CDK5-mediated Phosphorylation Of Sirt2 Contributes To Depressive-like Behavior Induced By Social Defeat Stress

Background:Major depressive disorder（MDD）,simply known as depression,is a common,severe and recurrent disorder worldwide.However,MDD research continues to face the major challenge that traditional animal models often do not have multiple validities,which may explain the paucity of novel therapeutic interventions targeting the molecular mechanisms of MDD.A newly-developed mouse model that utilizes social defeat stress（SDS）has shown perfect etiological,predictive,discriminative and face validity for depression studies.Repeated exposure to SDS causes a robust depression-like phenotype and high degree of molecular plasticity,providing a potential means to detect the neuropathological mechanisms underlying affective illnesses,such as depression.Histone deacetylases（HDACs）reportedly play an essential role in the mechanisms underlying depression.As class III HDACs,sirtuins have garnered attention for their function in psychiatric disorders.Among all sirtuins the expression of Sirt2 was the strongest in the brain and Sirt2 has been associated with many nervous system disorders.However,there is a paucity of literature on the role of Sirt2 in affective illnesses.The role of Sirt2 in depression remains unclear.The few previous research on the role of Sirt2 in depression is contradictory.These conflicting data suggest that the use of inhibitors may not be appropriate for Sirt2-based research because of the crosstalk among sirtuin subfamily members.Therefore,we employed Sirt2 knockout mice to explore the role of Sirt2 in SDS-induced depression mouse model.Methods:1.PCR was used to identify the genotype of Sirt2 knockout mice and the wildtype littermates.Western blotting was used to analyze the expression of Sirt2 protein in Sirt2^-/-mice.2.10-day repeated social defeat stress was adopted to establish the mouse model of MDD.Social interaction test,sucrose preference test,forced swimming test,elevated plus maze test and open field test were performed to assess the behaviors associated with depression.3.Western blotting was performed for the analysis of total Sirt2 protein level,cytoplasmic and nuclear Sirt2 protein levels,phosphorylated Sirt2 level,p25 level and phosphorylated CDK5 level.Immunofluorescence was used to assess the distribution of Sirt2 protein in nucleus and cytoplasm.4.A myristoylated membrane-permeabilising peptide（Sirt2-p）was developed from a12-amino acid fragment of the Sirt2 protein to specifically interfere with the phosphorylation of Sirt2 by CDK5 at serine residues 368 and 372.Sirt2-p or scrambled peptide（control）were intraperitoneally injected once a day before each SDS session.After the 10-day repeated SDS was completed,behavior tests and molecular experiments were performed.5.Then the VTA was targeted with viruses of rAAV-cfos-tTA-WPRE-pA and rAAV-TRE-tight-hM3D（Gq）-mCherry-WPRE-PA to selectively label the subpopulation of neurons activated during SDS in VTA.Under the management of doxycycline（Dox）diet,hM3D（Gq）-mCherry was selectively tagged in the c-Fos-expressing VTA neurons that were activated by microdefeat stress.6.The mice labelled with hM3D（Gq）-mCherry were manipulated by intraperitoneally injecting with CNO or NS,and two hours later social interaction test or molecular experiments were performed.Results:1.Behavior tests（sucrose preference test,forced swimming test,elevated plus maze test,open field test）associated with depression were performed to assess the responses of Sirt2^+/+mice and Sirt2^-/-mice,but no differences were found.After 10 day repeated social defeat stress,social interaction test,sucrose preference test,forced swimming test,elevated plus maze test,open field test were performed.We found Sirt2 knockout blocks SDS-induced depressive-like behavior.2.SDS-induced depression was established in C57BL/6 mice.Western blotting was used to assessed Sirt2 protein expression in the nuclei suggested to be the most closely associated with depression.No change of total Sirt2 protein levev was observed in these nuclei in SDS mice.However,nuclear Sirt2 protein levels in the amygdala were detected notably increased after SDS with western blotting and immunofluorescence.3.Total proteins of the amygdala were extracted for the assessment of phosphorylated Sirt2 level and activity of CDK5 in depressed mice.It was observed that phosphorylated Sirt2 level increased with elevated p25 and elevated phosphorylated CDK5 in the amygdala of SDS mice.4.A myristoylated membrane-permeabilising peptide（Sirt2-p）was developed to specifically interfere with the phosphorylation of Sirt2 by CDK5 at serine residues 368and 372.The infusion of Sirt2-p prevented SDS-induced behavioral responses in mice.Western blotting was used to assessed subcellular localization of Sirt2 protein.The mice infused with Sirt2-p did not exhibite SDS-induced Sirt2 nuclear translocation in the amygdala.5.The subpopulation of neurons activated during SDS in VTA were selectively label with hM3D（Gq）-mCherry.The expression of hM3D（Gq）-mCherry in the VTA was examined and significantly enhanced virus signals were observed in microdefeat mice when compared with the control groups.6.The mice injected with CNO exhibited the depressive-like behavior compared with the mice injected with NS.Moreover,total proteins in the amygdala were extracted and analysed with western blotting.The CNO injection led to an increase in p25 production and phosphorylated CDK5,while the mice injected with CNO exhibited Sirt2 nuclear translocation in the amygdala.Conclusions:1.Sirt2 knockout blocks SDS-induced depressive-like behavior.2.Phosphorylation of Sirt2 by CDK5 mediates Sirt2 nuclear translocation in the amygdala,contributing to SDS-induced depressive-like behavior.3.Specifically interfering phosphorylation of Sirt2 at serine residues 368 and 372 by CDK5 may be a new target for the treatment of psychiatric disorders,including depression.4.The molecular mechanism underlying VTA-amygdala modulation in depression may be the over activation of CDK5 and the resulting Sirt2 nuclear translocation in the amygdala.