PROJECT SUMMARY Aging is the most prominent nongenetic risk factor for age-associated diseases including late onset Alzheimer’s disease (AD) and related dementias. Accumulating evidence has implicated altered sphingolipid ceramide pathways in aging and neurodegenerative diseases. However, the cellular and molecular basis underlying ceramide aberration and how it contributes to aging-associated cognitive decline and AD pathogenesis remains poorly understood. Our preliminary data revealed aging-dependent accrual of ceramides in astrocytes and, to a less extent, microglia in brain regions known highly susceptible to aging- and AD-related functional deterioration. Lipidomic analysis of young and aged mouse brains uncovered specific upregulation of very long chain (VLC) ceramides with concomitant decreases in corresponding sphingomyelin molecules, suggesting aging-dependent activation of sphingomyelinase. Extracellular amyloid beta deposition induces robust astroglial and microglial activation and ceramide production in AD as well as in a mouse model of amyloidosis at even young ages. Moreover, we found significantly elevated acid sphingomyelinase (aSMase) activity in AD brains compared to age matched controls. Given that elevated astroglial ceramides increase the vulnerability of oligodendroglia to inflammatory cytokine released by microglia, that oligodendroglia/myelin damage and/or dysfunction has increasingly implicated in AD pathology, and that ceramides abnormally accumulate in the microglial lysosomal compartment, we hypothesize that dysregulated ceramide in glial cells is a previously unrecognized active driver in the progression of aging-related cognitive decline and amyloid pathology. The overarching goal of this project is to investigate cellular and molecular pathways leading to disruptions of sphingolipid ceramide homeostasis in physiological aging and amyloidosis conditions and to identify ceramide-mediated pathogenic pathways. Specifically, we will leverage the powerful sphingolipid targeted lipidomics and cell type-specific genetic manipulation of aSMase and VLC ceramide synthase CerS2 to (1) determine the effect of VLC ceramide on astrocyte function and paracrine impact on oligodendroglia and neurons including cellular damage and senescence; (2) investigate the aSMase-ceramide pathway in regulating microglial clearance of myelin debris and amyloid beta and inflammatory activation; and (3) employ novel conditional knockout and knockdown mice to determine the contribution of astroglial and microglial aSMase-ceramide pathway to disease progression in a mouse AD model of amyloidosis. This project shall generate new insights into aging- related dysregulation of the bioactive ceramide and provide a foundation for exploring the potential of interventions targeted at sphingolipids and neuroinflammation.