Manganese is an essential mineral, but chronic exposure to high amounts of it, such as is experienced by welders, is associated with symptoms of Parkinson’s disease. A hallmark of the disease is aggregation of the protein α-synuclein (αSyn), which is toxic to neurons. Harischandra et al. found that extracellular vesicles called exosomes isolated from serum collected from welders contained misfolded αSyn. In cell culture and mouse models, exposure to manganese or to isolated, manganese-induced exosomes promoted the transfer of αSyn between neurons and microglia, which induced inflammation and neuronal cell death. These findings further explain the role of manganese in neurodegenerative disease.

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 (Mn 2+ ) 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 Mn 2+ exposure. These exosomes were endocytosed through caveolae into primary microglial cells, thereby mounting neuroinflammatory responses. Furthermore, Mn 2+ -elicited exosomes exerted a neurotoxic effect in a human dopaminergic neuronal model (LUHMES cells). Moreover, bimolecular fluorescence complementation (BiFC) analysis revealed that Mn 2+ accelerated the cell-to-cell transmission of αSyn, resulting in dopaminergic neurotoxicity in a mouse model of Mn 2+ exposure. Welders exposed to Mn 2+ had increased misfolded αSyn content in their serum exosomes. Stereotaxically delivering αSyn-containing exosomes, isolated from Mn 2+ -treated αSyn-expressing cells, into the striatum initiated Parkinsonian-like pathological features in mice. Together, these results indicate that Mn 2+ exposure promotes αSyn secretion in exosomal vesicles, which subsequently evokes proinflammatory and neurodegenerative responses in both cell culture and animal models.

Hence, in this study, we assessed the effects of environmental Mn 2+ on αSyn aggregation, secretion and cell-to-cell transmission. To elucidate the mechanism of Mn 2+ -induced αSyn release, we followed a systematic approach from in vitro to ex vivo and finally in vivo experimental models and human samples to better understand the role of exosomes in cell-to-cell transmission of misfolded αSyn protein.

Emerging evidence from many neurodegenerative disorders, including synucleinopathies, now has expanded the notion of cell-to-cell transmission of misfolded proteins as a common mechanism for the onset and progression of these diseases ( 15 – 18 ). Although the exact mechanisms for protein aggregate spreading in the CNS still largely remain unknown, several models including exocytosis, cell injury, receptor-mediated endocytosis, tunneling nanotubes, and exosomal transmission have been proposed ( 7 ). Although genetic predisposition is an important risk factor in many familial cases of Parkinsonian syndromes, environmental exposure to certain metals, herbicides, or insecticides has been linked to the pathogenesis of these diseases ( 19 ). This includes the divalent metal manganese (Mn 2+ ) that humans are exposed to through contaminated air and drinking water, as well as the use of Mn 2+ -containing consumer and agricultural products. In trace amounts, Mn 2+ is essential for human health, but environmental exposure to high doses of Mn 2+ results in manganism, a debilitating movement disorder sharing many Parkinsonian features, although it may not represent clinical PD, because manganism lacks the classic response to levodopa and certain distinctive neurological symptoms ( 20 ). Occupational exposure to Mn 2+ -containing welding fumes has been linked to increased risk of Parkinsonism ( 21 – 24 ). Yet, despite its prevalence and thus potential risk to human health and the development of neurodegenerative disorders, the mechanisms by which Mn 2+ exerts its neurotoxic effects and its role in the prion-like propagation of αSyn aggregates are not well understood thus far.

Accumulating evidence indicates that extracellular αSyn becomes pathogenic by activating neuroinflammatory and neurodegenerative responses in vitro ( 9 , 10 ). The nature of the secretory mechanisms of αSyn remains elusive. However, studies have shown that neurons can secrete αSyn into the extracellular milieu through a brefeldin-A insensitive pathway involving exosome vesicles ( 6 , 11 ). Exosomes are nanoscale vesicles generated within the endosomal system and secreted upon fusion of multivesicular bodies with the plasma membrane. Originally, exosomes were thought to be molecular “garbage bags” associated with disposal of waste materials from cells. However, it was discovered that exosomes are more like molecular cargo vessels carrying key molecules that include microRNAs and proteins and, therefore, playing a role in cell-to-cell communication and disease propagation ( 9 , 12 – 14 ). Thus, understanding exosome biology can advance therapeutic and biomarker discoveries in many diseases including neurological diseases.

