Cited by 549Neuroinflammation and Neurodegeneration MechanismsParkinson's Disease Mechanisms and TreatmentsAutophagy in Disease and Therapy

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Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration

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Published version of a preprint

Abstract

Microglia maintain brain homeostasis by removing neuron-derived components such as myelin and cell debris. The evidence linking microglia to neurodegenerative diseases is growing; however, the precise mechanisms remain poorly understood. Herein, we report a neuroprotective role for microglia in the clearance of neuron-released α-synuclein. Neuronal α-synuclein activates microglia, which in turn engulf α-synuclein into autophagosomes for degradation via selective autophagy (termed synucleinphagy). Synucleinphagy requires the presence of microglial Toll-like receptor 4 (TLR4), which induces transcriptional upregulation of p62/SQSTM1 through the NF-κB signaling pathway. Induction of p62, an autophagy receptor, is necessary for the formation of α-synuclein/ubiquitin-positive puncta that are degraded by autophagy. Finally, disruption of microglial autophagy in mice expressing human α-synuclein promotes the accumulation of misfolded α-synuclein and causes midbrain dopaminergic neuron degeneration. Our study thus identifies a neuroprotective function of microglia in the clearance of α-synuclein via TLR4-NF-κB-p62 mediated synucleinphagy.

Overview

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TL;DR

Microglia engulf neuron-released α-synuclein and degrade it through selective autophagy, termed synucleinphagy, via TLR4–NF-κB–p62/SQSTM1 signaling. Disrupting microglial autophagy in mice expressing human α-synuclein increases misfolded α-synuclein and causes midbrain dopaminergic neuron degeneration.

Key findings

  • Neuron-released human α-synuclein (hα-Syn) is engulfed by microglia in vivo in AAV9-hα-Syn-injected mice and hα-Syn-Tg mice, accompanied by microglial activation and detection of hα-Syn in isolated microglia.
  • Ingested hα-Syn forms ubiquitin-, p62-, and GFP–LC3-positive puncta that peak at 6 h and decline by 24 h; clearance is blocked by SAR405, Bafilomycin A1, or Chloroquine, enhanced by Torin1, and abolished in Atg7- or Atg14-deficient microglia and p62-KO microglia.
  • hα-Syn-induced p62 upregulation requires TLR4 and NF-κB: Tlr4-KO, TAK-242, or ML-120B suppresses p62 induction and hα-Syn/ubiquitin puncta; hα-Syn prefers TLR4 over TLR2 for NF-κB activation in HEK293T reporter assays and does not induce TLR4 endocytosis or IRF3 signaling like LPS.
  • Microglia-specific Atg7 deficiency in hα-Syn mice increases detergent-insoluble hα-Syn, p-S129 α-synuclein-positive structures, and decreases TH-positive and Nissl-positive cells in the SNpc at 7 months after tamoxifen; hα-Syn-Tg mice alone showed no dopaminergic degeneration up to 12 months.

Methods

  • Mouse models included AAV9-GFP or AAV9-hα-Syn injection into SNpc, Thy-1 hα-Syn transgenic mice, Cx3cr1CreER-IRES-Eyfp, Atg7flox/flox, Atg14flox/flox, p62-KO, Tlr4-KO, and GFP–LC3 mice; primary microglia, adult microglia isolation, and HEK293T NF-κB-luciferase TLR assays were used.
  • Main analyses included RT-qPCR, western blot, immunostaining with confocal 3D reconstruction, electron microscopy, co-immunoprecipitation, brain fractionation into detergent-soluble/insoluble fractions, flow cytometry, and stereological counting of TH/Nissl-positive SNpc neurons.
  • Statistics used unpaired two-tailed Student’s t test or Mann–Whitney U test, one-way ANOVA with Newman–Keuls post hoc, or two-way ANOVA with Bonferroni post hoc; values are mean ± SEM.

Limitations

  • The paper states that the exact mechanism by which α-synuclein enters microglia remains unresolved, the specific E3 ligase for α-synuclein in microglia is unidentified, and the significance of other TLRs requires future study.
  • Evident from the text: mechanistic dissection relies heavily on cultured primary microglia and recombinant hα-Syn, while in vivo evidence uses overexpression models (AAV or Thy-1 transgenic hα-Syn) rather than endogenous α-synuclein; no human tissue or clinical data are presented.

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Registry

Crossref · 10.1038/s41467-020-15119-w

Cites

74 of 74 references verified0 citation errors · 0 not checkable

Statistics

51 tests recomputed · 0 with the reported and recomputed p on opposite sides of .05

