The Mechanism Of Microglial Exosomes On Hippocampal Neurogenesis In Depressed Rats

Background:Depression,one of the most common neuropsychiatric disorders,which is a severe medical and social problem all over the world.Its typically symptoms involving anhedonia,behavioral despair,low mood and pessimism seriously affect and reduce the quality of life in these individuals.Depression is a recurrent lifelong condition with a complicated pathogenesis.Current clinical antidepressant treatments have a poor prognosis,and newly developed drugs show a high failure rate in clinical trials.Therefore,it is urgent to find new targets for the treatment of depression.In recent years,clinical evidence has shown that neuroinflammation is a potential risk factor for depression,but the mechanism is unclear.Microglia are the main participants in the inflammatory response of the central nervous system.Previous series of studies conducted by our research group found that microglia were activated in depression-related brain regions,such as hippocampus and prefrontal cortex,accompanied by a decrease in new neurons in the dentate gyrus(DG)area of the hippocampus.Adult neurogenesis is considered to be an important participant in the maintenance of brainhomeostasis and disease occurrence,mainly regulated by factors such as genetic background,environmental stress,growth factors,and pathological damage,but its participation in the development of depression,especially whether the crosstalk between microglia and neural stem cells(or neurons)is involved in the regulation of neurogenesis is currently unclear.Based on this,with use of the chronic unpredictable mild stress(CUMS)-induced rat model of depression and various experimental methods or techniques such as behavior,molecular biology,virology,and brain patch clamp electrophysiology in this study to investigate whether microglia exosomes can regulate hippocampal neurogenesis in depressed rats,and to explore its role and mechanism in the pathogenesis of depression.Methods:(1)The rats were randomly divided into Control group(non-stressed)and CUMS group.After five weeks of chronic unpredictable stress stimulation in CUMS group rats,we first evaluated the behavior of rats subjected to CUMS as assessed in the sucrose preference,forced swim tests,open field test and elevated cross maze test.Immunofluorescence assay was used to detect the number of Nestin positive cells,Sox-2 positive cells and DCX positive cells in the hippocampal DG region of rats,as well as the activation status of microglia.(2)Collect and purify exosomes from rat serum of each group.The expression of exosomes or microglia-specific proteins markers such as CD63,CD81,MCT-1,CD14,IL-1β,CD13 were detected by Western bolt and flow cytometry.The exosomes secreted by LPS-treated BV2 microglia were collected and co-cultured with neural stem cells.The number of Ki67 positive neural stem cells,BrdU positive neural stem cells,DCX positive cells,βⅢ-Tubulin positive cells,MAP2 positive cells and NeuN positive cells were observed by immunofluorescence technique.(3)Exosomes were isolated from the serum of CUMS rats and subjected to miRNA high-throughput sequencing,then bioinformatics technology was used to search for differentially expressed miRNAs regulating neurogenesis.(4)A lentivirus knocking down miR-146a-5p was constructed and transfected into BV2 microglia cells.The secreted exosomes were collected for co-culture with neural stem cells.Immunofluorescence was used to observe the number of proliferation-related cells such as BrdU positive cells,as well as the number of DCX positive cells and pⅢ-Tubulin positive cells.(5)An AAV-miR-146a-5p virus was constructed to overexpress miR-146a-5p within the DG of normal rats or block miR-146a-5p in CUMS rats by bilateral stereotactic injection.Three weeks after virus infection,the rats were subjected to behavioral tests such as sucrose preference test and forced swimming test.Immunofluorescence was used to detect the changes of neurogenesis related indicators in the hippocampal DG region of rats.The expression of Krüppel-like transcription factor 4(KLF4)and its downstream related proteins CDKL5 and p-STAT3 were detected by Western blot.(6)An AAV adeno-associated virus(AAV)knocking down KLF4 was constructed and injected bilaterally into the DG of normal rats.Three weeks after infection,the rats were subjected to behavioral tests such as sucrose preference test and forced swimming test.Immunofluorescence was used to detect changes in neurogenesis-related indicators in the DG area of the hippocampus.Western blot was used to detect the expression of the downstream related protein CDKL5 and p-STAT3.