The Role Of Chaperone-mediated Autophagy In Neurotoxicity Induced By Alpha-Synuclein After METH Exposure

Background:Methamphetamine(METH)is one of the most commonly abused amphetamines in ice.Abuse of ice will cause varying degrees of damage to the vital organs and mental systems of the abuser.Numerous studies have shown that if long-term use of METH can destroy dopaminergic neuron terminals and kill nerve cells that produce other neurotransmitters,human brain cells will occur Neurodegenerative lesions similarly to patients with Parkinson's disease and Alzheimer's disease.Chaperone-mediated autophagy(CMA)is a selective catabolic pathway which allows to degrade specific cytosolic proteins which contains a pentapeptide targeting motif biochemically related KFERQ-like sequence,and this motif is selectively recognized by a constitutive chaperone,the chaperone heat shock cognate protein 70 kDa(hsc70).Hsc70 which resident in lysosomal(lys-hsc70)binds the substrate and targets to the lysosome membrane to lysosomal receptor lysosome-associated membrane protein type 2A(Lamp-2a),and hereafter translocate through the lysosomal membrane directly and degradation in the lysosomal lumen.Lamp-2a is considered to be the rate-limiting step of the CMA pathway,the expression level of Lamp-2a directly correlates with the activity of CMA,and CMA levels can be modulated through up-or downregulation of Lamp-2a expression.Hsc70 is not only the essential chaperone in the CMA translocation system to allows substrate protein entry into the lysosomal lumen,but also play a critical role in the neuronal death associated with neurodegenerative diseases since its significant downregulation both in PD and AD.Objectives:The objective of this study was to elucidate the relationship between changes in CMA activity levels and METH-induced high expression of a-Syn,abnormal aggregation,and neurotoxicity produced,and to investigate the effect of METH on the expression levels of key molecules of CMA pathway Lamp2a and Hsc70 and the effect of regulating CMA activity on METH-induced neurotoxicity.This study will prove that the regulation of CMA activity plays an important role in METH-induced a-Syn high expression,abnormal aggregation and neuronal damage,and the increase of CMA activity level can reduce the neurotoxicity induced by METH,and provides a certain theoretical basis and research direction for METH neurotoxicity molecular target therapeutic research.Method:1.To detect toxicity after METH treatment and changes in expression levels of key molecules Lamp2a and Hsc70 in CMA pathway.PC 12 cells were initially exposed to 0,1.0,2.0,2.5,3.0,and 4.0 mM METH,and SH-SY5Y cells were exposed to 0,0.5,1.0,1.5,2.0 and 2.5 mM METH and the prefrontal cortex and striatal neuronal culture of C57BL/6J mice embryos were isolated from pregnant C57 mice on 16-18 day pregnancy,and exposed to 0,0.2,0.4,0.6,0.8,and 1.0 mM METH to evaluate the expression of a-syn,pS129 ?-Syn,Lampl,Lamp2a and hsc70 with dose-dependence of METH by Western blot.2.To investigate the effect of silencing Lamp2a on CMA activity and METH-induced neurotoxicity in SH-SY5Y cells.SH-SY5Y cells were transfected with NC siRNA or Lamp-2a siRNA and then treated with or without METH(2.0 mM)for 24 h.Western blot and quantitative analyses were performed to evaluate the efficiency of Lamp-2a knockdown,and the expression of a-syn,phosphorylated a-syn and 5G4 and apoptosis markers PARP and cleaved-caspase 3 in SH-SY5Y cells.Cell apoptosis was evaluated with TUNEL staining.3.To investigate the effect of overexpression of Hsc70 on SHMA activity and METH-induced neurotoxicity in SH-SY5Y cells.SH-SY5Y cells were transfected with NC plasmid or Hsc70 overexpression plasmid and then treated with or without METH(2.0 mM)for 24 h.Western blot and quantitative analyses were performed to evaluate the efficiency of Lamp-2a knockdown,and the expression of a-Syn,phosphorylated a-Syn and 5G4 and apoptosis markers PARP and cleaved-caspase 3 in SH-SY5Y cells.Cell apoptosis was evaluated with TUNEL staining.Results:1.Meth increases expression of a-Syn and its abnormal aggregation forms in neurons and CMA activity was decreased.2.Silencing Lamp-2a expression increases the METH-induced neurotoxicity.3.Upregulating Hsc70 expression attenuates METH-induced neurotoxicity.Conclusion:In this study,we find that the expression of Lamp-2a is increased after METH exposure while Hsc70 expression levels do not significantly change in SY-SH5Y,PC 12 and primary neuronal cells.We also demonstrate that knockdown of Lamp-2a expression using synthetic siRNA sequences can increase METH-induced neurotoxicity with increased expression of a-Syn and its abnormal aggregation forms.At the same time,we find that upregulating Hsc70 expression by synthetic plasmid DNA can attenuate the neurotoxicity induced by METH with relieved a-Syn aggregation.These findings indicate that CMA plays a vital role in METH-induced toxicity and manipulation of CMA may represent a valuable potential therapeutic approach for METH-induced/-like neurotoxicity.