Synucleinopathies are characterized by the presence of cytoplasmic inclusions called Lewy bodies and neurites composed of α-synuclein (αSyn) and ubiquitin ( 1 ). Among them, Parkinson’s disease (PD) is the most common, marked by motor and nonmotor deficits and progressive degeneration of dopaminergic neurons projecting from the substantia nigra pars compacta (SNpc) to the striatum. Multiple system atrophy (MSA) and diffuse Lewy body disease (DLB) also belong to this group of disorders, with Lewy bodies found primarily in glial cells of the basal ganglia in MSA and in more diffuse areas of the cortex in DLB. Although the physiological functions of αSyn are poorly understood, evidence suggests that the accumulation of aberrant αSyn species exerts intracellular toxic effects in the central nervous system (CNS). The idea that αSyn can pathologically propagate throughout the CNS recently gained much attention with the finding of αSyn species in human plasma and cerebral spinal fluid (CSF) ( 2 , 3 ) and the host-to-graft propagation of αSyn-positive Lewy bodies in fetal ventral mesencephalic and embryonic nigral neurons transplanted in human patients with PD ( 3 , 4 ). Recent studies have suggested that intercellular transmission of αSyn aggregates is associated with the progression of PD ( 5 – 7 ) and MSA ( 8 ).

RESULTS

Mn2+ exposure up-regulates oligomeric αSyn secretion in exosomes Emerging evidence indicates that misfolded αSyn is a transmissible pathological agent responsible for the initiation and spread of Parkinsonian pathology (25–27). To investigate the effect of exposure to the neurotoxic metal Mn2+ on αSyn transmission and the underlying molecular mechanisms, we established an αSyn-expressing dopaminergic neuronal cell model (GFP_Syn) by stably transfecting MN9D mouse dopaminergic neuronal cells with a construct encoding N-terminal green fluorescent protein (GFP)–tagged human wild-type (WT) αSyn. A control cell line (GFP_EV) was also generated by stably transfecting cells with a pmaxFP-Green-N control vector. Immunocytochemical analyses indicated that >90% of the GFP_Syn cells were positive for GFP-tagged human αSyn and that all GFP_EV cells were positive for GFP (Fig. 1A). Western blots indicated a low abundance of endogenous αSyn in both stable cell lines and a greater abundance of GFP-tagged αSyn in GFP_Syn cells (Fig. 1B). 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. Next, we performed 3-(4, 5-dimethylthiazolyl-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)–based cytotoxicity assays to determine the sensitivity of naïve MN9D cells to Mn2+. The Mn2+ concentration required to kill 50% of MN9D cells (LC 50 ) in 24 hours was 1129 μM (fig. S1A). On the basis of this LC 50 and previously published doses for Mn2+ in dopaminergic neuronal cell lines (28, 29), we chose to use a low-dose (300 μM) Mn2+ for our subsequent studies. To evaluate whether αSyn was released from the cells, we analyzed the amount of secreted αSyn in the conditioned media after Mn2+ treatment in serum-free Dulbecco’s modified Eagle’s medium (DMEM). The medium was collected and concentrated using centrifugal concentrators together with bovine serum albumin (BSA; final concentration, 10 μg/ml) as an internal spiked control. Mn2+ treatment at 300 μM markedly enhanced the release of GFP-tagged αSyn into the extracellular milieu when compared to time-matched untreated cells (Fig. 1C and fig. S1B). We also immunoblotted the same membranes with an antibody against lactate dehydrogenase A (LDHA), an enzyme marker indicative of cellular toxicity (Fig. 1C and fig. S1C). Cytotoxicity after exposure to 300 μM Mn2+ was minimal in both GFP_Syn and GFP_EV cell groups, further confirming that αSyn protein detected in the culture media resulted from the actual release of αSyn and was not due to cytotoxicity. To further investigate the underlying molecular mechanisms of αSyn secretion and its relevance in the progression of neurodegenerative disorders, we further characterized the morphological features of the cargo behind αSyn secretion. Our analysis of differentially ultracentrifuged conditioned media through TEM indicated the presence of nanoscale exosomal vesicles morphologically similar to previously reported exosomes (9) in both vehicle- and Mn2+-treated samples (Fig. 1D). Further analysis of cell lysates, conditioned media, and exosomes through Western blot analysis revealed that αSyn was primarily enriched in exosome fractions after Mn2+ exposure (Fig. 1E). We also comprehensively assessed particle size and concentration in conjunction with protein analysis of purified exosomes to assess isolation efficacy and purity. Our Western blot analysis of isolated exosomes and whole-cell lysates showed the presence