Figures

Fig. 1 Neuron-released α-synuclein is engulfed by microglia in vivo. a, b, f, g Brain sections from AAV-GFP (n = 768 cells, six animals) and AAV-hα-Syn-injected mice (n = 986 cells, six animals) at 6 weeks post AAV injection (a, b) or from 10-month-old Non-Tg (n = 246 cells, three animals) and hα-Syn-Tg (n = 478cells, seven animals) mice (f, g) were stained for a marker of microglia, Iba-1, to visualize microglia morphology in the striatum. Reconstruction of microglia processes was generated by Filament-Tracer tool in Imaris software (a, e) and filament lengths (p < 0.0001) and the number of terminal points (p < 0.0001) were quantified (left panels in b and g). The number of microglia at the striatum was counted using ImageJ software (NIH, Bethesda, MD) (right panel in b (p = 6.59E-07) and g (p = 4.88E-05)). p-values were calculated by two-tailed Mann–Whitney U test (the left two panels) and unpaired two-tailed Student’s t test (the right panel). Scale bar, 10 µm. c CD45intermediate and CD11bhigh microglia were isolated from brains injected with AAV-hα-Syn (n = 3) and assayed for RT-qPCR using primers as indicated, compared with AAV-GFP (n = 3) at 4-weeks after AAV. p-values were calculated by unpaired two-tailed Student’s t test. d, h Pooled CD45intermediate and CD11bhigh microglia were prepared either from AAV-GFP (n = 3) and AAV-hα-Syn-injected mice (n = 3) (d) or Non-Tg (n = 4) and hα-Syn-Tg (n = 4) mice (h) brains at the indicated time points and lysed for W.B using α-synuclein antibodies (MJFR1 clone in d, 2F12 clone in h) and actin. e, i Brain sections from Cx3cr1CreER-IRES-Eyfp mice injected with AAV-hα-Syn were stained using anti-GFP/YFP antibody and human α-synuclein antibody at 6 weeks after AAV (e), and brain sections from 10-month-old Non-Tg and α-Syn-Tg mice were stained using Iba-1 antibody and human α-Synuclein antibody (i). 3D reconstruction of microglia and human α-synuclein was produced using Imaris software (Bitplane, right panel) as described in the “Methods" section. Scale bar, 10 µm. All values are reported as mean ± SEM. Data are representative of three independent experiments.
Fig. 1 Neuron-released α-synuclein is engulfed by microglia in vivo. a, b, f, g Brain sections from AAV-GFP (n = 768 cells, six animals) and AAV-hα-Syn-injected mice (n = 986 cells, six animals) at 6 weeks post AAV injection (a, b) or from 10-month-old Non-Tg (n = 246 cells, three animals) and hα-Syn-Tg (n = 478cells, seven animals) mice (f, g) were stained for a marker of microglia, Iba-1, to visualize microglia morphology in the striatum. Reconstruction of microglia processes was generated by Filament-Tracer tool in Imaris software (a, e) and filament lengths (p < 0.0001) and the number of terminal points (p < 0.0001) were quantified (left panels in b and g). The number of microglia at the striatum was counted using ImageJ software (NIH, Bethesda, MD) (right panel in b (p = 6.59E-07) and g (p = 4.88E-05)). p-values were calculated by two-tailed Mann–Whitney U test (the left two panels) and unpaired two-tailed Student’s t test (the right panel). Scale bar, 10 µm. c CD45intermediate and CD11bhigh microglia were isolated from brains injected with AAV-hα-Syn (n = 3) and assayed for RT-qPCR using primers as indicated, compared with AAV-GFP (n = 3) at 4-weeks after AAV. p-values were calculated by unpaired two-tailed Student’s t test. d, h Pooled CD45intermediate and CD11bhigh microglia were prepared either from AAV-GFP (n = 3) and AAV-hα-Syn-injected mice (n = 3) (d) or Non-Tg (n = 4) and hα-Syn-Tg (n = 4) mice (h) brains at the indicated time points and lysed for W.B using α-synuclein antibodies (MJFR1 clone in d, 2F12 clone in h) and actin. e, i Brain sections from Cx3cr1CreER-IRES-Eyfp mice injected with AAV-hα-Syn were stained using anti-GFP/YFP antibody and human α-synuclein antibody at 6 weeks after AAV (e), and brain sections from 10-month-old Non-Tg and α-Syn-Tg mice were stained using Iba-1 antibody and human α-Synuclein antibody (i). 3D reconstruction of microglia and human α-synuclein was produced using Imaris software (Bitplane, right panel) as described in the “Methods" section. Scale bar, 10 µm. All values are reported as mean ± SEM. Data are representative of three independent experiments.CC BY · PubMed Central
Fig. 2 Microglia-engulfed α-synuclein is degraded by autophagy. a, b, c Cultured primary microglia from WT mice (a and left panels of b) and GFP–LC3-transgenic mice (right panels of b) were treated 250 nM hα-Syn protein and stained with antibodies against human α-synuclein (MJFR1 clone, a and b), ubiquitin (a), or p62 (b). The number of hα-Syn/ubiquitin-positive puncta was quantified (c, n = 29, three independent experiments). In a, empty arrowheads indicate a portion of α-synuclein colocalized with ubiquitin-positive puncta and disappear in 24 h. Arrows represent a portion of α-synuclein not colocalized with ubiquitin. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(5,168) = 139.6, p = 6.43E-58, left panel; F(5,162) = 118.4, p = 3.14E-52. Scale bar, 10 µm; 2 µm in magnified boxes. d At 6 weeks post AAV-hα-Syn inoculation into GFP–LC3 mice, brain slices were fixed and stained with antibodies against human α-synuclein, GFP, and Iba-1 (left panel). 3D surface-rendering images of each protein in microglia were made with Imaris software (Bitplane, right panel). Scale bar, 10 µm; 1 µm in the magnified box. e Ultrastructure of puncta from 250 nM hα-Syn protein-treated cells for 6 h was observed by an electron microscope. Arrows indicate double-membrane structures. Scale bar, 500 nm in the left panel; 100 nm in the right panel. f, g Microglia were treated with 250 nM hα-Syn protein and SAR405 (f, n = 32), Bafilomycin A1 (f, n = 32), and Chloroquine (f, n = 30) or Torin (g, n = 38) was added. The number of hα-Syn/ubiquitin-positive puncta was quantified in the lower panel. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test (f, F(3,130)=39.88, p = 2.43E-18) and unpaired two-tailed Student’s t test (g, p = 5.38E-18). h, i Microglia cultured either from Atg7flox/flox mice (left panel) from Atg14flox/flox mice (right panel) with or without Cx3cr1Cre expression were assayed for W.B using antibodies against ATG7 or ATG14, p62, and LC3 I/II (h). Cells were treated with 250 nM hα-Syn protein for 24 h, and the number of hα-Syn/ubiquitin-positive puncta was quantified (I, n = 32, p < 0.0001 for Atg7; n = 35, p < 0.0001 for Atg14). p-values were calculated by two-tailed Mann–Whitney U test. All values are reported as mean ± SEM. Data are representative of at least three independent experiments.
