Phosphorylated Proteomics Study For The Rapid Antidepressant Effect Of Ketamine

Background and AimDepression is a type of mental and psychological disorder with long-term low mood as the main clinical feature.Ketamine,as a new rapid antidepressant,is the most important discovery in the field of psychotherapy in nearly half a century.Phosphorylation proteomics is widely used to detect the phosphorylation level of whole proteins.Therefore,the study combined mass spectrometry(MS)-based label-free quantitative phosphorylation proteomics,behavior,and pharmacology to provide a basis for the research on the key signaling pathways and molecular mechanism of depression and ketamine treatment.Methods1.Chronic unpredicted mild stress(CUMS)was used to prepare the model of depression.The antidepressant effect of ketamine was evaluated by intraperitoneal injection.2.Microdissection was used to rapidly extract the medial prefrontal cortex(mPFC)and nucleus accumbens(NAc).3.The phosphorylation levels of the mPFC and the NAc were detected by mass spectrometry(MS)-based label-free quantitative phosphorylation proteomics.4.The Omics Bean data analysis tool was used to analyze the phosphorylated proteomics data about Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)and protein-protein interaction(PPI).5.The KEGG database and literature were used to map the synaptic signaling pathways in which phosphorylated proteins were involved.6.The phosphorylation level of Csnk1a1(T321)was verified by western blot and the expression profile of Prkcg was detected by RNAscope.Results1.CUMS induced depression-like phenotypes in mice,namely weight loss,anhedonia,and behavioral despair.Significant remission of anhedonia was observed 1hour after intraperitoneal administration of ketamine,and significant remission of behavioral despair was observed 24 hours later.2.In the mPFC,209 and 128 differential phosphorylation sites were identified in the CUMS-induced group and ketamine-induced group,respectively.In the NAc,119 and 102 differential phosphorylation sites were identified in the CUMS-induced group and ketamine-induced group,respectively.3.As to signaling pathways,it is revealed that synapse is the common signaling pathways,including Glutamatergic synapse,GABAergic synapse,Cholinergic synapse,and Dopaminergic synapse.4.Between the CUMS-induced group and ketamine-induced group,there were 13 overlapping protein phosphorylation sites in the mPFC,and their phosphorylation levels can be reversed by ketamine.In the NAc,10 overlapping protein phosphorylation sites were reversed by ketamine.5.Western blot confirmed that the phosphorylation level of Csnk1a1(T321)was increased in the CUMS + saline group and reversed after ketamine treatment.6.Prkcg was expressed in both mPFC and NAc,with a stronger expression in the mPFC by RNAscope.Conclusion1.CUMS induces a depression-like phenotype in mice,and ketamine exerts an antidepressant effect.2.The identified phosphorylated proteins,such as PKC,VGCC and et al were involved in synaptic signaling pathways,further supporting the role of synaptic mechanisms in depression and predicting the signaling pathway mechanism of ketamine in the treatment of depression,which may provide the basis for developing new therapeutic strategies.3.CUMS induced changes in protein phosphorylation levels can be reversed after ketamine treatment,providing corresponding target proteins for further elucidating the molecular mechanism of depression and ketamine’s antidepressant effect.