TNF-α promotes α-synuclein propagation through stimulation of senescence-associated lysosomal exocytosis

Abstract

Cell-to-cell propagation of α-synuclein is thought to be the underlying mechanism of Parkinson's disease progression. Recent evidence suggests that inflammation plays an important role in the propagation of protein aggregates. However, the mechanism by which inflammation regulates the propagation of aggregates remains unknown. Here, using in vitro cultures, we found that soluble factors secreted from activated microglia promote cell-to-cell propagation of α-synuclein and further showed that among these soluble factors, TNF-α had the most robust stimulatory activity. Treatment of neurons with TNF-α triggered cellular senescence, as shown by transcriptomic analyses demonstrating induction of senescence-associated genes and immunoanalysis of senescence phenotype marker proteins. Interestingly, secretion of α-synuclein was increased in senescent neurons, reflecting acquisition of a senescence-associated secretory phenotype (SASP). Using vacuolin-1, an inhibitor of lysosomal exocytosis, and RNAi against rab27a, we demonstrated that the SASP was mediated by lysosomal exocytosis. Correlative light and electron microscopy and immunoelectron microscopy confirmed that propagating α-synuclein aggregates were present in electron-dense lysosome-like compartments. TNF-α promoted the SASP through stimulation of lysosomal exocytosis, thereby increasing the secretion of α-synuclein. Collectively, these results suggest that TNF-α is the major inflammatory factor that drives cell-to-cell propagation of α-synuclein by promoting the SASP and subsequent secretion of α-synuclein.

Fig. 1. Soluble factors secreted from activated microglia increase the propagation of α-synuclein.

a, b Effects of activated microglia on the cell-to-cell propagation of α-synuclein. BiFC-positive cells are indicated with arrows. Scale bar: 10 μm. b Quantification of the percentage of BiFC-positive cells in (a). c Levels of neuron-secreted α-synuclein. The amount of released α-synuclein was determined by sandwich ELISAs. d, e Effects of transcription factor inhibitors on propagation-promoting activity. BiFC-positive cells are indicated with arrows. Scale bar: 20 µm. e Quantification of the percentage of BiFC-positive cells in (d). Bay, Bay 11-7085; SR, SR11302; PMX, polymyxin B. f Screening of inflammatory factors (50 ng/ml) for propagation-promoting activity. g Levels of secreted TNF-α in cultures of the microglia treated with LPS or vehicle, measured by ELISAs. All data are presented as the mean ± SEM (*P < 0.05, ^#P < 0.05, **P < 0.005, ^##P < 0.005, ****P < 0.0001). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc comparison between groups (b, e) and Dunnett’s post hoc comparison between groups (f) or by two-tailed unpaired Student’s t test (c, g).

Fig. 2. TNF-α regulates α-synuclein propagation.

a, b Effects of TNF-α on the cell-to-cell propagation of α-synuclein. a Representative images in which α-synuclein propagation was observed (treated dose: 50 ng/ml). BiFC-positive cells are indicated with arrows. Scale bar: 20 µm. b Quantification of the relative percentages of BiFC-positive cells. c, d Effects of MgCM from cultures of the WT (TNF-α^+/+) or TNF-α^−/− microglia treated with LPS (MgCM-LPS) or DMSO (MgCM-control) on the propagation of α-synuclein. Arrows: BiFC-positive puncta. Scale bar: 20 µm. d Percentage of BiFC-positive cells in (c). e–i Effects of TNF-α on the spreading of α-synuclein in vivo. e Representative images of phosphorylated α-synuclein (pS129) staining in mouse brain sections at 20 weeks post-injection of α-synuclein^V40G multimers (or PBS) into the WT (TNF-α^+/+) or TNF-α^−/− mice. Scale bar: 100 μm. f–i Levels of pS129. Optical densities were measured in the motor cortex (f; WT + PBS, n = 8; KO + PBS, n = 6; WT + α-synuclein, n = 7; KO + α-synuclein, n = 8), cingulate cortex (g; WT + PBS, n = 8; KO + PBS, n = 6; WT + α-synuclein, n = 7; KO + α-synuclein, n = 8), rhinal cortex (h; WT + PBS, n = 8; KO + PBS, n = 7; WT + α-synuclein, n = 8; KO + α-synuclein, n = 8), and amygdala (i; WT + PBS, n = 8; KO + PBS, n = 7; WT + α-synuclein, n = 7; KO + α-synuclein, n = 8). j–l Effects of TNF-α on the accumulation of α-synuclein in neurons. j Representative western blot images of α-synuclein in Triton X-100-soluble (Tx-sol) and Triton X-100-insoluble (Tx-insol) fractions. The quantified region is indicated on the right side of the blot. The arrowhead indicates quantified α-synuclein in Tx-sol. The line includes the quantified area in Tx-insol. Quantification of α-synuclein in Tx-sol (k) and Tx-insol (l) fractions. m, n Effects of TNF-α on the secretion of α-synuclein in neurons. m Secreted α-synuclein in media, measured by ELISAs. n Effects of TNF-α-deficient microglia on the neuronal secretion of α-synuclein. Primary neurons were treated with conditioned media acquired from the WT or TNF-α-deficient microglia pretreated with LPS or vehicle. Data are expressed as the mean ± SEM (*P < 0.05, ^#P < 0.05 **P < 0.005, ^##P < 0.005, ***P < 0.0005, ****P < 0.0001, ^####P < 0.0001). Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc comparison between groups (b, k–m), two-way ANOVA with Bonferroni’s post hoc comparison between groups (d), and two-way ANOVA with Tukey’s post hoc comparison between groups (f–i, n).

