Project Summary Age is the single strongest predictive factor for Alzheimer's disease (AD). The vast majority of AD cases arise sporadically in the seventh decade of life or beyond, with no known etiology aside from advanced age. To understand cellular and molecular changes at the root of AD, studies need experimental methods that discrim- inate healthy from pathological aging. Until now, options for in vitro study of aged human neurons, the cell type devastated by AD, have been lacking. This proposal will use induced neurons (iNs), a method that generates neurons from fibroblasts by direct transdifferentiation, to study the cellular processes in neuronal aging and AD. Unlike induced pluripotent stem cell (iPSC)-derived neurons, which pass through a stem cell intermediate phase and become physiologically rejuvenated, iNs retain age-related features of their source fibroblasts. Gene expression, epigenetic marks, and cell metabolism in iNs correspond to the age of the donor. Using the iN system, this proposal will evaluate the interaction between two age-correlated factors associated with the AD brain: cellular senescence and inflammation. Preliminary data demonstrate that iNs from AD patients are more likely to show protein markers of senescence and upregulation of senescence-associated genes than iNs from healthy aged controls (CTL). In Aim 1, senescent and non-senescent cells from AD and CTL Iines will be examined by RNA-Seq and ATAC-Seq. These analyses will identify pre-existing differences in non-senescent AD neurons that differentiate them from CTL as well as specific components of cellular senescence that are unique to neurons, a cell type in which the existence of senescence remains controversial. In other tissues, senescent cells release inflammatory factors (collectively termed the senescence associated secretory pheno- type or SASP) that disrupt the local environment, contributing to age-related tissue dysfunction. In Aim 2, the ability of secreted factors from AD and CTL iNs to activate human astrocytes and microglia will be assessed. These experiments will generate a list of neuronal components of the SASP and identify which are contributors to the chronic inflammation that arises with AD. Finally, Aim 3 will examine cellular processes hypothesized to be upstream of senescence. Prior work and preliminary data show that AD and CTL iNs differ in their cellular metabolism and intracellular nucleotide pools. The nucleotide content of AD and CTL iNs will be compared, and small molecules will be used to manipulate components of nucleotide synthesis or salvage and test the resulting impact on senescence. These studies will reveal whether inherent differences in nucleotide metab- olism lead to increased rates of cellular senescence and contribute to the inflammatory response. As a whole, these studies will provide novel insights into how senescence and inflammation interact within the aged neuronal environment that is permissive to AD.