The Effect And Mechanism Of MANF In Depression

PART Ⅰ: THE CORRELATION OF MANF EXPRESSION WITH MDD PATIENTSObjective: To detect the expression levels of serum MANF in Drug-naive MDD(DN-MDD)patients and Drug-treatment MDD(DT-MDD)patients,and to explore the clinical value of serum MANF levels as a diagnostic marker of MDD.Methods: Peripheral blood serum were collected from DN-MDD and DTMDD inpatients and healthy control(HC)group.ELISA was used to detect the serum MANF levels,and the differences of MANF expression among the MDD and HC were compared.The correlation between serum MANF levels and MDD was statistically analyzed.Results:(1)Compared with the HC group,the expressions of serum MANF were significantly reduced in MDD group.But the levels of MANF in DTMDD group were significantly higher than DN-MDD group.(2)Serum MANF levels were negative correlated with HAMD scores in DN-MDD group(r=-0.414,P=0.007)by Pearson correlation analysis.(3)The ROC curve demonstrated that the area under the curve(AUC)of serum MANF protein in DN-MDD group was 0.955,and the AUC of DT-MDD group was 0.782.Conclusions: The levels of serum MANF protein are associated with MDD.Serum MANF expressions decrease in DN-MDD group,and DT-MDD group is higher than DN-MDD group,indicating that the serum MANF level would be increased after antidepressant treatment.The levels of serum MANF might be a potential biomarker for the diagnosis of DT-MDD patients.PART Ⅱ: THE EFFECT AND MECHANISM OF MANF IN DEPRESSION-LIKE BEHAVIORS IN MICEObjective: To detect the MANF expression in mice models of depression.To investigate the effect and further explore the underlying mechanism of MANF in depression.Methods:(1)The depression mice models induced by CSDS and CRS were established.Western Blot and RT-q PCR were used to determine the both levels of MANF protein and m RNA in hippocampus and PFC,and the ERSrelated proteins levels were also detected by Western Blot.(2)The mice were exposed to CSDS or CRS to induce depression-like behavior after stereotactic injection of AAV-Manf into the hippocampus,and the effects of overexpression of MANF on depression-like behavior were observed.(3)MANF interference virus AAV-sh Manf was injected stereotaxically into the hippocampus to observe whether knockdown of MANF expression affected the behaviors of mice;Subthreshold social defeat stress(SSDs)was added to further investigate the effect on the depression-like behaviors in mice.(4)Western blot was used to tested the levels of ERS and inflammation response-related proteins in the hippocampus,and RT-q PCR was used to determine the levels of inflammatory cytokines m RNA.Results:(1)The levels of MANF protein and m RNA in the hippocampus of CSDS susceptive mice and CRS mice were increased by Western Blot and RT-q PCR,and further detection showed that MANF was also increased in PFC.Meanwhile,the levels of ERS-marker proteins GRP78 and CHOP in the hippocampus and PFC were significantly increased.(2)AAV-Manf injection into hippocampus induced overexpression of MANF.CSDS and CRS treatment decreased mice’ sucrose preference rate,spontaneous activity and exploration behaviors of OFT,and increased the immobility time of TST and FST.The deficit was significantly ameliorated by overexpression of MANF.(3)MANF overexpression significantly inhibited the levels of CSDS and CRS-induced GRRP78 and GRP94 increase in mice hippocampal,and decreased the activation of PERK/ATF4/CHOP signaling pathway,as well as TLR4/My D88/NF-κB pathway–related proteins and inflammatory cytokines(IL-1β,IL-6 and TNF-α).(4)AAV silencing hippocampal MANF decreased MANF expression level,and depression-like behavioral tests showed that knockdown of MANF significantly increased the immobility time of TST,but there was not significant difference in other behavioral tests.Further knockdown of MANF combining with SSDS subthreshold stress enhanced the depression susceptibility of mice,and induced depression-like behaviors.(5)Knockdown of MANF expression combining with SSDS increased the protein levels of MANF,GRP78,GRP94 and PERK/ATF4/CHOP signaling pathways,induced the expression of TLR4/My D88/NF-κB pathway-related proteins,and also increased the levels of pro-inflammatory cytokines m RNA in hippocampus.Conclusion: MANF and ERS are associated with depression.Overexpression of hippocampal MANF can improve depression-like behavior,inhibit ERS and inflammatory response in depression mice.In contrast,knockdown of hippocampal MANF expression increases stress susceptibility and induce depression-like behavior in mice,resulting in ERS and inflammatory responses.It is suggested that MANF may affect the occurrence and development of depression by regulating ERS and inflammatory response.PART Ⅲ: THE MECHANISM OF MANF IN MICROGLIA TREATED WITH LPS AND TM IN VITROObjective: To establish ERS and inflammation of microglia models in vitro,and to investigate the mechanism of MANF in microglia cells through ERS and inflammation.Methods:(1)BV2 cells inflammatory model was induced by LPS and the ERS model of BV2 microglial was induced by a chemical ER stressor,tunicamycin(TM).(2)Recombinant MANF protein(rh MANF)was used to enhance the expression of MANF,and then the protective effect of rh MANF on both LPS and TM induced BV2 cell injury was observed.(3)Lentivirus transfection was used to interfere the MANF expression,and the effects of knockdown of MANF expression on ERS and inflammatory response of BV2 microglia were observed.(4)Western blot was used to tested the levels of ERS and inflammation response-related proteins in the hippocampus,and RT-q PCR was used to determine the levels of pro-inflammatory cytokines m RNA.Results:(1)In vitro,LPS and TM treatment induced increased expression of MANF protein in BV2 microglia,and also increased expression of ERS marker proteins GRP78 and CHOP.(2)Recombinant MANF significantly restored the expression levels of ERS-activating proteins GRRP78 and GRP94 in BV2 cells treated with LPS and TM,and inhibited the activation of the PERK/ATF4/CHOP signaling pathway.(3)rh MANF obviously reduced the levels of TLR4/ My D88/NF-κB pathway-related proteins,and pro-inflammatory cytokines in BV2 microglia treated with LPS and TM.(4)RT-q PCR analysis showed that lentiviral MANF interference virus LVsh Manf significantly reduced the expression of MANF gene in BV2 cells.(5)In vitro,knockdown of MANF increased the protein levels of GRP78,GRP94 and the PERK/ATF4/CHOP signaling pathway,and induced the expression of the TLR4/My D88/NF-κB pathway related proteins in BV2 cells.Meanwhile,knockdown of MANF increased the m RNA levels of inflammatory cytokines(IL-1β,IL-6 and TNF-α)in BV2 cells.CONCLUSIONS: Recombinant MANF can improve ERS and inflammatory response induced by LPS and TM in BV2 microglia,while silencing MANF expression can induce ERS and inflammatory response,which sugges that MANF affects microglia activation by regulating ERS and inflammatory response.