Stimulus-Driven Remodeling of Microglial Endocannabinoid Metabolism Illuminates ABHD12 Function in Immunity

Microglia, the brain's macrophages, dynamically reprogram their metabolism to acquire appropriate and unique states to coordinate neuroinflammatory responses. Lipid metabolism, such as endocannabinoid signaling, is increasingly acknowledged for playing a central role in regulating these states. However, the enzymatic activities underlying these processes remain poorly characterized. Here, we combine activity-based protein profiling (ABPP) with targeted lipidomics to map lipid metabolism across distinct pro- and anti-inflammatory microglial states. We reveal stimulus-dependent remodeling of endocannabinoid metabolism, identifying DAGLβ and ABHD12 as key enzymes with opposing activity patterns. Correlation analysis across the differential expression and activity levels in microglial states revealed DAGLβ activity aligns with transcript and protein abundance, while ABHD12 activity is uncoupled from expression levels indicative of post-translational regulation. To enable cellular activity profiling, we developed a tailored ABHD12 probe (LEI-612), which demonstrated that cellular ABHD12 activity inversely correlated with 2-AG levels. Pharmacological inhibition of ABHD12 with DO264 elevated 2-AG, reduced AA, and modulated cytokine release through dual mechanisms: CB2R-dependent suppression of TNF-α and PGE2-dependent regulation of IL-6. Our integrated chemical proteomics and lipidomics approach highlights the importance of activity-based profiling of enzymes in defining microglial states and uncover ABHD12 as a key regulator of 2-AG metabolism and microglial immune function.