of canonical exosome proteins, such as CD9, Alix, Flotillin-1, the diminished nuclear envelop marker Lamin, and the endoplasmic reticular protein GRP78, demonstrating a pure exosome preparation isolated using an ultrafiltration protocol (fig. S1D). Furthermore, using the NanoSight LM10, we visualized, counted, and measured the size of exosomes isolated from GFP_Syn cells in the presence and absence of Mn2+. The average diameter of exosomes isolated from control cells was comparable to that of Mn2+-treated exosomes (150.8 ± 7.05 nm to 148.6 ± 12.42 nm, respectively; Fig. 1F), indicating that Mn2+ exposure does not alter the size distribution of exosomes. These calculated sizes are consistent with previously published observations (6, 9). We detected significantly more exosomes in the Mn2+-treated cells than in vehicle-treated cells (fig. S1E), indicating that Mn2+ exposure significantly enhances exosome release. The exosomal surface membrane protein markers Alix and Flotillin-1 were readily detected in all exosome samples (Fig. 1G). We observed more αSyn-GFP protein in the exosomes isolated from Mn2+-exposed cells than from untreated cells as determined by Western blot analysis (Fig. 1G), indicating that Mn2+ increases the amount of αSyn in exosomal cargos. Similar results were obtained by quantitative enzyme-linked immunosorbent assay (ELISA) analysis (fig. S1F). To ensure that the enhanced exosome release was not driven by the GFP fluorescence tag, we explored the exosome release profile using a different protein tag based on a poly(His) affinity tag-bound αSyn protein. Naïve MN9D cells were transfected with either a 6× His-tagged human WT αSyn–bearing plasmid or an empty 6× His plasmid. As described in previous experiments, cells were then treated with Mn2+, and exosomes were isolated from conditioned medium. Using a dual-color Western blot analysis, we readily detected the release of His-tagged αSyn as seen by colocalization of fluorescence secondary antibodies corresponding to 6× His (green) and human αSyn (red) (fig. S1G). Furthermore, isolated exosomes exhibited the expected morphology (fig. S1H), size profiles (fig. S1I), and concentration (fig. S1J), consistent with our observations of exosomes isolated from Mn2+-treated GFP_Syn cells. The existence of αSyn oligomers in biological fluids and in exosomal fractions isolated from cultured cells (6, 11) has been well characterized. Therefore, using conformation-specific antibodies against prefibrillar oligomers (30) and specifically prefibrillar αSyn species (fig. S1K), we sought to determine whether misfolded αSyn proteins accumulate in exosomes of Mn2+-stimulated cells. When compared to exosomes isolated from vehicle-treated cells, accumulations of prefibrillar oligomers detected by A11 antibody noticeably increased in Mn2+-stimulated GFP_Syn exosomes but not in Mn2+-stimulated GFP_EV exosomes (Fig. 1G, bottom panel). Our slot blot analysis using a newly developed αSyn antibody against filament readily detected an increased level of αSyn filament in Mn2+-stimulated exosomes isolated from GFP_Syn cells (Fig. 1G, bottom panel), confirming that oligomeric protein accumulation resulted from Mn2+-induced αSyn protein misfolding. Furthermore, we measured misfolded αSyn oligomer abundance in these exosomes using the highly sensitive, thioflavin T (ThT)–based αSyn fibril formation assay. In this microplate-based misfolded protein seeding assay, exosomes isolated from Mn2+- or vehicle-stimulated GFP_Syn and GFP_EV cells, serving as the seeds presumably with trace amounts of αSyn fibrils, are added to a recombinant human αSyn substrate and repeatedly agitated. By first optimizing the assay using different concentrations of synthetically aggregated αSyn as seed, we found that the onset of amyloid fibril formation, which increases fluorescence intensity when ThT binds to aggregates, directly correlated to αSyn fibril seed density (fig. S1L). This correlation has previously been used to quantify the aggregation kinetics of two major forms of amyloid-β peptides and transmissible spongiform encephalopathy (TSE)–associated forms of prion protein (31, 32). Here, we found that Mn2+-stimulated GFP_Syn cell–derived exosomes (hereafter referred to as “αSyn exosomes”) underwent nucleation-dependent seeded aggregation at a significantly higher rate than vehicle-stimulated αSyn exosomes (Fig. 1H). However, we did not observe a marked increase in ThT readout in GFP_EV-derived exosomes (hereafter referred to as “GFP exosomes”) treated with either Mn2+ or vehicle (Fig. 1H). Collectively, our data suggest that Mn2+ exposure increases the number of αSyn-containing exosomes released and up-regulates the aggregated αSyn protein cargo packaged into these exosomes.

