The Role And Mechanism Of Neural Stem Cell-Specific NRBF2 In Chronic Stress-Induced Depressive-Like Behaviors

Objective:Hippocampal neurogenesis impairment is one of the pathological mechanisms underlying major depressive disorder（MDD）.Autophagy is essential for maintaining cellular stress response and homeostasis,and the knockout of autophagy-related genes leads to self-renewal and developmental abnormalities in neural stem cells.Numerous studies have revealed that autophagy deficiency is involved in the pathogenesis of MDD and that autophagy is a potential target of antidepressants.Nuclear receptor binding factor 2（NRBF2）,a crucial component of autophagy-associated PIK3C3/VPS34-containing class III phosphatidylinositol 3-kinase complex,enhances ATG14L-linked VPS34 kinase activity to positively promote autophagy.Recent single-cell RNA sequencing data indicate that NRBF2 is expressed in the adult neural stem cells（a NSCs）of dentate gyrus（DG）.However,whether and how cell type-specific NRBF2 regulates adult hippocampal neurogenesis in chronic stress-induced depression remains unclear.Here,our study investigated the role and mechanism of a NSCs-specific NRBF2 in chronic stress-induced depressive-like behavior in mice.Methods:Chronic social defeat stress,chronic unpredictable stress,and chronic exposure to corticosterone with behavioral tests were performed to establish mouse models of depression.The a NSCs from DG were cultured in vitro.Enzyme-linked immunosorbent assay was performed to detect the activity of kinase.Co-immunoprecipitation and western blotting were used to examine the protein levels.Autophagic vesicles were observed by transmission electron microscopy.Immunofluorescence was used to detect the changes of adult hippocampal neurogenesis.The intrinsic excitability of newborn neurons was measured by electrophysiological recordings.NRBF2 knockout mice and Nestin-Cre ER^T2transgene mice were constructed,and the mechanism of a NSCs-specific NRBF2 was determined by combining pharmacological and genetic intervention methods,proteomics,and phosphorylated proteomics.Results:（1）Chronic stress induced depressive-like behavior in mice and led to reduced expression of NRBF2 and autophagy impairment in the DG.（2）Chronic social defeat stress impairs the adult hippocampal neurogenesis.（3）Overexpression of NRBF2 in DG improved autophagy deficiency,adult hippocampal neurogenesis impairment,and depressive-like behavior induced by chronic stress.Temozolomide blocked NRBF2-mediated antidepressant effects in mice via inhibiting adult hippocampal neurogenesis;（4）NRBF2knockout mice displayed neural stem cell autophagy deficiency,adult hippocampal neurogenesis impairment and depressive-like phenotype,which were improved by conditional knock-in NRBF2 to a NSCs in DG.（5）Deficiency of NRBF2 in a NSCs inhibited the self-renewal and neurogenesis of a NSCs,leading to the reduced activity of VPS34kinase and impaired autophagic flux.（6）Injection of VPS34 specific inhibiter SAR405 to DG promoted adult hippocampal neurogenesis impairment and the susceptibility of mice to stress,while overexpression of NRBF2 in a NSCs ameliorated SAR405-induced autophagy deficiency and proliferative inhibition in a NSCs.（7）Conditional knockdown of NRBF2 in a NSCs led to depressive-like behavior and adult hippocampal neurogenesis impairment.Specifical overexpression of NRBF2 in DG a NSCs ameliorated adult hippocampal neurogenesis impairment and depressive-like behavior induced by chronic stress.（8）NRBF2 deletion in DG disrupted the network of neurogenesis-related proteins in DG,while conditional overexpression of NRBF2 in DG a NSCs alleviated the neurogenesis-related protein network disruption caused by chronic stress.Conclusion:NRBF2-dependent autophagic deficiency in a NSCs mediates the chronic stress-induced defective adult hippocampal neurogenesis and depressive-like behavior.Specific silencing of NRBF2 in a NSCs led to the autophagy deficiency of neural stem cells,the disrupted neurogenesis-associated protein networks,and adult hippocampal neurogenesis impairment,which subsequently induced the depressive-like behavior.Specific overexpression of NRBF2 in the DG a NSCs improved the neurogenesis impairment and depressive-like behavior of both chronic social defeat stress-exposure mice and NRBF2 knockout mice,providing a new insight for the treatment of MDD.