Sustained Amyloid Degradation Induces Lysosomal Exhaustion and Lipid-Driven Microglial Dysfunction in Alzheimer's Disease - PROJECT SUMMARY Alzheimer's disease (AD) affects over 6 million Americans, costs $300 billion annually, and global prevalence is projected to reach 150 million by 2050. Genome-wide association studies consistently implicate microglia as central regulators of AD risk, yet more than a century after Alois Alzheimer described lipid-laden glia surrounding plaques, we still do not understand why amyloid-clearing microglia accumulate lipid droplets (LDs) or how this metabolic failure drives neurodegeneration. Using human pluripotent stem cell (hPSC)-derived microglia, we discovered that large fibrillar Aβ aggregates—unlike monomers or oligomers—trigger digestive exophagy (DE), a specialized clearance mechanism for substrates too large to internalize through canonical phagocytosis. Our preliminary data demonstrate that sustained DE progressively depletes lysosomal hydrolases, disrupts lipid catabolism, and drives LD accumulation enriched in neutral lipids including esterified arachidonic acid. Concurrently, we observe reduced prostaglandin biosynthesis and altered eicosanoid profiles, indicating that arachidonic acid normally mobilized for immune signaling becomes sequestered within LDs. In our engineered Aβ-plaque-on-dish neuronal-microglial coculture system, neuronal injury occurs exclusively at plaque-neurite interfaces where DE-active, LD-laden microglia cluster; reframing AD neurodegeneration as a spatially restricted consequence of clearance-driven metabolic collapse rather than amyloid burden alone. Our integrated experimental platform combines scalable 2D hPSC-microglia for temporal proteomic and lipidomic profiling, CRISPR-engineered genetic perturbations, human forebrain organoids with transplanted microglia, and neuron- microglia-astrocyte tricultures to dissect these mechanisms across progressively complex tissue contexts. This project tests the central hypothesis that DE-mediated Aβ clearance drives lysosomal exhaustion, which is required for LD accumulation, and that this LD-rich state disrupts prostaglandin-dependent immune competence and causes focal neuronal injury. In Aim 1, we will define the mechanistic link between Aβ clearance and microglial metabolic collapse by disrupting DE genetically (SYK and PLCG2 knockouts) and pharmacologically, quantifying LD kinetics, lipid composition, and lysosomal function across hPSC-microglia, organoids, and tricultures, and testing whether DE blockade protects neurons at plaque interfaces. In Aim 2, we will determine how LD accumulation impairs prostaglandin-mediated immune signaling using microglia with genetically tunable LD content (LD-high: ATGL knockout; LD-low: ACSL1 knockout), measuring prostaglandins, eicosanoid precursors, cytokine output, and chemotactic capacity, and testing whether arachidonic acid supplementation rescues immune dysfunction independent of LD burden. By establishing digestive exophagy and lysosomal exhaustion as targetable metabolic checkpoints linking aggregate clearance to lipid-driven immune failure, this work will reveal new therapeutic strategies to preserve microglial neuroprotection in Alzheimer's disease.
