S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia

Abstract

Dysregulation of autophagic pathways leads to accumulation of abnormal proteins and damaged organelles in many neurodegenerative disorders, including Parkinson's disease (PD) and Lewy body dementia (LBD). Autophagy-related dysfunction may also trigger secretion and spread of misfolded proteins, such as α-synuclein (α-syn), the major misfolded protein found in PD/LBD. However, the mechanism underlying these phenomena remains largely unknown. Here, we used cell-based models, including human induced pluripotent stem cell-derived neurons, CRISPR/Cas9 technology, and male transgenic PD/LBD mice, plus vetting in human postmortem brains (both male and female). We provide mechanistic insight into this pathologic pathway. We find that aberrant S-nitrosylation of the autophagic adaptor protein p62 causes inhibition of autophagic flux and intracellular buildup of misfolded proteins, with consequent secretion resulting in cell-to-cell spread. Thus, our data show that pathologic protein S-nitrosylation of p62 represents a critical factor not only for autophagic inhibition and demise of individual neurons, but also for α-syn release and spread of disease throughout the nervous system.SIGNIFICANCE STATEMENT In Parkinson's disease and Lewy body dementia, dysfunctional autophagy contributes to accumulation and spread of aggregated α-synuclein. Here, we provide evidence that protein S-nitrosylation of p62 inhibits autophagic flux, contributing to α-synuclein aggregation and spread.

Keywords: Lewy body dementia; Parkinson's disease; autophagy; p62; α-synuclein.

Figure 1.

S-Nitrosylation of p62 in PD/LBD models. A, B, S-Nitrosylation of exogenous and endogenous p62 by the NO donor SNOC. HA-p62 transfected SH-SY5Y cells (A) or untransfected SH-SY5Y cells (B) were exposed to 100 μm freshly prepared SNOC or old SNOC (from which NO had been dissipated). After 20 min, cell lysates were subjected to the biotin-switch assay. The “ascorbate minus” sample served as a negative control. SNO-p62 and total (input)-p62 detected by immunoblot with anti-p62 antibody. C, S-Nitrosylation of p62 by endogenously generated NO. SH-SY5Y cells exposed to 1 μm rotenone (Rot) in the presence of 1 mm l-arginine and subjected to biotin-switch assay. D, Ratio of SNO-p62/input p62 (lanes 1 and 3). Data are mean ± SEM; n = 3. **p < 0.01, Student's t test. E, S-Nitrosylation of p62 in transgenic PD/LBD mouse model. Brain lysates from 4-month-old control (WT) or human Thy1 promoter-driven α-syn-overexpressing mice subjected to biotin-switch assay. F, Ratio of SNO-p62/input p62 from WT or α-syn-overexpressing mice. Data are mean ± SEM; n = 4 mice in each group. *p < 0.05, Student's t test. G, S-Nitrosylation of p62 in hiPSC-DA neurons. A53T mutant α-syn or isogenic control (corr) hiPSC-DA neurons in the presence or absence of 1 μm rotenone were subjected to the biotin-switch assay. The corr and A53T blots are derived from the same gel. H, Ratio of SNO-p62/input p62 (lanes 1, 3, and 5). For each histogram, data are mean ± SEM; n = 3. **p < 0.01, ***p < 0.001, ANOVA with Tukey's correction.

Figure 2.

Non-nitrosylatable p62(C331A) mutant mimics SNO-p62 to increase LC3 binding. A, SH-SY5Y cells were transfected with HA-p62 and GFP-LC3. After 1 d, cells were exposed to 200 μm old or fresh SNOC, and 30 min later, lysed and immunoprecipitated with anti-HA antibody. Total cell lysates (Input) and immunoprecipitates (IP) were probed with anti-GFP and anti-HA antibodies on immunoblots. “Heavy chain” shows presence of antibody. B, Coimmunoprecipitated GFP-LC3 levels normalized to immunoprecipitated HA-p62. Data are mean ± SEM; n = 3. **p < 0.01, Student's t test. C, SH-SY5Y cells were transfected with C-terminal HA-tagged WT p62 or mutant p62(C331A). Cells were exposed to 100 μm old or fresh SNOC and biotin-switch assay performed 30 min later. D, Ratio of SNO-p62/input p62 for C-terminal WT HA-p62 or mutant p62(C331A) transfected SH-SY5Y cells. Data are mean ± SEM; n = 3. ***p < 0.001 by Student's t test. E, SH-SY5Y cells were transfected with HA-tagged WT p62 or mutant p62(C331A). The next day, cells were exposed to 200 μm old or fresh SNOC; 30 min later, lysates were prepared and immunoprecipitated with anti-HA antibody. Total cell lysates (Input) and immunoprecipitates (IP) were immunoblotted with anti-GFP and anti-HA antibodies. F, Coimmunoprecipitated GFP-LC3 levels normalized to immunoprecipitated HA-p62. Data are mean ± SEM; n = 3. *p < 0.05, ANOVA with Tukey's correction. G-I, Homology model of the p62-LC3 complex. G, Overall structure of the p62-LC3 complex. Homology modeling of the p62 fragment (magenta) was docked to the crystal structure of LC3 (gray [2ZJD]). A network of Asp-Arg interactions (shown as dotted lines with distance between the atoms shown in Å) is present around p62(Cys331); these interactions facilitate binding of p62 to LC3 (H) or p62(Ala331) (I). See Extended Data Figures 2-1 and 2-2.

Figure 3.

