title: TNF compromises lysosome acidification and reduces α-synuclein degradation via autophagy in dopaminergic cells.
abstract: Tumor necrosis factor-α (TNF) is increasingly implicated as a critical pro-inflammatory cytokine involved in chronic inflammation and neurodegeneration of Parkinson's disease (PD). However, the cellular and molecular events that lead to dopaminergic neuron degeneration are not fully understood. In this study, we demonstrated that microglia-released and recombinant TNF disrupted α-synuclein (α-SYN) degradation and caused its accumulation in PC12 cells and midbrain neurons. At subtoxic doses, recombinant TNF was found to increase the number of LC3 puncta dots and LC3II protein level, associated with the increases of P62 protein level. Inhibition of lysosomal degradation with Bafilomycin A1 pretreatment abrogated the TNF-induced elevation in LC3II protein level whereas autophagy inhibitor 3-methyladenine did not affect it. Moreover, TNF led to a marked increase in the number of yellow LC3 dots with a marginal elevation in red-only dots in RFP-GFP-tandem fluorescent LC3 (tf-LC3) transfected PC12 cells, implying the impairment in autophagic flux. Furthermore, TNF treatment reduced lysosomal acidification, as LysoTracker Red fluorescence and LysoSensor fluorescence shift from blue to yellow was markedly decreased in TNF-treated PC12 cells. Co-treatment with mammalian target of rapamycin kinase complex 1 (mTORC1) inhibitor PP242, which activated transcription factor EB (TFEB) signaling and lysosome biogenesis, partially rescued the accumulation of α-SYN in PC12 cells and midbrain neurons. Taken together, our results demonstrated that at subtoxic levels, TNF was able to impair autophagic flux and result in α-SYN accumulation by compromising lysosomal acidification in dopaminergic cells. This may represent a novel mechanism for TNF-induced dopaminergic neuron degeneration in PD.
authors.affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China.
authors.forename: Mei-Xia
authors.initials: MX
authors.lastname: Wang
authors.affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China.
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Cheng
authors.affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Jin
authors.affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China.
authors.forename: Yu-Lan
authors.initials: YL
authors.lastname: Cao
authors.affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China; Beijing Key Laboratory for Parkinson's Disease, Beijing 100053, China.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Yang
authors.affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China.
authors.forename: Jian-Da
authors.initials: JD
authors.lastname: Wang
authors.affiliation: Institute of Neuroscience, Soochow University, Suzhou 215123, China; Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: Institute of Neuroscience, Soochow University, Suzhou 215123, China.
authors.forename: Fen
authors.initials: F
authors.lastname: Wang
authors.affiliation: Institute of Neuroscience, Soochow University, Suzhou 215123, China; Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China. Electronic address: hulifang@suda.edu.cn.
authors.forename: Li-Fang
authors.initials: LF
authors.lastname: Hu
authors.affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China; Beijing Key Laboratory for Parkinson's Disease, Beijing 100053, China. Electronic address: liucf@suda.edu.cn.
authors.forename: Chun-Feng
authors.initials: CF
authors.lastname: Liu
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D059290:Dopaminergic Neurons / Q000187:drug effects / Q000378:metabolism*; D004622:Embryo, Mammalian; D005260:Female; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology*; D008636:Mesencephalon / Q000166:cytology; D016716:PC12 Cells; D011247:Pregnancy; D011687:Purines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012844:Sincalide / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D007211:Indoles; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D052118:Lysosomal-Associated Membrane Protein 1; D011687:Purines; D014409:Tumor Necrosis Factor-alpha; D051844:alpha-Synuclein; C572919:PP242; D012844:Sincalide
keywords: Autophagy; Dopaminergic cells; Lysosome acidification; TNF; α-Synuclein