Manganese promotes the aggregation and prion-like cell-to-cell exosomal transmission of α-synuclein

Abstract

The aggregation of α-synuclein (αSyn) is considered a key pathophysiological feature of certain neurodegenerative disorders, collectively termed synucleinopathies. Given that a prion-like, cell-to-cell transfer of misfolded αSyn has been recognized in the spreading of αSyn pathology in synucleinopathies, we investigated the biological mechanisms underlying the propagation of the disease with respect to environmental neurotoxic stress. Considering the potential role of the divalent metal manganese (Mn²⁺) in protein aggregation, we characterized its effect on αSyn misfolding and transmission in experimental models of Parkinson's disease. In cultured dopaminergic neuronal cells stably expressing wild-type human αSyn, misfolded αSyn was secreted through exosomes into the extracellular medium upon Mn²⁺ exposure. These exosomes were endocytosed through caveolae into primary microglial cells, thereby mounting neuroinflammatory responses. Furthermore, Mn²⁺-elicited exosomes exerted a neurotoxic effect in a human dopaminergic neuronal model (LUHMES cells). Moreover, bimolecular fluorescence complementation (BiFC) analysis revealed that Mn²⁺ accelerated the cell-to-cell transmission of αSyn, resulting in dopaminergic neurotoxicity in a mouse model of Mn²⁺ exposure. Welders exposed to Mn²⁺ had increased misfolded αSyn content in their serum exosomes. Stereotaxically delivering αSyn-containing exosomes, isolated from Mn²⁺-treated αSyn-expressing cells, into the striatum initiated Parkinsonian-like pathological features in mice. Together, these results indicate that Mn²⁺ exposure promotes αSyn secretion in exosomal vesicles, which subsequently evokes proinflammatory and neurodegenerative responses in both cell culture and animal models.

Figure 1:. Mn upregulates exosomal release of oligomeric αSyn.

(A) Immunofluorescence of stably expressed GFP-fused human αSyn (red) in GFP_Syn M9ND cells and GFP fluorescence (green) in both control GFP_EV and human αSyn-expressing GFP_Syn cells. Hoechst dye stained the nuclei (blue). Magnification, 60X. Scale bar, 10 μm. (B) Western blots (WBs) of GFP_Syn and GFP_EV cells for human αSyn (~45 kDa) in GFP_Syn cells and endogenous mouse αSyn (18 kDa). (C) Representative WBs of conditioned medium from cells in (B), control or exposed to Mn (300 μM), for GFP-fused αSyn and LDHA. (D) TEM to examine the morphology of secreted exosomes from GFP_Syn cells. Scale bar, 100 nm. (E) Western blot analysis for αSyn abundance in MN9D cells, conditioned media and exosomes. (F) Representative NanoSight Particle tracking, indicating size and concentration of exosomes from GFP_Syn cells, from vehicle-stimulated (red) and Mn-stimulated (blue) cells. (G) WBs for GFP-fused human αSyn in exosomes from GFP_Syn and GFP_EV cells. Exosome-positive markers flotillin-1 and Aip-1/Alix were enriched in both cell types. Slot blotting (SS) of exosome lysates indicates A11-positive oligomeric proteins and fibrillar αSyn in Mn-stimulated exosomes. (H) RT-QuIC of Mn-stimulated or vehicle-stimulated exosomes from GFP_Syn and GFP_EV cells to assess the abundance of misfolded αSyn.

Figure 2:. Mn-stimulated exosomes promote neuroinflammatory responses.

(A) Immunofluorescence analysis of primary microglial cells (IBA1; red color) exposed to exosomes (GFP; green color). Hoechst dye stained the nuclei (blue). Magnification, 60X; scale bar, 10 μm. Amoeboid and pseudopodic morphology of primary microglial cells exposed to Mn-stimulated αSyn exosomes was visually assessed (lower images). (B to D) Representative Western blots (B) and densitometry (C and D) assessing IBA-1 and iNOS abundance after exposure to Mn-stimulated αSyn exosomes, as a measure of their potential to promote neuroinflammatory responses in vitro. Data are mean ± SEM (*p≤0.05, **p<0.01 by one-way ANOVA with Tukey’s post-test) of five independent experiments. (E to H) Pro-inflammatory cytokine release upon exosome treatment was quantified using Luminex bead-based cytokine assays. Data are mean ± SEM (**p<0.01, ***p<0.001 by one-way ANOVA with Tukey’s post-test) of four individual experiments performed in 8 replicates.

Figure 3:. Microglia internalize Mn-stimulated αSyn exosomes through caveolin-1-mediated endocytosis.

(A) Immunofluorescence analysis of the chemical inhibition of Mn-stimulated αSyn exosome uptake. The left column represents merged images of IBA-1 immuno-positive microglia (red) and PKH67-labeled exosomes (green), middle column represents effective uptake/inhibition of PKH67-labeled exosomes (green), and the right column represents the 3D surface reconstruction generated by Imaris software. Magnification, 60X. Scale bar, 10 μm. (B to D) Inhibition of pro-inflammatory cytokine release quantified using Luminex bead-based cytokine assays. Data are mean ± SEM (*p≤0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Tukey’s post-test) of four individual experiments each performed with 8 technical replicates. (E) Effective inhibition of nitric oxide release from genistein and Dynasore (each 50 μM)-treated WTMC cells observed through Griess assay. Data are mean ± SEM (***p<0.001, ns = not significant) of four individual experiments performed in 8 replicates. (F to I) Assessment of pro-inflammatory cytokine release upon treatment of caveolin-1– or clathrin–knockdown (Cav1-KD and CLTC-KD, respectively) primary murine microglial cells with Mn-stimulated αSyn exosomes, quantified using Luminex bead-based cytokine assay. Data are mean ± SEM (**p<0.01, ***p<0.001 by one-way ANOVA with Tukey’s post-test) of four individual experiments performed in 8 replicates.

