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

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 Δex7/8 mice, a genetically accurate CLN3 disease model. Microglia were isolated from young and old Cln3 Δex7/8 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 Δex7/8 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. ### Competing Interest Statement The authors have declared no competing interest. * NCLs : Neuronal ceroid lipofuscinoses IF : immunofluorescence IHC : immunohistochemistry min minutes BSA : Bovine Serum Albumin NHS : Normal Horse Serum PBS : Phosphate buffered saline RT : Room temperature O/N : Overnight HBSS : Hank’s Balanced Salt Solution FBS : Fetal bovine serum NGS : Normal Goat Serum LFQ : Label-free quantification GOTERM\_CC\_FAT : gene ontology enrichment for cellular component GOTERM\_BP\_FAT : gene ontology enrichment for biological process PFA : Paraformaldehyde LD : Lipid droplet MACS : magnetic-activated cell sorting subunit c Subunit c of the mitochondrial ATP synthase TEM : Transmission electron microscopy ER : Endoplasmic reticulum LC/MS : liquid chromatography/mass spectrometry GO : Gene Ontology CNS : Central nervous system NPC : Nieman Pick Type C disease ALS : amyotrophic lateral sclerosis