SARS-CoV-2 promotes microglial synapse elimination in human brain organoids

  1. Samudyata
  2. Ana O. Oliveira
  3. Susmita Malwade
  4. Nuno Rufino de Sousa
  5. Sravan K. Goparaju
  6. Jessica Gracias
  7. Funda Orhan
  8. Laura Steponaviciute
  9. Martin Schalling
  10. Steven D. Sheridan
  11. Roy H. Perlis
  12. Antonio G. Rothfuchs
  13. Carl M. Sellgren

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Abstract

Neuropsychiatric manifestations are common in both the acute and post-acute phase of SARS-CoV-2 infection, but the mechanisms of these effects are unknown. In a newly established brain organoid model with innately developing microglia, we demonstrate that SARS-CoV-2 infection initiate neuronal cell death and cause a loss of post-synaptic termini. Despite limited neurotropism and a decelerating viral replication, we observe a threefold increase in microglial engulfment of postsynaptic termini after SARS-CoV-2 exposure. We define the microglial responses to SARS-CoV-2 infection by single cell transcriptomic profiling and observe an upregulation of interferon-responsive genes as well as genes promoting migration and synapse engulfment. To a large extent, SARS-CoV-2 exposed microglia adopt a transcriptomic profile overlapping with neurodegenerative disorders that display an early synapse loss as well as an increased incident risk after a SARS-CoV-2 infection. Our results reveal that brain organoids infected with SARS-CoV-2 display disruption in circuit integrity via microglia-mediated synapse elimination and identifies a potential novel mechanism contributing to cognitive impairments in patients recovering from COVID-19.

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  1. Version published to 10.1038/s41380-022-01786-2
  2. Version published to 10.1101/2021.07.07.451463v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.07.07.451463: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Ethics: All individuals signed a written informed consent before participating in the study, as approved by the Institutional Review Board of Partners HealthCare (Boston, MA, USA) and the Regional Ethical Review Boards in Stockholm, Sweden.
    IRB: Ethics: All individuals signed a written informed consent before participating in the study, as approved by the Institutional Review Board of Partners HealthCare (Boston, MA, USA) and the Regional Ethical Review Boards in Stockholm, Sweden.
    Sex as a biological variableVero E6 cells: Vero E6 (ATCC-CRL-1586) cells were maintained in Dulbecco’s modified eagle medium (DMEM, Cytiva) supplemented with 5 % heat-inactivated fetal bovine serum (FBS, Cytiva), 100 U/mL penicillin and 100 μg/mL streptomycin (Cytiva). iPSC reprogramming: Two healthy human iPSC lines (males) were obtained from the MGH Neurobank.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: All fibroblasts and iPSCs were screened and found negative for Mycoplasma and stained positive for pluripotency markers like octamer-binding transcription factor 4 (POU domain, class 5, transcription factor 1) and TRA-1– 60.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blocked cryosections were incubated with the respective primary antibodies, diluted in blocking solution and incubated overnight at 4°C: anti-ACE2 (rabbit, Nordicbiosite ASJ1B6222, 1:100), anti-β-tubulin (mouse, Promega G712A, 1:500), Cleaved caspase-3 (rabbit, Cell Signaling, 1:300), anti-PAX6 (mouse, Developmental Studies Hybridoma Bank, 1:100), anti-SOX10 antibody (goat, R&D AF2864, 1:50), anti-OLIG2 (goat, R&D AF2418, 1:100), anti-GFAP (mouse, Sigma G3893, 1:100), anti-IBA1 (goat, Abcam AB5076, 1:100), anti-IBA1 (rabbit, Wako 019-19741, 1:100) and SARS-CoV-2 (2019-nCoV) Nucleoprotein / NP Antibody (rabbit, Nordicbiosite 158-40143, 1:200).
    anti-ACE2
    suggested: (Enzo Life Sciences Cat# ALX-804-722-C100, RRID:AB_11180102)
    anti-β-tubulin
    suggested: None
    Cleaved caspase-3
    suggested: None
    anti-PAX6
    suggested: None
    anti-SOX10
    suggested: None
    anti-OLIG2
    suggested: (Rockland Cat# 200-401-E03, RRID:AB_11183021)
    anti-GFAP
    suggested: (Sigma-Aldrich Cat# G3893, RRID:AB_477010)
    anti-IBA1 (goat, Abcam AB5076
    suggested: (Abcam Cat# ab5076, RRID:AB_2224402)
    anti-IBA1
    suggested: (FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504)
    SARS-CoV-2 (2019-nCoV) Nucleoprotein /
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 cells: Vero E6 (ATCC-CRL-1586) cells were maintained in Dulbecco’s modified eagle medium (DMEM, Cytiva) supplemented with 5 % heat-inactivated fetal bovine serum (FBS, Cytiva), 100 U/mL penicillin and 100 μg/mL streptomycin (Cytiva). iPSC reprogramming: Two healthy human iPSC lines (males) were obtained from the MGH Neurobank.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Image quantification: Immunofluorescence images were analyzed using the CellProfiler software to measure and classify cells according to the expression of the selected markers.
    CellProfiler
    suggested: None
    Doublets were identified using scDblFinder package and removed with caution.
    scDblFinder
    suggested: None
    Filtered count matrices were merged and analyzed downstream using the Seurat package.
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    Differential expression testing and functional interpretation: We performed differential gene expression analyses on each cluster across conditions on log-normalized values using the MAST package in R.
    MAST
    suggested: (MAST, RRID:SCR_016340)
    Significant DEGs were used to perform GO term overrepresentation analysis using the enrichR package.
    enrichR
    suggested: (Enrichr, RRID:SCR_001575)
    Gene set enrichment analysis (GSEA) was performed on ranked DEGs using fgsea package for pathway analysis (KEGG, GO:BPdatabase).
    Gene set enrichment analysis
    suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199)
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Intercellular interaction: Cellular crosstalk was inferred via ligand-receptor pair expression using CellphoneDB package (version 2) in python.
    CellphoneDB
    suggested: (CellPhoneDB, RRID:SCR_017054)
    python
    suggested: (IPython, RRID:SCR_001658)

    Results from OddPub: Thank you for sharing your code.

    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: 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

    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.

  4. Version published to 10.1101/2021.07.07.451463v1 on bioRxiv