The Role Of Circadian Clock Gene Bmal1 In A MPTP-Induced Mouse Model Of Parkinson’s Disease

Objective: Bmal1 is one of the most important central clock genes of circadian rhythm.Circadian rhythm dysfunction is one of the non-motor symptoms in Parkinson’s disease(PD).However,the role of circadian rhythm disorders in Parkinson’s disease is poorly understood.This study aims to investigate whether the deletion of Bmal1 aggravates microglial-mediated neuroinflammation to accelerate the dopaminergic cell death in Parkinson’s disease.Methods:In vitro,inflammation factors(IL-1β,TNF-α,IL-6,i NOS)was detected in BV2 cells transfected with Bmal1 si RNA after treated with LPS or MPP+ by real-time RT-PCR.Dopaminergic neurons were treated different supernatant of microglia and BV2 cell by Immunofluorescence.The expression of related proteins in the signal pathway of NFκB was measured by western blotting。In vivo,6-month-old Bmal1 knockout male mice(Bmal1-/-)were injected 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)or saline.Rotarod test and openfield test were used to evaluate motor function.Metabolic cage monitoring system was used to detect changes circadian rhythms in mice.Western blotting was used to detect the TH protein level of striatum.The inflammatory factors IL-1β and TNF-α were detected by real-time RT-PCR.HPLC was used to determine dopamine(DA)and DOPAC levels in striatum.Immunohistochemistry was used to examine dopaminergic neurons,microglia cell,and astrocyte in substantia nigra compacta(SNc).Results:In vitro,compared to control group,BV2 cells transfected with Bmal1 si RNA showed obvious inflammatory reaction,accompanied by the increase of IL-1β and TNF-α(P<0.01).The neurites of dopaminergic neurons were shorten with treated LPS or MPP+,however,dopaminergic neurons of transfected with Bmal1 si RNA reduced further compared to control group(P<0.05).Compared to control group,P-IKK was increased with LPS treatment for 15min(P<0.05).Also,P-IκBα and P-P65 were increased with LPS treatment for 30 min(P<0.01).In vivo,the time on the rod was significantly reduced in MPTP-treated Bmal1-/-mice in rotarod test(P<0.05).The activitys in the three dimensions of X,Y,and Z were increased(P<0.05),while food intake,water intake,and calorie consumption decreased,suggesting that the loss of the Bmal1 caused circadian rhythm disturbance.Compared to the MPTP-treated Bmal1+/+ mice,the number of DA neurons in striatum and SNc significantly decreased(P<0.05).DA and DOPAC concentration also reduced in striatum(P<0.05)in MPTP-treated Bmal1-/-mice.Conversely,significantly increased m RNA levels of IL-1β and TNF-α in striatum(P<0.05),and activated microglial and astrocyte in SNc were observed in MPTP-treated Bmal1-/-mice.It suggested that the loss of Bmal1 gene aggravated the inflammatory response and may be involved in the occurrence of PD.Conclusion: Neuroinflammation arises from the absence of Bmal1,exacerbating the MPTP-induced dopaminergic cell death in the progression of Parkinson’s disease.