Mn2+-stimulated exosomes promote neuroinflammatory responses Although exosomes play a major role in many physiological and pathological processes, the exosome-cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Feng and colleagues (33) showed that exosomes are taken up more efficiently by phagocytic cells than by nonphagocytic cells, which suggests that phagocytic processes facilitate exosome uptake. This is particularly important in microglia, which are the resident macrophages in the CNS and whose phagocytic capabilities make them the first and primary active immune defense. Moreover, aberrant activation of glial cells and associated proinflammatory cytokines is increased in neurodegenerative diseases (3, 34, 35) and in experimental models of PD (36). Therefore, we exposed primary murine microglia to either vehicle- or Mn2+-stimulated exosomes to study whether Mn2+-stimulated exosomes have any role in neuroinflammatory processes. We added purified exosomes to primary microglia and allowed their cellular internalization to occur for 24 hours at 37°C. Our immunocytochemical analysis with an anti–ionized calcium binding adaptor molecule 1 (IBA-1) and an anti-GFP antibody revealed GFP-positive punctate structures inside the microglial cells, indicating efficient exosomal internalization. However, only microglia exposed to Mn2+-stimulated αSyn-containing exosomes exhibited a pronounced amoeboid morphology resulting from the activation and formation of diverse surface protrusions, such as blebs and filopodia, similar to those of other phagocytic cells (Fig. 2A). The expression of IBA-1 and inducible nitric oxide synthase (iNOS), as revealed by Western blot analysis, increased significantly in cells treated with Mn2+-induced αSyn-containing exosomes in contrast to cells receiving vehicle-stimulated αSyn-containing exosomes, further confirming a distinct activation of microglia and subsequent inflammatory nitrative stress (Fig. 2, B to D). Supporting these observations, the release of proinflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin-12 (IL-12), IL-1β, and IL-6, from microglia was significantly increased upon exposure to Mn2+-stimulated αSyn-containing exosomes, compared to vehicle-stimulated αSyn-containing exosomes or GFP control exosomes (Fig. 2, E to H). These data collectively indicate that Mn2+-stimulated αSyn-containing exosomes are biologically active and capable of activating microglial cells and inducing the release of proinflammatory cytokines, which may further contribute to the inflammatory process. 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.

Microglia internalize Mn2+-stimulated αSyn exosomes through caveolin-1–mediated endocytosis The endocytic process in mammalian cells involves multiple mechanisms depending on the host cell type, as well as cargo type and fate. So far, different modes of endocytosis seem to be responsible for the uptake of exosomes by both phagocytic and nonphagocytic cells (33, 37, 38). The previously described mechanisms of classical endocytosis include clathrin-dependent endocytosis, macropinocytosis, and clathrin-independent endocytic pathways (such as caveolae-mediated uptake associated with lipid rafts in the plasma membrane). However, the mechanisms by which exosomes interact with recipient cells such as microglia and how exosomes are sorted after entry into these cells remain unclear. Therefore, we used a WT mouse microglial cell line (WTMC), which has morphology and surface marker expression that are highly similar to those of primary microglia (39), to determine which endocytic pathway microglia use to take up exosomes. As the initial pharmacological approach, we treated WTMC with various inhibitors of endocytosis, including dynasore, which binds dynamin to inhibit both caveolae- and clathrin-dependent endocytosis; (N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of macropinocytosis; and chlorpromazine and genistein, which inhibit clathrin- and caveolin-mediated endocytosis, respectively (37, 40). To better visualize exosomal vesicles, the cell-derived exosomes were prelabeled with the green fluorescent dye PKH67, which is stably incorporated into lipid regions of the vesicle membrane, and then incubated with WTMC cells. Confocal microscopy revealed efficient internalization of the vehicle- and Mn2+-stimulated αSyn-containing exosomes by the WTMC (fig. S2A). The three-dimensional (3D) surface reconstruction images generated by Imaris software revealed the homogeneous internalization of exosomes by the microglial cells and the activated microglial morphology upon internalization of Mn2+-stimulated, but not of vehicle-stimulated, αSyn-containing exosomes (fig. S2A). Next, we pretreated WTMC with one of the endocytosis inhibitors, chlorpromazine (5 μM), genistein (50 μM), EIPA (10 μM), or dynasore (50 μM) (40), for 60 min at 37°C. Subsequently, Mn2+-stimulated PKH67-labeled αSyn-containing exosomes were added, and incubation was continued for 24 hours. Confocal microscopy (Fig. 3A) indicated successful inhibition (80 to 90%) of exosome uptake by dynasore and genistein, whereas EIPA and chlorpromazine were unable to effectively inhibit (50 to 60%) exosome uptake. Therefore, given its clathrin independence and dynamin dependence during internalization, exosome uptake in our microglial cell cultures was primarily controlled through caveolae-dependent endocytosis. In a parallel experiment, we cotreated primary microglial cells with Mn2+-stimulated αSyn-containing exosomes and the endocytosis inhibitors to further analyze the production of proinflammatory cytokines and nitrite. Similarly, dynasore and genistein significantly attenuated the production of the proinflammatory cytokines TNFα, IL-1β, and IL-6 in response to Mn2+-stimulated αSyn exosomes, whereas chlorpromazine and EIPA did so only marginally or not at all (Fig. 3, B to D). Dynasore, genistein, and EIPA, but not chlorpromazine, significantly reduced nitrite production (Fig. 3E). Thus, these data indicate that the primary uptake of Mn2+-stimulated αSyn-containing exosomes by microglia involves a caveolae-dependent endocytotic pathway. 