Research ArticleNeurodegeneration

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

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Sci. Signal.  12 Mar 2019:
Vol. 12, Issue 572, eaau4543
DOI: 10.1126/scisignal.aau4543
  • Fig. 1 Mn2+up-regulates 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_empty vector (EV) and human αSyn–expressing GFP_Syn cells. Hoechst dye stained the nuclei (blue). Magnification, 60×. Scale bar, 10 μm. (B) Western blots of GFP_Syn and GFP_EV cells for human αSyn (~45 kDa) in GFP_Syn cells and endogenous mouse αSyn (18 kDa). (C) Representative Western blots of conditioned medium from cells in (B), control or exposed to Mn2+ (300 μM), for GFP-fused αSyn and LDHA. (D) Transmission electron microscopy (TEM) to examine the morphology of secreted exosomes from GFP_Syn cells. (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 Mn2+-stimulated (blue) cells. (G) Immuoblots (IBs) 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 Mn2+-stimulated exosomes. Ab, antibody. (H) RT-QuIC of Mn2+-stimulated or vehicle-stimulated exosomes from GFP_Syn and GFP_EV cells to assess the abundance of misfolded αSyn. Data are representative of six experiments.

  • Fig. 2 Mn2+-stimulated exosomes promote neuroinflammatory responses.

    (A) Immunofluorescence analysis of primary microglial cells (IBA-1; red) exposed to exosomes (GFP; green). Hoechst dye stained the nuclei (blue). Magnification, 60×. Scale bar, 10 μm. Amoeboid and pseudopodic morphology of primary microglial cells exposed to Mn2+-stimulated αSyn exosomes was visually assessed (bottom images). Veh, Vehicle. (B to D) Representative Western blots (B) and densitometry (C and D) assessing IBA-1 and iNOS abundance after exposure to Mn2+-stimulated αSyn exosomes, as a measure of their potential to promote neuroinflammatory responses in vitro. Data are means ± SEM [*P ≤ 0.05 and **P < 0.01 by one-way analysis of variance (ANOVA) with Tukey’s posttest] of five independent experiments. (E to H) Proinflammatory cytokine release upon exosome treatment was quantified using Luminex bead-based cytokine assays. Data are means ± SEM (**P < 0.01 and ***P < 0.001 by one-way ANOVA with Tukey’s posttest) of four individual experiments performed in eight replicates.

  • Fig. 3 Microglia internalize Mn2+-stimulated αSyn exosomes through caveolin-1–mediated endocytosis.

    (A) Immunofluorescence analysis of the chemical inhibition of Mn2+-stimulated αSyn exosome uptake. The left column represents merged images of IBA-1–immunopositive microglia (red) and PKH67-labeled exosomes (green), the 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, 60×. Scale bar, 10 μm. (B to D) Inhibition of proinflammatory cytokine release quantified using Luminex bead-based cytokine assays. Data are means ± SEM (*P ≤ 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s posttest) of four individual experiments each performed with eight technical replicates. (E) Effective inhibition of nitric oxide release from genistein- and dynasore-treated (50 μM each) WTMC cells observed through Griess assay. Data are means ± SEM (***P < 0.001; ns, not significant) of four individual experiments performed in eight replicates. (F to I) Assessment of proinflammatory cytokine release upon treatment of caveolin-1 or clathrin-knockdown (KD) (Cav1-KD and CLTC-KD, respectively) primary murine microglial cells with Mn2+-stimulated αSyn exosomes, quantified using Luminex bead-based cytokine assay. Untrt, untreated; Chlo, Chlorpromazine; Geni, Genistein; Dyna, Dynasore. Data are means ± SEM (**P < 0.01 and ***P < 0.001 by one-way ANOVA with Tukey’s posttest) of four individual experiments performed in eight replicates.

  • Fig. 4 Mn2+-induced cell-to-cell transmission of αSyn oligomers.

