PROJECT SUMMARY The pathogenesis of Alzheimer's disease (AD) remains elusive. Inheritance of the apolipoprotein (APO) E4 allele is the strongest genetic risk factor for sporadic late onset AD, whereas the APOE2 allele is protective and the most common APOE3 allele is neutral. While the mechanisms by which APOE4 modifies the risk of AD are not fully elucidated, compelling evidence indicates that the pathogenic effects of APOE4 are mediated by lipid- related pathways. Integrative multi-omics studies have consistently demonstrated the strong association of lipid pathways with AD phenotypes and that APOE4 disrupts intra/intercellular lipid homeostasis in cellular, organoid, and animal models as well as postmortem brain tissue from individuals carrying different APOE alleles with or without AD. Intriguingly, emerging evidence suggests that specific species of lipids/metabolic alterations in subcellular organelles, in particular mitochondria, lead to neurotoxicity and neurodegeneration. Mitochondria are the powerhouse of cells and provide the energy to sustain vital cellular functions. Notably, brain contains two major populations of mitochondria, the synaptic mitochondria that originate from the synaptic bouton of neurons and the non-synaptic mitochondria that originate from neuronal and glial cell bodies. Lipidomic analysis indicates that synaptic and non-synaptic mitochondria have distinct lipid profiles that regulate compartmental energy metabolism in the brain. Importantly, dysfunction of mitochondria, especially synaptic mitochondria, is well established as one of the earliest deficits in the progression of AD. APOE4 has been associated with increased impairment of mitochondrial structure and function compared with APOE3 in various models and human patients. However, whether APOE genotypes regulate the lipidome of mitochondria during brain aging and whether the dynamic changes of mitochondrial lipidomes affect the progression of AD are unknown. We hypothesize that mitochondrial lipidomic dynamics and its interaction with APOE4 drive pathogenic brain aging and AD. Three independent yet interrelated specific aims are proposed to test the hypothesis, using both mouse models and human brains and a combination of behavioral and pathological approaches, coupled with innovative targeted and unbiased cellular, molecular technologies, including lipidomics, transcriptomics, and brain clearing and 3D imaging. Aim 1 is to assess the impact of APOE isoforms on mitochondrial lipidomic dynamics associated with brain aging in humanized APOE4 and APOE3 mice. Aim 2 is to elucidate the relationship between mitochondrial lipidomic dynamics and the progression of cognitive deficits and amyloid pathology in APP/PS1 mice. Aim 3 is to define the interaction of mitochondrial lipidome with different APOE isoforms and its relation to cognitive function and AD pathology in human brains. The results are expected to uncover the impact of mitochondrial lipidomic dynamics and i...