Effect Of Rasd2 In CUMS-induced Depression-like Behavior And Its Potential Mechanism

Background:Depression is a psychological disease characterized by sadness,loss of interest or pleasure,low self-worth,disturbed sleep or appetite,poor concentration,and etc.Depression causes a heavy emotional and financial burden for patients and their families.Therefore,the studies of depression have become an urgent public health issue.Clinical trials have shown alterations in depressed patients with decreased levels of dopamine and its metabolites,and there is a positive correlation between depression and stress.Symptoms of depression such as lack of motivation,lack of pleasure and decreased socialization suggest that the reward system is involved in the pathophysiology of depression.Changes in DRD2 levels in the reward system are closely associated with depression pathogenesis,and activation of DRD2 may exert an antidepressant-like effect,as well as enhance the efficacy of interventions with other antidepressant drugs.In our previous study investigating the mechanism of antidepressant-like effects of acute fasting,transcriptomic analysis revealed that acute fasting induced changes in Rasd2 expression in the PFC.Further studies showed that Rasd2 modulates DRD2-mediated antidepressant-like effects in ovariectomized mice.Furthermore,a transcriptomic analysis of mice exposed to chronic mild swimming stress found that stress can alter the expression of Rasd2 in the PFC,these suggesting that Rasd2 is likely involved in the pathogenesis of stress-induced depression.Rasd2 is a member of the RASD subfamily and is mainly enriched in the striatum.Rasd2 mainly regulates the dopamine receptor signaling pathways in the central nervous system and can act as a modulator of dopamine transmission by modulating the striatal c AMP/PKA signaling pathway.In addition,Rasd2 regulates the Akt/m TOR signaling cascade and is a key bridge between the PI3 K and Akt pathways.However,the effect of Rasd2 in stress-induced depression and its potential mechanisms remain unreported.Objective:In this study,we used chronic-unpredictable mild stress(CUMS)mice as a depression model and used behavioral test,western bolt,immunofluorescence and DREADDs technique to explore the effects of Rasd2 on depression-like behavior in CUMS mice and its related mechanisms regulated by dopamine from the cellular-molecular level to the circuit level,which provide new molecular targets for the treatment of depression.Methods:Firstly,we constructed depression model mice by CUMS.The effects of CUMS on depression-like behaviors were investigated by behavioral tests(open field test,sucrose preference test,forced swimming test and tail suspension test).The effects of CUMS on corticosterone levels in serum and c-Fos expression in depression-related brain regions were investigated by enzyme-linked immunosorbent assay and immunohistochemistry,respectively,to verify the success of depression model construction.Subsequently,the effects of CUMS on Rasd2 expression in stress-related brain regions were investigated by immunohistochemistry.The molecular mechanism of Rasd2 to improve the depression-like behavior in CUMS mice was explored by western blot and co-immunoprecipitation.Finally,the effect of CUMS on c-Fos expression in the projective neurons was investigated by fluorescence gold retrograde tracing and immunofluorescence.Next,Rasd2 overexpression/knockdown mice were constructed by microinjection of a lentivirus mediating Rasd2 expression in the NAc core,and the effects of Rasd2overexpression/knockdown on depression-like behavior in CUMS mice were investigated by behavioral tests.The changes of Rasd2,Glu A1 and DRD1/DRD2-c AMP-PKADARPP-32 and PI3K-Akt-m TORC1 signaling pathway-related protein expressions in NAc and m PFC were investigated by western blot.Immunofluorescence was used to explore the colocalization of Rasd2 and DRD2 in the NAc.We applied DREADDs to selective activate/inhibit the Pr L glutamatergic inputs to the NAc core,and investigated the behavioral changes of mice by behavioral tests.The effects of Pr L-NAc core circuit activation/inhibition on Rasd2-positive neurons and DRD2-positive neurons activation were explored by immunofluorescence.Finally,based on Drd2-cre transgenic mice,we constructed mice with specific Rasd2 overexpression on DRD2 expressing neurons in the Pr L-NAc core circuit and investigated the effects of