Single cell analysis core

PROJECT SUMMARY Alzheimer’s disease (AD) is a devastating neurodegenerative disease for which the largest single risk factor is age. At present, there are no effective treatments for this disease. Because AD exacts such an enormous economic and emotional toll on patients and their families, there is an urgent need to develop new approaches to understand and manage AD. Core D is significant in the context of this program project because it provides a new approach to understanding how aging, through the accumulation of a small population of senescent cells, might drive the neuronal degeneration associated with AD. Single-cell profiling allows us to characterize rare subpopulations of cells, such as senescent cells, that might escape detection by conventional analyses of bulk populations. For decades, we inferred the presence and properties of senescent cells by profiling gene expression using homogenized tissues, cells or even entire organisms. This approach led to many discoveries, although increasingly with diminishing returns as we began to appreciate the role that small populations of cells can have on surrounding cells, entire tissues and/or the systemic milieu. To enable the planned projects in this PPG, Core D will develop four specific aims. In AIM 1, we will characterize cell size and senescent gene expression profiles of astrocytes, microglia, and neurons induced to senescence in culture, and determine changes in chromatin accessibility via ATAC-seq. We hypothesize that induced cellular senescence results in a heterogeneous array of cell sizes and that senescent cells of different sizes have different gene expression profiles. AIM 2 will determine the extent to which nuclei isolated from senescent and non-senescent astrocytes, neurons, and microglia reflect the gene expression pattern of intact single cells induced to senesce. Based on a handful of studies using other non-senescent cell types, we hypothesize that nuclei isolated from these cells will, to a large extent, reflect the gene expression pattern in intact single cells. AIM 3 will develop and apply a new technology – spatial transcriptomics -- for in vivo validation of single-cell senescent signatures from Aims 1 and 2. We will develop this technology to study the interplay between neurons and senescent astrocytes. In AIM 4, we will apply methods and workflows developed in Aims 1–3 to each of the paradigms being tested in Projects 1–3. Having established specific protein (Core C) and gene expression signatures for different cell types as a result of senescence in aims 1 and 2, this final aim will use a novel validation method to confirm our unique single cell expression signatures in tissue sections or cell models where applicable in each project. Overall our Core will provide powerful new approaches to better understand the role of senescence in multiple model systems of AD at the level of the single cell.