ABSTRACT In the past three years, we made great progress in understanding the role of mitochondrial (mt) DNA metabolism in NLRP3 inflammasome activation. Importantly, we found that NLRP3 itself is activated on binding of oxidized (Ox) mtDNA, whose generation depends on new mtDNA synthesis which is stimulated on TLR engagement. TLR signaling results in induction of CMPK2, which catalyzes the rate limiting step in the biosynthesis of dCTP, a precursor for mtDNA synthesis. We recently discovered that Ox-mtDNA needs to be cleaved by the endonuclease Fen1 and transported/leaked to the cytoplasm, where NLRP3 is located, via the mitochondrial inner membrane (MIM) MPTP pore and the mitochondrial outer membrane (MOM) VDAC channel. Macrophage exposure to different NLRP3 activators results in rapid Ca2+ influx into mitochondria, as well as perturbations to the electron transfer chain that result in mtROS production. Whereas Ca2+ influx into mitochondria leads to pore and channel opening, mROS lead to oxidation of newly synthesized mtDNA before it is cleaved by Fen1 to 600 bp fragments that leak into the cytoplasm. Another important player in mtDNA metabolism is OGG1, a baseexcision repair enzyme that removes oxidized deoxyguanosine from mtDNA, whose insufficiency greatly increases Alzheimer’s disease (AD) risk. We found that OGG1 ablation in macrophages enhances NLRP3 inflammasome activation, whereas mitochondrially targeted OGG1 inhibits NLRP3 inflammasome activation. We therefore we plan to study the involvement of mtDNA metabolism in AD pathogenesis, using the accelerated 5xFAD model crossed with ApoE4 knockin mutant mice. These mice will be analyzed over the course of disease development and progression for CMPK2, NLRP3, ASC, caspase-1, OGG1 and Fen1 expression and presence of Ox-mtDNA and mature IL-1β in their circulation, cerebrospinal fluid, and brain tissue homogenates. Microglia (MG) from these mice will be isolated at different time points and analyzed for presence of ASC specs, indicative of NLRP3 inflammasome assembly, presence of cytosolic mtDNA and signs of MPTP opening and VDAC oligomerization. We will determine whether crossing of 5xFAD/ApoE4 mice with mtOgg1Tg mice results in reduced abundance of cytoplasmic mtDNA, Ox-mtDNA and IL-1β and whether this parallels the amelioration of neurodegeneration and cognitive loss. Conversely, we will delete OGG1 in MG of 5xFAD/ApoE4 mice to determine whether this results in accelerated development of neurodegeneration. We will also cross 5xFAD/ApoE4 and Cmpk2ΔMG mice, in which the Cmpk2 gene was ablated with MG-specific Cre and determine whether this also results in disease amelioration. According to the results of the above experiments, 5xFAD/ApoE4 mice will be treated with newly developed CMPK2 and Fen1 inhibitors and will be evaluated for attenuation of neurodegeneration and cognitive loss. Human AD tissue will also be examined for CMPK2 and Fen1 expression as well as presenc...