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Synthetic alpha-synuclein fibrils cause mitochondrial impairment and selective dopamine neurodegeneration in part via iNOS-mediated nitric oxide production

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Abstract

Intracellular accumulation of α-synuclein (α-syn) are hallmarks of synucleinopathies, including Parkinson’s disease (PD). Exogenous addition of preformed α-syn fibrils (PFFs) into primary hippocampal neurons induced α-syn aggregation and accumulation. Likewise, intrastriatal inoculation of PFFs into mice and non-human primates generates Lewy bodies and Lewy neurites associated with PD-like neurodegeneration. Herein, we investigate the putative effects of synthetic human PFFs on cultured rat ventral midbrain dopamine (DA) neurons. A time- and dose-dependent accumulation of α-syn was observed following PFFs exposure that also underwent phosphorylation at serine 129. PFFs treatment decreased the expression levels of synaptic proteins, caused alterations in axonal transport-related proteins, and increased H2AX Ser139 phosphorylation. Mitochondrial impairment (including modulation of mitochondrial dynamics-associated protein content), enhanced oxidative stress, and an inflammatory response were also detected in our experimental paradigm. In attempt to unravel a potential molecular mechanism of PFFs neurotoxicity, the expression of inducible nitric oxide synthase was blocked; a significant decline in protein nitration levels and protection against PFFs-induced DA neuron death were observed. Combined exposure to PFFs and rotenone resulted in an additive toxicity. Strikingly, many of the harmful effects found were more prominent in DA rather than non-DA neurons, suggestive of higher susceptibility to degenerate. These findings provide new insights into the role of α-syn in the pathogenesis of PD and could represent a novel and valuable model to study DA-related neurodegeneration.

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Acknowledgements

This work was supported by the DSF Charitable Foundation, the Consolidated Anti-Aging Foundation, the National Institutes of Health (NS059806, NS095387, ES020718, ES020327), the Blechman Foundation, and the American Parkinson Disease Association. The authors would like to thank Dr. Simon Watkins (Center for Biologic Imaging, University of Pittsburgh, PA, USA) for the use of the motorized stage system and Evan Howlett for technical assistance.

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Author notes

  1. Victor Tapias

    Present address: Department of Neurology and Neuroscience, Weill Cornell Medicine, 525 East 68th Street, New York, NY, 10065, USA

Authors and Affiliations

  1. Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 1300 York Ave, New York, NY, 10065, USA

    Victor Tapias

  2. Department of Neurology, University of Pittsburgh, Pittsburgh, PA, 15260, USA

    Xiaoping Hu, Laurie H. Sanders & J. Timothy Greenamyre

  3. Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA, 15260, USA

    Xiaoping Hu, Laurie H. Sanders & J. Timothy Greenamyre

  4. Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA

    Kelvin C. Luk & Virginia M. Lee

  5. Pittsburgh VA Healthcare System, Pittsburgh, PA, 15206, USA

    J. Timothy Greenamyre

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Contributions

VT contributed to the design and conceptualization of the study and complete data collection, analysis and interpretation of data, drafting and revision of the manuscript. XH carried out the cell culture. KCL synthesized the PFFs and contributed to interpretation of the data and revision of the manuscript. LHS performed the mitochondrial respiration assays. VML assisted with manuscript revision. JTG participated in the design and conceptualization of the study, data interpretation, and revision of the manuscript.

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Correspondence to Victor Tapias.

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The authors declare that there are no actual or potential conflicts of interest, including any financial, personal, or other relationships with people or organizations during the development of the work submitted.

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18_2017_2541_MOESM1_ESM.jpg

Fig. S1 (related to Fig. 1). α-Syn localization and nature. Confocal microscopy imaging at 300x magnification revealed that intracellular α-syn inclusions (C; α-syn pSer129, red) occurred specifically in DA neurons (A; TH+, blue) rather than astrocytes (B; GFAP, green). Scale bar = 10 µm (JPEG 2804 kb)

18_2017_2541_MOESM2_ESM.jpg

Fig. S2 (related to Fig. 3). α-Syn fibrils decrease protein levels of synaptic proteins in dopamine neurons. (A-D) Series of high-resolution confocal micrographs at 100x depicting VAMP2 (red) and synaptophysin (green) immunoreactivity. (E) Barogram showing a strong reduction in VAMP2 immunofluorescence in PFFs and rotenone-treated DA neurons. (F) Quantitative analysis of VAMP2 protein levels in non-DA neurons. (G-H) Bar graph comparing the levels of synaptophysin in DA and non-DA neurons. Data are mean ± S.E.M. from two separate experiments done in triplicate for each culture condition. *** p < 0.001, ** p < 0.01 and * p < 0.05 vs VEH + VEH. ### p < 0.001, ## p < 0.01 and # p < 0.05 compared to PFFs + VEH. ** p < 0.01 compared to VEH + ROT (one-way ANOVA followed by Newman–Keuls multiple comparisons test). Scale bar = 20 μm (JPEG 6355 kb)