Fig. 2 Microglia-engulfed α-synuclein is degraded by autophagy. a, b, c Cultured primary microglia from WT mice (a and left panels of b) and GFP–LC3-transgenic mice (right panels of b) were treated 250 nM hα-Syn protein and stained with antibodies against human α-synuclein (MJFR1 clone, a and b), ubiquitin (a), or p62 (b). The number of hα-Syn/ubiquitin-positive puncta was quantified (c, n = 29, three independent experiments). In a, empty arrowheads indicate a portion of α-synuclein colocalized with ubiquitin-positive puncta and disappear in 24 h. Arrows represent a portion of α-synuclein not colocalized with ubiquitin. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(5,168) = 139.6, p = 6.43E-58, left panel; F(5,162) = 118.4, p = 3.14E-52. Scale bar, 10 µm; 2 µm in magnified boxes. d At 6 weeks post AAV-hα-Syn inoculation into GFP–LC3 mice, brain slices were fixed and stained with antibodies against human α-synuclein, GFP, and Iba-1 (left panel). 3D surface-rendering images of each protein in microglia were made with Imaris software (Bitplane, right panel). Scale bar, 10 µm; 1 µm in the magnified box. e Ultrastructure of puncta from 250 nM hα-Syn protein-treated cells for 6 h was observed by an electron microscope. Arrows indicate double-membrane structures. Scale bar, 500 nm in the left panel; 100 nm in the right panel. f, g Microglia were treated with 250 nM hα-Syn protein and SAR405 (f, n = 32), Bafilomycin A1 (f, n = 32), and Chloroquine (f, n = 30) or Torin (g, n = 38) was added. The number of hα-Syn/ubiquitin-positive puncta was quantified in the lower panel. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test (f, F(3,130)=39.88, p = 2.43E-18) and unpaired two-tailed Student’s t test (g, p = 5.38E-18). h, i Microglia cultured either from Atg7flox/flox mice (left panel) from Atg14flox/flox mice (right panel) with or without Cx3cr1Cre expression were assayed for W.B using antibodies against ATG7 or ATG14, p62, and LC3 I/II (h). Cells were treated with 250 nM hα-Syn protein for 24 h, and the number of hα-Syn/ubiquitin-positive puncta was quantified (I, n = 32, p < 0.0001 for Atg7; n = 35, p < 0.0001 for Atg14). p-values were calculated by two-tailed Mann–Whitney U test. All values are reported as mean ± SEM. Data are representative of at least three independent experiments.CC BY · PubMed Central
Fig. 3 p62 mediates sequestration and degradation of α-synuclein. a Microglia were treated with 250 nM hα-Syn protein for the indicated time and assayed for W.B using antibodies against p62 and human α-synuclein. The levels of p62 protein were quantified in the lower panel. n = 3 over three independent experiments. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(3,8) = 13.44, p = 0.0017. b After treatment of 250 nM hα-Syn protein for the indicated time, the levels of p62 mRNA were examined by RT-qPCR. p62 mRNA was normalized to Actin mRNA. n = 6 over three independent experiments. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(3,20) = 35.43, p = 3.39E-08. c The absence of p62 protein was confirmed in microglia obtained from p62-KO mice by W.B. d, e WT and p62-KO microglia were treated with 250 nM hα-Syn protein for 6 h, and stained with antibodies against human α-synuclein and ubiquitin (d). The number of hα-Syn/ubiquitin-positive puncta was quantified (e). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. F(1,139)=272.5, p = 1.44E-34 for hα-Syn-genotype interaction; F(1,139) = 315.3, p = 1.45E-37 for hα-Syn; F(1,139)=303.7, p = 8.81E-37 for genotype. Scale bar, 10 µm. f–h The number of p62 puncta was quantified in microglia at striatum of brains either from AAV-hα-Syn-injected mice (n = 3, f, g, p < 0.0001) or from hα-Syn-Tg mice (n = 5, h, p < 0.0001) compared with AAV-GFP-injected mice (n = 3) and non-Tg mice (n = 3), respectively. p-values were calculated by two-tailed Mann–Whitney U test. Scale bar, 10 µm. i At 6 weeks post AAV-hα-Syn administration into Cx3cr1CreER-IRES-Eyfp mice, brain slices were stained with antibodies against GFP/YFP, human α-synuclein, p62 (left panel), and ubiquitin (right panel). Scale bar, 10 µm; 1 µm in the magnified box. j Brain sections from 10-month-old hα-syn-Tg mice were fixed and stained using antibodies against human α-synuclein, Iba-1, and p62. Scale bar, 10 µm; 1 µm in the magnified box. k, l, m Human α-synuclein were immunoprecipitated from AAV-hα-Syn-injected brain (k), hα-Syn-Tg brain (l), and cultured microglia treated with 250 nM hα-Syn protein for 6 h (m). Arrows indicate the high molecular weight of p62. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.
