Does the SARS-CoV-2 Spike Receptor-Binding Domain Hamper the Amyloid Transformation of Alpha-Synuclein after All?

The study addresses whether the SARS-CoV-2 spike receptor-binding domain (RBD) affects alpha-synuclein (aSyn) aggregation into amyloid fibrils, a process implicated in Parkinson’s disease (PD). COVID-19 infection and vaccination have been linked to neurological effects, and S1 (containing RBD) can cross the blood–brain barrier. Prior studies on full-length S-protein showed no clear effect on aSyn aggregation, while modeling suggested RBD could interact with amyloidogenic proteins. Considering that RBD may circulate during infection and vaccination (and is the sole antigen in some vaccines), the authors hypothesized that RBD interacts with aSyn and may influence its amyloid transformation. They aimed to combine molecular dynamics (MD) modeling with biochemical and cell-based assays to evaluate RBD’s impact on aSyn aggregation and resulting cytotoxicity.

The paper reviews reports on neurological complications of COVID-19 and vaccination, and potential links with neurodegenerative diseases, including PD. S1 is known to cross the blood–brain barrier in mice. Previous experimental work suggested full-length SARS-CoV-2 S-protein did not bind aSyn by immunoprecipitation nor affect its aggregation, whereas N-protein could accelerate aSyn amyloid formation. Computational studies proposed that RBD can interact with aSyn, beta-amyloid precursor or peptide, and prion protein. The authors highlight the need to investigate RBD specifically, given its presence in circulation (as S1 fragment) and use in RBD-based vaccines, and the limitations of static docking without MD for intrinsically disordered aSyn.

- Proteins and reagents: Recombinant human ACE2-Fc, SARS-CoV-2 Spike RBD (Arg319–Phe541), anti-RBD (5308), anti-aSyn (LB509); Thioflavin T (ThT), Congo Red, ANS, o-phenylenediamine, MTT; culture reagents and SH-SY5Y cells. - Alpha-synuclein expression/purification: Full-length WT aSyn expressed in E. coli with silent Tyr136 codon change to prevent cysteine misincorporation. Acid precipitation at pH 2.8, neutralization, ammonium sulfate salting to 40% saturation; stored as concentrated suspension. Concentration by A280 (ε0.1% = 0.412). - Fibril preparation: aSyn precipitate dialyzed to PBS pH 7.4; diluted to 28 µM (0.4 mg/mL) with or without RBD at 2.8 or 5.6 µM. Incubated in glass tubes (0.3 mL) at 37°C, 600 rpm for up to 52 h. Aliquots taken for ThT fluorescence; turbidity at A400 monitored. - ThT fluorescence assay: 10 µL aliquots mixed with 100 µL of 35 µM ThT; incubated 10 min at 20°C; excitation 430 nm, emission 485 nm; measured in triplicate. - Intrinsic Trp and ANS fluorescence: Spectra recorded at 20°C in 3 mm path cuvettes. Trp excitation 295 nm, emission 300–400 nm. ANS added at 50× molar excess; excitation 365 nm, emission 400–600 nm; 60 min incubation in dark. - Congo Red binding: Dye added at 10:1 molar ratio to protein in PBS pH 4.0; 10 min incubation; absorption spectra recorded (3 mm path) with buffer subtraction. - Modified ELISA for complex detection: ACE2-Fc (10 µg/mL, 100 µL PBS) adsorbed to plate 1 h, 20°C; washed with PBST. RBD and aSyn pre-incubated 1 h at 20°C at 1:1 (1.1 mg/mL each), then applied (10 µg/mL mix). Detection with anti-RBD or anti-aSyn primary antibodies and HRP-conjugated secondary; developed with o-phenylenediamine/H2O2; OD492 read. - Circular dichroism: Far-UV CD (190–250 nm) of 20 µM RBD, 20 µM aSyn, and equimolar mixture in 10 mM potassium phosphate pH 7.4 at 20°C; 0.1 mm cuvette; five scans averaged. - Cell culture and MTT cytotoxicity: SH-SY5Y cultured in DMEM/F12 with 10% FBS, 1% GlutaMAX, 1% pen/strep at 37°C, 5% CO2. Cells seeded 15,000/well; treated for 24 h with 0.8 µM aSyn monomers, 0.8 µM aSyn fibrils (formed 24 h at pH 4.0), 0.26 µM RBD, or mixture of fibrils formed in presence of 5.6 µM RBD (final 0.8 µM aSyn, 0.26 µM RBD). MTT final 0.375 mg/mL for 4 h; dissolved in DMSO; absorbance at 570 nm (ref 630 nm). - Docking and molecular dynamics: Structures used: aSyn micelle-bound (PDB 1XQ8) and fibril (PDB 2N0A); SARS-CoV-2 S-RBD (PDB 6M0J). Docking via HDOCK; interactions analyzed by PDBsum; Kd predicted by PPA-Pred. MD simulations with GROMACS 2018, CHARMM36m, TIP3P water; energy minimization; equilibration (200 ps NVT, 200 ps NPT at 310 K, 1 atm with restraints); production runs at 310 K, 1 atm with v-rescale thermostat and Parrinello-Rahman barostat, PME electrostatics (12 Å cutoff), 2 fs timestep; 100 ns for monomer complex and 200 ns for fibril complex. Interactions (hydrogen bonds, salt bridges, non-bonded contacts) quantified over trajectories.

