Mechanisms Of Circadian Proteins In Depression And Neuroinflammation

PART I Tyrosine hydroxylase down-regulation after loss of Abelson helper integration site 1(AHI1)promotes depression via the circadian clock pathway in miceAim: The decrease of neurotransmitters in the brain is an important pathological feature of depression patients,while the levels of serotonin(5-HT)and dopamine(DA)in the Ahi1 konck out(KO)mice are significantly reduced.We attempt to explore the signal pathway of AHI1(Abelson helper integration site 1)involving in regulation of neurotransmitters.Methods: Depression-like behaviors were measured by tail suspension test(TST)and forced swim test(FST).m RNA levels of the key enzymes and transporters involved in production or release of DA and 5-HT in mice were measured by quantitative RT-PCR(q RT-PCR).Immunochemistry and immunofluorescence were applied to examine the tyrosine hydroxylase(TH)neuron number and activity in mouse ventral tegmental area(VTA).Western blot and q RT-PCR were performed to examine the expression levels of related genes after knockdown or over-expression of AHI1 in N2 a cells.Dual luciferease assay was used to examine the transcriptional activity.The interaction between AHI1 and RORα was identified by co-immunoprecipition assay and immunofluorescence assay.SR8278 was infused into the mouse midbrain(VMB)through stereotaxic apparatus.Results: Ahi1 KO mice showed a significant increase of immobility as compared to controls in TST and FST.q RT-PCR and Western blot assay showed that TH m RNA and protein levels but not other detected genes were decreased in Ahi1 KO mouse midbrains.Stereological analysis revealed that there is no difference of TH-positive neuron numbers in ventral tegmental area(VTA)between Ahi1 KO mice and the littermate controls using diaminobenzidine(DAB)staining.However,immunofluorescence staining showed that the TH fluorescence intensity was significantly decreased in VTA of Ahi1 KO mice compared with the littermate controls.In Neuro-2a(N2a)cells,knockdown of Ahi1 by two targeted si RNAs significantly decreased both TH m RNA and TH protein levels.Meanwhile,overexpression of exogenous EGFP-AHI1 markedly increased TH protein levels.Overexpression of EGFP-AHI1 in cells dramatically increased TH promoter-driven luciferase reporter activity.TH protein levels are significant lower in Ahi1 KO mice midbrain than littermate controls at both CT00 and CT12.In Ahi1 KO mouse midbrains,REV-ERBα protein and m RNA levels were dramatically increased compared with the littermate controls,however,NURR1 protein levels kept unchanged.Knockdown of Rev-erα increases TH protein levels in cells.We next cloned a mutant TH promoter that lacks REV-ERBα binding site and investigated the effect of AHI1 on its activity.Interestingly,EGFP-AHI1 lost the ability to influence the mutant TH promoter activity.REV-ERBα protein levels are increased in Ahi1 KO mice midbrain compared with littermate controls at both CT00 and CT12.In Bmal1 knockdown cells,increase of TH and decrease of REV-ERBα protein and m RNA levels were observed.BMAL1/CLOCK dramatically repressed TH promoter activity,and BMAL1/CLOCK had no significant effect on the mutant TH promoter lacking REV-ERBα binding site,indicated by reporter gene assays.Increase of REV-ERBα and decrease of TH protein levels induced by Ahi1 knockdown were completely eliminated by BMAL1 deficiency.TH levels were dramatically increased in VTA in Ahi1 KO mice after SR8278 administration.In addition,SR8278 administration significantly improved the performance of Ahil KO mice in TST,but did not significantly affect the control mice.Conclusion: AHI1 regulates TH transcriptional expression through circadian RORα/BMAL1/REV-ERBα pathway,to participate in mood and behavior regulation.Loss of AHI1 leads to an increase of BMAL1/REV-ERBα that negatively regulates TH expression and DA biosynthesis,which contributes to mouse depression behaviors.PART II Pharmacological activation of REV-ERBα represses LPS-induced microglial activation through NF-κB pathwayAim: REV-ERBα is a core circadian clock component,which plays a crucial role in maintaining immune functions,however,its role in neuroinflammation and the molecular mechanism are largely unclear.Here,we demonstrate the anti-neuroinflammatory effects by pharmacological activation of REV-ERBα with its agonist GSK4112.Methods: We used microglial cell line,primary microglia and animal model to investigate the effect of REV-ERBα agonist GSK4112 in vitro and in vivo.BV2 cells,a mouse microglial cell line,were pretreated with GSK4112,followed by treatment of lipopolysaccharide(LPS).Primary microglia were extracted from newborn mouse cortex and prepared.Primary microglia were treated like BV2 cells.GSK4112 and LPS were infused into the mouse midbrain(VMB)through stereotaxic apparatus.MTT assay was performed to detect the cell viability.Western blot and q RT-PCR were performed to examine the expression levels of related genes.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of cytokines in the supernatants of BV2 cells.Immunofluorescence assay was applied to examine the activity of microglia and neuron,and the distribution of p65.Subcellular fractionation assay was used to detect the nuclear translocation of p65.Luciferase reporter assay was performed to detect the transcriptional activity.Results: We found that the enhanced activity of REV-ERBα could diminish LPS-induced microglial activation in vitro and in vivo.In BV2 cells,a pretreatment with GSK4112 significantly blocked LPS-induced inflammatory protein and m RNA expressions,as well as cytokines interleukin 6(IL-6)and tumor necrosis factor α(TNFα)release.A pretreatment with GSK4112 also inhibited LPS-induced inflammatory response in primary microglia.In vivo,microinfusion of GSK4112 into mouse midbrains remarkably diminished LPS-induced microglial activation.GSK4112 inhibited LPS-induced the phosphorylation and nuclear translocation of p65,a nuclear factor κB(NF-κB)subunit,thereby suppressing microglial activation.Moreover,GSK4112-mediated neuroinflammation inhibition protected neuron from microglia-induced cellular damage in vitro and in vivo.Our findings suggest that an enhanced REV-ERBα activity inhibits microglial activation,providing neuroprotection effect.Conclusion: In the present study,we demonstrated that pharmacological activation of REV-ERBα suppresses LPS-induced microglial activation both in vitro and in vivo,which is mediated by NF-κB pathway.In addition,neuroinflammation inhibition by REV-ERBα agonist GSK4112 protects neurons from LPS-mediated damage.