Project Summary/Abstract The largest risk factor for developing chronic disease, including neurodegenerative diseases, such as Alzheimer’s disease (AD), is age. There is now abundant evidence that aging processes can be driven by DNA damage, which is ubiquitous and a cause of many adverse cell fates, such as apoptosis and cellular senescence. A major pathologic consequence of DNA damage and its erroneous repair is DNA mutation, from base substitutions to very large chromosomal alterations. With the emergence of advanced single-cell technology it has recently been shown that mutations accumulate in neurons during human aging at a frequency that is higher in brains affected by neurodegeneration. This is in keeping with earlier findings using cytogenetic methods indicating increased aneuploidy associated with Alzheimer’s disease. We have recently shown that increased aneuploidy induces cellular senescence, including the senescence-associated secretory phenotype (SASP). Clearance of senescent cells has shown beneficial effects on various aspects of AD disease progression implicating cellular senescence as an emerging and important cell fate in the biology of age-related neurodegeneration. Recent work suggests that cellular processes involving non-neuronal cells (NeuN-negative cells) significantly contribute to the pathology of AD in both humans and mouse models. Studying normative aging in the mouse we identified a significant accumulation of aneuploidy in NeuN-negative cells isolated from the cerebral cortex, but not from the cerebellum of old mice. In this application we propose to build on our observations to test the hypothesis that NeuN-negative cells in the brain are particularly susceptible to age related accumulation of aneuploidy and large-scale genomic instability promoting senescence. Genomic instability-induced senescent NeuN-negative cells could fail to accomplish their neuronal nursing functions and/or acquire neurotoxic properties and be particularly detrimental for AD progression. To test our hypothesis, in Aim 1 we will establish the genomic landscape of cells from the human cortex and hippocampus during normal aging and AD. Using newly developed, highly sensitive single cell-based assays including multiple displacement amplification (SCMDA) and multicolor interphase DNA-RNA- FISH (iDR-FISH) to measure aneuploidy and senescence in situ we will compare NeuN-negative and NeuN- positive cells from healthy aged donors to age-matched AD patients and young adult controls. This will establish a comprehensive analysis of aneuploidy and/or other forms of genomic instability during human aging in brain regions and cell types associated with AD and AD-type dementias. In Aim 2 using primary cells as well as induced human pluripotent stem cells (hiPSCs) from late-onset AD patients and disease-free controls, we will study the cell non-autonomous effects of aneuploidy-induced senescence on neurons using co-culture models. Two small molecules that...