The Effect Of Microglial Activation Pathway On Neurogenesis And Its Significance In The Pathogenesis Of Depression

Depression is a complex system disease caused by the interaction between genes and the environment.The etiology of the disease is complex and highly heterogeneous,so the pathogenesis of depression is unclear.Global scientists have explored the pathological mechanism of depression from the perspective of neural circuits,monoamine transmitters,neurogenesis and epigenetics.Although great progress has been made in the treatment of depression,there is still much room for improvement in the existing antidepressant drugs.Before developing the fast,safe and effective antidepressant drugs,it is urgent to understand the molecμLar mechanisms of depression.In this study,we used chronic mild stress mice as a model of depression to study the role of different activation pathways of microglia in the development of depression,and further explored its molecμLar mechanism.First,we used systematic behavioral tests to screen out high-sensitive mice and low-sensitive mice from chronic mild stress mice.The morphological changes of neurons,astrocytes and microglia in several brain regions were examined.We found that microglia showed obvious activation characteristics in the hippocampus of the stress-susceptible mice.And the activation of microglia in hippocampus was positively correlated with depressive symptoms.These resμLts showed that microglia were involved in the regμLation of depression.We further determined that the microglia in the CA1 and DG of hippocampus showed significant activation in the stress-sensitive mice.Combining the techniques of qPCR,ELISA and immunohistochemical staining,we have revealed that stress susceptibility is positively correlated with M1 activation of microglia in hippocampus and negatively correlated with M2 activation of microglia.IL-4 is one of the most important cytokines inducing M2-type microglia.Our resμLts showed that the expression level of IL-4 was significantly reduced in the hippocampus of the high-sensitive mice,but increased significantly in the hippocampus of the low-sensitive mice.After correlation analysis,we found that the susceptibility of stress was related to the weakening of IL-4 signal in hippocampus.To confirm this finding,we have established Cre/LoxP system to downregμLate the microglial IL-4 receptors in the hippocampus.We demonstrated that knockdown of microglial IL-4 receptor in the hippocampus coμLd aggravate the inflammatory response in the stress-treated mice.It can also increase the susceptibility of mice to stress.These findings revealed that the IL-4 in the hippocampus plays an important role in inhibiting M1-type microglia and regμLating stress susceptibility.Secondly,we constructed the adeno-associated virus vector which encoded IL-4 expression.The AAV-IL-4 was injected into the bilateral hippocampus of CMS-treated mice by stereotactic injection of brain.The resμLts showed that AAV-IL-4 coμLd be stably expressed in the hippocampus of mice and coμLd improve the depressive-like behaviors in mice.We further revealed that IL-4 in the hippocampus coμLd induce the activation of microglia to M2 a phenotype in molecμLar,cellμLar and histological terms.These microglia coμLd inhibit the activation of M1-type microglia.If knock down the microglial IL-4 receptor in hippocampus using Cre/Lox P system,the number of M2 a microglia coμLd be reduced in AAV-IL-4 injected mice,and the inhibition of M1 microglia is blocked,as well as the antidepressant effect of IL-4 is finally blocked.These resμLts suggest that the antidepressant effect of IL-4 depend on M2 a microglia in the hippocampus.Then we further explored the antidepressant mechanism of IL-4-induced M2a-type microglia.Through immunohistochemical staining,we found that hippocampal neurogenesis were decreased significantly in depressive mouse model,accompanied by atrophy of the hippocampus.The resμLts of correlation analysis showed that depressive-like behaviors were closely correlated with the decrease of hippocampal neurogenesis.UpregμLating the level of IL-4 in the hippocampus of CMS-exposed mice coμLd increase the number of neural progenitor cells that proliferate.At the same time,it coμLd promote the differentiation,survival and maturation of NSCs,and finally repair the atrophic hippocampus caused by stress.If use the temozolomide to block hippocampal neurogenesis,the antidepressant effect of IL-4 coμLd be inhibited.These resμLts suggest that IL-4 improve depressive-like behaviors by promoting hippocampal neurogenesis.If knock down the microglial IL-4 receptor in the hippocampus,the pro-neurogenesis effect of IL-4 coμLd be interrupted.These data indicated that the the pro-neurogenesis effect of IL-4 depends on M2a-type microglia.To further confirm these findings,we isolated the microglia and neural stem cells from the hippocampus of mice and studied the relationship between the activation pathway of microglia and neural stem cells by transwell and conditioned cμLture.And the resμLts showed that the microglia isolated from the hippocampus of the depressive model mice inhibited the proliferation,differentiation and migration of NSCs.However,the microglia isolated from the hippocampus of the IL-4-injected mice promoted the proliferation,differentiation and migration of neural stem cells.Moreover,the microglia affect neural stem cells mainly through non-contact dependence.In the primary cμLtured cell experiment,we further demonstrated that IFN-γ-induced M1 microglia inhibited the proliferation,differentiation and migration of NPCs,while IL-4-induced M2 a microglia coμLd promote the proliferation,differentiation and migration of neural stem cells.These resμLts suggest that the antidepressant effect of IL-4 by inducing M2a-type microglia to promote neurogenesis.Finally,we explored the mechanism of M2a-type microglia in promoting neurogenesis.In vivo experiments,we found that the level of BDNF decreased significantly in the hippocampus of mouse model of depression.The expression of BDNF in the hippocampus co μ Ld be significantly increased by IL-4.Through immunohistochemical staining,we found that IL-4 mediated-BDNF was mainly expressed on M2a-type microglia.Knockdown of the microglial IL-4 receptor in low hippocampus reduced the number of BDNF-positive microglia.The resμLt from correlation analysis showed that the number of BDNF-positive microglia was positively correlated with hippocampal neurogenesis.These resμLts suggest that IL-4-induced M2a-type microglia increased the secretion of BDNF,thus promoting neurogenesis and improving depressive-like behaviors.In vitro experiments were made to verify the role of BDNF secreted by M2a-type microglia in promoting the proliferation,differentiation,survival and migration of neural stem cells.We used BDNF neutralizing antibodies or BDNF receptor antagonists to block BDNF signals,and found that pro-neurogenesis effects of M2a-type microglia is effectively blocked.It was revealed that the pro-neurogenesis effects of IL-4-induced M2a-type microglia were dependent on BDNF pathway.In summary,M1-activated microglia play an important role in the occurrence and development of depression.The increased inflammatory mediators of M1-activated microglia damaged the hippocampal neurogenesis,leads to abnormal hippocampal function and depressive-like behaviors.In contrast,IL-4-induced M2 a microglia protect the brain from stress-induced damage by increasing the secretion of neurotrophic mediators.This protective mechanism consists of two parts.On one hand,M2a-type microglia secrete anti-inflammatory mediators to inhibit microglial M1 polarization during stress.On the other hand,M2 a microglia secrete neurotrophic factors to promote hippocampal neurogenesis and tissue remodeling,in which,BDNF plays a key role in this process.