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. Next, using primary murine microglial cultures, we confirmed the prominent role of caveolin-1–mediated endocytosis in microglial uptake of αSyn-containing exosomes. For this, we used fluorescently labeled transferrin and the cholera toxin B subunit (ctxB), which are widely recognized as ligands exclusively internalized via clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively, in several cell types (40, 41). Primary microglial cells were pretreated with chlorpromazine or genistein as described above for 60 min at 37°C. At the end of the incubation, cells were cotreated for 24 hours at 37°C with one of either two combinations: Alexa Fluor 555–labeled transferrin and PKH67-labeled exosomes (fig. S2B) or Alexa Fluor 555–labeled ctxB and PKH67-labeled exosomes (fig. S2C). Although chlorpromazine treatment significantly inhibited Alexa Fluor 555–conjugated transferrin uptake, it only moderately inhibited the uptake of PKH67-labeled exosomes or Alexa Fluor 555–conjugated ctxB (fig. S2B). Cells treated with genistein exhibited 90 to 100% inhibition of both Alexa Fluor 555–conjugated ctxB and PKH67-labeled exosome uptake (fig. S2C). Genistein, however, did not inhibit microglial uptake of transferrin. Therefore, our data suggest that caveolin-mediated endocytosis is the primary facilitator for the recognition and internalization of neuronal exosomes in a microglial cell model. To further rule out the possible nonspecific effects of pharmacological/chemical inhibitors, we next used CRISPR-Cas9 nuclease RNA-guided genome editing to individually KD caveolin-1 or clathrin in the WTMC to validate our experimental results involving chemical inhibition of endocytosis. The selective gene silencing of caveolin-1 and clathrin in WTMC was confirmed with Western blotting (fig. S2D). In Luminex magnetic bead–based cytokine analysis, the release of the proinflammatory cytokines IL-6, IL-12, TNFα, and IL-1β was reduced significantly by exposing clathrin-KD cells to Mn2+-stimulated αSyn exosomes in contrast to control microglial cells (Fig. 3, F to I). A further reduction in αSyn exosome–stimulated proinflammatory cytokine release occurred in caveolin-1–KD cells. In contrast, we did not observe changes in anti-inflammatory cytokine IL-10 (fig. S2E). Therefore, microglial internalization of exosomes derived from αSyn-expressing dopaminergic neuronal cells depends on multiple mechanisms, particularly the involvement of caveolin-1–dependent endocytosis.

Mn2+-stimulated αSyn exosomes induce neuronal cell death in vitro After establishing the role of Mn2+-stimulated αSyn exosomes in activating neuroinflammatory processes in microglia, we expanded our experiments to evaluate whether the exosomes play a role in neurodegeneration. For this purpose, we established a neuron-glia mixed culture system using primary microglial cells and a differentiated human dopaminergic neuronal model referred to as Lund human mesencephalic (LUHMES) cells (fig. S3A). Because LUHMES cells can be differentiated into morphologically and biochemically mature postmitotic dopamine-like neurons, they are widely used as an in vitro model system for dopaminergic neurotoxicity (42). A Transwell cell culture system enabled us to mimic their CNS environment by culturing pure microglial and neuronal cells separately but in close proximity within the same, shared culture media. Specifically, pure primary microglia were grown on porous upper inserts, whereas differentiated LUHMES cells were grown on a coverslip in the bottom well of the chamber (fig. S3A). Thus, exposing the microglial cells to Mn2+-stimulated αSyn exosomes enabled us to observe significant Mn2+-stimulated αSyn exosome–mediated cytotoxicity or apoptosis, as indicated by increased caspase-3 activity in differentiated LUHMES cells (fig. S3B). In contrast, GFP exosomes or vehicle-stimulated αSyn exosomes did not significantly increase caspase-3 activity, indicating that Mn2+-stimulated αSyn exosome–mediated cell death resulted from the combined effects of increased inflammation and oligomeric proteins packaged in Mn2+-stimulated αSyn exosomes. Exosome uptake readily occurred in exosome-treated LUHMES cells as evidenced by GFP-immunoreactive punctate structures inside the neuronal cells (fig. S3C). Immunolabeling of the LUHMES cells with neuron-specific class II β-tubulin (Tuj1) confirmed the fully differentiated postmitotic nature of the LUHMES cells, as described previously (42). Collectively, our data indicate that Mn2+-stimulated αSyn exosomes could initiate neuronal apoptosis through activation of neuroinflammatory processes in microglia.

