The Protective Mechanism Of GM1 Ganglioside In Parkinson’s Disease: α-Synuclein Autophagic Degradation

Aims One of the main pathological features of Parkinson’s disease(PD)is accumulation of Lewy bodies in dopaminergic neurons.Most of α-synuclein(α-syn)is expressed in brain and spinal cord,and is the main component of Lewy bodies.Aberrant a-syn can affect the release of neurotransmitters and the expression or activity of tyrosine hydroxylase(TH),and further cause a series of toxic damage such as oxidative stress of neurons.Autophagy is a normal physiological process for cells to eliminate abnormal organelles,proteins and other components,and has certainly protective effect on diseases of central nervous system.Therefore,it is a potential method to treat Parkinson’s disease by enhancing autophagy to eliminate aberrant a-syn.Monosialotetrahexosylganglioside(GM1 ganglioside)can be used clinically for the treatment of Parkinson’s disease.Both clinical and animal experiments have proved that GM1 ganglioside can relieve the progression of Parkinson’s disease.Previous studies have shown that GM1 ganglioside can promote the clearance of a-syn,and its mechanism may be related to autophagy.Based on previous studies,we explored the relationship between GM1 ganglioside and autophagy and a-syn clearance,and determined the target protein of GM1 ganglioside in autophagy pathway.Methods (1)The mechanism of GM1 ganglioside promoting the degradation of a-syn.Western blot technique was used to screen the best action concentration and time of GM1 in PC12α-syn A53T inducible cells.The effect of lysosome function on the degradation of a-syn was studied by CQ.The effects of GM1 gangliosides on the expression of α-syn and autophagy related proteins ULK1,p-ULK1Ser555,p62,and LC3 were detected by Western blot in PC 12α-syn A53T inducible cells.Comparing the effect of autophagy inhibitor 3-MA with autophagy activator rapamycin,the relationship between autophagy promoted by GM1 ganglioside and a-syn clearance was determined.Immunofluorescence and laser scanning confocal microscopy were used to investigate the colocalization of GM1,LC3 and a-syn,and to further verify whether that GM1 ganglioside promoted the autophagic degradation of a-syn.(2)The target protein of GM1 ganglioside in autophagy signaling pathway.Using ACTP tool prediction and molecular docking,we predict GM1 potential targets.Q-PCR technology was used to detect the expression of ATG13 mRNA in SH-SY5Y cells after giving GM1 ganglioside.The effects of GM1 ganglioside on the expression of ATG13 were investigated by immunofluorescence and laser scanning confocal microscopy.The effects of GM1 ganglioside on the interaction between ATG13 and ULK1 were detected by immunoprecipitation and Western blot,to determine whether GM1 could promote the formation of ULK1 complex by activating ATG13.Finally,The colocalization of GM1 ganglioside and ATG13 was investigated by immunofluorescence and laser scanning confocal microscopy,and the targeting effect of GM1 ganglioside on ATG13 was further determined.Results (1)The induction of Doxycycline in PC12α-SynA53T inducible cells for 12 hours,and then 40 μM GM1 for 24 hours can significantly reduce the expression of α-syn,increase the expression of TH,and increase the ratio of LC3 II and LC3 I,so selecting 40 μM for 24 hours of administration.CQ can inhibit the degradation of a-syn by GM1,indicating that the effect of GM1 is related to lysosome function.PC 12α-syn A53T inducible cells were induced by doxycycline,and the expression of a-syn was significantly up-regulated(P<0.01).The α-syn could be significantly declined(P<0.01)after the GM1 ganglioside treatment,but autophagy inhibitor 3-MA could significantly reversed the effect of GM1 treament(P<0.01).GM1 ganglioside affected the expression of autophagy-related proteins.Compared with the DOX group,GM1 ganglioside can increase the ratio of LC3 Ⅱ and LC3 I(P<0.01),reduce the autophagy cargo protein p62(P<0.01),increase the expression of ULK1(P<0.01)and the expression of the p-ULKlSer555 protein(P<0.01).The autophagy inhibitor 3-MA can reverse the expression of the above protein on the condition of GM1 ganglioside treament.immunofluorescence and laser scanning confocal microscopy were used to observe the PC 12α-syn A53T inducible cells induced by doxycycline and GM1 ganglioside treatment,LC3 and a-syn were obviously colocated,indicating that GM1 ganglioside promote the autophagosome engulfing a-syn.GM1 ganglioside with FITC fluorescent labeling conbined by chemical reaction and laser confocal 3D imaging function can be used to determine the specific distribution of GM1-FTTC.The result revealed that GM1-FITC can be used to trace the GM1 ganglioside in the cell.Utiliaing GM1-FITC,the spatial relationship between GM1 ganglioside and a-syn or LC3 in cells was further investigated by immunofluorescence technique.The results showed that there was obvious colocalization between GM1-FITC and a-syn or LC3.The above results indicate that the clearance effect of GM1 ganglioside on a-syn is related to its promotion of autophagy.GM1 ganglioside may be directly involved in the autophagosome engulfing α-syn.(2)Molecular docking results showed that ATG13 may be a GM1 target protein in autophagy signaling pathway.After given GM1 ganglioside in SH-SY5Y cells,the expression of ATG13 mRNA were increased,but there was no significant difference(P>0.05).The expression of ATG13 was up-regulated and the interaction between ATG13 and ULK1 was enhanced after treatment by GM1 ganglioside.The activation of GM1 ganglioside to ATG13 eventually promoted the formation of ULK1 complex.The results of immunofluorescence showed that GM1 ganglioside increase the expression and aggregation of ATG13.Laser confocal scanning showed that GM1 ganglioside had obvious colocalization with ATG13,indicating that there was interaction between GM1 and ATG13.The above results indicate that GM1 can increase the expression of ATG13,increase its binding to ULK1 protein,and ultimately activate the downstream autophagy pathway.Conclusion (1)GM1 ganglioside can eliminate abnormal a-syn,promote autophagy,enhance and participate the autophagosome engulfing a-syn.(2)GM1 ganglioside targets ATG13,and enhances its binding with ULK1,thereby promoting the autophagic degradation of α-syn.