Effects Of Hippocampal Protein Succinylation On Energy Metabolism And Neuronal Plasticity In Mouse Model Of Depression

BackgroundMajor depressive disorder(MDD)is a mood disorder characterized by low mood,slow thinking and decreased exercise.Its pathogenesis is complex.Recent studies have found that in depressive patients,in the central nervous system or periphery,there is abnormal energy metabolism.Mitochondria are the places where glucose,lipoid and protein are ultimately oxidized to release energy.Studies have shown that mitochondrial dysfunction in MDD can lead to changes in neurotransmitter production,synaptic plasticity and nerve regeneration,even cause the loss of neurons,which are closely related to MDD.Succinylation is a newly discovered post-translational modification of proteins,mainly occurring in mitochondria,which is involved in the regulation of key enzyme activities in a variety of cellular metabolic processes,such as glycolysis,TCA,and scavenging of reactive oxygen species.It has been found that protein succinylation is involved in the pathological process of various nervous system diseases such as Alzheimer’s disease,Parkinson’s disease and cerebral ischemia.However,whether succinylation is associated with depression has not been reported.Pyruvate dehydrogenase complex(PDHC)is an important rate-limiting enzyme in mitochondrial energy metabolism.It catalyzes the conversion of pyruvate into acetyl coenzyme A(ACA)in mitochondria,maintaining the dynamic balance between glycolysis and the tricarboxylic acid cycle.Regulation of glucose metabolism and the production of ATP.Since almost all of the ACA in the brain is derived from pyruvate,the loss of PDHC function often leads to the dysfunction of mitochondrial energy metabolism in the brain and the damage of the nervous system.However,it is not clear whether the PDHC is modified by succinylation and the enzyme activity is changed in MDD.ObjectiveTo elucidate the effects of succinylation modification on brain energy metabolism and neuronal plasticity in depression,and provide a new perspective for the pathogenesis and treatment of depression.MethodsChronic unpredictable mild stress(CUMS)depression model was established with C57BL/6J mice.During this process,the body weight of the mice was weighed once a week.After 8 weeks of the model was established,the behavioral tests of the open field,elevated plus maze,forced swimming and tail suspension test were performed.The levels of ATP and ACA in hippocampus and mitochondria and lactic acid(LA)in hippocampus were detected by enzymatic linked immunosorbent assay(ELISA).The succinylation level of hippocampal protein and PDHC was analyzed by protein modification quantitative omics.PDH activity was detected by enzyme activity detection kit.Immunohistochemical staining(IHC)and Western blot were used to detect the expression of postsynaptic dense protein 95(PSD95)and synaptophysin(SYP)in the hippocampus and prefrontal cortex.In vitro,the expression of dessuccinylase SIRT5 in human neuroblastoma cell line SH-SY5 Y was knocked down by lentiviral vector.Purinomycin was used for screening and retinoic acid(RA)was used to induce differentiation.The succinylation levels of total protein and mitochondrial protein were detected by Western blot.The effect of succinylation modification on mitochondrial energy metabolism was detected by ELISA.Cell protein and mitochondrial protein PDHC were enriched by immunoprecipitation(IP),and the succinylation level of PDHC was detected by Western blot.The changes of PDH activity were detected by enzyme activity kit.Immunofluorescence(IF)and Western blot were used to detect the changes of neuronal plasticity.Results1.Established a mouse model of depressionCompared with control group,the body weight of CUMS model mice showed a decreasing trend.In the OFT,the total travel distance and the travel time spent in the central area decreased significantly.In the EPM test,the time spent in the open arm was significantly reduced.In the TST and FST,the immobility time of mice was significantly up-regulated.The results of weight change and behavior tests indicated that the animal model of depression was established successfully.2.The level of succinylation was upregulated in the hippocampus of CUMS model mice(1)The results of quantitative omics analysis of succinylated modification showed that compared with the Control group,the modification level of 184 sites in the CUMS group was up-regulated,and 49 sites was down-regulated(1.2 times as the change threshold P<0.05).(2)Bioinformatics analysis showed that most of the proteins up-regulated by succinylation were related to mitochondrial energy metabolism.3.Changes of energy metabolism in the brain of CUMS model miceELISA results showed that the production of ATP and ACA in the hippocampus and its mitochondria was significantly reduced,while the production of LA in the hippocampus was increased.4.The succinylation level of PDHC in the hippocampus of CUMS model mice was up-regulated,and PDH activity was decreased(1)Bioinformatics analysis showed that the levels of succinylated PDHC(including E1/PDH,E2 and E3 subunits)in hippocampal tissues of depressed mice increased.(2)The results of enzyme activity test showed that the PDH activity in the hippocampal mitochondria of the depressed model mice was significantly reduced.5.Changes of neuronal plasticity in the brain of CUMS model miceImmunohistochemical staining and Western blot analysis showed that the expressions of PSD95 and SYP in prefrontal cortex and hippocampus were significantly decreased in the depressed group.6.Lentivirus transfected SH-SY5 Y cell line and knocked down the expression of SIRT5(1)After the lentiviral vector was transfected into the cells,the qRT-PCR results showed that the expression level of SIRT5 m RNA in the cells was significantly reduced.Western blot results showed that the expression of SIRT5 protein was decreased after lentivirus transfection,indicating that lentivirus transfection was effective.(2)After RA induced cell differentiation,the cells were found to be fusiform and polygonal with elongated protrusion and interconnection.The expression level of Neu N in cells was significantly increased.7.The effect of knock down SIRT5 on succinylation levelTotal cell proteins and mitochondrial proteins were extracted,and succinylated pan-antibody was used to detect the results by Western blot.The results showed that the succinylated levels of both cell and mitochondrial proteins were significantly increased after knocking down SIRT5.8.Influence of SIRT5 knockdown on energy metabolism of neuronsELISA results showed that the expressions of ATP and ACA in SIRT5 cells and mitochondria were significantly decreased after knockdown.9.The effect of SIRT5 knockdown on PDHC succinylation level and PDH activity(1)PDHC was enriched by immunoprecipitation,and the results showed that the succinylation level of PDHC in cells and mitochondria was significantly up-regulated after SIRT5 knockdown.(2)The results of enzyme activity test showed that mitochondrial PDH activity decreased significantly after SIRT5 knockdown.10.Influence of SIRT5 knockdown on neuroplasticityImmunofluorescence and Western blot results showed that the expression of PSD95 and SYP in SIRT5 knockdown cells decreased significantly.ConclusionsThe elevated level of succinylation in the hippocampus of depression model mice may lead to the decreased activities of key enzymes such as PDHC,affect the aerobic metabolism of glucose and the generation of ATP in the brain,and lead to the down-regulation of mitochondrial energy metabolism and the decrease of neuronal plasticity,which may be related to the occurrence of depression.