Anionic Nanoplastic Contaminants Promote Parkinson’s Disease-Associated α-Synuclein Aggregation

Recent studies have identified increasing levels of nanoplastic pollution in the environment. Here we find that anionic nanoplastic contaminants potently precipitate the formation and propagation of α-synuclein protein fibrils through a high-affinity interaction with the amphipathic and non-amyloid component (NAC) domains in α-synuclein. Nanoplastics can internalize in neurons through clathrin-dependent endocytosis, causing a mild lysosomal impairment that slows the degradation of aggregated α-synuclein. In mice, nanoplastics combine with α-synuclein fibrils to exacerbate the spread of α-synuclein pathology across interconnected vulnerable brain regions, including the strong induction of α-synuclein inclusions in dopaminergic neurons in the substantia nigra. These results highlight a potential link for further exploration between nanoplastic pollution and α-synuclein aggregation associated with Parkinson’s disease and related dementias.


Fig. S1. Validation of monomeric and α-synuclein fibril seeds.
A) Representative SDS-PAGE and Coomassie stain of selected eluted fractions from boiled bacterial lysates with untagged human α-synuclein protein processed through HiPrep Q HP 16/10 columns.
B) Representative negative stain electron microscopy of purified α-synuclein monomer and fibril seeds (generated by sonication of full length fibrils to the terminal stage).Monomer protein was screened via TEM to detect possible oligomers or larger aggregates.Processed sonicated fibril seed length was consistent with light-scattering diameter estimations for the stumpy fibril particles.Very small stumpy fibril particles are exceptionally stable under physiological conditions according to past analyses (24).Scale bar, 100 nm.C) Group analysis of the propensity of α-synuclein fibrils to bind nanoplastic particles after 30 min RTe incubation in PBS, or PBS supplemented with 400 nM NaCl (i.e., high-salt), or 1% DMSO in water, measured by light-scatter.Each group mean (column) represents an average of 10 acquisitions with error bars indicating SEM.B) Representation of docking box (purple, 46 Å) used for the Induced Fit Docking method to introduce nanoplastics to α-synuclein monomeric protein.
C) Snapshots of neutral nanoplastic and α-synuclein (ribbon colored by residue index) complex, D) cationic nanoplastic and α-synuclein complex, or E) α-synuclein alone across MD simulations, at 100 ns intervals.Nanoplastics depicted as space fill, showing polar hydrogens only; white = hydrogen, pink = carbon, blue = nitrogen.α-Synuclein is depicted in ribbons and colored by residue index.Of note, neutral and cationic nanoplastics have different associations, fewer contacts, and weaker interactions with αsynuclein as compared to anionic nanoplastic.F) RMSD plots of MD-simulated nanoplastics confirming simulations converge within 10 ns.
RMSDs were calculated for heavy atoms of each ligand using frame 1 as a reference.G) RMSD plots of nanoplastic/α-synuclein complexes and α-synuclein alone confirming simulations converge within 200 ns.RMSDs were calculated for indicated atoms of the complexes and α-synuclein alone using frame 1 as a reference.C-alphas is the RMSD of only the α-carbons of α-synuclein's backbone, Side Chains is the RMSD of only the heavy atoms of the residue side chains.Ligand with respect to (wrt) Ligand is the RMSD calculated for the heavy atoms of the nanoplastic in the α-synuclein complex.
Simulations converge within 200 ns.A) Representative frame of anionic nanoplastic and α-synuclein complex, with individual blow ups highlighting plastic-protein surface interactions.Carboxylates associate with areas of positively charged densities to form strong polar bonds while the hydrophobic backbone of the nanoplastic occupies regions rich with nonpolar residues.The surface of α-synuclein bound to anionic nanoplastic becomes free of solvent water.Anionic nanoplastic is depicted as ball-and-stick, showing polar hydrogens only; pink = carbon, red = oxygen.α-synuclein backbone depicted as cartoon ribbon colored by residue index; residues are ball-and-stick showing polar hydrogens only; white = hydrogen, grey = carbon, blue = nitrogen, red = oxygen; 50 % transparent molecular surface colored by residue charge, blue = positive, grey = neutral, red = negative B) A representative web of Van der Waals (VDW) contacts shows tight association of the anionic plastic in the protein complex.Shown is a matched frame (as in Figure 2D), but now indicating VDW interactions.Anionic nanoplastic forms a web of good VDW contacts with α-synuclein.Green dashed lines represent good VDW contacts, whereas orange dashed lines represent rough VDW contacts, and red designates bad VDW contacts.Increases in SPR intensity after iterative cycles of immobilization, highlighted in gray, indicates the effects of conjugation of α-synuclein to the chip with 200 -1000 RU, an index generally compatible with sensitive kinetic measurements.C) Columns depict final immobilization as shown in B) for full length α-synuclein, αsynuclein fibrils, and the two N-terminal truncated variant proteins.D) Group analysis histograms of changes in SPR binding intensity upon injection of anionic and E) cationic nanoplastic particles for interaction with full length α-synuclein, and the truncated 36-140 and 60-140 variant proteins.The rate of binding in SPR measurements was calculated with Biacore T200 Evaluation Software as SPR RU at the end of the analyte injection, with subtracted blank reference values.F) Titration-based SPR curves of immobilized α-synuclein fibrils with injection of nanoplastic particles demonstrates anionic (left panel), but not cationic nanoplastic, can interact tightly with α-synuclein.Extracted average K d values from three independent reactions with indicated SEM and X 2 , calculated with heterogeneous analyte model fit on Biacore T200 software.Binding analyses of both fibrils (middle) and full length monomeric α-synuclein protein (right panel) to cationic nanoplastic particles were marked as negative in the fitting process.While addition of cationic nanoplastic to fibrils immobilized on the chips results in spikes of intensity during injection steps, dissociation cycling leads to immediate return to the initial RU levels, indicative of weak and nonspecific interaction.G) Binding analysis of cationic nanoplastic to 36-140 (left) and 60-140 (right) α-synuclein variants measured by SPR kinetic analysis.H) Representative SPR sensorgrams of interactions between immobilized full length αsynuclein (left), truncated 36-140 protein (middle), and truncated 60-140 protein (right) and anionic or cationic nanoplastic at 15 nM.Anionic nanoplastic particle injection shows specific association and dissociation profiles exclusively in the presence of conjugated full length α-synuclein, while positively charged particles demonstrate strong associations followed by rapid dissociations that highlight weak and non-specific interactions.Truncated 36-140 α-synuclein shows weak association with cationic particles and no response with anionic nanoplastic.Likewise truncated 60-140 protein demonstrates no significant binding to anionic or cationic nanoplastic particles.A) A representative Airyscan confocal image from mouse primary hippocampal neurons (DIV 7) simultaneously exposed to both FITC-labeled nanoplastics and Alexa-647labeled human α-synuclein fibrils.Neurons were immuno-stained at 24 hours after the addition of the particles.The orthogonal view (left panel) from sequential Z-stacks is shown.Pink line is the intersection of y,z; Yellow line is x,z; Cyan line is x,y.Cyan box is the x,y plane; Pink box is the y,z plane; Yellow box is the x,z plane.Scale bar represents 10 μm.The reconstructed 3D view is shown in the right panel.
B) Representative images from widefield microscopy of neurons with 1 nM of either anionic or cationic polystyrene beads added for 2 hours prior to the addition of 66.7 pM pHrodolabeled α-synuclein fibrils.Cells were washed two times with Hank's Balanced Salt Solution (HBSS) to help eliminate non-internalized particles prior to the cultures stained with Hoechst.Further, the dye pHrodo-Red should not fluoresce at all until internalized in cells into an acidified vesicle.An analysis of >50 images analyzed for each condition suggests the nanoplastic addition does not change the uptake of α-synuclein fibrils into neurons.Scale bar, 200 μm  600°C (e.g., polystyrene).A zoomed retention time for the pyrolyzates is shown highlighting peaks associated with styrene ions.All six brain samples were positive for styrene ions.Methods used for this analysis were based on a previous method of the analysis of whole blood samples from healthy controls for the presence of polystyrene nano-and microplastic polystyrene detection (8).More details for this method can be found in Supplementary Methods.***Free dyes were removed from the preparations with desalting columns prior to usage.All concentrated nanoplastics were maintained in 0.1% tween-20 to prevent aggregation of the particles prior to usage in experiments.