Figure 4:. Mn-induced cell-to-cell transmission of αSyn oligomers.

(A and B) Confocal microscopy assessing BiFC for control and Mn-treated V1S/SV2 co-cultures. Magnification, 60X; scale bar, 10 μm. As a control, cells transfected with V1S-alone and SV2-alone (B) did not fluoresce. (C) Exosomal αSyn abundance detected in the conditioned media from V1S/SV2 co-cultures. (D) Representative FACS scatter plots assessing BiFC-positive cells in vehicle- and Mn-treated S1V/SV2 co-transfection. (E) FACS analysis of BiFC-positive cells transfected with S1V, SV2, or both in control and Mn-treated cultures. Data are mean ± SEM of four experiments performed in duplicates; **p<0.01 by one-way ANOVA with Tukey’s post-test. (F) VenusYFP epifluorescence in SNpc. VenusYFP fluorescence (green, high-magnification inset) colocalized with SNpc TH-immunostaining (red). Hoechst dye stained nuclei (blue). Magnification, 60X; scale bar, 10 μm. Diagram illustrates injection (mm from bregma) of AAV8-V1S and AAV8-SV2. (G) Highest VenusYFP epifluorescence in Mn-exposed animals, localized via BiFC epifluorescence overlay. (H) Increased BiFC fluorescence in Mn-exposed mice. Data are mean ± SEM from 7 animals/group; *p≤0.05 by Student’s t-test. (I to K) Representative movement tracks (I), number of movements (J), and total distance traveled (K) of control and Mn-exposed mice. Data are mean ± SEM of ≥12 animals per group; *p≤0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Tukey’s post-test. (L and M) DAB-based detection (L) and stereological counting (K) of TH-positive neurons in coronal SNpc sections from control and Mn-exposed mice. Images (L) are representative, at 2X magnification; arrows indicate loss of TH-positive neurons in Mn-treated mice. Data (M) are mean ± SEM from 7 animals per group; **p<0.01 and ***p<0.001 by one-way ANOVA with Tukey’s post-test. (N) Horizontal activity of the mice described and analyzed in (I to K).

Figure 5:. Mn exposure promotes exosome release in αSyn-A53T transgenic animals and αSyn oligomer transmission in humans.

(A and B) Concentration (A) and representative NanoSight Particle tracking size distribution plot (B) of serum exosomes isolated from αSyn-A53T transgenic and WT rats exposed to Mn (15 mg/kg body weight per day) or vehicle for 30 days (n = 7 rats per group). *p≤0.05 by Kruskal-Wallis with Dunn’s multiple comparison test. (C and D) Scatter plots of total serum αSyn concentration (C) and total serum exosome concentration (D) measured by αSyn ELISA and NanoSight, respectively (ns = not significant; P = 0.2855 and 0.6472, by Student’s t-test, respectively). Data are mean ± SEM of 8 welders and 10 control human samples. (E and F) RT-QuIC assay comparing exosomes isolated from welders and control humans. Blue and red shaded areas (E) represent SEM of the mean ThT fluorescence for welder and control samples; (F) analysis of relative mean ThT fluorescence intensity in the groups. Data are from n= 10 samples per group; ***p<0.001 by Student’s t-test (G and H) Scatter plots (G) of the densitometry analysis of the dot blots (H) assessing misfolded αSyn content in welder-derived and control individual-derived serum exosomes. Data are mean ± SEM of n ≥ 7 samples each; ns = not significant by Student’s t-test.

Figure 6:. Mn-stimulated αSyn exosomes induce Parkinson-like motor deficits in non-transgenic mice.

(A) Diagram illustrating route and coordinates (mm from bregma) of stereotaxic exosome injections (coronal view). Exosomes were inoculated into the left hemisphere at subdural depths, indicated using a single needle tract. (B and C) Open-field behavior analysis measured using VersaMax apparatus, assessing stereotypy counts (B) and movement time (C) in C57BL/6 mice injected with exosomes isolated from Mn- or vehicle-treated GFP_EV and GFP_Syn cells. Data are mean ± SEM of n ≥12 animals per group; *p≤0.05; ns = not significant by one-way ANOVA with Tukey’s post-test. (D) Immunohistological analysis of phosphorylated αSyn (pSyn129/81A), p62 immunoreactivity, and primary microglial cells (IBA1) in brain tissue from mice injected with Mn-stimulated αSyn exosomes. Corresponding brain regions shown in far-left panel. Magnification, 40X; scale bar, 25 μm. (E) Amphetamine-induced rotation test. The graph shows net scores for ipsilateral rotational asymmetry (number and direction of rotations) induced by amphetamine 180 days post-lesioning in mice receiving vehicle- or Mn-stimulated GFP or αSyn exosomes.