Background: Microglia are the primary brain cell type regulating neuroinflammation and they are important for healthy aging. Genes regulating microglial function are associated with an increased risk of neurodegenerative disease. Loss-of-function mutations in CLN3, which encodes an endolysosomal membrane protein, lead to the most common childhood-onset form of neurodegeneration, featuring early-stage neuroinflammation that long precedes neuronal cell loss. How loss of CLN3 function leads to this early neuroinflammation is not yet understood. Methods: Here, we have comprehensively studied microglia from Cln3∆ex7/8 mice, a genetically accurate CLN3 disease model. Microglia were isolated from young and old Cln3∆ex7/8 mice for downstream molecular and functional studies. Results: We show that loss of CLN3 function in microglia leads to classic age-dependent CLN3-disease lysosomal storage as well as an altered morphology of the lysosome, mitochonodria and Golgi compartments. Consistent with these morphological alterations, we also discovered pathological proteomic signatures implicating defects in lysosomal function and lipid metabolism processes at an early disease stage. CLN3-deficient microglia were unable to efficiently turnover myelin and metabolize its associated lipids, showing severe defects in lipid droplet formation and significant accumulation of cholesterol, phenotypes that were corrected by treatment with autophagy inducers and cholesterol lowering drugs. Finally, we observed reduced myelination in aging homozygous Cln3∆ex7/8 mice suggesting altered myelin turnover by microglia impacts myelination in the CLN3-deficient brain. Conclusion: Our results implicate a cell autonomous defect in CLN3-deficient microglia that impacts the ability of these cells to support neuronal cell health. These results strongly suggest microglial targeted therapies should be considered for CLN3 disease.

Loss of CLN3 in microglia leads to impaired lipid metabolism and myelin turnover