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

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).

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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 (LC₅₀) in 24 hours was 1129 μM (fig. S1A). On the basis of this LC₅₀ 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.

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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.

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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.

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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.

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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.

Mn²⁺-stimulated αSyn exosomes induce Parkinson-like motor deficits in mice

The accumulation of misfolded proteins and associated behavioral deficits is fundamental pathogenic process in the progression of PD (46). Furthermore, evidence from recent in vitro and in vivo studies suggests that misfolded proteins play a central role in a variety of neurodegenerative disorders by functioning as “seeds” for progressive protein misfolding and aggregation processes, much like prions (58, 59). Thus far, we have shown that Mn²⁺-stimulated exosomes contain prefibrillar αSyn oligomers that potentiate neuroinflammatory and degenerative responses in vitro. As a next step, we directly investigated whether Mn²⁺-stimulated αSyn exosomes carry disease-associated prefibrillar αSyn oligomers, which can seed and propagate pathology in vivo. We injected C57BL/6 mice with exosomes isolated from Mn²⁺- or vehicle-treated GFP_EV and GFP_Syn cells. About 4 × 10⁸ to 5 × 10⁸ of exosomal particles (total exosome proteins, ~8 μg) from each treatment group were stereotaxically injected into one side of the striatum (Fig. 6A), and neurobehavioral deficits were monitored over time. The behavioral performance of animals injected with αSyn exosomes tended to worsen when tested again at 180 days after inoculation. Specifically, mice receiving Mn²⁺-stimulated αSyn exosomes tended to be more hypoactive, showing reduced exploratory locomotor activity as measured by stereotypy counts (Fig. 6B) in an open-field locomotor test. However, some behavioral deficits, such as movement time (Fig. 6C), were not statistically significant, perhaps because exosome injections were done unilaterally, and the observed αSyn-containing proteinaceous inclusions were primarily localized in cortical-striatal regions proximal to the injection site. PD has long been recognized as an asymmetric disorder, with unilateral motor symptom onset as one of its cardinal features, especially in patients classified as early-stage PD (60–62). The etiology of these asymmetric motor symptoms remains unclear, although several hypotheses involving lateralized vulnerability, an index of abnormal protein deposition, have been advanced (60, 61). To test for a possible behavioral bias arising from exosome-induced behavioral asymmetry, we used the amphetamine-induced rotation test. Our data indicated increased ipsilateral movements in the Mn²⁺-stimulated αSyn exosome–injected mice, thus indicating unilateral CNS damage in these animals, when compared to mice receiving vehicle-stimulated αSyn exosomes or vehicle- and Mn²⁺-stimulated GFP exosomes (Fig. 6D). Although the mechanism for the unilateral behavioral bias encountered here is not completely understood, it likely involves an increase in neurodegenerative processes induced by ipsilaterally localized, αSyn-containing proteinaceous inclusions.

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To confirm in vivo αSyn misfolding, we examined whether Mn²⁺-stimulated αSyn exosomes lead to the deposition of αSyn-containing proteinaceous inclusions. Histological analyses revealed Ser¹²⁹-phosphorylated αSyn (pSyn129) and p62-immunoreactive cytoplasmic inclusions in mice injected with Mn²⁺-stimulated αSyn exosomes (Fig. 6E) but not in mice injected with vehicle-stimulated αSyn exosomes or vehicle- and Mn²⁺-stimulated GFP exosomes (fig. S5, A to F). Furthermore, our dual staining with pSyn129 and microglia marker IBA-1 reveals microglial cells with larger cell bodies and shorter processes around the pSyn129-positive neurons, indicating that Mn²⁺-stimulated αSyn exosomes triggered an inflammatory response in the brain. In contrast, animals injected with vehicle-stimulated αSyn exosomes resulted in no pSyn129 pathology in cortical regions (fig. S5A) and minimal pathology around the striatal injection site (fig. S5D). Mice receiving vehicle-stimulated GFP exosomes (fig. S5, B and E) or Mn²⁺-stimulated GFP exosomes (fig. S5, C and F) did not exhibit any protein inclusions or GFP-immunopositive structures, suggesting that the GFP protein may have been cleaved off and degraded. Collectively, our results indicate that Mn²⁺-stimulated, αSyn-containing exosomes can initiate certain Parkinsonian symptoms and propagate αSyn misfolding and aggregation in mice. Collectively, our experimental results have important implications pertaining to the prion-like, cell-to-cell transmission of aggregated proteins in progressive neurodegenerative disorders.