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Fig. S3 (related to Fig. 6). α-Syn fibrils downregulate dopamine protein content in a time-dependent fashion. (A-C) Representative immunoblots depicting the expression of TH+, MAP2, and β-Actin. Densitometric analysis revealed that PFFs did not exhibit any deleterious effect following 4 days post-addition (A1). A lack of pathogenicity was also observed 7 days post PFFs administration when neurons were treated with PFFs; however, a slight decrease in TH+ content was detected when PFFs were combined with rotenone (B1). By 14 days post PFFs addition, a considerably reduction in TH+ protein levels was found after exposure to PFFs alone or in combination with rotenone (C1). PFFs did not produce adverse effects on non-DA neurons (A2, B2, and C2). β-Actin was used for loading control normalization. Results were normalized to percentage neuron survival of vehicle control cells. Values are expressed as mean optical densitometry ± S.E.M. from four independent cultures run in triplicate. **** p < 0.0001, *** p < 0.001, ** p < 0.01and * p < 0.05 compared to VEH + VEH. #### p < 0.0001 and ### p < 0.001 vs PFFs + VEH. * p < 0.05 relative to VEH + ROT (one-way ANOVA followed by Newman-Keuls multiple comparisons test) (JPEG 1296 kb)

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Fig. S4 (related to Fig. 7). α-Syn fibrils enhance mitochondrial ROS levels. Barograms depicting representative MitoSox red traces from single identified neurons. The rate O •−2 of production was significantly higher following PFFs treatment (B) relative to control group (A). Combined exposure of PFFs and rotenone heightened the toxic effects of rotenone (C and E vs D and F). Measurements were conducted at least three times for all conditions from three independent neuron cultures. On an average, ~ 30-40 neurons per condition were investigated (JPEG 1991 kb)

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Fig. S5 (related to Fig. 7). α-syn fibrils exposure does not alter mitochondrial mass content. Both qualitative and quantitative analyses of confocal micrographs (100x and 300x) did not show any variation in TOM20 immunoreactivity between groups in DA (I) and non-DA (J) neurons. Scale bars = 20 μm at low magnification and 10 μm at high magnification (JPEG 4386 kb)

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Fig. S6 (related to Fig. 8). Detection of 3-nitrotyrosine in mitochondrial proteins. (A-D) To determine the subcellular localization of the upregulated amounts of protein tyrosine nitration after PFFs exposure, primary neurons were fixed and stained with 3-NT and TOM20 antibodies. (E-F) Pearson’s coefficient between 3-NT and TOM20 in both DA and non-DA neurons. Quantitative correlational analysis did not reach statistical significance among groups after PFFs exposure, suggesting that increased 3-NT levels arise from the nucleus and/or cytosol rather than mitochondrion. Data from three independent experiments in triplicate were pooled and presented as the average mean fluorescence intensity ± S.E.M. An average of ~ 25-30 TH+ and 450-550 MAP2 neurons were examined. * p < 0.05 vs VEH + VEH (one-way ANOVA followed by Newman-Keuls multiple comparisons test). Scale bar = 5 μm (JPEG 5172 kb)

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Fig. S7 (related to Fig. 9). α-Syn fibrils do not activate microglial cells. Data analysis revealed that although chronic exposure to rotenone induced important alterations in the morphology of Iba1 cells, no statistical important changes were detected in morphological activation (A), number of Iba1-positive cells (B), and overall area occupied by microglial cells (C) following PFFs treatment. Data from three independent cultures in triplicate were combined to determine means ± S.E.M. ** p < 0.01 relative to VEH + VEH. ## p < 0.01 vs PFFs + VEH (one-way ANOVA followed by Newman-Keuls multiple comparisons test) (JPEG 1092 kb)

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Tapias, V., Hu, X., Luk, K.C. et al. Synthetic alpha-synuclein fibrils cause mitochondrial impairment and selective dopamine neurodegeneration in part via iNOS-mediated nitric oxide production. Cell. Mol. Life Sci. 74, 2851–2874 (2017). https://doi.org/10.1007/s00018-017-2541-x

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