Rasd2 overexpression on DRD2 expressing neurons in the Pr L-NAc core circuit on CUMS-induced depression-like behavior by immunofluorescence and behavioral tests.Results:CUMS induced a decrease in sucrose preference,an increase in immobility time in the tail suspension test and forced swim test in mice,as well as a decrease in climbing time and swimming time in the forced swim test,but had no effect on the total travel distance,crossing,rearing and number of entries into the central area in the open field test.In addition,CUMS induced an increase in serum corticosterone levels and a decrease in c-Fos expression in NAc(core and shell),m PFC(Cg1,Pr L and IL)and HP(CA1,CA2,CA3 and DG),and these results suggest that the CUMS depressed mice were successfully constructed.In the immunohistochemical study,CUMS reduced Rasd2 expression in mice NAc(core and shell)and m PFC(Cg1 and IL).Similarly,western blot study showed that CUMS induced a decrease in Rasd2,m TORC1 and DRD2 expression and an increase in DARPP-32 and DRD1 expression in NAc,as well as a decrease in Rasd2,Glu A1 and m TORC1 expression and an increase in DARPP-32 expression in m PFC.The results of Co-immunoprecipitation assays showed that,CUMS decreased the interaction between Rasd2 and DRD2.Finally,retrograde neural tracing showed that the upstream nuclei of NAc core by fluorescent gold and found that CUMS induced a decrease in neuronal activation in the circuit,which revealed that CUMS induced depression-like behavior in mice by inhibiting the Pr L-NAc core circuit.Next,we found that Rasd2 overexpression in the NAc core increased sucrose preference in CUMS mice,decreased immobility time in the tail suspension test and forced swimming test,decreased climbing time and increased swimming time in the forced swimming test,whereas Rasd2 knockdown had no such effects after CUMS.In addition,Rasd2 overexpression or knockdown had no effect on the total travel distance,crossing,rearing and number of entries into the central area in the open field test.Western blot results showed that Rasd2 overexpression increased the expression of Glu A1 in the m PFC and decreased the expression of DARPP-32 in the NAc.Rasd2 overexpression activated DRD2-c AMP-PKA-DARPP-32 and PI3K-Akt-m TORC1 signaling pathways in the NAc,and also activated PI3K-Akt-m TORC1 signaling pathway in the m PFC,while knockdown of Rasd2 only inhibited PI3K-Akt-m TORC1 signaling pathway in the NAc.In addition,Rasd2 overexpression increased the co-localization of Rasd2 and DRD2 in the NAc core of CUMS mice.In the further studies,it was found that selective activation of the Pr L glutamatergic inputs to the NAc core alleviated depression-like behavior in CUMS mice,as evidenced by increased sucrose preference,decreased immobility time(tail suspension test and forced swimming test),and increased swimming time.Activation or inhibition of the Pr L-NAc core circuit did not affect the total travel distance,crossing,rearing and number of entries into the central area in the open field test.Activation or inhibition of the Pr L-NAc core circuit induced increased or decreased activation of Rasd2-positive neurons and DRD2-positive neurons in the NAc core,respectively.Finally,Rasd2 overexpression in DRD2 expressing neurons of Pr L-NAc core circuit increased DRD2 co-localization with Rasd2 in the NAc core and increased sucrose preference,decreased immobility time in the tail suspension test and forced swimming test,and also increased climbing time and swimming time in the forced swimming test.Conclusions:In summary,our study reveals that CUMS reduces Rasd2 expression in the NAc and m PFC of mice and induces depression-like behavior by inhibiting the Pr L-NAc core circuit.And,Rasd2 overexpression in NAc core has an alleviating effect on depression-like behavior in CUMS mice,which is mediated by DRD2-c AMP-PKA-DARPP-32 and PI3K-Akt-m TORC1 signaling pathways.In addition,the activation of Pr L glutamatergic inputs to the NAc core can produce antidepressant-like effects by Rasd2-positive neurons and DRD2-positive neurons activation in the NAc core.This study also found that Rasd2 overexpression in the DRD2 expressing neurons can modulate the Pr L-NAc core circuit and further alleviate depression-like behaviors.This study highlights the importance of Rasd2 in improving stress-induced depression-like behavior and provides a theoretical basis for the development of antidepressant drugs.