Study Of The Inhibitory Effect Of Insulin-like Growth Factor-1 On MPTP/MPP~+-Induced Autophagy In Dopaminergic Neurons

A mounting body of evidence indicated that autophagy was one of the pathogenesis of Parkinson’s disease(PD).Autophagosome accumulated in the brain tissues of PD patients after postmortem.Excessive activation of autophagy resulted in the death of dopaminergic neurons.Therefore,regulation the balance of the cell autophagy function may provide a new target for the treatment of PD.Insulin growth factor-1(IGF-1)is a polypeptide hormone which is synthesized mainly in the liver and can be transported across the blood-brain barrier.Circulating IGF-1 level progressively declines with age.The age-related decrease of IGF-1 appears to be linked to the pathogenesis of neurodegenerative diseases such as PD and Alzheimer’s disease(AD).IGF-1 could protect dopaminergic neuron through anti-apoptosis and antioxidant stress both in vivo and in vitro.G protein-coupled estrogen receptor(GPER)is a seven-transmembrane G protein-coupled receptor which locates on the cell membrane and plasma membrane.There was functional interaction between IGF-1/IGF-1R and GPER in regulating the growth of cancer cells in mesothelioma and lung cancer.Therefore,in the nigrostriatal system,we speculate that IGF-1 might protect dopaminergic neurons through regulating the cell autophagy and the mechanism might be related to IGF-1R and GPER mediated signaling pathways.Objective:1.To investigate whether IGF-1 could protect dopaminergic neurons through regulating the dysregulation of autophagy;2.To investigated the target and molecular mechanism of IGF-1 in inhibiting excessive autophagy.Methods:PD mice model was established by intraperitoneal injection of MPTP and PD cellular model was prepared by MPP~+treatment.IGF-1,IGF-1R antagonist JB-1 or GPER antagonist G15 were injected into the lateral ventricle through stereotaxic technique.Rotarod test,pole test and open filed test were used to detect the behavior of mice.In order to explore molecular mechanism of the neuroprotective effect of IGF-1 through regulating autophagy,the content of dopamine(DA)were measured by high performance liquid chromatography(HPLC).Cell viability was detected by MTT method.Western blot was used to detect the markers of autophagy.Results:1.Mechanism study of the neuroprotective effects of IGF-1 against MPTP-induced dopaminergic neuron damage(1)The results of rotarod test and open field test showed that MPTP significantly inhibited the movement of mice(P<0.001,P<0.05).IGF-1 significantly improved dyskinesia and significantly increased the movement distance of mice(P<0.01),which could be blocked by JB-1 or G15(P<0.05,P<0.01).The results of pole test showed that IGF-1 significantly shortened the total time to descend the pole(t-turn)and the time to orient down(t-total)of mice(P<0.01,P<0.05).These effects could be blocked by JB-1(P<0.05,P<0.01).While G15 treatment could only partly but not significantly block these effects.(2)HPLC results showed that IGF-1 obviously improved MPTP-induced decrease of DA content in striatum compared with MPTP group(P<0.05).This effect could be blocked by JB-1 and G15(P<0.05).(3)The results of autophagy-associated protein LC3-Ⅱand P62 showed that IGF-1 could inhibit MPTP-induced up-regulation of LC3-Ⅱand down-regulation of P62(P<0.01,P<0.001),which could be blocked by JB-1 and G15(P<0.01,P<0.05).(4)IGF-1 could counteract MPTP-induced decrease of TH protein expressions and the increase ofα-synuclein protein expression(P<0.001,P<0.05).These effects could be blocked by JB-1 and G15(P<0.01,P<0.05).2.Mechanism study of IGF-1 against MPP~+-induced excessive autophagy in SH-SY5Y cells(1)The results of MTT showed that IGF-1 significantly resisted MPP~+neurotoxicity and increased cell viability(P<0.05),which could be blocked by JB-1 and G15(P<0.01).(2)IGF-1 could inhibit MPP~+-induced up-regulation of LC3-Ⅱ(P<0.05)and down-regulation of P62(P<0.05).JB-1 and G15 both blocked these effects of IGF-1(P<0.05,P<0.001,P<0.01).(3)Fluorescence microscopy results showed that the expression of LC3 increased in MPP~+injury group,IGF-1 could inhibit the increase of LC3,the expressions of LC3 increased both in JB-1 and G15 group comparing with IGF-1+MPP~+group..(4)Western blot analysis of TH andα-synuclein protein expressions showed that IGF-1could reverse MPP~+-induced decrease of TH protein expression and the increase ofα-synuclein protein expression compared with MPP~+group(P<0.01,P<0.05).These effects could be blocked by JB-1 and G15(P<0.01,P<0.05).(5)MPP~+significantly inhibited the phosphorylation levels of Akt and mTOR(P<0.01).IGF-1 could reverse these effects of MPP~+(P<0.05,P<0.01)which could be antagonized by JB-1 and G15(P<0.01,P<0.05).Conclusion:IGF-1 inhibits MPTP/MPP~+-induced neuronal excessive autophagy and protects DA neurons.GPER is involved in the neuroprotective effect of IGF-1.The potential mechanism may be related to the IGF-1R/PI3k/Akt-m TOR signaling pathway.