The Role And Mechanism Of Neurobeachin In Stress-induced Depression-like Behaviors In Mice

Part I The role of Neurobeachin in chronic social defeat stress-induceddepression-like behaviors in miceObjective: Dysfunction of synaptic plasticity is considered to be the neurobiological mechanisms of major depressive disorder(MDD).Neurobeachin(Nbea)has an important function in regulating synaptic plasticity.When Nbea expression is reduced,it will result in impaired synaptic plasticity.However,the function of Nbea in depression is still unknown.Here,we used the model of chronic social defeat stress(CSDS)to investigate the intrinsic relationship between Nbea expression and depression-like behavior in mice.Methods: Using the CSDS model to distinguish susceptible and resistant mice by the social interaction text and sucrose preference text.Nbea protein level in the susceptible and resistant mice was examined by western blot in the hippocampus,prefrontal cortex,anterior cingulate cortex,amygdala,nucleus accumbens,and lateral habenular;glutamatergic synaptic function in susceptible mice was evaluated by patch clamp recording;Nbea RNAi virus and Nbea C terminal overexpression virus were stereotactic injection to study depressive-like behavior of mice.The role of fluoxetine on Nbea protein expression and the depression-like behavior was researched.Results:(1)Brain regions screening revealed that the expression of Nbea protein in the hippocampal was decreased in susceptible mice(Control group: 1.00±0.07,Susceptible group: 0.79±0.10,Resilient group: 1.09±0.04,P<0.05),Nbea protein expression in other brain regions did not change.Further studies found that the expression of Nbea in the dorsal hippocampus was decreased(P<0.05).(2)Patch clamp recordings revealed the impaired excitatory synaptic transmission in the dorsal hippocampus.The frequency of m EPSC in susceptible mice was significantly reduced(P<0.05),the amplitude of m EPSCwas decreased(P<0.001).(3)Knockdown of Nbea in dorsal hippocampal combining with subthreshold stress increased depression vulnerability and reduce the social interaction rate of mice(P<0.05),as well as the sucrose preference rate(P<0.01).(4)Overexpression of Nbea C-terminal can reverse the social interaction(P<0.001)and surcrose preference of susceptible mice(P<0.05).(5)Intraperitoneal injection of fluoxetine(20 mg/kg)for two weeks increased protein expression of Nbea in the dorsal hippocampus(P<0.05),and reversed the social interaction(P<0.001)and immobile time of susceptible mice(P<0.001).Conclusion: We demonstrated the relationship between Nbea protein expression and the depressive-like behavior induced by chronic defeat stress.The decrease in Nbea expression promotes vulnerability of depression.On the contrary,the increase in the expression of Nbea may improve the depression-like behavior.Part II The role and mechanism of Neurobeachin in stress vulnerabilityof miceObjective: Depression vulnerability is an intrinsic feature of mice.In basic situation,enhanced function of depression vulnerability genes or reduced expression of stress tolerance genes will lead to increased vulnerability to depression.Based on the results of previous studies,we hypothesized that Nbea is a stress-tolerant gene,which could regulate depression vulnerability in mice.In order to study the role and mechanism of Nbea in stress-induced depression vulnerability,we started this part of the research.Methods: Anxiety-and depression-like behaviors were used to distinguish depressionvulnerability in mice after subthreshold stress;Nbea RNAi virus were stereotactic injection to study anxiety-and depression-like behavior;the changes in glutamate receptor-associated subunits and GABAA receptor-related subunits were detected by western blot;glutamatergic synapse function was recorded by patch clamp;dendritic spine imaging and three-dimensional imaging were performed to study dendritic spine density changes.Bioinformatics database was used to analyze the functional proteins which interaction with Nbea.Results:(1)In the basal condition,knockdown of Nbea decreased the time in the central area of the open field test(P<0.05),the distance to open arms(P<0.05)and time in the elevated plus-maze test(P<0.05).(2)Knockdown of Nbea expression combining with subthreshold stress reduced ratio of social interaction(P<0.01)and sucrose preference(P<0.001).(3)Knockdown of Nbea expression combining with subthreshold stress reduced the expression of NR1 subunit(P<0.001),NR2 A subunit(P<0.001)and NR2 B subunit(P<0.001)(4)Knockdown of Nbea expression accompanied with subthreshold stress reduced the expression of Glu A1 subunit(P<0.001)(5)Subthreshold stress significant decreased the expression of GABAA receptor γ2 subunit(P<0.05).(6)Knockdown of Nbea expression together with subthreshold stress reduced the frequency(GFP+stress group: 3.02±0.57,Nbea-KD+stress group: 1.93±0.43,P=0.18)and amplitude of m EPSC(GFP+stress group: 17.82±1.65,Nbea-KD+stress group: 12.23±1.27,P<0.05).(7)Knockdown of Nbea expression accompanied with subthreshold stress reduced the total dendritic spine density(P<0.001)and mushroom-like dendritic spines(P<0.05)(8)Using bioinformatics database to discover Nbea interacting functional proteins,SAP102 and GABARAP.Kncokdown of Nbea reduced the expression level of SAP102(P<0.05)and GABARAP(P<0.05)induced by subthreshold stress.Conclusion: Knockdown of Nbea expression in dorsal hippocampus results in dysfunctionof glutamate receptors and GABAA receptors,reduction of dendritic spine density in mushroom-type and increase in stress vulnerability in mice.When the expression level of Nbea is decreased,the regulatory effect on SAP102 is weakened,resulting in impaired glutamate receptor function;and the regulatory effect on GABARAP is also weakened,resulting in impaired GABAA receptor function.