Fig. 3. TNF-α induces neuronal senescence.

a Heatmap representing the expression levels (log2 read count number) of DEGs with upregulated (fold change >1.3) or downregulated (fold change <0.7) expression after treatment with TNF-α or vehicle (n = 5 per group). b The top 12 enriched KEGG terms for the 118 DEGs in the TNF-α-treated neurons. c Simplified networks of significantly enriched GO terms. Each term is statistically significant (Benjamini–Hochberg correction <0.05). The nodes (colored circles) display significantly enriched parent GO terms; the edges show the overlapping genes between terms; and the size of the node represents the number of enriched genes. d Enrichment plot of DEGs in the TNF-α-treated versus vehicle-treated neurons (FDR q-value <0.005). e Heatmap of the expression levels of 22 DEGs related to the JAK-STAT senescence pathway. f High confidence protein–protein interaction network of the 22 DEGs related to the JAK-STAT senescence pathway, constructed using STRING. g Heatmap of the log2-fold changes of the 22 DEGs related to the JAK-STAT senescence pathway. h–k Expression of CDKN1A (h), TP53 (i), JAK1 (j), and STAT1 (k), measured by RT-qPCR. l Immunofluorescence analysis of p21 protein in primary neurons treated with TNF-α or vehicle. m Quantification of relative p21 fluorescence in the nucleus. n C12FDG staining representing the accumulation of SA-β-gal. Nuclei were stained with DAPI (blue). o Quantification of C12FDG levels. p–r SASP genes GROα (p), IL-6 (q), and IL-1α (r), measured by RT-qPCR. All data are presented as the mean ± SEM (*P < 0.05, **P < 0.005, ***P < 0.0005). Statistical significance was determined by two-tailed unpaired Student’s t test (h–k, m, o, p–r).

Fig. 4. Neuronal senescence modulates α-synuclein propagation.

a–d Secretion of α-synuclein during neuronal aging. a Representative western blot images of α-synuclein in Triton X-100-soluble (Tx-sol) and Triton X-100-insoluble (Tx-insol) fractions and in culture media (Media). Asterisks indicate nonspecific antibody binding. The arrowhead indicates the quantified α-synuclein. Quantification of α-synuclein in Tx-sol (b), Tx-insol (c), and media (d). e–h Inhibition of p21 ameliorated senescence-induced secretion of α-synuclein. e Representative western blot images of α-synuclein in Tx-sol, Tx-insol, and media. Asterisks indicate nonspecific antibody binding. The arrowhead indicates the quantified α-synuclein. Quantification of α-synuclein in Tx-sol (f), Tx-insol (g), and media (h). The levels of α-synuclein in cell extracts and media were normalized to the values of β-actin and secretogranin II (SGII), respectively (n = 3). i, j Effects of UC2288 (p21 inhibitor) on the cell-to-cell propagation of α-synuclein. BiFC-positive cells are indicated with arrowheads. Scale bar: 20 µm. j Quantification of BiFC-positive cells (n = 3, minimum 500 cells per experiment). k–n Inhibition of p21 reduced TNF-α-induced secretion of α-synuclein. k Representative western blot images of α-synuclein in the Tx-sol and Tx-insol fractions. The arrowhead indicates the quantified α-synuclein in Tx-sol. The bar on the right side of the blot indicates the quantified area in Tx-insol. Quantification of α-synuclein in the Tx-sol (l) and Tx-insol (m) fractions. n Secreted α-synuclein, measured by ELISAs. All data are presented as the mean ± SEM (*P < 0.05, ***P < 0.0005, ****P < 0.0001). Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc comparison between groups (b–d), two-way ANOVA with Tukey’s post hoc comparison between groups (f–h, l–n), or two-tailed unpaired Student’s t test (j).

