SARS-CoV-2 infection and viral fusogens cause neuronal and glial fusion that compromises neuronal activity

  1. Ramón Martínez-Mármol
  2. Rosina Giordano-Santini
  3. Eva Kaulich
  4. Ann-Na Cho
  5. Magdalena Przybyla
  6. Md Asrafuzzaman Riyadh
  7. Emilija Robinson
  8. Keng Yih Chew
  9. Rumelo Amor
  10. Frédéric A. Meunier
  11. Giuseppe Balistreri
  12. Kirsty R. Short
  13. Yazi D. Ke
  14. Lars M. Ittner
  15. Massimo A. Hilliard

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Abstract

Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca 2+ imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.

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  1. Version published to 10.1126/sciadv.adg2248
  2. ScreenIT

    SciScore for 10.1101/2021.09.01.458544: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Animal ethics and mouse strains: All experimental procedures using animals were conducted under the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific purposes, and were approved by the University of Queensland Animal Ethics Committee (2019/AE000243).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After blocking, the neurons were incubated with the primary antibodies to GFP (#AB16901, Merck Millipore), mCherry (#ab167453, Abcam) and anti-MAP2 (#188004, Synaptic Systems), diluted in primary antibody solution (1% BSA in PBS) overnight at 4 °C.
    GFP
    suggested: (Millipore Cat# AB16901, RRID:AB_90890)
    anti-MAP2
    suggested: None
    They were then washed with PBS and incubated with the secondary antibodies Alexa Fluor 488 goat anti-chicken (#A-11039, ThermoFisher Scientific),
    anti-chicken
    suggested: (Molecular Probes Cat# A-11039, RRID:AB_142924)
    , Alexa Fluor 647 goat anti-guinea pig (#A21450, ThermoFisher Scientific) and DAPI (#62248, ThermoFisher Scientific) in the secondary antibody solution (5% horse serum in PBS) for 1 h at room temperature.
    anti-guinea pig
    suggested: (Molecular Probes Cat# A-21450, RRID:AB_141882)
    Experimental Models: Organisms/Strains
    SentencesResources
    Wildtype (WT, C57Bl/6 background) mice were maintained on a 12 h light/dark cycle and housed in a PC2 facility with ad libitum access to food and water.
    C57Bl/6
    suggested: None
    Previously generated transgenes used in this study include: vdEx1266 [Pmec-4::p15 5ng/μl]; vdEx1268 [Pmec-4::p15 5ng/μl].
    vdEx1266
    suggested: None
    vdEx1268
    suggested: None
    Recombinant DNA
    SentencesResources
    The Pmec-4::p15 plasmid was constructed by subcloning p15 between Msc I and Nhe I.
    Pmec-4::p15
    suggested: None
    The CMV::p15 plasmid was generated by subcloning p15 into the pmaxCloning™ vector (Lonza, # VDC-1040) between Hind III and Not I.
    CMV::p15
    suggested: None
    pmaxCloning™
    suggested: None
    The CMV::SARS-CoV-2-S-2P plasmid was generated by introducing two prolines at the 986 (K986P) and the 987 (V987P) positions of the SARS-CoV-2-S gene of the pCMV14-3X-Flag-SARS-CoV-2 S plasmid.
    CMV::SARS-CoV-2-S-2P
    suggested: None
    Mutations were done using the QuikChangeII Site-Directed Mutagenesis Kit (p15Δ21/22 forward primer 5’-CCACCGCCAAATGCTTTTGTTGAAAGCAGTTCTACTG-3’; p15Δ21/22 reverse primer 5’-CAGTAGAACTGCTTTCAACAAAAGCATTTGGCGGTGG-3’; SARS-CoV-2-S-2P forward plasmid 5’-CCTGAGTCGCCTTGATCCGCCGGAAGCTGAAGTTC-3’; SARS-CoV-2-S-2P reverse plasmid 5’-GAACTTCAGCTTCCGGCGGATCAAGGCGACTCAGG-3’).
    5’-CCTGAGTCGCCTTGATCCGCC GGAAGCTGAAGTTC-3’; SARS-CoV-2-S-2P
    suggested: None
    The Kaede-N1 plasmid was a gift from Michael Davidson (Addgene plasmid # 54726; http://n2t.net/addgene:54726; RRID:Addgene_54726) 36.
    detected: RRID:Addgene_54726)
    The mito-mPA-GFP plasmid was a gift from Richard Youle (Addgene plasmid # 23348; http://n2t.net/addgene:23348; RRID:Addgene_23348) 37.
    mito-mPA-GFP
    suggested: None
    detected: RRID:Addgene_23348)
    The pCMV14-3X-Flag-SARS-CoV-2 S plasmid was a gift from Zhaohui Qian (Addgene plasmid # 145780; http://n2t.net/addgene:145780; RRID:Addgene_145780) 38.
    detected: RRID:Addgene_145780)
    The pcDNA3.1-SARS2-Spike plasmid was a gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032; RRID:Addgene_145032) 39.
    detected: RRID:Addgene_145032)
    The SARS-CoV-2 S-6P plasmid was a gift from Jason McLellan (Addgene plasmid # 154754; http://n2t.net/addgene:154754; RRID:Addgene_154754) 30.
    S-6P
    suggested: None
    detected: RRID:Addgene_154754)
    The pcDNA3.1-hACE2 plasmid was a gift from Fang Li (Addgene plasmid # 145033; http://n2t.net/addgene:145033; RRID:Addgene_145033)
    detected: RRID:Addgene_145033)
    All injection mixes had a total concentration of 100 ng/μl and contained the transgene of interest, empty pSM plasmid as a filler, and a co-injection marker for the identification of transgenic animals.
    pSM
    suggested: None
    Software and Algorithms
    SentencesResources
    Both systems were controlled by Zeiss Zen Black software.
    Zen Black
    suggested: (Black Zen software, RRID:SCR_018163)
    The animals were imaged with a Zeiss Axio Imager Z1 microscope equipped with a Photometrics camera (Cool Snap HQ2), and analyzed using Metamorph software (Molecular Devices) and FIJI-ImageJ.
    Metamorph
    suggested: None
    Image acquisition was performed using SlideBook 6.0 (3I, Inc) and processed using FIJI-ImageJ.
    SlideBook
    suggested: (SlideBook , RRID:SCR_014300)
    Statistical analysis: Results were analyzed statistically using GraphPadPrism software (GraphPad Software, Inc).
    GraphPad
    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 did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.09.01.458544v1 on bioRxiv