The Effects Of Ykt6 On Autophagy-lysosome Pathway In Experimental Models Of Parkinson’s Disease

Objective:Soluble N-ethylmaleimide-sensitive factor attachment protein receptors(SNARE)are associated with Parkinson’s disease(PD).This study aimed to investigate the alterations of SNARE protein Ykt6 both in vivo and in vitro PD models.The role of Ykt6 on the autophagy-lysosome pathway in experimental model of PD was investigated to explore the possible mechanism.Methods:(1)SH-SY5Y cells were treated with different concentrations of MPP+(0.25-2 mM)or rotenone(100-800 nM)for 48 h to establish a cell model of PD.The cell viability was detected by the MTT assay.The protein expression of Ykt6 in SH-SY5Y cells was detected via Western blotting and immunofluorescence,then the mRNA level of Ykt6 in SH-SY5Y cells was determined by RT-PCR.(2)C57BL/6J mice were treated with an intraperitoneal injection of MPTP or rotenone and overexpressed A53T to establish a model of PD in vivo.The locomotion ability of the mice was evaluated by rotarod test,balance beam test,and pole test.The mice were sacrificed,and the striatum and substantia nigra was isolated.Western blotting was used to measure the expression level of Ykt6 and tyrosine hydroxylase(TH).(3)SH-SY5Y cells were transfected with Ykt6 siRNA using GP-transfect-Mate,and the knock efficiency was verified by the Western blots.Cell viability was detected by the MTT assay,cell membrane integrity was detected by the lactate dehydrogenase(LDH)assay,the flow cytometry was used to analyze the apoptosis rate,and expression of apoptosis-related protein cleaved-caspase3 was determined by Western blotting.RT-PCR and Western blotting were used to investigate the transcription and protein levels of α-synuclein(a-syn).The protein changes of the lysosomal membrane-associated protein1(Lampl)were detected by Western blotting,and the lysosomes were stained by Lyso-Tracker Red(LYT)to assess the lysosomal damage.(4)The expression of Lampl in models of PD was measured by Western blotting and immunofluorescence.SH-SY5Y cells were transfected with Ykt6 siRNA using GP-transfect-Mate,then exposed to MPP+,Western blotting was used to investigate the levels of a-syn,Lampl,cathepsin D(CTSD),microtubule-associated protein light chain 3(LC3),and p62.LYT and acridine orange(AO)were used to stain the lysosomes and the acidic vesicles,respectively.Nuclear protein was extracted to detect the expression of nuclear transcription factor EB(TFEB).SH-SY5Y cells were transfected with Ykt6 plasmid,the protein expression of LC3,Lampl,α-syn,and TFEB were detected.Results:(1)MPP+and rotenone could significantly reduce the protein and mRNA levels of Ykt6.The protein expression of Ykt6 was decreased in MPTP,rotenone and overexpressed A53T animal models.(2)Knocking down Ykt6 decreased SH-SY5Y cell viability,increased LDH release level and apoptosis rate,and increased the protein expression of cleaved-caspase3.Knockdown of Ykt6 had no effect on the transcription level of a-syn in SH-SY5Y cells,whereas Ykt6 deficiency promoted the accumulation ofα-syn protein.Ykt6 knockdown also reduced the expression of Lamp 1 and decreased lysosomal fluorescence.(3)In SH-SY5Y cells,silencing Ykt6 followed by exposure to MPP+increased the accumulation of α-syn,reduced the protein expression of Lampl and CTSD,weakened the fluorescence intensity of lysosomal and AO,increased the expression of p62 protein,decreased the expression of TFEB in the nucleus.Ykt6 increased the protein expression of LC3 and Lampl,decreased the accumulation of α-syn,and promoted the nuclear translocation of TFEB in MPP+-treated cells.Conclusion:Ykt6 protein expression was decreased in the PD models.Knockdown of Ykt6 in SH-SY5Y cells induced cellular apoptosis.Silencing Ykt6 followed by exposure to MPP+exacerbated lysosomal dysfunction.Ykt6 increased the levels of Lampl and TFEB,thereby playing an protection effect on the autophagy-lysosome pathway.