Fig. S2 .
Fig. S2.Cup-abraded environmental polystyrene nanoplastic in α-synuclein aggregation.A) Workflow of nanoplastic production from plastic cups.Microplastics from polystyrene plastic cups generated by grinding were sonicated to break larger aggregates and then filtered (0.2-micron) to enrich nanoplastic particles.B) Representative light scattering plot and TEM of abraded nanoplastics contaminants from a polystyrene cup.Arrowheads indicate particles with sizes consistent with light scattering analysis (upper panel, blue).Scale bar, 200 nm.C) Representative TEM images of 70 μM human α-synuclein (monomeric) shaken at 37 °C in absence (top) or presence of 1 nM (estimated, Zeta nanoparticle tracking analysis) polystyrene cup nanoplastic contaminants for 6 days.Bounding boxes show magnified representative fibrils and nanoplastic-tethered particle interactions.Bars = 500 nm and 100 nm for magnified inset.

Fig. S3 .
Fig. S3.Structures and molecular dynamics time frames, ligand RMSDs, and complex RMSDs that highlight the differential effects of anionic nanoplastics on α-synuclein folding.

Fig. S5 .
Fig. S5.Validation of N-term truncated α-synuclein recombinant proteins.A) N-truncated α-synuclein variants 36-140 and 60-140 were prepared similarly to full length protein.Representative SDS-PAGE and Coomassie stain analysis of eluted fractions demonstrate pure protein preparations selected for further purification and concentration.Variant 36-140 shows lower molecular weight as expected, with a negative charge of -11.2 at pH 7.5, while 60-140 has a negative charge of -12.2 at pH 7.5.B) Representative dynamic light scattering (DLS) histograms of purified 36-140 (left) and 60-140 (right), plotted relative to the mass distribution-predicted molecular weight, demonstrating monomeric (i.e., non-aggregated) protein.

Fig. S6 .
Fig. S6.Surface plasmon resonance analysis showing activation, immobilization, deactivation, and the necessity of negatively-charged nanoplastic for interaction with the amphipathic domain in α-synuclein.

Fig. S7 .
Fig. S7.Characterization of primary neurons via tau and NeuN immunostaining shows high neuronal purity and high neuronal tau-positive process complexity.

Fig. S9 .
Fig. S9.Endocytosis inhibitors in neurons do not result in acute lysosomal acidification differences within the 2-hour timeframe used here for analysis.

FGP25C*
Nanoplastic particles were purchased from Lab261, Inc. ** S.E.M.s are calculated from at least triplicate independent measures using dynamic light scattering.