TNF Disrupts α-synuclein Degradation Via Autophagosome-Lysosome Pathway

Objective:To investigate the effect of TNF on α-synuclein degradation and to explore the potential role of autophagy-lysosome pathway involved in this processs.Methods:TNF-α was used to treat PC12 cells. The contents of TNF-α and IL-1β in LPS- or vehicle- treated microglia conditioned medium were determined with ELISA kits. The cell viability was determined with a CCK-8 assay. Western blot method was used to examine the protein expressions of α-synuclein, LC3 II, p62 and LAMP1. GFP-LC3 were transiently transfected into PC12 cells. α-synuclein and p62 m RNA levels were detected by reverse transcription-PCR(rt-PCR). The co-localization of autophagy-related proteins LC3 and lysosomal marker lamp1 was observed by immunofluorescence staining under confocol microscope. Lysotracker staining was used to detect the lysosome acidification. The Lyso Sensor Yellow/Blue dextran was used to detect and quantify the Lysosomal p H.Results:The content of IL-1β in Microglia conditioned medium after LPS treatment was about(320±67.8) pg/ml, and the content of TNF was about(1400±93.2) pg/ml, which were both significantly higher than the control(P<0.01). Within anti-TNF antibody were incubated with conditioned media, the content of supernatant TNF-α were significantly reduced in conditioned medium, and IL-1β showed no significant change. The PC12 cells were treated by the conditioned media and the role of supernatant samples after each treatment, α-synuclein levels of conditional media group was significantly higher;PC12 cell viability was concentration-dependent decreased by TNF-α(0,1,5,10, 50, 100 and 500 ng/ml), α-synuclein protein levels of TNF-α showed a concentration-dependent increased and then decreased, m RNA levels of α-synuclein and TNF-α concentrations had no correlation; after the proteasome inhibitor MG132 inhibited UPS pathway, the protein levels of α-synuclein did not change significantly compared with the control group, when using autophagy blocker CQ inhibited ALP pathway, the protein levels of α-synuclein increased significantly compared with the control group; the protein levels of LC3- and p62 appeared synchronization concentration-dependent increased after Ⅱ TNF-αtreatment, and the m RNA p62 did not appear to change, after overexpression LC3, free GFP protein levels decreased with the effect of TNF-α; after TNF-α treatment, GFP-LC3(green fluorescence) and LAMP1(red fluorescence) in PC12 cells can be completely colocalization, Lyso Tracker fluorescence intensity decreased, lysosomal p H rised;m TORC1 inhibitor PP242 allows lysosomal acidification, the up-regulation of TFEB and LAMP1, lysosomal p H decreased, promote degradation of α-synuclein.Conclusion:TNF is thought the main pro-inflammatory cytokine released by activated microglias.The α-synuclein accumulation induced by TNF in PC12 cells results from damaged autophagy and lysosomal dysfunction. PP242 rescued the dysfunction of autophagosome-lysosome pathway and α-synuclein accumulation.