Fig. 5. Senescence-associated secretion of α-synuclein is regulated by lysosomal exocytosis.

a Levels of secreted β-hexosaminidase during neuronal senescence (n = 9). b Effects of vacuolin-1 on the senescence-induced secretion of β-hexosaminidase (n = 3). c Correlation between α-synuclein and β-hexosaminidase secretion during senescence (n = 3). d Effects of UC2288 (p21 inhibitor) on senescence-induced secretion of β-hexosaminidase (n = 3). e Correlation between α-synuclein and β-hexosaminidase secretion in the presence or absence of UC2288 (n = 3). f–i Inhibition of lysosomal exocytosis reduced senescence-induced secretion of α-synuclein. f Representative western blot images of α-synuclein in the Triton X-100-soluble (Tx-sol) fraction, Triton X-100-insoluble (Tx-insol) fraction, and culture media (Media). The arrowhead indicates the quantified synuclein. Asterisks indicate nonspecific antibody binding. Quantification of α-synuclein in Tx-sol (g), Tx-insol (h), and media (i). j Correlation between α-synuclein and β-hexosaminidase secretion in the presence or absence of vacuolein-1 (n = 3). k Subcellular localization of α-synuclein. Green, BiFC-positive α-synuclein aggregates; red, LAMP1; blue, nuclei. Scale bar: 20 μm. Lower panels 1 & 2: magnification of areas bounded by squares in upper panels. l CLEM analysis of BiFC-positive structures. Lower panels iv & vi: magnifications of the two BiFC puncta (1 and 2) in Panels i, ii & iii, respectively. Scale bar: 4 μm. Panels v & vii: magnifications of BiFC puncta 1 and 2. m Immunoelectron microscopy analysis of α-synuclein-positive lysosome-like structures. Blue arrowheads indicate α-synuclein; red arrows indicate CD63 (upper panel) and LAMP1 (lower panel). Scale bar: 200 nm. n–r Effects of RAB27a knockdown on α-synuclein secretion. n, o RAB27a knockdown efficiency. Quantification of RAB 27a in (o) (n = 3). p Representative western blot images of α-synuclein in the Tx-sol and Tx-insol fractions. The bar on the right side of the blot indicates the quantified area in the Tx-insol fraction. q Quantification of α-synuclein in the Tx-insol fraction (n = 3). r Quantification of α-synuclein in culture media by ELISAs (n = 3). s, t Effect of RNAi-mediated RAB27a knockdown on α-synuclein propagation in C. elegans. s Venus fluorescence in the pharynx, representing the extent of cell-to-cell propagation of α-synuclein in this C. elegans model. Quantification of Venus fluorescence at Day 2. More than fifty worms for each line were analyzed (n = 3). u–w Effects of vacuolin-1 on α-synuclein propagation. u Representative images of BiFC in passages 1, 3, and 5 in the presence or absence of vacuolin-1. Arrows indicate BiFC puncta. Scale bar: 20 μm. v Quantification of the BiFC-positive cells in (u) (n = 3; minimum 1000 cells per experiment). w Quantification of BiFC fluorescence (secreted α-synuclein) in culture media (n = 3). The levels of α-synuclein and RAB27a in cell extracts were normalized to those of β-actin, and the levels of proteins in media were normalized to those of secretogranin II (SGII). All data are presented as the mean ± SEM (*P < 0.05, **P < 0.005 ***P < 0.0005, ^###P < 0.0005, ****P < 0.0001, ^####P < 0.0001). Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc comparison between groups (a), two-way ANOVA with Tukey’s post hoc comparison between groups (b, d, g–i, v, w), or two-tailed unpaired Student’s t test (o, q, r, t).

Fig. 6. TNF-α-induced SASP mediates secretion of α-synuclein through lysosomal exocytosis.

a–d Inhibition of lysosomal exocytosis reduced TNF-α-induced secretion of α-synuclein. a Representative western blot images of α-synuclein in Triton X-100-soluble (Tx-sol) and Triton X-100-soluble-insoluble (Tx-insol) fractions. The arrowhead indicates the quantified α-synuclein in Tx-sol. The line on the right side of the blot indicates the quantified area in Tx-insol. Quantification of α-synuclein in the Tx-sol (b) and Tx-insol (c) fractions. d Secreted α-synuclein, measured by ELISAs. e, f Effects of vacuolin-1 (e) and UC2288 (f) on TNF-α-induced secretion of β-hexosaminidase. All data are expressed as the mean ± SEM (*P < 0.05, **P < 0.005, ^####P < 0.0001). Statistical significance was determined by two-way ANOVA with Tukey’s post hoc comparison between groups (b–f).

Fig. 7. The SASP of neurons induced by TNF-α regulates α-synuclein propagation through lysosomal exocytosis.

Activated microglia secrete soluble factors, including TNF-α, which induce neuronal senescence. The SASP of senescent neurons increases α-synuclein secretion through lysosomal exocytosis. The α-synuclein thus secreted is not only transferred to connected neurons as part of cell-to-cell aggregate propagation but also activates glial cells to sustain inflammatory responses.