(7)An AAV adeno-associated virus with knockdown or overexpression of circANKS1B was constructed and stereotaxically injected into the hippocampal DG region of rats.Three weeks after infection,the rats were subjected to behavioral tests such as sucrose preference test and forced swimming test.Immunofluorescence was used to detect the changes of neurogenesis related indicators.Western blot and RT-PCR were used to detect the expressions of miR-146a-5p and its downstream related proteins KLF4,CDKL5 and p-STAT3.Results:(1)After 5 weeks of CUMS exposure,the percentage of sucrose consumption in the sucrose preference test was significantly reduced,the immobility time in the forced swimming test was significantly prolonged and there was no significant difference in the number of crossings in the open field test.Both the retention time ratio and entry number ratio of the open arm were significantly reduced in the elevated cross maze test,in these CUMS-exposed rats compared with that of the Control group.Compared with Control group,the numbers of Nestin positive cells,Sox-2 positive cells and DCX positive cells in the hippocampal DG area of CUMS rats were significantly reduced,and the microglia were significantly increased with amoeba-like activation.These results suggested that chronic unpredictable mild stress induced depression-like behavior,activated microglia and inhibited neurogenesis in the hippocampal of rats.(2)The results of Western blot and flow cytometry showed the high expressions of the exosomal marker proteins,CD63 and CD81,were present in in serum exosomes of CUMS rats.Meanwhile,high expression levels of the microglia-specific proteins TMEM119,CD13,MCT-1,CD 14,CD11b and IL-1β,suggested that the increased exosomes in peripheral blood of depressive-like rats were mainly derived from microglia.Immunofluorescence results showed that the co-culture of LPS-treated microglia-derived exosomes with neural stem cells significantly reduced the number of Ki67-positive cells,BrdU positive cells,DCX positive cells,βⅢ-Tublin positive cells,MAP2 positive cells and NeuN positive cells.These results suggested that microglia-derived exosomes could inhibit the proliferation and differentiation of neural stem cells.(3)Sequencing results showed that various miRNAs were significantly dysregulated in samples from CUMS versus Control rats.Microglia-specific miR-146a-5p was significantly upregulated in serum exosomes of depressed rats.Notably,miR-146a-5p was identified as being most closely related to signaling pathways regulating pluripotency of stem cells,suggesting that miR-146a-5p in serum exosomes may be a key molecule regulating neurogenesis.(4)Immunofluorescence results showed that the exosomes derived from these BV2 microglia which were treated with the lentiviral knocking down miR-146a-5p have no inhibitory effect on the number of BrdU positive cells,DCX positive cells and βⅢ-Tubulin positive cells,suggesting that exosomes derived from microglia could deliver miR-146a-5p to neurons to regulate the proliferation and differentiation of neural stem cells.Microglial exosomes regulated the proliferation and differentiation of neural stem cells through their enriched miR-146a-5p.(5)Overexpression of miR-146a-5p in the hippocampal DG area of normal rats could induce depression-like behaviors.The number of neural stem cells and new neurons in the hippocampal DG region was reduced,accompanied by a decrease in the expression of KLF4 and CDKL5,as well as an increase in the expression of p-STAT3.Knockdown of miR-146a-5p in the hippocampal DG region of depressive-like rats could improve depressive-like behavior,reduce the number of neural stem cells and new neurons,increase the expression of KLF4 and CDKL5,and decrease the expression of p-STAT3.(6)Knockdown of KLF4 in the hippocampal DG region of normal rats resulted in depression-like phenotype and the number of newly developed cells within the hippocampal DG region were reduced.In addition,protein levels of CDKL5 were significantly decreased,while phosphorylation levels of STAT3 was greatly increased.(7)Knockdown of circANKS1B in the DG region of the normal rats induced depression-like symptoms and decreased newborn neurons,accompanied with increased expression of miR-146a-5p,decreased expression of KLF4 and CDKL5,and increased expression of p-STAT3.Overexpression of circANKS1B in the DG region of the hippocampus of CUMS rats could reverse depressive-like behavior and increase the number of newborn neurons,accompanied with a decrease in the expression of miR-146a-5p,an increase in the expression of KLF4 and CDKL5,as well as the decreased expression of p-STAT3.Conclusion:(1)Chronic unpredictable mild stress induced depression-like symptoms,which were accompanied with the activation of microglia and the inhibition of hippocampal DG neurogenesis in rats.(2)Exosomes in serum of CUMS rats were mainly derived from microglia,which could significantly inhibit the proliferation and differentiation of neural stem cells.(3)Microglia-derived exosomes containing miR-146a-5p inhibited the proliferation of neural stem cells and the differentiation into mature neurons,resulting in a decrease in the number of new neurons.(4)MiR-146a-5p directly targeted KLF4 and regulated neurogenesis in rat hippocampal DG region by inhibiting the KLF4/CDKL5 pathway.(5)CircANKS1B participated in the regulation of hippocampal neurogenesis and the pathogenesis of depression by inhibiting the miR-146a-5p/KLF4 signaling pathway.