Fig. 3 p62 mediates sequestration and degradation of α-synuclein. a Microglia were treated with 250 nM hα-Syn protein for the indicated time and assayed for W.B using antibodies against p62 and human α-synuclein. The levels of p62 protein were quantified in the lower panel. n = 3 over three independent experiments. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(3,8) = 13.44, p = 0.0017. b After treatment of 250 nM hα-Syn protein for the indicated time, the levels of p62 mRNA were examined by RT-qPCR. p62 mRNA was normalized to Actin mRNA. n = 6 over three independent experiments. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(3,20) = 35.43, p = 3.39E-08. c The absence of p62 protein was confirmed in microglia obtained from p62-KO mice by W.B. d, e WT and p62-KO microglia were treated with 250 nM hα-Syn protein for 6 h, and stained with antibodies against human α-synuclein and ubiquitin (d). The number of hα-Syn/ubiquitin-positive puncta was quantified (e). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. F(1,139)=272.5, p = 1.44E-34 for hα-Syn-genotype interaction; F(1,139) = 315.3, p = 1.45E-37 for hα-Syn; F(1,139)=303.7, p = 8.81E-37 for genotype. Scale bar, 10 µm. f–h The number of p62 puncta was quantified in microglia at striatum of brains either from AAV-hα-Syn-injected mice (n = 3, f, g, p < 0.0001) or from hα-Syn-Tg mice (n = 5, h, p < 0.0001) compared with AAV-GFP-injected mice (n = 3) and non-Tg mice (n = 3), respectively. p-values were calculated by two-tailed Mann–Whitney U test. Scale bar, 10 µm. i At 6 weeks post AAV-hα-Syn administration into Cx3cr1CreER-IRES-Eyfp mice, brain slices were stained with antibodies against GFP/YFP, human α-synuclein, p62 (left panel), and ubiquitin (right panel). Scale bar, 10 µm; 1 µm in the magnified box. j Brain sections from 10-month-old hα-syn-Tg mice were fixed and stained using antibodies against human α-synuclein, Iba-1, and p62. Scale bar, 10 µm; 1 µm in the magnified box. k, l, m Human α-synuclein were immunoprecipitated from AAV-hα-Syn-injected brain (k), hα-Syn-Tg brain (l), and cultured microglia treated with 250 nM hα-Syn protein for 6 h (m). Arrows indicate the high molecular weight of p62. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.CC BY · PubMed Central
Fig. 4 α-synuclein-induced p62 upregulation requires TLR4. a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number of hα-Syn/ubiquitin-positive puncta was quantified (c). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In (a), F(3,16) = 3.381, p = 0.0443 for hα-Syn-genotype interaction; F(3, 6) = 11.46, p = 0.0003 for hα-Syn; F(1,16) = 43.09, p = 6.51E-06 for genotype. In b, F(3,40) = 3.199, p = 0.0334 for hα-Syn-genotype interaction; F(3,40) = 8.326, p = 0.0002 for hα-Syn; F(1,40) = 21.77, p = 3.41E-05 for genotype. In c, F(2,174) = 12.56, p = 8.03E-06 for hα-Syn-genotype interaction; F(2,174) = 91.61, p = 6.64E-28 for hα-Syn; F(1,174) = 24.89, p = 1.46E-06 for genotype. d, e Cells were pretreated with TAK-242 and treated with 250 nM hα-Syn protein. The protein levels of p62 and the number of hα-Syn/ubiquitin-positive puncta were determined by W.B (d, n = 4) and immunostaining (e, n = 35), respectively. p-values were calculated by unpaired two-tailed Student’s t test (d, p = 0.0001) and one-way ANOVA with Newman–Keuls post hoc test (e, F(2,92) = 156.7, p = 2.35E-30). f Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4-KO mice injected with AAV-hα-Syn brains. Scale bar, 10 µm. g The number of p62 puncta was quantified in microglia at striatum of WT mice (n = 5) and Tlr4-KO mice (n = 5). p-values were calculated by two-tailed Mann–Whitney U test. h Microglia were treated with either 250 nM hα-Syn protein (red, n = 6) or 50 ng/ml LPS (blue, n = 7 for 0–6 h, n = 6 for 9 h) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. In hα-Syn protein treatment, F(3,20)=35.43, p = 3.39E-08 for p62, F(3,20) = 6.655, p = 0.0027 for Il-1b, F(3,20) = 8.907, p = 0.0006 for Tnf, F(3,20) = 5.104, p = 0.0087 for Infb1; In LPS treatment, F(3,23) = 19.53, p = 1.61E-06 for p62, F(3,23) = 4.643, p = 0.0111 for Il-1b, F(3,23) = 12.09, p = 5.92E-05 for Tnf, F(3,23)=35.86, p = 7.68E-09 for Infb1. i After treating 250 nM hα-Syn protein or 50 ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. j After treating hα-Syn protein (n = 7) or LPS (n = 7) for 30 min to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(2,18) = 15.10, p = 0.0001. k Microglia were pretreated with Dynasore (n = 3) for indicated concentration and treated with 250 nM hα-Syn protein for 3 h. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.
Fig. 4 α-synuclein-induced p62 upregulation requires TLR4. a, b, c Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein and assayed for W.B (a, n = 3), RT-qPCR (b, n = 6) or for immunostaining (c, n = 30). Band intensities were quantified in the right panel of a. The number of hα-Syn/ubiquitin-positive puncta was quantified (c). p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In (a), F(3,16) = 3.381, p = 0.0443 for hα-Syn-genotype interaction; F(3, 6) = 11.46, p = 0.0003 for hα-Syn; F(1,16) = 43.09, p = 6.51E-06 for genotype. In b, F(3,40) = 3.199, p = 0.0334 for hα-Syn-genotype interaction; F(3,40) = 8.326, p = 0.0002 for hα-Syn; F(1,40) = 21.77, p = 3.41E-05 for genotype. In c, F(2,174) = 12.56, p = 8.03E-06 for hα-Syn-genotype interaction; F(2,174) = 91.61, p = 6.64E-28 for hα-Syn; F(1,174) = 24.89, p = 1.46E-06 for genotype. d, e Cells were pretreated with TAK-242 and treated with 250 nM hα-Syn protein. The protein levels of p62 and the number of hα-Syn/ubiquitin-positive puncta were determined by W.B (d, n = 4) and immunostaining (e, n = 35), respectively. p-values were calculated by unpaired two-tailed Student’s t test (d, p = 0.0001) and one-way ANOVA with Newman–Keuls post hoc test (e, F(2,92) = 156.7, p = 2.35E-30). f Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4-KO mice injected with AAV-hα-Syn brains. Scale bar, 10 µm. g The number of p62 puncta was quantified in microglia at striatum of WT mice (n = 5) and Tlr4-KO mice (n = 5). p-values were calculated by two-tailed Mann–Whitney U test. h Microglia were treated with either 250 nM hα-Syn protein (red, n = 6) or 50 ng/ml LPS (blue, n = 7 for 0–6 h, n = 6 for 9 h) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. In hα-Syn protein treatment, F(3,20)=35.43, p = 3.39E-08 for p62, F(3,20) = 6.655, p = 0.0027 for Il-1b, F(3,20) = 8.907, p = 0.0006 for Tnf, F(3,20) = 5.104, p = 0.0087 for Infb1; In LPS treatment, F(3,23) = 19.53, p = 1.61E-06 for p62, F(3,23) = 4.643, p = 0.0111 for Il-1b, F(3,23) = 12.09, p = 5.92E-05 for Tnf, F(3,23)=35.86, p = 7.68E-09 for Infb1. i After treating 250 nM hα-Syn protein or 50 ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. j After treating hα-Syn protein (n = 7) or LPS (n = 7) for 30 min to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(2,18) = 15.10, p = 0.0001. k Microglia were pretreated with Dynasore (n = 3) for indicated concentration and treated with 250 nM hα-Syn protein for 3 h. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.CC BY · PubMed Central