Direct detection of Mn2+-induced cell-to-cell transmission of αSyn oligomers in vitro and in vivo To further clarify the role of Mn2+ in cell-to-cell transmission of αSyn aggregates, we adopted an assay based on bimolecular fluorescence complementation (BiFC), which has been successfully applied to assess protein oligomerization, protein-protein interaction, and cell-to-cell transmission in in vitro and in vivo models (5, 6, 43). For this assay, human WT αSyn is fused to either the N-terminal (V1S) or C-terminal (SV2) fragment of the Venus protein, which is an improved variant of GFP (fig. S4A). These two chimeras alone are not able to complement Venus fluorescence, which only occurs when the split Venus moieties fused with αSyn are brought together and covalently linked. V1S and SV2 constructs were individually transfected to MN9D cells, which were then cocultured (Fig. 4A). Fluorescence resulting from dimerization or oligomerization of the V1S and SV2 fusion proteins (44) during cell-to-cell transfer of αSyn was visualized using BiFC (Fig. 4, A and B). The Mn2+-treated V1S/SV2 coculture system exhibited a visually greater BiFC signal when compared to the vehicle-treated V1S/SV2 coculture using confocal microscopy analysis (Fig. 4A), indicating that Mn2+ stimulation enhances cell-to-cell transmission of αSyn. To better understand the cargo mechanism of αSyn transmission, we used isolated exosomes found in conditioned medium collected from V1S and SV2 individually transfected cultures, as well as V1S/SV2 coculture systems. Western blot analysis of purified exosomes readily detected the αSyn immunopositive V1S and SV2 protein fragments and the exosomal surface marker Alix in exosome lysates (Fig. 4C), implying exosomes as a possible cargo mechanism for cell-to-cell transmission of αSyn. Furthermore, when V1S and SV2 BiFC constructs were individually transfected into MN9D cells, neither cells fluoresced (Fig. 4B, two left panels). However, once cells were cotransfected with V1S and SV2 (two right panels), αSyn-αSyn interactions (6) reconstituted the Venus fluorescent protein (Fig. 4B). Moreover, the formation of reconstituted Venus fluorescent protein sharply increased in Mn2+-treated cotransfected cells (Fig. 4B, two right panels), confirming that Mn2+ exposure enhances αSyn aggregation. 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. To study the nature of αSyn species visualized by the BiFC assay, total cell lysates were separated with Western blotting and immunoblotted with an anti-ubiquitin antibody. As expected, cells cotransfected with V1S and SV2 followed by Mn2+ treatment accumulated both as high–molecular weight polyubiquitinated proteins, indicating that Mn2+ enhanced protein oligomerization when compared with vehicle-treated cells (fig. S4B). Using αSyn and GFP antibodies, we also detected discrete bands corresponding to Venus-link-αSyn (V1S) and αSyn-Venus (SV2) protein expression and their N-terminal Venus fluorescent tag (fig. S4B). To ensure that αSyn oligomerization and transmission were not driven by the Venus fluorescent moieties, we adopted another protein complementation assay based on a luciferase assay system consisting of the two fusion constructs αSyn-hGLuc1 (S1) and αSyn-hGLuc2 (S2) as described elsewhere (6, 44). While this assay uses the same principle as the BiFC assay, Gaussia princeps luciferase only reconstitutes with S1 and S2 protein interaction, allowing direct monitoring of αSyn-αSyn protein interactions in their normal cellular environment. Cotransfection of S1 and S2 constructs showed about fivefold higher luciferase activity relative to the background signal from cells transfected with either S1 or S2 plasmids alone. Furthermore, exposure of S1 and S2 cotransfected cells to Mn2+ significantly increased luciferase activity relative to vehicle-treated S1 and S2 cotransfected cells (fig. S4C). These data are consistent with our fluorescent-based BiFC assay results and support our finding that Mn2+ exposure induces misfolded αSyn species. Last, cells cotransfected with V1S and SV2 and treated with either Mn2+ or vehicle for 24 hours were fixed and processed for flow cytometry analysis to further confirm that Mn2+ promotes cell-to-cell transmission of oligomeric αSyn. Because the reconstituted Venus fluorescent protein formed in the BiFC experiment matures at 37°C as a strong fluorescent signal, we used fluorescence-activated cell sorting (FACS) to contrast GFP-positive cell populations exposed to Mn2+ or vehicle treatments (Fig. 4D). Our FACS analysis shows significantly more GFP-positive cells in Mn2+-exposed cells than in the vehicle-treated control group (Fig. 4E). Consistent with our BiFC assay, we did not detect