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Overexpression of Human E46K Mutant α-Synuclein Impairs Macroautophagy via Inactivation of JNK1-Bcl-2 Pathway

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An Erratum to this article was published on 01 June 2014

Abstract

Parkinson’s disease (PD) is pathologically characterized by selective loss of dopaminergic neurons in the midbrain and the existence of intracellular protein inclusions termed Lewy bodies, largely composed of α-synuclein. Genetic studies have revealed that rare point mutations in the gene encoding α-synuclein including A30P, A53T, and E46K are associated with familial forms of PD, indicating a pathological role for mutant α-synuclein in PD etiology. However, the mechanisms underlying the neuronal toxicity of mutant α-synuclein are still to be elucidated. Growing evidence has suggested a deleterious effect of mutant α-synuclein on the autophagy-lysosome pathway. In this study, we discovered that overexpression of human E46K mutant α-synuclein impaired macroautophagy in mammalian cells. Our data showed that overexpression of E46K mutant α-synuclein impaired autophagy at an early stage of autophagosome formation via the c-Jun N-terminal kinase 1 (JNK1)-Bcl-2 but not the mammalian target of rapamycin (mTOR) pathway. Overexpressed E46K mutant α-synuclein inhibited JNK1 activation, leading to a reduced Bcl-2 phosphorylation and increased association between Bcl-2 and Beclin1, further disrupting the formation of Beclin1/hVps34 complex, which is essential for autophagy initiation. Furthermore, overexpression of E46K mutant α-synuclein increased the vulnerability of differentiated PC12 cells to rotenone treatment, which would be partly due to its inhibitory effects on autophagy. Our findings may shed light on the potential roles of mutant α-synuclein in the pathogenesis of PD.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China Grants (no. 81274122, 81102831, 81073078, 81373997, 81373510), the Special Purpose for New Drug Development (2012ZX09301002-004, 2012ZX09103101-006), the Studies on Structure and function of Bioactive Substances from Natural Medicines (IRT1007), the Beijing Natural Science Foundation (7131013), the Research Fund for the Doctoral Program of Higher Education of China (2012110613000), the Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (no. BZ0150), the PUMC Youth Fund, and the Fundamental Research Funds for the Central Universities. The authors thank Professor ZW Hu for presenting CHO cells.

Conflict of Interest

The authors report no biomedical financial interests or potential conflicts of interest.

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Authors and Affiliations

  1. State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, I Xiannongtan Street, Xicheng District, 100050, Beijing, People’s Republic of China

    Jia-Qing Yan, Yu-He Yuan, Yan-Na Gao, Ju-Yang Huang, Kai-Li Ma, Yan Gao, Wan-Qing Zhang & Nai-Hong Chen

  2. Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China

    Yan-Na Gao & Wan-Qing Zhang

  3. Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Beijing, People’s Republic of China

    Yan-Na Gao

  4. Shanxi University of Traditional Chinese Medicine, Taiyuan, People’s Republic of China

    Xiao-Feng Guo

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  1. Jia-Qing Yan
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Fig. S1

Overexpression of WT α-synuclein has no effects on autophagy. (a) Stable PC12 cells overexpression of GFP, WT and E46K mutant α-synuclein were lysted. The detergent-soluble and detergent-insoluble fractions were subjected to SDS-PAGE. (b) HEK293 cells were transfected with WT, E46K mutant α-synuclein and an empty vector for 48 h. Cells were harvested and the detergent-soluble and detergent-insoluble fractions were subjected to SDS-PAGE. (c) Stable PC12 cells and HEK293 cells transfected with WT and E46K mutant α-synuclein were lysted and immunoblotted for LC3. (d) PC12 cells stably overexpressing WT α-synuclein-GFP were starved or not in EBSS for 4 h, then lysted and subjected to SDS-PAGE for analysis of the indicated proteins. (e) HEK293 cells were transfected with WT α-synuclein or an empty vector for 48 h followed by 4 h incubation with or not with EBSS. Indicated proteins were analyzed using western blotting. Data were shown as means ± SD for three independent experiments performed in triplicate. * P < 0.05, ** P < 0.01 compared with controls, # P < 0.05, ## P < 0.01 compared with WT α-synuclein by Two-tailed Student’s t- test. (GIF 103 kb)

High Resolution Image (TIFF 39,033 kb)

Fig. S2

Overexpression of E46K mutant α-synuclein impairs ubiquitin-dependent degradation and enhances CMA activity. (a) PC12 cells stably overexpressing E46K mutant α-synuclein or GFP were lysted and immunoblotted for ubiquitin. (b) PC12 cells stably overexpressing E46K mutant α-synuclein or GFP were lysted. The detergent-insoluble fraction was solubilized and subjected to SDS-PAGE. (c) Immunostaining of ubiquitin in PC12 cells stably overexpressing E46K mutant α-synuclein and GFP. Images were acquired by a confocal microscopy. Scale bar: 10 μm. (d) PC12 cells stably overexpressing E46K mutant α-synuclein-GFP were treated with 10 μg/ml cycloheximide for the indicated periods, then lysted and subjected to immunoblotting for p53. (e) PC12 cells stably overexpressing E46K mutant α-synuclein-GFP were treated with 10 μg/ml cycloheximide for the indicated periods, then lysted and subjected to immunoblotting for GAPDH. Data were shown as means ± SD for three independent experiments performed in triplicate. * P < 0.05, ** P < 0.01 by Two-tailed Student’s t- test. (GIF 107 kb)

High Resolution Image (TIFF 46,478 kb)

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Yan, JQ., Yuan, YH., Gao, YN. et al. Overexpression of Human E46K Mutant α-Synuclein Impairs Macroautophagy via Inactivation of JNK1-Bcl-2 Pathway. Mol Neurobiol 50, 685–701 (2014). https://doi.org/10.1007/s12035-014-8738-1

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  • DOI: https://doi.org/10.1007/s12035-014-8738-1

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