Fig. 5 NF-κB mediates α-synuclein-TLR4 signaling and p62 induction. a WT microglia were treated with 250 nM hα-Syn protein for the indicated time and assayed for W.B using antibodies as indicated. The levels of proteins were quantified in the right panel (n = 6). p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(4,24) = 9.196, p = 0.0001 for p-NF-κB, F(4,24) = 4.530, p = 0.0072 for IkB, F(4,24) = 0.8644, p = 0.4994 for p-ERK1/2, F(4,24) = 1.369, p = 0.2741 for p-JNK, F(4,24) = 5.372, p = 0.0031 for p-p38. b, c Cells were pretreated with ML-120B, an IKK-2 inhibitor, or SB203580 and SB202190, two different p38 inhibitors, for indicated concentrations before 250 nM hα-Syn protein treatment, and assayed for W.B (b, n = 3–5, F(3,12) = 3.750, p = 0.0413) or immunostaining (c, n = 33, F(4,160) = 56.21, p = 1.53E-29). The levels of proteins were quantified in the right panel. The number of hα-Syn/ubiquitin-positive puncta was counted and quantified (c). p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. d–f Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein for 3 h with indicated concentration without (d, n = 6) or with (e (n = 6), f (n = 4)) TLR2-blocking antibody (T2.5), and assayed for RT-qPCR using primers for Il-1b (left panel, F(3,40) = 20.70, p = 2.95E-08 for hα-Syn-genotype interaction; F(3,40) = 69.43, p = 6.55E-16 for hα-Syn; F(1,40) = 69.57, p = 2.74E-10 for genotype) and Tnf (right panel, F(3,40) = 10.12, p = 4.32E-05 for hα-Syn-genotype interaction; F(3,40) = 21.73, p = 1.64E-08 for hα-Syn; F(1,40) = 34.36, p = 7.39E-07 for genotype). p-values were calculated by two-way ANOVA with Bonferroni post hoc test (wt vs ko in d), one-way ANOVA with Newman–Keuls post hoc test (non-treated vs. treated in d, e; F(3,20) = 22.11, p = 1.46E-06 for Il-1b, F(3,20) = 11.88, p = 0.0001 for Tnf), and unpaired two-tailed Student’s t test (f). g–i HEK293T cells were transfected with a luciferase vector that expresses luciferase under the promoter containing NF-κB-binding elements, and various TLRs. Then, cells were treated hα-Syn protein with indicated concentration (g (n = 9), h (n = 9–12), i (n = 9)), 50 ng/ml LPS (i), or Pam3CSK4 (i) for 24 h without (g, h) or with (i) 10 µg/ml TLR2-blocking antibody (T2.5) and control IgG. p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In g, F(4,80) = 19.63, p = 2.74E-11 for interaction; F(4,80) = 27.31, p = 2.66E-14 for hα-Syn; F(1,80) = 47.95, p = 9.90E-10 for protein overexpression. In h, F(5,126) = 6.070, p = 4.54E-05 for interaction; F(5,126) =6.070, p = 4.54E-05 for hα-Syn; F(1,126) = 73.83, p = 2.77E-14 for protein overexpression. In (i), F(3,64) = 16.98, p = 3.19E-08 for interaction; F(3,64) = 177.4, p = 5.90E-31 for stimuli; F(1,64) = 17.47, p = 9.03E-05 for antibody. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.
Fig. 5 NF-κB mediates α-synuclein-TLR4 signaling and p62 induction. a WT microglia were treated with 250 nM hα-Syn protein for the indicated time and assayed for W.B using antibodies as indicated. The levels of proteins were quantified in the right panel (n = 6). p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(4,24) = 9.196, p = 0.0001 for p-NF-κB, F(4,24) = 4.530, p = 0.0072 for IkB, F(4,24) = 0.8644, p = 0.4994 for p-ERK1/2, F(4,24) = 1.369, p = 0.2741 for p-JNK, F(4,24) = 5.372, p = 0.0031 for p-p38. b, c Cells were pretreated with ML-120B, an IKK-2 inhibitor, or SB203580 and SB202190, two different p38 inhibitors, for indicated concentrations before 250 nM hα-Syn protein treatment, and assayed for W.B (b, n = 3–5, F(3,12) = 3.750, p = 0.0413) or immunostaining (c, n = 33, F(4,160) = 56.21, p = 1.53E-29). The levels of proteins were quantified in the right panel. The number of hα-Syn/ubiquitin-positive puncta was counted and quantified (c). p-values were calculated by one-way ANOVA with Newman–Keuls post hoc test. d–f Microglia obtained from WT mice and Tlr4-KO mice were treated with hα-Syn protein for 3 h with indicated concentration without (d, n = 6) or with (e (n = 6), f (n = 4)) TLR2-blocking antibody (T2.5), and assayed for RT-qPCR using primers for Il-1b (left panel, F(3,40) = 20.70, p = 2.95E-08 for hα-Syn-genotype interaction; F(3,40) = 69.43, p = 6.55E-16 for hα-Syn; F(1,40) = 69.57, p = 2.74E-10 for genotype) and Tnf (right panel, F(3,40) = 10.12, p = 4.32E-05 for hα-Syn-genotype interaction; F(3,40) = 21.73, p = 1.64E-08 for hα-Syn; F(1,40) = 34.36, p = 7.39E-07 for genotype). p-values were calculated by two-way ANOVA with Bonferroni post hoc test (wt vs ko in d), one-way ANOVA with Newman–Keuls post hoc test (non-treated vs. treated in d, e; F(3,20) = 22.11, p = 1.46E-06 for Il-1b, F(3,20) = 11.88, p = 0.0001 for Tnf), and unpaired two-tailed Student’s t test (f). g–i HEK293T cells were transfected with a luciferase vector that expresses luciferase under the promoter containing NF-κB-binding elements, and various TLRs. Then, cells were treated hα-Syn protein with indicated concentration (g (n = 9), h (n = 9–12), i (n = 9)), 50 ng/ml LPS (i), or Pam3CSK4 (i) for 24 h without (g, h) or with (i) 10 µg/ml TLR2-blocking antibody (T2.5) and control IgG. p-values were calculated by two-way ANOVA with Bonferroni post hoc test. In g, F(4,80) = 19.63, p = 2.74E-11 for interaction; F(4,80) = 27.31, p = 2.66E-14 for hα-Syn; F(1,80) = 47.95, p = 9.90E-10 for protein overexpression. In h, F(5,126) = 6.070, p = 4.54E-05 for interaction; F(5,126) =6.070, p = 4.54E-05 for hα-Syn; F(1,126) = 73.83, p = 2.77E-14 for protein overexpression. In (i), F(3,64) = 16.98, p = 3.19E-08 for interaction; F(3,64) = 177.4, p = 5.90E-31 for stimuli; F(1,64) = 17.47, p = 9.03E-05 for antibody. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.CC BY · PubMed Central
