Summary/Abstract of Parent Grant As advanced age is the most significant risk factor for Alzheimer’s disease (AD), targeting detrimental age-related processes may lead to effective therapies. Mitochondrial dysfunction and pro-inflammatory signaling are each thought to be key drivers of aging and AD. However, a clear understanding of the connections between mitochondrial homeostasis, immune signaling and aging remains elusive. Mitochondrial DNA (mtDNA) is normally kept within the mitochondria. However, under conditions of mitochondrial stress or damage, mtDNA can be released into the cytosol thus encountering cytosolic DNA sensors and activating pro- inflammatory responses. Cytosolic mtDNA has been reported in the brains of AD patients and cellular models of AD, but, whether cytosolic mtDNA can be targeted for therapeutic intervention in AD has not been determined. Moreover, fundamental questions remain regarding the occurrence and role of cytosolic mtDNA in aging and age-related health decline. In preliminary work, we find that aging leads to a striking decline in mitochondrial autophagy and a concurrent accumulation of cytosolic mtDNA, which is linked to proinflammatory signaling in different organ systems of Drosophila including the brain. Critically, we have discovered that adult-onset, neuron-specific silencing of EYA, a molecule involved in sensing cytosolic DNA, dampens inflammatory signaling in the aged brain and extends lifespan. In addition, we have developed genetic approaches to reduce cytosolic mtDNA, via increased lysosomal DNase activity, in aged flies. These findings provide an important first step towards understanding the mechanistic interplay between cytosolic mtDNA, immune signaling and healthspan. Here, we propose to build upon these groundbreaking findings by exploring three broad questions: 1) What are the mechanistic relationships between mitochondrial homeostasis, cytosolic mtDNA, and aging? 2) Does cytosolic mtDNA and associated pro-inflammatory signaling drive aging and age-related health decline? 3) Can modulating cytosolic mtDNA and associated pro- inflammatory signaling counteract Alzheimer’s disease pathogenesis? The work described herein will bring about fundamental knowledge towards our understanding of the mechanisms of aging and AD-related pathology. Our findings may also lead to novel therapeutic approaches to counteract aging, AD and related dementias.