PROJECT SUMMARY Recent studies showed that cellular senescence plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Senescent cells are detected in the brains of AD patients and mouse models of AD. Selective elimination of senescent cells genetically or pharmacologically reduces neurodegenerative pathologies and ameliorates cognitive deficits in AD mouse models. However, the underlying mechanism by which senescence contributes to AD pathogenesis is not known. It is also unclear which brain cell types are the primary drivers of senescence- dependent neurodegeneration. The goal of this project is to gain a mechanistic understanding of the role of senescence in AD pathogenesis. Cellular senescence is highly heterogeneous, and the characteristic features of senescent cells vary significantly upon various senescence-inducing stimuli, cell types and the stages of senescence. Despite the heterogeneity of senescence, various senescence-inducing stimuli converge on two major senescence pathways, the p53/p21 and the p16/pRb pathways, which can independently activate senescence. In this proposal, we will dissect these two senescence pathways separately to uncover their functional consequence in the cellular pathogenesis of AD using human induced pluripotent stem cell (iPSC)- based AD models. Patient-derived iPSCs have revolutionized disease modeling and AD-related cell pathologies have been recapitulated in neurons differentiated from AD-iPSCs. It has been suggested that neurons in AD patients are prone to senescence and senescent neurons constitute most senescent cells in the brains of AD patients. Further, genetic regulation and physiological consequence of senescence in post-mitotic cells such as neurons are greatly understudied, as most studies focus on cells capable of proliferation. Towards our goal, we have generated two inducible systems to activate the p53/p21 and the p16 pathways independently in human iPSCs in a controllable and synchronized manner. The inducible approach allows temporal control for isogenic comparisons of the same cell populations over the course of differentiation of iPSCs to neurons with and without the activation of a specific senescence pathway. This strategy minimizes experimental heterogeneity associated with differentiated cultures and provides a tighter control for inter-line variations. In Aim 1, we will focus on the p53/p21 pathway activated by telomere dysfunction and its impact on senescence and AD cellular pathogenesis in differentiated cortical neurons and cerebral organoids. In Aim 2, we will investigate the role of the p16 pathway in senescence and AD cellular pathogenesis in neurons and cerebral organoids. This project will produce broadly valuable cellular resources for senescence and AD research and provide new insight into senescence’s role in AD pathogenesis.