The Role Of Hippocampal CRMP2 In CUMS-induced Depression-like Behavior In Mice

Depression is a common mental illness,which not only brings serious physical and psychological burden to the patients,but also brings huge economic loss to the society.Its pathogenesis is often considered to be closely related to biological and social factors,such as genes and stress.Although no gene has been identified that directly causes depression,stress may act as a risk factor,making genetically susceptible individuals more likely to develop depression.In preclinical studies,chronic mild unpredictable stress(CUMS)is commonly used as an animal model of depression.The stressors in this model are relatively mild,and they can mimic various stress that is encountered by human beings in daily life,and make animals produce depression-like behaviors.The neuroplasticity hypothesis on the pathogenesis of depression shows that the adjustment of plasticity in different brain regions during the process of environmental adaptation of the nervous system plays an important role in the pathological process of depression.Hippocampus is an important structure of limbic system,which is related to emotion regulation,learning and memory.Many studies have shown that depression is closely related to changes in hippocampal plasticity.CA3 is a stress-sensitive region and an important part of information transmission in the hippocampus.The plasticity of dendritic spines of CA3 pyramidal neurons is changed in response to different stress processes.Therefore,it is important to study the plasticity of dendritic spine in CA3 brain region.CRMP2 is a member of the collapsing response mediator protein family.It is widely expressed in the nervous system,especially in the hippocampus.It has no enzymatic activity,and it possibly can be molecular scaffolds or adapters.It promotes the growth of dendrites and axons,Ca²⁺ homeostasis,neurotransmitter release and other processes,and is associated with a variety of neurological diseases.Its alteration in mRNA levels have been found in both depressed patients by RNA-seq and animal models of depression.Currently,the relationship between CRMP2 and the CUMS-induced depression-like behavior is not very clear.Therefore,the aim of this study was to answer the following questions:Are depression-like behaviors induced by CUMS in mice accompanied by changes in expression of hippocampal CRMP2?Can this change be reversed by antidepressant drugs?Do mice exhibit depression-like behaviors when endogenous CRMP2 is decreased in the hippocampal CA3 pyramidal neurons?What are the possible mechanisms underlying these changes?To answer these questions,I performed the first experiment in which the mice were divided into four groups,these animals received CUMS for 5 weeks and the antidepressant treatments(fluoxetine and ketamine).Depression-like behaviors induced by CUMS were evulated by behavioral tests including sucrose preference test,tail suspension test,forced swimming test,open field and elevated cross maze experiments,Western blot was used to detect the changes of CRMP2 expression in the hippocampal region.The second experiment was to determine whether reduction of endogenous CRMP2 induces depression-like behaviors or alter susceptibility of animals to stress.Adeno-associated virus(AAV)encoding sh-CRMP2-EGFP or control shRNA-EGFP was injected into the hippocampal CA3 area of animals using stereotactic technique and behavioral changes in these animals were evulated after AAV injection.After behavioral tests,the animals were divided into four groups:control groups with control shRNA or CRMP2 shRNA and CUMS groups with control shRNA or CRMP2 shRNA.The sucrose preference test was conducted on each group every seven days,until the sucrose consumption was significantly reduced in one group,and other behavioral tests were continued to be performed.After behavioral tests,the dendritic spines in the hippocampus of each group were labeled with a gene gun and were imaged with a confoclmicroscopy.Western blot was used to detect the changes in the expression of glutamate receptors and synaptic-related proteins in the hippocampus.The results show:1.Depression-like behaviors in mice induced by 5-week CUMS are associated with a decrease in levels of CRMP2 protein in the hippocampus,and the antidepressant drug fluoxetine and ketamine alleviated depression-like behaviors or reversed the change in CRMP2 expression.Western Blot analysis showed that the expression of CRMP2 protein in the CUMS group was significantly decreased,while fluoxetine and ketamine could reverse the alterations of CRMP2 expression in different degrees.2.Immunofluorescence staining and Western Blot verified that AAV-shRNA-EGFP was successfully expressed after stereotaxic injection and expression of CRMP2 shRNA caused a decrease in the expression of CRMP2 protein in the hippocampus.3.The decreased expression of CRMP2 in the hippocampal CA3 did not directly cause depression-like behavior in mice.Compared with the mice that received control shRNA,decreased levels of CRMP2 did not induce depression-like behaviors and showed no memory defects in the new object recognition test.4.The decreased expression of CRMP2 in the hippocampal CA3 made animals being more sensitive to CUMS to develop depression-like behavior after stress.5.CUMS-induced epression-like behaviors and companying decrease in the leves of CRMP2 protein in the hippocampus were accompanied by the decreased spine density in apical dendrites of hippocampal CA3 pyramidal neurons and the decreased expression of glutamate receptor GluN2B protein.The density of the dendritic spines in the hippocampal CA3 apical dendrites was decreased significantly in mice that received AAV-CRMP2 shRNA and the mice received AAV-control shRNA after CUMS.The density of the mushroom spines in the mice received AAV-CRMP2 shRNA significantly decreased after CUMS,while the density of the mushroom spines of the mice received AAV-control shRNA virus did not show a significant change.Meanwhile,Western blot results showed that the expression of NMDA receptor subunit GluN2B decreased significantly after CUMS in mice received AAV-CRMP2 shRNA,while the similar changes did not occur in mice that received AAV-control shRNA.The results of this study demonstrated that decreased CRMP2 expression in the mice hippocampal CA3 area caused an increase in susceptibility to CUMS to develop depression-like behaviors,which were accompanied by changes in dendritic spine density and the levels of synaptic proteins including GluN2B.These results can enhance our understanding of depression and pave the path for developing novel approaches for treating this disease.