultimately be further validated by imputation from available data bases and using hiPSCs to generate specific cell types for validating transcriptional analyses. Our overarching goal in this Supplement is to apply the described technologies and new informatic approaches to a sufficient number of archived specimens to permit formulating the actual transcription factors and pathways that distinguish initiation of the AD process in specific CNS cell types.

PROJECT NARRATIVE We propose to use simultaneous sn RNA-seq and ATAC-seq to extend our original Specific Aims to uncover the enhancer program underlying the aging events in each CNS cell type. This approach will permit us to examine the central question as to whether the altered enhancer and transcriptome changes in AD in each cell type represents a trajectory distinct from the normal aging-related alterations in these cell types, identifying the actual transcription factors and pathways that distinguish earliest events of the AD process in specific CNS cell types.