    (A and B) Confocal microscopy assessing BiFC for control and Mn2+-treated V1S/SV2 cocultures. Magnification, 60×. Scale bars, 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 cocultures. (D) Representative FACS scatter plots assessing BiFC-positive cells in vehicle- and Mn2+-treated S1V/SV2 cotransfection. (E) FACS analysis of BiFC-positive cells transfected with S1V, SV2, or both in control and Mn2+-treated cultures. Data are means ± SEM of four experiments performed in duplicates; **P <0.01 by one-way ANOVA with Tukey’s posttest. ND, not detected. (F) VenusYFP epifluorescence in SNpc. VenusYFP fluorescence (green, high-magnification inset) colocalized with SNpc tyrosine hydroxylase (TH)–immunostaining (red). Hoechst dye–stained nuclei (blue). Magnification, 60×. Scale bar, 10 μm. Diagram illustrates injection (millimeters from the bregma) of AAV8-V1S and AAV8-SV2. AP, anterior posterior; ML, medial lateral; DV, dorsal ventral. (G) Highest VenusYFP epifluorescence in Mn2+-exposed animals, localized via BiFC epifluorescence overlay. (H) Increased BiFC fluorescence in Mn2+-exposed mice. Data are means ± SEM from seven animals per group; *P ≤ 0.05 by Student’s t test. (I to L) Representative movement tracks (I), number of movements (J), total distance traveled (K), and horizontal activity (L) of control and Mn2+-exposed mice. Data are means ± SEM of ≥12 animals per group; *P ≤ 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s posttest. (M and N) Diaminobenzidine (DAB)–based detection (M) and stereological counting (N) of TH-positive neurons in coronal SNpc sections from control and Mn2+-exposed mice. Images are representative, at 2× magnification; arrows indicate loss of TH-positive neurons in Mn2+-treated mice. Data are means ± SEM from seven animals per group; **P < 0.01 and ***P < 0.001 by one-way ANOVA with Tukey’s posttest.

  • Fig. 5 Mn2+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 Mn2+ (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 comparisons test. (C and D) Scatterplots of total serum αSyn concentration (C) and total serum exosome concentration (D) measured by αSyn ELISA and NanoSight, respectively (P = 0.2855 and 0.6472, respectively, by Student’s t tests). Data are means ± 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) Scatterplots (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 means ± SEM of n ≥ 7 samples each, by Student’s t test.

  • Fig. 6 Mn2+-stimulated αSyn exosomes induce Parkinson-like motor deficits in nontransgenic mice.

    (A) Diagram illustrating route and coordinates (millimeters from the bregma) of stereotaxic exosome injections (coronal view). Exosomes were inoculated into the left hemisphere at subdural depths, indicated using a single needle tract. Ctx, Cortex; Str, Striatum. (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 Mn2+- or vehicle-treated GFP_EV and GFP_Syn cells. Data are means ± SEM of n ≥ 12 animals per group; *P ≤ 0.05, by one-way ANOVA with Tukey’s posttest. (D) Amphetamine-induced rotation test. The graph shows net scores for ipsilateral rotational asymmetry (number and direction of rotations) induced by amphetamine 180 days after lesioning in mice receiving vehicle- or Mn2+-stimulated GFP or αSyn exosomes. (E) Immunohistological analysis of phosphorylated αSyn (pSyn129/81A), p62 immunoreactivity, and primary microglial cells (IBA-1) in brain tissue from mice injected with Mn2+-stimulated αSyn exosomes. Corresponding brain regions are shown in far-left panel. Magnification, 40×. Scale bar, 25 μm.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/572/eaau4543/DC1

    Fig. S1. Mn2+ increases exosomal αSyn release.

    Fig. S2. Exosomes were internalized through caveolin-mediated endocytosis.

    Fig. S3. GFP_Syn cell–derived exosomes induce neuronal cell death in vitro.

    Fig. S4. Visualization of cell-to-cell transmission of αSyn through protein fragment complementation assays.

    Fig. S5. Vehicle-stimulated GFP_Syn cell–derived exosomes result in minimal αSyn pathology in vivo.

  • This PDF file includes:

    • Fig. S1. Mn2+ increases exosomal αSyn release.
    • Fig. S2. Exosomes were internalized through caveolin-mediated endocytosis.
    • Fig. S3. GFP_Syn cell–derived exosomes induce neuronal cell death in vitro.
    • Fig. S4. Visualization of cell-to-cell transmission of αSyn through protein fragment complementation assays.
    • Fig. S5. Vehicle-stimulated GFP_Syn cell–derived exosomes result in minimal αSyn pathology in vivo.

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