PROJECT SUMMARY The retinal pigmented epithelium (RPE) is an amitotic, long-lived cell type that supports healthy visual function for numerous decades. The aging retina displays pathologic features that are exaggerated in a complex disease called age-related macular degeneration (AMD). Progressive degeneration of the aging RPE is a hallmark of geographic atrophy, a late-stage of AMD that causes vision loss. We recently discovered an excess abundance of cytosolic complementary DNA derived from the SINE retrotransposon element (RTE) Alu family in the RPE of human eyes with geographic atrophy and that cytosolic Alu cDNAs are highly cytotoxic to the RPE in animal and human cell-based studies. These Alu cDNAs are produced via reverse transcription of Alu RNAs by the long- interspersed nuclear element-1 (L1). We also found that Alu cDNAs trigger the cytosolic escape of mitochondrial DNA (mtDNA), and together these two cytosolic DNAs collaborate, in mechanistically unclear ways, to activate a multifaceted innate immune response that results in cell death. To gain a better understanding of the combinatory impact of Alu cDNAs and mtDNA and their causal contribution to aging in the retina, we will (1) create temporal, spatial, and sequence topographic maps of Alu cDNA subfamilies and mtDNA in young, healthy aged, and AMD-affected human eyes; (2) probe the stimuli for and mechanisms of Alu cDNA generation in aging by investigating whether L1 regulators influence Alu cDNA production and retinal toxicity; and (3) elucidate the pathway of mitochondrial dysfunction triggered by Alu cDNA and the noncanonical inflammatory pathways triggered by Alu cDNA and mtDNA joint activity that are responsible for retinal cell death. This proposal is responsive to RFA-AG-23-015 as it will provide mechanistic insights into how cytosolic Alu cDNAs and mtDNA mediate interactions of aging hallmarks and modulate healthspan by mapping their sequence and expression during aging, determining the regulat