GFP-positive cells when transfected with either V1S or SV2. Thus, using multiple experimental approaches, we demonstrate that Mn2+ exposure promotes cell-to-cell transmission of αSyn in our cell culture system. Once we established the effect of Mn2+ on cell-to-cell transmission of oligomeric αSyn cell culture models, we attempted to further confirm our findings using animal models of Mn2+ toxicity. We used an in vivo protein complementation approach consisting of co-injecting adeno-associated virus (AAVs) encoding αSyn fused to the N- or C-terminal half of Venus fluorescent protein (43). Thirty days after stereotaxically co-injecting AAV8-V1S and AAV8-SV2 into the SNpc of C57BL/6 mice (fig. S4D), animals were exposed to either vehicle or Mn2+ (15 mg/kg per day) via oral gavage once daily for another 30 days (fig. S4D). Two additional control groups were injected with either AAV8-V1S or AAV8-SV2 virus to exclude the possibility of nonspecific fluorescence from one-half of the VenusYFP protein, and another group was injected with AAV8-CBA–VenusYFP as a positive control for the experiment. At 60 days after viral injection, the VenusYFP fluorescence that had colocalized in TH-positive cells was visible in the SNpc of animals injected with AAV8-CBA–VenusYFP, confirming our injection target and the expression of VenusYFP epifluorescence (Fig. 4F). To determine whether Mn2+ exposure promotes αSyn oligomerization and pathogenesis in vivo, we used the Kodak In-Vivo FX Image Station to study VenusYFP expression and localization in vehicle-treated and Mn2+-exposed mice. Using MATLAB, we captured and converted whole-brain fluorescent images into heat maps, which were then superimposed on white-light reference images to show anatomical localization of VenusYFP fluorescence (Fig. 4G). Quantification of fluorescent intensities indicates that Mn2+ promoted V1S and SV2 protein-protein interactions resulting in αSyn oligomerization, which increased about 350% in Mn2+-exposed animals when compared to vehicle-treated animals (Fig. 4H). Control animals injected with either AAV8-V1S or AAV8-SV2 alone did not express any VenusYFP fluorescence on the injected side, demonstrating that the fragmented Venus protein lacks background fluorescence. Because our in vivo study of αSyn-mediated neurotoxicity targeted the SNpc, we also assessed the locomotor behavioral performance of V1S- and SV2-cotransduced and nontransduced mice exposed to Mn2+ via oral gavage. Nontransduced and transduced mice were age-matched littermates, and the Mn2+ or vehicle exposures were conducted simultaneously. After the 30-day Mn2+ treatment paradigm, we measured locomotor performance using a computerized infrared activity monitoring system (VersaMax, AccuScan), which quantifies animal movement variables based on infrared beam breaks. Representative maps of the locomotor movements of vehicle- and Mn2+-treated nontransduced (no injection) and transduced (AAV8-V1S + AAV8-SV2) mice suggest that Mn2+ decreased movements in both experimental groups and that viral-transduced mice exhibited greater Mn2+-induced movement deficits (Fig. 4I). Mn2+ markedly decreased the total number of movements (Fig. 4J), total distance traveled (Fig. 4K), and horizontal activity (Fig. 4L) in transduced mice compared to vehicle-treated animals. Our results suggest that αSyn misfolding mediates neurotoxicity and impairs locomotor behavior and that Mn2+ exposure augments behavioral deficits. Next, we examined nigral dopaminergic neuronal viability after a 30-day Mn2+ exposure period in both AAV8-V1S + AAV8-SV2–cotransduced and nontransduced animals. Coronal sections through the SN were immunostained for TH and visualized by DAB (Fig. 4M). Dopaminergic neuronal loss was evaluated using unbiased stereology of TH-immunoreactive neurons on both the ipsilateral and contralateral sides. DAB staining and stereological counting of TH+ neurons revealed severe loss of nigral dopaminergic neurons, especially in the SNpc and substantia nigra pars lateralis (SNpl) of Mn2+-treated AAV8-V1S + AAV8-SV2–cotransduced animals relative to vehicle controls (Fig. 4N). These observations show that Mn2+-induced cell-to-cell transmission of oligomeric αSyn promotes nigral dopaminergic neurodegeneration in vivo. In contrast, Mn2+-exposed nontransduced animals showed no significant loss of TH+ neurons when compared to their vehicle control animals. Overall, these results, together with the abovementioned whole-brain imaging of the cell-to-cell transmission of αSyn, strongly demonstrate that exposure to the environmental neurotoxicant Mn2+ can augment the progression of cell-to-cell transmission of oligomeric αSyn in vivo, resulting in dopaminergic neuronal degeneration.