Fig. 6 Autophagy-deficient microglia cause α-synuclein accumulation. a Experimental plan to test the roles of microglia-specific Atg7-deficiency in AAV-hα-Syn mice. b After 6 weeks after AAV-hα-Syn, brains from Atg7flox/flox mice (n = 4) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox mice (n = 3) were homogenized and fractionated into a detergent-soluble fraction and detergent-insoluble fraction and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP. Actin were used as loading controls. Arrow indicates the EYFP band. c Experimental plan to test the roles of microglia-specific Atg7-deficiency in hα-Syn-Tg mice. d, e At 7 months after tamoxifen treatment, brain slices were fixed and stained with antibodies against human α-synuclein, p62, and Iba-1. Representative 3D reconstruction pictures of microglia containing p62 and human α-synuclein in the striatum of Atg7flox/flox mice and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox mice bred with hα-Syn-Tg mice were produced using Imaris (Bitplane, d). The number of p62 puncta or p62-positive/hα-Syn-positive puncta was quantified in microglia at striatum of Atg7flox/flox; hα-Syn-Tg mice (n = 5) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 5) (e). p-values were calculated by two-tailed Mann–Whitney U test. Scale bar, 10 µm. f At 7 months after tamoxifen treatment, brains from Atg7flox/flox; hα-Syn-Tg mice (n = 7) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 6) were homogenized and fractionated into detergent-soluble fraction and detergent-insoluble fraction, and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP for confirming the presence of Cx3cr1CreER-IRES-Eyfp. Band intensities were quantified in the lower panel. p-values were calculated by unpaired two-tailed Student’s t test. g–j p-S129-α-synuclein-positive structures were visualized in brain sections from Atg7flox/flox; hα-Syn-Tg mice (n = 7) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 6) through DAB staining method (g, i), and quantified (h, j). p-values were calculated by unpaired two-tailed Student’s t test. Scale bar, 100 µm. All values are reported as mean ± SEM. Data are representative of at least three independent experiments.
Fig. 6 Autophagy-deficient microglia cause α-synuclein accumulation. a Experimental plan to test the roles of microglia-specific Atg7-deficiency in AAV-hα-Syn mice. b After 6 weeks after AAV-hα-Syn, brains from Atg7flox/flox mice (n = 4) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox mice (n = 3) were homogenized and fractionated into a detergent-soluble fraction and detergent-insoluble fraction and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP. Actin were used as loading controls. Arrow indicates the EYFP band. c Experimental plan to test the roles of microglia-specific Atg7-deficiency in hα-Syn-Tg mice. d, e At 7 months after tamoxifen treatment, brain slices were fixed and stained with antibodies against human α-synuclein, p62, and Iba-1. Representative 3D reconstruction pictures of microglia containing p62 and human α-synuclein in the striatum of Atg7flox/flox mice and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox mice bred with hα-Syn-Tg mice were produced using Imaris (Bitplane, d). The number of p62 puncta or p62-positive/hα-Syn-positive puncta was quantified in microglia at striatum of Atg7flox/flox; hα-Syn-Tg mice (n = 5) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 5) (e). p-values were calculated by two-tailed Mann–Whitney U test. Scale bar, 10 µm. f At 7 months after tamoxifen treatment, brains from Atg7flox/flox; hα-Syn-Tg mice (n = 7) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 6) were homogenized and fractionated into detergent-soluble fraction and detergent-insoluble fraction, and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP for confirming the presence of Cx3cr1CreER-IRES-Eyfp. Band intensities were quantified in the lower panel. p-values were calculated by unpaired two-tailed Student’s t test. g–j p-S129-α-synuclein-positive structures were visualized in brain sections from Atg7flox/flox; hα-Syn-Tg mice (n = 7) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 6) through DAB staining method (g, i), and quantified (h, j). p-values were calculated by unpaired two-tailed Student’s t test. Scale bar, 100 µm. All values are reported as mean ± SEM. Data are representative of at least three independent experiments.CC BY · PubMed Central
Fig. 7 Autophagy-deficient microglia promote neurodegeneration. a, b At 7 months after tamoxifen treatment, brains from Atg7flox/flox; hα-Syn-Tg mice (n = 7) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 6) were fixed and stained using an antibody against tyrosine hydroxylase (TH) using DAB method (a). TH-positive cells and Nissl-positive cells in the SNpc area were counted (b). p-values were calculated by unpaired two-tailed Student’s t test. Scale bar, 100 µm. c, d At 12 months after tamoxifen treatment, TH-positive cells and Nissl-positive cells were counted in SNpc area of Atg7flox/flox (n = 6) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox (n = 6) brains. e Hypothetical model of microglial synucleinphagy. All values are reported as mean ± SEM.
Fig. 7 Autophagy-deficient microglia promote neurodegeneration. a, b At 7 months after tamoxifen treatment, brains from Atg7flox/flox; hα-Syn-Tg mice (n = 7) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox; hα-Syn-Tg mice (n = 6) were fixed and stained using an antibody against tyrosine hydroxylase (TH) using DAB method (a). TH-positive cells and Nissl-positive cells in the SNpc area were counted (b). p-values were calculated by unpaired two-tailed Student’s t test. Scale bar, 100 µm. c, d At 12 months after tamoxifen treatment, TH-positive cells and Nissl-positive cells were counted in SNpc area of Atg7flox/flox (n = 6) and Cx3cr1CreER-IRES-Eyfp; Atg7flox/flox (n = 6) brains. e Hypothetical model of microglial synucleinphagy. All values are reported as mean ± SEM.CC BY · PubMed Central

