Molecular Mechanism Of DRD1 And NMDA Receptor Regulation Of DARPP-32 Phosphorylation Into Nuclear Chromatin Remodeling In Mental Behavioral Disorders Caused By Ketamine Abuse

Objective:Ketamine is a derivative of phenylcyclohexylpiperidine,which is used as a surgical anesthetic and a rapid antidepressant in clinics.Because of its strong addiction,it is easy to cause abuse under non-medical conditions.As a non-competitive channel blocker of NMDA receptor,the anesthetic and analgesic properties of ketamine are usually attributed to the direct inhibition of NMDA receptor.However,some studies have shown that ketamine can exert its life function by upregulating the expression of DRD1.As an intracellular regulatory factor,DARPP-32 phosphorylation is regulated by a variety of neurotransmitters and is an important intracellular signal integration molecule..DRD1 can activate c AMP/PKA/DARPP-32 for signal transduction,and DARPP-32 can also be regulated by NMDA receptor/Ca²⁺.The change of DARPP-32 phosphorylation state will regulate the phosphorylation level of histone H3 through PP1,and then regulate the modification of transcription.Therefore,in this study,we intend to establish a mouse model of ketamine abuse to study whether DRD1 and NMDA receptors regulate DARPP-32 phosphorylation/dephosphorylation and whether they are involved in mental disorders caused by ketamine abuse.Methods:To establish four groups of mouse models:normal saline control group,ketamine abuse group,DRD1 agonist group and DRD1 antagonist treatment group,and observe the behavior of mice in open field,water maze,Barnes maze and forced swimming test.The distribution and expression levels of NMDAR1,DRD1,PKA,PP2B,p-Thr34DARPP-32,DARPP-32 and PP1 in the prefrontal cortex and hippocampus of the four groups of model mice were detected by Western blot and immunohistochemical fluorescence techniques.The level of m RNA transcriptome in the hippocampus of each group was detected by RNA-seq,and the difference of chromatin opening in each group was detected by ATAC-seq.HT22 cells treated with gradient ketamine were established,and the expression of DRD1,PKA,PP2B,p-Thr34 DARPP-32,DARPP-32,p-Thr75DARPP-32,p-Ser97 DARPP-32,PP1,Histone-H3 and p-Ser10 Histone-H3 in HT22 cells was detected by Western blot technique,and the interaction of DARPP-32 protein in HT22cells was determined by immunoprecipitation.Results:Behavioral results showed that ketamine abuse could increase spontaneous activity,impair spatial memory ability and abnormal depression-like mental behavior in mice.Injection of DRD1 agonist alone could produce similar results to ketamine.At the same time,DRD1 antagonist could alleviate or reverse the behavioral changes induced by ketamine.Immunofluorescence results showed that DRD1 and NMDA receptors were co-located in the prefrontal cortex and hippocampus of mice.The results of Western blot experiment showed that ketamine and DRD1 agonists could increase the expression of DRD1,PKA,PP2A,DARPP-32 and p-Thr34 DARPP-32 in prefrontal cortex and hippocampus of mice,DRD1 antagonists could reverse the increase of DRD1 expression induced by ketamine,ketamine could induce the increase of NMDA receptor expression,but DRD1 agonists and antagonists had no effect on NMDA receptor expression.Sequencing of m RNA transcriptional group and enrichment of KEGG and GO pathway in mouse hippocampal tissue showed that ketamine upregulated the expression of DRD1/c AMP/PKA pathway and DARPP-32 in mouse hippocampus.ATAC-seq sequencing of hippocampal tissue of mice showed that the chromatin opening of ketamine group was different from that of control group.In HT22 cells of mouse hippocampal neurons treated with 0-1000μM ketamine for24 hours,Western blot results showed that ketamine increased the expression of DRD1,PKA and p-Thr34 DARPP-32 in a dose-dependent manner,p-Thr75 DARPP-32 decreased in a dose-dependent manner,and p-Ser97 DARPP-32 had no obvious change trend.After the nuclear and cytoplasmic separation of HT22 cells treated with 100μM ketamine,it was found that p-Thr34 DARPP-32 phosphorylation increased and accumulated into the nucleus.Protein interaction between DARPP-32 and PP1 was found in co-immunoprecipitation assay.Conclusion:1.The increased expression of DRD1 and NMDA receptors is involved in mental and behavioral disorders caused by ketamine abuse.2.DRD1/PKA and NMDA receptor/PP2B signal pathway jointly regulate the changes of DARPP-32 phosphorylation and nuclear cytoplasmic distribution.3.The increased phosphorylation of threonine residue at the 34th position of DARPP-32 and its aggregation into the nucleus affect the transcription by affecting the activity of PP1 and increasing the phosphorylation of histone H3.