The Function Of Cadm1 In The Regulation Of Neuronal Metabolism And Microglial Immunity

BackgroundCadm1 is a cell membrane protein mainly enriched in the central nervous system.It is expressed in neurons and neuroimmune cells,and related to body metabolism.Our previous studies have showed that inhibition of Cadm1 expression in excitatory neurons not only increases metabolism and energy expenditure,but also enhances sensitivity to insulin and leptin.As the main endogenous immune cells in the brain,microglia play an important role in immune monitoring and immune defense and maintain the homeostasis of neuronal microenvironment.However,the functions of Cadm1 in neurons and microglia,as well as the mechanism of Cadm1-mediated regulation of the nervous system and obesity,remain to be clarified.Objective1.To study the effect of Cadm1 on neuronal leptin and insulin signaling pathway.2.To investigate the possible mechanism of Cadm1 regulating metabolic homeostasis in microglia inflammatory response.Methods1.N2 a cells were cultured in different glucose concentration medium,and western blot and q PCR analysis the expression of Cadm1;si Cadm1 was transfected into N2 a cells for 48 h,the effects of Cadm1 inhibition in N2 a cells on insulin and leptin signaling pathways were detected by western blot and q PCR.2.Wild-type（WT）at 8-10 weeks were intraperitoneally injected with 0.75mg/g leptin,Western blot analysis the expression of Cadm1 and other related proteins in hypothalamus and hippocampus;Immunofluorescence analysis the changes of POMC neurons and microglia in hypothalamus.3.Hippocampal neuron HT22 cells were cultured in the conditioned medium of BV2 cells transfected with si Cadm1 for 48 h,and western blot analysis the proteins expression of apoptosis related signal pathway.4.Leptin and lipopolysaccharide stimulated BV2 cells,western blot and q PCR analysis the expression of Cadm1 and inflammatory cytokines;After transfection of si Cadm1 into BV2 cells for 48 h,western blot and q PCR analysis the effects of Cadm1 inhibition on leptin and lipopolysaccharide induced cytokine release and inflammation related signaling pathways.Results1.Western blot and q PCR showed that the expression of Cadm1 is inhibited in N2 a cells after increasing glucose concentration;Inhibition of Cadm1 in N2 a cells activates the Jak2/STAT signaling pathway,and suppresses insulin induced AKT/GSK3β signaling activation.2.Western blot showed that leptin decreased the expression of Cadm1 in hypothalamus and hippocampus of mice;Immunofluorescence showed that POMC neurons and microglia in hypothalamus of mice were activated after leptin treatment,the branching processes of microglia were shortened and thickened,and the expression of CD68 increased.3.HT22 cells cultured in BV2 cell conditioned medium transfected with si Cadm1 increased the expression of apoptosis suppressor protein Bcl2.4.Western blot and q PCR showed that leptin and lipopolysaccharide inhibited the expression of Cadm1 in BV2 cells;The knockdown of Cadm1 in BV2 microglia,leptininduced IL1β and TNFα release,and lipopolysaccharide-induced IL6,TNFα,IFNβ,IL1β,iNOS and IL18 release decreased,and NFκB,AKT and Stat1 phosphorylation levels were inhibited.Conclusion1.Cadm1 was regulated by glucose metabolism and negatively regulated leptin signaling pathway activation,while the knockdown of Cadm1 inhibited insulin signaling.2.The inhibition of Cadm1 in microglia through down-regulating AKT/NFκB/Stat1 signaling pathway to improve inflammation,and is beneficial to reduce microgliamediated neurotoxicity.