Verification

Claims the paper makes

77 Own result · 32 Own interpretation · 14 Partly supported · 10 No full text of the sources · 7 Supported · 7 Own method · 4 Mixed support · 3 Couldn’t confirm · 2 Supported so far · accuracy not yet measured

Show all 113 claims

  • Probably not a substantial claim. 27
  • It may state two separate claims as one. 19
43 of 43
  • The paper relies on this without support.

    The high molecular weight p62 species observed by co-immunoprecipitation is oligomeric p62.

    “supporting that oligomeric p62 directly binds and recruits hα-Syn into autophagosomes.”
  • The paper relies on this without support.

    Direct evidence for autophagy in selective degradation of α-synuclein is entirely lacking.

    “Direct evidence for autophagy in selective degradation of α-synuclein is entirely lacking.”
  • The paper relies on this without support.

    Whether microglia take part in the degradation of α-synuclein remains unclear.

    “whether or not microglia, the prototypical scavenger cell in the brain, take part in the degradation of α-synuclein remains unclear.”
  • The paper relies on this without support.

    The exact mechanism for the clearance of internalized α-synuclein remains unclear.

    “However, the exact mechanism for the clearance of internalized α-synuclein remains unclear.”
  • The paper relies on this without support.

    The two mouse models used (AAV9-hα-Syn and hα-Syn-Tg) faithfully model neuron-released α-synuclein relevant to human synucleinopathies.

    “we employed two mouse models, both of which express wild-type (WT) human α-synuclein (hα-Syn) in a neuron-specific manner.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Statistics that don’t add up

Each reported p-value recomputed from its test statistic and degrees of freedom.

1 of 1

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • Doesn’t add upF(3, 6) = 11.46reported p = .0003 · recomputed p = .007

    “F(3, 6) = 11.46, p = .0003”

  • ConsistentF(5, 168) = 139.6reported p = 6.43e-58 · recomputed p < .001

    “F(5,168) = 139.6, p = 6.43e-58”

  • ConsistentF(5, 162) = 118.4reported p = 3.14e-52 · recomputed p < .001

    “F(5,162) = 118.4, p = 3.14e-52”

  • ConsistentF(3, 130) = 39.88reported p = 2.43e-18 · recomputed p < .001

    “F(3,130)=39.88, p = 2.43e-18”

  • ConsistentF(3, 8) = 13.44reported p = .0017 · recomputed p = .002

    “F(3,8) = 13.44, p = .0017”

  • ConsistentF(3, 20) = 35.43reported p = 3.39e-8 · recomputed p < .001

    “F(3,20) = 35.43, p = 3.39e-08”

  • ConsistentF(1, 139) = 272.5reported p = 1.44e-34 · recomputed p < .001

    “F(1,139)=272.5, p = 1.44e-34”

  • ConsistentF(1, 139) = 315.3reported p = 1.45e-37 · recomputed p < .001

    “F(1,139) = 315.3, p = 1.45e-37”

  • ConsistentF(1, 139) = 303.7reported p = 8.81e-37 · recomputed p < .001

    “F(1,139)=303.7, p = 8.81e-37”

  • ConsistentF(3, 16) = 3.381reported p = .0443 · recomputed p = .044

    “F(3,16) = 3.381, p = .0443”

  • ConsistentF(1, 16) = 43.09reported p = .00000651 · recomputed p < .001

    “F(1,16) = 43.09, p = 6.51e-06”

  • ConsistentF(3, 40) = 3.199reported p = .0334 · recomputed p = .033

    “F(3,40) = 3.199, p = .0334”

  • ConsistentF(3, 40) = 8.326reported p = .0002 · recomputed p < .001

    “F(3,40) = 8.326, p = .0002”

  • ConsistentF(1, 40) = 21.77reported p = .0000341 · recomputed p < .001

    “F(1,40) = 21.77, p = 3.41e-05”

  • ConsistentF(2, 174) = 12.56reported p = .00000803 · recomputed p < .001

    “F(2,174) = 12.56, p = 8.03e-06”

  • ConsistentF(2, 174) = 91.61reported p = 6.64e-28 · recomputed p < .001

    “F(2,174) = 91.61, p = 6.64e-28”

  • ConsistentF(1, 174) = 24.89reported p = .00000146 · recomputed p < .001

    “F(1,174) = 24.89, p = 1.46e-06”

  • ConsistentF(2, 92) = 156.7reported p = 2.35e-30 · recomputed p < .001

    “F(2,92) = 156.7, p = 2.35e-30”

  • ConsistentF(3, 20) = 35.43reported p = 3.39e-8 · recomputed p < .001

    “F(3,20)=35.43, p = 3.39e-08”

  • ConsistentF(3, 20) = 6.655reported p = .0027 · recomputed p = .003

    “F(3,20) = 6.655, p = .0027”

  • ConsistentF(3, 20) = 8.907reported p = .0006 · recomputed p < .001

    “F(3,20) = 8.907, p = .0006”

  • ConsistentF(3, 20) = 5.104reported p = .0087 · recomputed p = .009

    “F(3,20) = 5.104, p = .0087”

  • ConsistentF(3, 23) = 19.53reported p = .00000161 · recomputed p < .001

    “F(3,23) = 19.53, p = 1.61e-06”

