The Correlation Research Of The Lysosomal Degradation Pathway And The Proteasomal Degradation Pathway For α-synuclein Protein

Objective To investigate the effect and mechanism of the lysosome dysfunction and the proteasome dysfunction in Parkinson disease (PD).Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor, were treated by lysosome inhibitor E64 and proteasome inhibitor lactacystin. Cell viability was measured by MTT assay; Flow cytometry was used to detect cell apoptosis; Protein aggregation was detected by the double labeling imunofluorescence staining of Thioflavine S andα-synuclein; the apoptosis of the double labeling cells was examined by Hoechst33258 staining.Results The cell viability declined in a concentration-dependent manner while the apoptosis ratio increased when treated with the lysosome inhibitor E64, the proteasome inhibitor lactacystin and lactacystin+E64 in turn; E64 and lactacystin inducedα-synuclein protein aggregate and lead to the formation of eosinophilic intracytoplasmic inclusions, which was immunoreactive toα-synuclein and Thioflavine S. with 7.94±0.97% and 20.33±2.4%; Theα-synuclein protein aggregation became more significant(36.77±3.5%)when the two inhibitors were used by combination(P<0.05); Some inclusion-harboring cells revealed positive Hoechst33258 staining(17.29±1.54%).Conclusions The lysosome dysfunction and the proteasome dysfunction play an important role in the metabolism ofα-synuclein, inclusions formation and dopaminergic neuronal death. Objective To investigate the pathway and mechanism ofα-synuclein in lysosomal degradation pathway.Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor. PC12 cells were treated by Rotenone to makeα-synuclein model. Then PC12 cells were treated by different lysosomal inhibitors E64, 3-MA, BafA1. Protein aggregation was detected by the double labeling of Thioflavine S andα-synuclein for imunofluorescence staining.Results E64 inducedα-synuclein protein aggregate and lead to the formation of eosinophilic intracytoplasmic inclusions, which was immunoreactive toα-synuclein and Thioflavine S. with 15.36±0.85% (P<0.05). Theα-synuclein protein aggregation was not significant (7.65±0.46%,8.72±0.39%)when the macroautophagy inhibitors 3-MA and microautophgy inhibitors BafA1 were used (P>0.05).Conclusions The chaperone—mediated autophagy pathway play an important role in the metabolism ofα-synuclein, inclusions formation and dopaminergic neuronal death.