CKLF induces microglial activation via triggering defective mitophagy and mitochondrial dysfunction.

ABSTRACT

Although microglial activation is induced by an increase in chemokines, the role of mitophagy in this process remains unclear. This study aimed to elucidate the role of microglial mitophagy in CKLF/CKLF1 (chemokine-like factor 1)-induced microglial activation and neuroinflammation, as well as the underlying molecular mechanisms following CKLF treatment. This study determined that CKLF, an inducible chemokine in the brain, leads to an increase in mitophagy markers, such as DNM1L, PINK1 (PTEN induced putative kinase 1), PRKN, and OPTN, along with a simultaneous increase in autophagosome formation, as evidenced by elevated levels of BECN1 and MAP1LC3B (microtubule-associated protein 1 light chain 3 beta)-II. However, SQSTM1, a substrate of autophagy, was also accumulated by CKLF treatment, suggesting that mitophagy flux was reduced and mitophagosomes accumulated. These findings were confirmed by transmission electron microscopy and confocal microscopy. The defective mitophagy observed in our study was caused by impaired lysosomal function, including mitophagosome-lysosome fusion, lysosome generation, and acidification, resulting in the accumulation of damaged mitochondria in microglial cells. Further analysis revealed that pharmacological blocking or gene-silencing of mitophagy inhibited CKLF-mediated microglial activation, as evidenced by the expression of the microglial marker AIF1 (allograft inflammatory factor 1) and the mRNA of proinflammatory cytokines (Tnf and Il6). Ultimately, defective mitophagy induced by CKLF results in microglial activation, as observed in the brains of adult mice. In summary, CKLF induces defective mitophagy, microglial activation, and inflammation, providing a potential approach for treating neuroinflammatory diseases.Abbreviation 3-MA 3-methyladenine; AIF1 allograft inflammatory factor 1; ANOVA analysis of variance; BAF bafilomycin A1; BSA bovine serum albumin; CCCP carbonyl cyanide m-chlorophenyl hydrazone; cGAMP cyclic GMP-AMP; CGAS cyclic GMP-AMP synthase; CKLF/CKLF1 chemokine-like factor 1; CNS central nervous system; DMEM Dulbecco's Modified Eagle Medium; DNM1L dynamin 1 like; GAPDH glyceraldehyde-3-phosphate dehydrogenase; GFP green fluorescence protein; IRF3 interferon regulatory factor 3; IgG immunoglobulin G; LAMP1 lysosomal-associated membrane protein 1; LAPTM4A lysosomal-associated protein transmembrane 4A; MAP1LC3B microtubule-associated protein 1 light chain 3 beta; Mdivi-1 mitochondrial division inhibitor 1; mRFP monomeric red fluorescent protein; mtDNA mitochondrial DNA; MTORC1 mechanistic target of rapamycin kinase complex 1; OPTN optineurin; PBS phosphate-buffered saline; PCR polymerase chain reaction; PINK1 PTEN induced putative kinase 1; PLL poly-L-lysine; PRKN parkin RBR E3 ubiquitin protein ligase; qPCR quantitative polymerase chain reaction; ROS reactive oxygen species; SQSTM1 sequestosome 1; TBK1 TANK-binding kinase 1; TFEB transcription factor EB; VDAC voltage-dependent anion channel.