SARS-CoV-2 D614 and G614 spike variants impair neuronal synapses and exhibit differential fusion ability

  1. Chiung-Ya Chen
  2. Yu-Chi Chou
  3. Yi-Ping Hsueh

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) exhibits two major variants based on mutations of its spike protein, i.e., the D614 prototype and G614 variant. Although neurological symptoms have been frequently reported in patients, it is still unclear whether SARS-CoV-2 impairs neuronal activity or function. Here, we show that expression of both D614 and G614 spike proteins is sufficient to induce phenotypes of impaired neuronal morphology, including defective dendritic spines and shortened dendritic length. Using spike protein-specific monoclonal antibodies, we found that D614 and G614 spike proteins show differential S1/S2 cleavage and cell fusion efficiency. Our findings provide an explanation for higher transmission of the G614 variant and the neurological manifestations observed in COVID-19 patients.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.12.03.409763: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal experiments were performed with the approval of the Academia Sinica Institutional Animal Care and Utilization Committee and in strict accordance with its guidelines and those of the Council of Agriculture Guidebook for the Care and Use of Laboratory Animals.
    RandomizationTen randomly selected non-overlapping areas of each sample were captured using a confocal microscope (LSM 700, Zeiss) equipped with a 20×/NA 0.8 (Plan-Apochromat) objective lens and Zen acquisition and analysis software (Zeiss).
    BlindingExperiments were performed blind by relabeling the samples with the assistance of other laboratory members.
    Power Analysisnot detected.
    Sex as a biological variableFor SARS-CoV-2 spike RBD antibody generation, only male BALB/c mice were used.
    Cell Line AuthenticationAuthentication: Cotransfected GFP was used to outline neuronal morphology.

    Table 2: Resources

    Antibodies
    SentencesResources
    Recombinant protein purification and antibodies: BL21(DE3) Escherichia coli bacteria carrying pMAL-c2x-RBD or pGEX-4T1-RBD were grown in LB media with Ampicillin.
    Ampicillin
    suggested: None
    Anti-SARS2-RBD mouse monoclonal antibodies (SA07 and SA15) were generated as described previously (Chen et al., 2011).
    Anti-SARS2-RBD
    suggested: None
    The HSP90 rabbit polyclonal antibody was a gift from Dr. Chung Wang (Liou and Wang, 2005).
    HSP90
    suggested: None
    The following commercialized antibodies were also used in this study: HA (rabbit, C29F4, Cell Signaling), HA (mouse, 16B12, abcam), Myc (mouse, 9B11, Cell Signaling), GFP (chicken, Abcam), calretinculin (rabbit, ThermoFisher), SARS-CoV/SARS-CoV-2 Spike S2 (mouse, 1A9, GeneTex), anti-mouse-horseradish peroxidase (HRP) (Jackson Lab), anti-rabbit-HRP (GE Healthcare), and anti-mouse/rabbit/chicken-Alexa 488/555 (Invitrogen).
    HA
    suggested: (Thermo Fisher Scientific Cat# 26183-A555, RRID:AB_2610625)
    GFP
    suggested: None
    anti-mouse-horseradish peroxidase (HRP
    suggested: None
    anti-rabbit-HRP
    suggested: (Kindle Biosciences Cat# R1006, RRID:AB_2800464)
    anti-mouse/rabbit/chicken-Alexa
    suggested: None
    Expression of human ACE2 protein was confirmed by immunoblotting using ACE2 antibody.
    ACE2
    suggested: None
    After blocking with 3% BSA in PBS, 1 ug/ml of anti-RBD antibodies (SA07 and SA15) and GST antibody in blocking buffer were added to the wells.
    anti-RBD
    suggested: None
    SA15
    suggested: None
    GST
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture, transfection, and immunoblotting: HEK293T, Neuro-2A, and COS1 cells were cultured in DMEM with 10% FBS plus penicillin and streptomycin at 37 oC and 5% CO2.
    Neuro-2A
    suggested: None
    COS1
    suggested: None
    Cell fusion: Spike (D614)-HA or Spike (G614)-HA was co-transfected with GFP into HEK293T cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals: WT C57BL/6 and BALB/c mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    For primary neuronal cultures, E16-17 C57BL/6 mouse embryos of both sexes were used.
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    Immunofluorescence images of cultured neurons at DIV6 were visualized with a fluorescence microscope (Axioimage M2; Zeiss) equipped with a 20×/NA 0.8 (Plan-Apochromat) objective lens and acquired using a cooled charge-coupled device camera (Rolera EM-C2; QImaging) with Zen software (Zeiss).
    Zen
    suggested: None
    The images were processed using Photoshop (Adobe) with minimal adjustment of brightness or contrast applied to the entire images.
    Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    Quantitation of neuronal morphology was performed using ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analysis: Statistical analysis were performed using GraphPad Prism 8 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 19, 22, 23, 24, 26 and 28. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.12.03.409763v1 on bioRxiv