The impact of coronavirus infection and vaccination on the nervous system is increasingly recognized, ranging from olfactory dysfunction to cognitive disorders. However, the relationship between COVID-19 and major neurodegenerative diseases like Alzheimer's and Parkinson's disease remains less understood. SARS-CoV-2 is hypothesized to worsen Parkinson's disease by influencing alpha-synuclein's pathological transformation, mitochondrial function, and dopamine levels. Increased vulnerability of Parkinson's patients to COVID-19 has also been reported. The SARS-CoV-2 spike protein undergoes proteolysis, releasing the S1 subunit, which contains the RBD. The S1 subunit, and specifically the RBD, is capable of crossing the blood-brain barrier. Prior research analyzed interactions of full-length spike protein with alpha-synuclein, but the RBD's effects on synuclein aggregation remain unclear; full-length S-protein showed no effect on aggregation. Molecular modeling suggested RBD interaction with alpha-synuclein, but experimental evidence is limited. The complexity of alpha-synuclein's conformational dynamics (unstructured monomer vs. structured oligomer/fibril) poses challenges for molecular modeling. Therefore, this study aimed to experimentally and computationally investigate the RBD's interaction with different forms of alpha-synuclein and assess its impact on amyloid transformation.

Several studies have explored the neurological effects of COVID-19 and its vaccines, highlighting various cognitive and sensory impairments. The association between SARS-CoV-2 infection and neurodegenerative diseases like Parkinson's and Alzheimer's is an emerging field of research, with limited data currently available due to the pandemic's recent onset. Existing evidence points to potential mechanisms by which SARS-CoV-2 could exacerbate Parkinson's disease, including direct effects on alpha-synuclein aggregation, mitochondrial dysfunction, and dopamine depletion. Furthermore, the increased susceptibility of Parkinson's disease patients to COVID-19 suggests a complex interplay between the virus and the disease's pathogenesis. Prior studies have examined the interaction of the full-length SARS-CoV-2 spike protein with alpha-synuclein, with inconclusive results regarding the impact on aggregation. The use of RBD-containing vaccines highlights the need to understand the specific effects of the RBD fragment on alpha-synuclein.

This study employed a multifaceted approach combining molecular modeling and various biochemical techniques. Recombinant human alpha-synuclein was expressed in E. coli and purified. Alpha-synuclein fibrils were prepared with or without RBD. Amyloid aggregation was assessed using Thioflavin T (ThT) fluorescence, turbidity measurements, Congo Red binding assay, and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. Protein-protein interactions were examined using a modified ELISA with ACE2-Fc as a capture agent for RBD. Circular dichroism (CD) spectroscopy analyzed secondary structure changes. Molecular modeling utilized the HDOCK server for protein-protein docking of RBD with monomeric and fibrillar alpha-synuclein, followed by molecular dynamics simulations using GROMACS. Cytotoxicity of the resulting fibrils was evaluated using MTT assay on SH-SY5Y neuroblastoma cells.

Molecular docking and dynamics simulations revealed stable interactions between RBD and both monomeric and fibrillar alpha-synuclein. The predicted dissociation constant for the RBD-monomeric alpha-synuclein complex was 7.26 × 10⁻¹⁰ M, indicating strong binding. Immunochemical assays confirmed the RBD-alpha-synuclein complex formation. Spectral analysis (Trp and ANS fluorescence) indicated conformational changes upon complex formation, while CD spectroscopy showed no alteration in secondary structure. Importantly, the presence of RBD completely blocked alpha-synuclein amyloid fibril formation, as assessed by ThT fluorescence and Congo Red binding. Although RBD did not prevent disordered aggregation (turbidity measurements), the resulting aggregates displayed significantly reduced cytotoxicity compared to conventional alpha-synuclein fibrils in SH-SY5Y neuroblastoma cells.

The findings strongly suggest that the RBD of the SARS-CoV-2 spike protein interacts with alpha-synuclein, but does not promote amyloid formation; rather, it inhibits it. The mechanism might involve hindrance of conformational changes necessary for amyloid formation, inhibition of oligomer formation, or "capping" of fibril ends. The RBD's interaction with alpha-synuclein does not appear to affect its interaction with ACE2. These results contrast with some studies suggesting a link between SARS-CoV-2 proteins and enhanced alpha-synuclein aggregation. The observed inhibition of amyloidogenesis by RBD highlights the potential for diverse effects of different viral proteins on amyloid processes. These findings could have significant implications for understanding the potential effects of COVID-19 and its vaccines on the risk of neurodegenerative diseases, particularly Parkinson's disease. The use of RBD-based vaccines may be safer in the context of amyloidogenic protein interactions compared to vaccines incorporating other viral proteins.

This study demonstrates that the SARS-CoV-2 RBD interacts with alpha-synuclein, but importantly, prevents its amyloid transformation and reduces the cytotoxicity of resulting aggregates. This suggests that RBD-based vaccines may not exacerbate synucleinopathies. Further research should investigate the mechanisms of this interaction and the effects of other SARS-CoV-2 proteins on amyloidogenic processes. Future vaccine development should consider the potential impact of vaccine components on amyloidogenic protein interactions.

The study primarily focused on in vitro experiments. The findings might not fully translate to the in vivo environment due to factors such as immune responses and other complex biological interactions. The study focused on a specific concentration range of RBD and alpha-synuclein, and additional experiments at varying concentrations are needed to fully define the interaction dynamics. The cytotoxicity assay was performed on one cell line, and additional cell lines should be tested for broader generalizability. Long-term effects and in vivo studies are necessary to completely assess the implications for neurodegenerative disease development.