Knock-in p62(C331A) mutant inhibits autophagic flux. A, WT or mutant p62(C331A) knock-in cells exposed to 400 nm BafA1 for 4 h were lysed and immunoblotted with anti-p62, anti-LC3, and anti-actin antibodies. B, C, Immunoblot quantification of relative p62 expression levels normalized to actin (B), and LC3-II/LC3-I ratio (C). Data are mean ± SEM; n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ANOVA with Tukey's correction. D, Representative confocal images of WT or mutant p62(C331A) in knock-in cells taken 6 h after prestaining with 1 μm DALGreen and 0.1 μm DAPRed dye for 30 min. Scale bar, 20 µm. E, Quantification of DALGreen and DAPRed puncta. Data are mean ± SEM; n = 3. *p < 0.05 (total puncta, WT vs C331A) by Student's t test. **p < 0.01 (DAPRed puncta, WT vs C331A) by ANOVA with Tukey's correction. F, Representative TEM images showing ultrastructure of autophagic vacuoles in WT (boxed areas; i, ii) or mutant p62(C331A) knock-in cells (boxed areas; iii, iv). Arrowhead indicates autolysosome. Arrows indicate autophagosomes. See Extended Data Figures 3-1, 3-2, and 3-3.

Figure 4.

Mutant p62(C331A) increases α-syn and p62/LC3 secretion. A, Immunoblots for p62, LC3, and Flotillin1 in supernatants or EVs obtained after ultracentrifugation from CM collected from V5-α-syn-transfected WT p62 or mutant p62(C331A) knock-in cells. LC3-I was the predominant form in supernatants, while LC3-II was the major form in EVs. B, C, V5-α-syn levels in EVs (B) and supernatants (C) measured by ELISA. Data are mean ± SEM; n = 4. *p < 0.05, Student's t test. D, Vesicle flow cytometry of CM (concentrated with 100 kDa cutoff filter) from mNeonGreen-tagged human α-syn (mNG-α-syn)-transfected WT p62 or mutant p62(C331A) knock-in cells. mNG-α-syn⁺ events were determined using a gate set on a bivariate plot of diameter versus mNG fluorescence for untransfected WT cell-derived EVs. E, Quantification of mNG-α-syn-positive EVs by flow cytometry. Inset, Immunoblot of CM used for flow cytometry. Data are mean ± SEM; n = 3. *p < 0.05, Student's t test. F, G, α-syn levels by ELISA in EVs (F) and supernatants (G) ultracentrifuge fractions of conditioned media from isogenic control or A53T hiPSC-DA neuronal cultures in the presence or absence of 1 mm l-NAME. Data are mean ± SEM; n = 3. *p < 0.05, **p < 0.01, Sidak's correction. See Extended Data Figures 4-1, 4-2, and 4-3.

Figure 5.

p62(C331A) mutant increases cell-to-cell transmission of α-syn. A, Schematic diagram of dual-cell BiFC system. In this assay, the hemi-Venus-α-syn (V1S) construct expresses α-syn conjugated to the N-terminal fragment of Venus, and α-syn-hemi-Venus (SV2) expresses α-syn conjugated to the C-terminal fragment of Venus. Upon α-syn dimerization/aggregation, the combination of V1S and SV2 emits green fluorescence. B, Scheme of analysis of α-syn cell-to-cell spread using the BiFC system. V1S-transfected WT or mutant p62(C331A) knock-in cells were plated on transwell inserts (donor cells), and SV2-transfected WT cells were plated on coverslips (recipient cells). One day after transfection, the donor and recipient cells were incubated for 3 d. C, Representative BiFC images (top panels) plus Hoechst staining (bottom panels). Scale bar, 20 µm. D, E, Quantification of green Venus-α-syn puncta (D) and relative intensity (E). Data are mean ± SEM; n = 3. *p < 0.05, **p < 0.01, Student's t test.

Figure 6.

S-Nitrosylation of p62 in human LBD brains and schema for α-syn cell-to-cell spread. A, S-Nitrosylation of human LBD and control brains. Human brain tissues with relatively short postmortem times (described in Extended Data Fig. 6-1) were subjected to biotin-switch assay. B, Ratio of SNO-p62/input p62 from control or LBD brains. Data are mean ± SEM; n = 5 brains in each group. **p < 0.01, Student's t test. C, Combined data for ratio of SNO-p62/Input p62 from Figures 1D, F, H and 6B (asterisks are from the original figures and retain their original meaning; vertical dotted line at “1” indicates the control for each condition from data shown in each of the other figures listed above). D, Schema of mechanism for S-nitrosylated p62 increasing cell-to-cell transmission of α-syn. Cytoplasmic misfolded α-syn can be directly released via nonclassical exocytosis or exophagy, yielding free α-syn in the extracellular medium (Ejlerskov et al., 2013). Alternatively, misfolded α-syn can be relegated to multivesicular bodies in the cell and then released into the extracellular space via enriched exosomes or EVs (Jang et al., 2010; Alvarez-Erviti et al., 2011; Danzer et al., 2012; Lee et al., 2013). SNO-p62 enhances the release of misfolded α-syn via both of these pathways by enhancing protein buildup because of blockade of autophagy at the stage of autolysosome formation. Released α-syn is then internalized by neighboring cells, and can seed endogenous α-syn to form aggregates (Luk et al., 2009; Nonaka et al., 2010). EV-mediated uptake occurs through direct endocytosis of the vesicle (Lee et al., 2008), while EV-independent uptake of α-syn may be mediated by binding to a receptor on the cell surface of the cell with subsequent endocytosis and endosome formation of the complex (Mao et al., 2016). See Extended Data Figure 6-1.