Mn2+ exposure promotes exosome release in αSyn-A53T transgenic animals and correlates with αSyn oligomer transmission in humans Having shown that exposing virally transduced mice to Mn2+ induces cell-to-cell transmission of oligomeric αSyn and dopaminergic neurodegeneration, we then evaluated the effect of Mn2+ exposure on serum exosome release in αSyn transgenic and littermate control rats. In this study, we used a newly developed BAC (bacterial artificial chromosome) transgenic rat model of PD expressing the A53T mutation of αSyn, which is known to cause early-onset autosomal dominant PD in humans. The mutant human αSyn transgene protein was expressed at an amount ~42-fold above that of endogenous αSyn in transgenic rats (45). The A53T mutation was not detectable in WT Sprague-Dawley control rats. Both nontransgenic and transgenic rats were treated with Mn2+ as described above, and their blood was collected through cardiac puncture at study termination. Serum separation and exosome isolation were carried out as described, and total serum exosome numbers were counted using the NanoSight particle analyzer. Mn2+-challenged αSyn-A53T transgenic rats produced significantly higher concentrations of exosomes than did either vehicle-treated transgenic rats (P = 0.0035) or nontransgenic rats (P = 0.0082) (Fig. 5A). Furthermore, Mn2+ exposure did not alter exosome size but only increased the number of exosomes released (Fig. 5B). Therefore, exosomes may act not only as a means of cell-to-cell communication during increased intracellular stress conditions but also as a cargo mechanism for secretion and cell-to-cell transmission of harmful or unwanted cellular proteins. 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. It has been proposed that exogenously added misfolded αSyn serves as nucleation seeds for propagating aggregate-initiated polymerization of αSyn in in vitro and in vivo models of PD (15–17, 46). Because exosomes are well recognized as one of the potential mechanisms mediating cell-to-cell transmission of cytosolic protein aggregates (47), and given the strong interaction between metal exposure and PD (24, 48–50), we undertook an exploratory study of the effects of Mn2+ exposure on αSyn transmission in humans. As a group, welders are at risk of prolonged exposures to environmental levels of metals, including Mn2+, that can be neurotoxic (21, 29, 51). We compared the exosomal αSyn content in serum from welders exposed to Mn2+ fumes (age, 26 to 65 years; mean, 46 ± 11.2 years; n = 8 individuals; serum obtained within 90 days of the study) to that found in serum from healthy controls (age, 28 to 73 years; mean, 49 ± 11.0 years; n = 10 individuals) with no history of welding [see details in (22) for the first description of the subjects, as well as (23)]. Serum exosomes were isolated as described above, and total αSyn concentrations in these exosomes were analyzed using a commercially available, highly sensitive luciferase-based αSyn ELISA kit. Total αSyn cargo in the serum exosomes did not differ (P = 0.2855) between welders and controls (Fig. 5C). We also counted total exosome numbers using NanoSight particle analysis. Contrary to our previous observations with transgenic cells and rats, the exosome counts of welders did not differ significantly from that of controls (Fig. 5D). One possible explanation accounting for this result is that the extent of Mn2+ exposure experienced by these welders was not sufficient to alter their serum exosome numbers or total αSyn cargo. Although the outcome we observed in humans did not directly support changes observed in transgenic cells and rats, widely varying αSyn abundance in peripheral blood and CSF samples has been reported in patients with PD relative to healthy controls (2, 52, 53). In the αSyn model of neuron injury, β-sheet–rich soluble oligomers are considered more toxic than monomers (54, 55). Therefore, we measured the abundance of αSyn oligomers in these human exosomes using the αSyn fibril formation assay. Blank and baseline–corrected average kinetic traces for seeded αSyn fibrillar formation assays revealed a significantly different lag phase between the averaged traces of welder exosome samples and those of control individuals. The lag-phase duration was determined from the point where the ThT fluorescence intensity first reached the amyloid detection threshold (Fig. 5E), defined as 5 SDs of the fluorescence intensity of the first 10 hours for the blank (3.66) sample. SEM lag phase was calculated via bootstrap with replacement protocol in MATLAB. The calculated lag phases for control and welder exosomes were 75.5 and 42.5 hours, respectively. Furthermore, we calculated the final fluorescence intensity to compare two kinetic traces by averaging the raw ThT fluorescence of the last 10 data points of each trace (Fig. 5F). Mean ThT fluorescence intensities for controls and welders differed significantly (P < 0.001), indicating that exosomes isolated from welders have a higher seeding capacity and misfolded αSyn protein content compared to exosomes isolated from healthy controls. This observation was further validated with dot blot analysis with antibody against fibrillar αSyn (Fig. 5, G and H). Together, our data indicate that environmental exposure to neurotoxic metals, such as Mn2+, increases the abundance of misfolded protein cargo in circulating exosomes. However, given the limitations associated with sample isolation and collection, it must be noted that blood-derived exosomes are an indirect measure of cell-to-cell transmission in the CNS. As such, it is unclear whether the results shown here are due to exosome clearance from the blood or are exosomes released by affected neurons into the systemic circulation. Nevertheless, previous reports indicate the presence of CNS-derived αSyn in plasma exosomes in patients with PD (56) and increased phosphorylated tau and amyloid-β (Aβ) proteins in blood-derived exosomes in patients with Alzheimer’s disease (57), further suggesting a role of exosomes in disease pathogenesis.