  • ConsistentF(3, 23) = 4.643reported p = .0111 · recomputed p = .011

    “F(3,23) = 4.643, p = .0111”

  • ConsistentF(3, 23) = 12.09reported p = .0000592 · recomputed p < .001

    “F(3,23) = 12.09, p = 5.92e-05”

  • ConsistentF(3, 23) = 35.86reported p = 7.68e-9 · recomputed p < .001

    “F(3,23)=35.86, p = 7.68e-09”

  • ConsistentF(2, 18) = 15.1reported p = .0001 · recomputed p < .001

    “F(2,18) = 15.10, p = .0001”

  • ConsistentF(4, 24) = 9.196reported p = .0001 · recomputed p < .001

    “F(4,24) = 9.196, p = .0001”

  • ConsistentF(4, 24) = 4.53reported p = .0072 · recomputed p = .007

    “F(4,24) = 4.530, p = .0072”

  • ConsistentF(4, 24) = 0.8644reported p = .4994 · recomputed p = .499

    “F(4,24) = 0.8644, p = .4994”

  • ConsistentF(4, 24) = 1.369reported p = .2741 · recomputed p = .274

    “F(4,24) = 1.369, p = .2741”

  • ConsistentF(4, 24) = 5.372reported p = .0031 · recomputed p = .003

    “F(4,24) = 5.372, p = .0031”

  • ConsistentF(3, 12) = 3.75reported p = .0413 · recomputed p = .041

    “F(3,12) = 3.750, p = .0413”

  • ConsistentF(4, 160) = 56.21reported p = 1.53e-29 · recomputed p < .001

    “F(4,160) = 56.21, p = 1.53e-29”

  • ConsistentF(3, 40) = 20.7reported p = 2.95e-8 · recomputed p < .001

    “F(3,40) = 20.70, p = 2.95e-08”

  • ConsistentF(3, 40) = 69.43reported p = 6.55e-16 · recomputed p < .001

    “F(3,40) = 69.43, p = 6.55e-16”

  • ConsistentF(1, 40) = 69.57reported p = 2.74e-10 · recomputed p < .001

    “F(1,40) = 69.57, p = 2.74e-10”

  • ConsistentF(3, 40) = 10.12reported p = .0000432 · recomputed p < .001

    “F(3,40) = 10.12, p = 4.32e-05”

  • ConsistentF(3, 40) = 21.73reported p = 1.64e-8 · recomputed p < .001

    “F(3,40) = 21.73, p = 1.64e-08”

  • ConsistentF(1, 40) = 34.36reported p = 7.39e-7 · recomputed p < .001

    “F(1,40) = 34.36, p = 7.39e-07”

  • ConsistentF(3, 20) = 22.11reported p = .00000146 · recomputed p < .001

    “F(3,20) = 22.11, p = 1.46e-06”

  • ConsistentF(3, 20) = 11.88reported p = .0001 · recomputed p < .001

    “F(3,20) = 11.88, p = .0001”

  • ConsistentF(4, 80) = 19.63reported p = 2.74e-11 · recomputed p < .001

    “F(4,80) = 19.63, p = 2.74e-11”

  • ConsistentF(4, 80) = 27.31reported p = 2.66e-14 · recomputed p < .001

    “F(4,80) = 27.31, p = 2.66e-14”

  • ConsistentF(1, 80) = 47.95reported p = 9.9e-10 · recomputed p < .001

    “F(1,80) = 47.95, p = 9.90e-10”

  • ConsistentF(5, 126) = 6.07reported p = .0000454 · recomputed p < .001

    “F(5,126) = 6.070, p = 4.54e-05”

  • ConsistentF(5, 126) = 6.07reported p = .0000454 · recomputed p < .001

    “F(5,126) =6.070, p = 4.54e-05”

  • ConsistentF(1, 126) = 73.83reported p = 2.77e-14 · recomputed p < .001

    “F(1,126) = 73.83, p = 2.77e-14”

  • ConsistentF(3, 64) = 16.98reported p = 3.19e-8 · recomputed p < .001

    “F(3,64) = 16.98, p = 3.19e-08”

  • ConsistentF(3, 64) = 177.4reported p = 5.9e-31 · recomputed p < .001

    “F(3,64) = 177.4, p = 5.90e-31”

  • ConsistentF(1, 64) = 17.47reported p = .0000903 · recomputed p < .001

    “F(1,64) = 17.47, p = 9.03e-05”

51 of 51

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

1 of 1

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

1 of 1

Citations

Cites

1 retracted

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Metadata

Authors
Choi, I., Zhang, Y., Seegobin, S. P., Pruvost, M., Wang, Q., Purtell, K., Zhang, B., & Yue, Z.
Affiliations
Icahn School of Medicine at Mount Sinai (US)
Corresponding author
Yue, Zhenyu
Venue
Nature Communications
Published
2020-03-13
Volume
11
Issue
1
Pages
1386-1386
ISSN
2041-1723 (ISSN-L)
OpenAlex
W3011306000
Cited by
549
References
78
Citations by year
2026: 71 · 2025: 112 · 2024: 97 · 2023: 75 · 2022: 100 · 2021: 69 · 2020: 25
FWCI
22.04
Type
journal article
Language
en
Open access
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NS060123, P50NS094733)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
Keywords
microglia, alpha-synuclein, selective autophagy, TLR4, NF-κB signaling, p62, neurodegeneration, dopaminergic neuron loss, neuroprotection, autophagy receptors, Parkinson's disease
MeSH terms
Animals, Autoantigens, Autophagy, Brain, Disease Models, Animal, Female, Humans, Mesencephalon, Mice, Inbred C57BL, Signal Transduction, NF-kappa B, Microglia, Mice, Knockout, Neurodegenerative Diseases, Toll-Like Receptor 4, Mice, alpha-Synuclein, HEK293 Cells, Dopaminergic Neurons

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-10-15, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-10-15 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Cited sources are checked with a language model’s help (gpt-6-sol).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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