7.8-dihydroxyflavone Ameliorates Motor Deficits Via Regulating Autophagy In MPTP-induced Mouse Model Of Parkinson’s Disease

Objective:Parkinson’s disease(PD)is a neurodegenerative disease characterized by the formation of Lewy bodies in the brain and loss of dopaminergic neurons in the substantia nigra and diminished dopamine content in the striatum.Recent reports show that 7.8-dihydroxyflavone(DHF),a Trk B agonist,attenuates theα-synuclein deposition and ameliorates motor deficits in PD model mice.However,the underlying mechanism is unclear.Reports show that misfolding and aggregation ofα-synuclein plays a central role in the pathogenesis of PD and autophagy is confirmed to be the major way to degradeα-synuclein aggregations.In this study,we investigated whether autophagy is involved in the clearance ofα-synuclein and the signaling pathway through which DHF exerts therapeutic effects.Methods:(1)Establishment of mice model of Parkinson’s disease:1.5-month-old C57BL/6 male mice were intraperitoneally injected with MPTP at a dose of 30mg/kg/d,once a day for 5 days.(2)1.5-month-old C57BL/6male mice were randomly divided into 5 groups:control group,MPTP group,MPTP+DHF group,MPTP+CQ group,and MPTP+DHF+CQ group.DHF(5 mg/kg,i.p.)and chloroquine(50 mg/kg,i.p.)were injected once a day during the whole process of experiments.Three behavioral tests including rotarod test,pole test and wire suspension test were conducted after drug administration,and the brain tissues were removed for immunohistochemical staining and western blot analysis after behavioral tests.TH(tyrosine hydroxylase)immunostaining was used to determine the number of dopaminergic neurons in the substantia nigra and striatum.the expression of TH,α-synuclein,p-Trk B and t-Trk B in striatum and midbrain of mice in different groups were detected by Western blot.(3)Establishment of cell model of Parkinson’s disease:N2A cells were treated with MPP~+at concentration of 500u M for 24h.(4)N2A cells were divided into control group,MPP~+group and MPP~++DHF group.cells were treated with DHF at concentration of 25u M for 24h.Immunofluorescence assay and western blot were conducted to detect whether DHF induced autophagy in N2A cells after treatment.(5)N2A cells were divided into control group,MPP~+group,MPP~++DHF group,MPP~++CQ group,MPP~++DHF+CQ group,MPP~++DHF+CQ+3-MA group,in which cells were treated with CQ at 50u M for 4h and 3-MA at 30m M for 24h.LC3 and P62 were detected by western blot to determine the effect of DHF on autophagy flux after treatment.(6)Trk B antagonist ANA-12 was used to determine whether the DHF-induced autophagy was dependent on Trk B activation.(7)U0126 and U73122 were used in N2A cells to determine the signaling pathway involved in DHF-induced autophagy.Results:(1)In rotarod test,mice in MPTP treated group spent significantly less time on the rod compared with controls(CTR),and DHF treatment(5 mg/kg,i.p.)prolonged the latency on the rod.However,the beneficial effect of DHF was diminished by autophagy inhibitor chloroquine(CQ)treatment.(2)In pole test,the mice in MPTP group spent much more time on descending to the floor compared with CTR,and DHF treatment significantly shortened the descending time.Similar to the findings in rotarod test,chloroquine markedly increased the descending time.(3)In wire suspension test,DHF treatment shortened the time to reach the platform in MPTP treated mice,while chloroquine treatment made it back to MPTP group level.(4)MPTP administration significantly increasedα-synuclein accumulation and decreased TH expression in both the striatum and midbrain,DHF treatment restoredα-synuclein accumulation and TH expression to control level,the effects of DHF on increasing TH expression and reducingα-synuclein accumulation were prevented by chloroquine treatment.(5)The number of TH-positive neurons in the substantial nigra(SN)was significantly decreased after MPTP treatment.The administration of DHF markedly blocked the loss of TH-positive neurons,while its protective effect was completely prevented by chloroquine treatment.Similarly,DHF treatment increased TH-positive terminals in the striatum of MPTP treated mice,while chloroquine blocked these changes.(6)Immunofluorescence experiments showed that LC3 puncta was significantly increased following DHF treatment in MPP~+treated cells.Western blot analysis showed that the LC3II and beclin-1 were decreased,and the expression of P62 was increased in MPP~+group,while DHF treatment restored the expression of P62,LC3II and beclin-1 to control levels.(7)Chloroquine treatment increased LC3II accumulation and the magnitude of LC3II accumulation was more significant in MPP~++DHF group than in MPP~+group,besides,the LC3II accumulation was inhibited by 3-MA.(8)ANA-12 inhibited the autophagy induced by DHF.(9)U0126 mimicked DHF induced autophagy in MPP~+treated N2A cells.(10)U73122 has no effect on DHF induced autophagy.Conclusions:(1)Autophagy inhibitor chloroquine abrogates the protective effects of DHF on motor deficits in MPTP induced mouse model of PD.(2)Autophagy inhibitor chloroquine prevents the effects of DHF on the restoration of TH and the decrease ofα-synuclein accumulation.(3)Autophagy inhibitor chloroquine prevents the neuroprotective effects of DHF on dopaminergic neurons.(4)DHF restores impaired autophagy in cell model of PD.(5)DHF induced autophagy is Trk B dependent in PD cell model.(6)DHF promotes autophagy through ERK-LKB1-AMPK pathway.