The Regulation And Underlying Mechanisms Of Microglia Autophagy On α-synuclein-triggered Neuroinflammation

Objective:Neuroinflammation and autophagy dysfunction have been shown to be involved in the pathogenesis of Parkinson’s disease.A lot of studies focused on and identified the impairments in dopamine neuron autophagy activity and its regulation.Few studies were designed to explore whether autophagy in microglia contributes to the pathogenesis of Parkinson’s disease,especially the inflammatory mechanism of this disorder.Therefore,this dissertation focuses on the regulation and underlysing mechanism of microglial autophagy on α-synuclein-triggered neuroinflammation.Methods:C57BL/6 mouse primary microglia and BV2 cell line were applied in this study.Human recombinant α-synuclein protein,at different concentrations(2.5 、 5 and 10 mg/ml),was used to treat BV2 or primary cultured microglial cells,and the protein expression levels of several autophagy-related proteins or their regulators such as LC3 II,P62,Beclin1 and mTOR were examined by immunoblotting following α-synuclein treatment for different periods(1h,3h,6h).The culture suppernatants of PC12 cells overexpressing wild-type α-synuclein were harvested and transferred into BV2 or primary microglia.The autophagy and autophagic flux changes were studied.The effect of α-synuclein on autophagy was studied in the presence of the lysosomal inhibitor Baf A1 or the mTOR inhibitor Rapamycin.We used autophagy-related gene Atg5 siRNA and autophagy inducer Rapamycin to modulate microglia autophagy and examine the potential effect of autophagy onα-synuclein-induced inflammation,by detecting the transcription and translation of several inflammatory-related molecules such as TNF-α,IL-1β and CD206 with quantitative PCR and ELISA assays in microglia treated with α-synuclein for 12 h or 24 h.Microglial phagocytosis change was tested using immune fluorescence combined with flow cytometric detection of microspheres.The expression of intracellular inflammatory corpuscle-associated proteins including Caspase-1 and NLRP3 following the treatments with autophagy regulators were studied by immunoblotting assay.Results: 1.α-synuclein inhibits autophagic activation in microgliaFollowing recombinant α-synuclein treatment,the autophagy-related protein LC3-II in BV2 and primary microglia,decreased in dose and time-dependent manners,while P62 protein expression increased in dose and time-dependent manners.P62 transcription did not change.To mimic and test the effect of neuron-secreted α-synuclein on microglia autophagy,the culture supernatant of PC12 cells overexpressing wild-type α-synuclein(α-synuclein conditioned medium)were harvested and applied to treat BV2 or primary microglia cells.similar results were obtained.However,the changes of LC3-II and P62 expressions disappeared when the α-synuclein in the culture supernatant was neutralized with the antibody against α-synuclein.Moreover,α-synuclein conditioned medium reduced LC3-II but enhanced P62 proteins even in the presence of the lysosome inhibitor Baf A1 and autophagy inducer Rapamycin.These results suggest that α-synuclein inhibits autophagic activation in microglia.2.mTOR signaling may be involved in α-synuclein inhibition on autophagyWhen the human recombinant α-synuclein protein or α-synuclein conditioned medium was applied to treat microglia,the expressions of autophagy-related proteins including ATG5,ATG7 or Beclin1 did not change.However,the phosphorylation levels of mTOR and its downstream molecule p70S6 K increasedand AMPK phosphorylation decreased.In addition,Akt phosphorylation increased,JNK and ERK phosphorylation levels did not change.These results suggest that α-synuclein may inhibit autophagy by activating mTOR and regulating its upstream signaling molecules such as Akt and AMPK.3.Microglial autophagy regulates neuroinflammationBy use atg5 siRNA or autophagy inducer Rapamycin to enhance or inhibit autophagic activity of microglia,The quantatative PCR and ELISA assay results showed that microglia autophagy activity was able to regulate neuroinflammatory responses,as Rapamycin cotreatment inhibited the transcription and generation of several pro-inflammatory cytokines such as TNF-α,IL-6 and IL-1βin α-synuclein-treated cells but increased the expression of the anti-inflammatory molecule CD206.By contrast,atg5 siRNA treatment enhanced the expression of these pro-inflammatory cytokines but reduced the anti-inflammatory molecule CD206 expression.Moreover,atg5 knockdown alone increased the expression of proinflammatory cytokines also increased significantly,along with the decrease of CD206 expression.4.Microglial autophagy modulates phagocytosisFurthermore,the results showed that microglia autophagy has an impact on the phagocytosis activity.Rapamycin was found to inhibit the microglia phagocytosis induced by α-synuclein.But,autophagy inhibition using atg5 siRNA enhanced its phagocytosis function.Conclusion:Taken together,the observations show that human recombinant α-synuclein protein monomer,as well as neuron-secretedα-synuclein,suppressed the autophagy activity possibly by activating mTORsignaling,accompanied by the inflammatory response in microglia.Microglial autophagy participates in the regulation of microglia inflammation and phagocytosis.