- Molecular modeling: Docking followed by MD showed a stable RBD–monomeric aSyn complex over 100 ns. After MD, the number of hydrogen bonds increased to 9 and nonpolar contacts to 155; new salt bridges formed (e.g., aSyn Lys32/Lys60 to RBD Asp389/Asp427). Predicted Kd ≈ 7.26 × 10^−10 M, indicating high affinity. For the aSyn oligomer/fibril model (2N0A), RBD formed tighter interactions after 200 ns MD with 2 salt bridges and 15 hydrogen bonds; RBD binding involved regions overlapping the ACE2-interacting face initially but shifted laterally during MD without precluding spike binding. The aSyn oligomer/fibril compacted with increased interchain interactions during MD. - Complex formation (experiment): Modified ELISA using immobilized ACE2-Fc captured RBD and co-captured aSyn from mixtures, detected by anti-RBD and anti-aSyn antibodies, confirming RBD–aSyn complex formation while preserving RBD–ACE2 interaction (ANOVA p < 0.01). - Spectroscopy: Mixing aSyn with RBD altered RBD intrinsic Trp fluorescence (indicative of environmental change) and increased ANS fluorescence intensity/shift, consistent with exposure/alteration of hydrophobic sites upon complex formation. CD spectra of aSyn, RBD, and their mixture indicated no significant change in secondary structure upon binding. - Amyloid aggregation assays: ThT fluorescence kinetics showed that RBD (2.8–5.6 µM) effectively abrogated the amyloid transformation of 28 µM aSyn under shaking at 37°C, despite continued aggregation indicated by turbidity (A400) increases, suggesting promotion of amorphous aggregation rather than fibril formation. - Congo Red and ANS validation: Congo Red absorption spectra showed ~20 nm red shift with standard aSyn fibrils, but minimal changes when aggregates formed in the presence of RBD, supporting reduced amyloid character. ANS spectra changes characteristic of amyloid were prevented by RBD. - Cytotoxicity: Conventional aSyn fibrils reduced SH-SY5Y viability by ~50% after 24 h exposure. RBD alone (0.26 µM) showed no cytotoxicity. Fibrils produced in the presence of RBD (5.6 µM during fibrillization; final 0.26 µM) were significantly less cytotoxic than conventional aSyn fibrils (ANOVA: * p < 0.05; ** p < 0.01).

Computational and experimental data concur that RBD binds aSyn without disrupting RBD–ACE2 association, consistent with modeling that the primary aSyn-binding sites do not fully overlap the ACE2 interface. Trp fluorescence changes support proximity of RBD tryptophans to aSyn helices upon binding. The inhibition of amyloid fibrillization by RBD, despite continued amorphous aggregation, suggests mechanisms such as impaired conformational transitions required for nucleation, inhibition of oligomer formation, or capping of fibril ends that prevents elongation. Prior work indicated full-length S-protein had minimal effect on aSyn aggregation, whereas N-protein can accelerate aSyn amyloid formation; the present findings indicate that RBD specifically does not promote and instead inhibits aSyn amyloidogenesis. In the context of vaccines and circulating viral proteins, these results argue that RBD-based antigens are unlikely to exacerbate synucleinopathies and may even counter amyloid formation, whereas N-protein exposure could pose risk. Given the prolonged preclinical phase of amyloid diseases, in vitro assessments of vaccine protein interactions with amyloidogenic proteins should complement safety evaluations. The absence of amyloidogenic peptides within the RBD sequence (compared to amyloidogenic segments identified in full-length S) further supports potential safety of RBD-only vaccine designs.

RBD of the SARS-CoV-2 spike protein interacts with both monomeric and amyloid forms of alpha-synuclein. Modeling suggests stronger binding to the amyloid form than to the monomer. Experimentally, RBD prevents the amyloid transformation of alpha-synuclein, redirecting aggregation toward amorphous forms, and produces aggregates with reduced amyloid signatures and significantly lower cytotoxicity to SH-SY5Y cells. These findings refute the notion that the spike RBD promotes aSyn amyloidogenesis and support RBD-based vaccination as comparatively safe regarding synucleinopathy risk. The presence of RBD in the body should not provoke synucleinopathies, while other viral proteins (e.g., N-protein) may contribute to pro-amyloid effects. Future work should extend to in vivo models, other amyloidogenic proteins, and detailed mechanistic studies of RBD’s inhibitory action on fibrillization.

The study is in vitro and relies on computational modeling plus biochemical assays without in vivo validation. Only the RBD fragment and alpha-synuclein were examined; effects of other viral proteins (e.g., N-protein) or full-length S under physiological conditions were not directly tested. The mechanistic basis of inhibition (e.g., nucleation vs elongation blockade) was not resolved. Long-term kinetics and polymorphism of fibrils formed in the presence of RBD were not structurally characterized. Generalizability to human pathophysiology and chronic exposure remains uncertain.