SARS-CoV-2 Proteins Interact with Alpha Synuclein and Induce Lewy Body-like Pathology In Vitro

Abstract

Growing cases of patients reported have shown a potential relationship between (severe acute respiratory syndrome coronavirus 2) SARS-CoV-2 infection and Parkinson's disease (PD). However, it is unclear whether there is a molecular link between these two diseases. Alpha-synuclein (α-Syn), an aggregation-prone protein, is considered a crucial factor in PD pathology. In this study, bioinformatics analysis confirmed favorable binding affinity between α-Syn and SARS-CoV-2 spike (S) protein and nucleocapsid (N) protein, and direct interactions were further verified in HEK293 cells. The expression of α-Syn was upregulated and its aggregation was accelerated by S protein and N protein. It was noticed that SARS-CoV-2 proteins caused Lewy-like pathology in the presence of α-Syn overexpression. By confirming that SARS-CoV-2 proteins directly interact with α-Syn, our study offered new insights into the mechanism underlying the development of PD on the background of COVID-19.

Keywords: Parkinson’s disease; SARS-CoV-2; alpha-synuclein; nucleocapsid protein; spike protein.

Figure 1

SARS-CoV-2 proteins’ interaction with α-Syn by protein–protein docking. (A) The protein–protein docking result of S_RBD (PDB ID: 1XQ8) and α-Syn. (A-1) Docking model showing the interaction of S_RBD (brown) and α-Syn (yellow). (A-2) Detail of the interacting residue of S_RBD and α-Syn. (A-3) Pie chart shows the key interaction of residues between S_RBD and α-Syn. (B) The protein–protein docking result of N_CTD (PDB ID: 6WJI) and α-Syn. (B-1) Surface diagram of N_CTD (brow) and α-Syn (yellow) complex model. (B-2) Detail of an interacting residue of N_CTD and α-Syn. (B-3) Pie chart shows the key interaction of residues between N_CTD and α-Syn. (C) The protein–protein docking result of N_NTD (PDB ID: 6VYO) and α-Syn. (C-1) Docking model of interaction of N_NTD and α-Syn. (C-2) Detail of an interacting residue of N_NTD and α-Syn. (C-3) Pie chart shows the key interaction of residues between N_NTD and α-Syn. Key interactions between residues are presented as dotted lines. The key interactions are color coded as follows: salt bridge (red), disulfide bonds (yellow), hydrogen bonds (blue), and non-bonded contacts (orange). The number of lines indicates the potential number of bonds. For non-bonded contacts, the width of the striped line indicates the number of potential contacts.

Figure 2

α-Syn directly interacts with SARS-CoV-2 proteins in HEK293 cells. (A) The distribution and location of SARS-CoV-2 proteins and α-Syn in HEK293 cells, as analyzed by confocal microscopy. DAPI was used to stain nuclei. (B) Interaction between endogenous SARS-CoV-2 S protein and α-Syn in HEK293 cells. Cell lysates of HEK293 cells co-transfected with S protein and α-Syn were prepared and used for Co-IP. The coimmunoprecipitates were analyzed by Western blotting with anti-α-Syn. (C) Interaction between endogenous SARS-CoV-2 N protein and α-Syn in HEK293 cells. Cell lysates of HEK293 cells co-transfected with N protein and α-Syn were prepared and used for Co-IP. The coimmunoprecipitates were analyzed by Western blotting with anti-α-Syn.

Figure 3

Elevated expression of α-Syn by SARS-CoV-2 proteins in HEK293 cells. (A) qRT-PCR analysis of the expression of α-Syn in HEK293 cells overexpressing S and N proteins. GAPDH was used as a loading control (n = 3). (B) Two days following transfection with pCMV3-S or pCMV3-N plasmids, the total protein from the HKE293 cells was extracted and detected by anti-SARS-CoV-2 S protein and anti-SARS-CoV-2 N protein. (C) Immunoblotting was performed using Syn1, an antibody that recognizes total α-Syn. GAPDH was used as a loading control. (D) The cells transfected with plasmids pCMV3-S or pCMV3-N for 48 h were fixed with paraformaldehyde containing 0.1% Triton X-100. Then, anti-α-Syn was detected by indirect immunofluorescence. DAPI was used to stain nuclei. (E) Western blotting detected “soluble” and “insoluble” fraction with an antibody against α-Syn (Syn1). p value represents results of One-way ANOVA. * p < 0.05; ** p < 0.01. “ns” means no significant difference.

Figure 4

The SARS-CoV-2 proteins cause Lewy-like pathology in HEK293 cells overexpressing α-Syn. (A) Seventy-two hours after HEK293 cells overexpressing α-Syn had been transfected with pCMV3-S or pCMV3-N plasmids, they were fixed and stained with anti-α-Syn aggregate (5G4). Expression of aggregated α-Syn was observed using fluorescence confocal microscopy. (B) Seventy-two hours after HEK293 cells overexpressing α-Syn had been transfected with pCMV3-S or pCMV3-N plasmids, the cells were fixed and stained with anti-pS129-α-Syn. The results were observed under fluorescence confocal microscopy. (C) The total protein from the HKE293 cells was extracted and detected by anti-pS129-α-Syn. The expression levels were assessed by ImageJ. * p < 0.05.