The human brain vasculature shows a distinct expression pattern of SARS-CoV-2 entry factors

  1. Moheb Ghobrial
  2. Jason Charish
  3. Shigeki Takada
  4. Taufik Valiante
  5. Philippe P. Monnier
  6. Ivan Radovanovic
  7. Gary D. Bader
  8. Thomas Wälchli

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

A large number of hospitalized COVID-19 patients show neurological symptoms such as ischemic- and hemorrhagic stroke as well as encephalitis, and SARS-CoV-2 can directly infect endothelial cells leading to endotheliitis across multiple vascular beds. These findings suggest an involvement of the brain- and peripheral vasculature in COVID-19, but the underlying molecular mechanisms remain obscure. To understand the potential mechanisms underlying SARS-CoV-2 tropism for brain vasculature, we constructed a molecular atlas of the expression patterns of SARS-CoV-2 viral entry-associated genes (receptors and proteases) and SARS-CoV-2 interaction partners in human (and mouse) adult and fetal brain as well as in multiple non-CNS tissues in single-cell RNA-sequencing data across various datasets. We observed a distinct expression pattern of the cathepsins B (CTSB) and -L (CTSL) - which are able to substitute for the ACE2 co-receptor TMPRSS2 - in the human vasculature with CTSB being mainly expressed in the brain vasculature and CTSL predominantly in the peripheral vasculature, and these observations were confirmed at the protein level in the Human Protein Atlas and using immunofluorescence stainings. This expression pattern of SARS-CoV-2 viral-entry associated proteases and SARS-CoV-2 interaction partners was also present in endothelial cells and microglia in the fetal brain, suggesting a developmentally established SARS-CoV-2 entry machinery in the human vasculature. At both the adult and fetal stages, we detected a distinct pattern of SARS-CoV-2 entry associated genes’ transcripts in brain vascular endothelial cells and microglia, providing a potential explanation for an inflammatory response in the brain endothelium upon SARS-CoV-2 infection. Moreover, CTSB was co-expressed in adult and fetal brain endothelial cells with genes and pathways involved in innate immunity and inflammation, angiogenesis, blood-brain-barrier permeability, vascular metabolism, and coagulation, providing a potential explanation for the role of brain endothelial cells in clinically observed (neuro)vascular symptoms in COVID-19 patients. Our study serves as a publicly available single-cell atlas of SARS-CoV-2 related entry factors and interaction partners in human and mouse brain endothelial- and perivascular cells, which can be employed for future studies in clinical samples of COVID-19 patients.

Article activity feed

  2. Version published to 10.1101/2020.10.10.334664v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.10.10.334664: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All the procedures performed with the use of samples obtained from patients were approved by the Institutional Research Ethics Review Board of the University Health Network (
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Immunofluorescence staining of TMPRSS2, CTSB, and CTSL in endothelial- and perivascular cells of the human adult brain vasculature: Fixed, Cryo-embedded, normal human temporal lobe of adult brain slices were cut in 40-µm thick sections, using a cryotome (Leica Cryostat 1720 Digital Cryotome), and submitted for single and double staining with the following antibodies (Abs): pig pAb anti-CD31 (
    TMPRSS2
    suggested: None
    anti-CD31
    suggested: None
    Software and Algorithms
    SentencesResources
    Illustration of the results was generated using Seurat (v.3.1.5).
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    The top 50 genes in each dataset were characterized based on GeneCards information and gene ontology classes from the Gene Ontology database.
    GeneCards
    suggested: (GeneCards, RRID:SCR_002773)
    The resulting pathways are ranked using NES, and FDR q-value, p-values are reported in the GSEA output reports.
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)
    Highly related pathways were grouped into a theme and labeled by AutoAnnotate (version 1.3) in Cytoscape (Version 3.7.0) and EnrichmentMap (version 3.3) was used to plot the pathways.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    EnrichmentMap
    suggested: (EnrichmentMap, RRID:SCR_016052)
    Z-stacks of optical planes (maximum intensity projections) and single optical planes were recorded and analysed by Zeiss Zen software.
    Zeiss Zen
    suggested: None

    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: We detected the following sentences addressing limitations in the study:
    We are aware of the limitations of these analyses, as for instance sparse cell types might be lacking or underrepresented/under-detected due to their low abundance, technical limitations related to isolation protocols, or bioinformatic analyses including technical/computational dropout effects. Thus, the specificity is high (meaning positive results are highly reliable) whereas the sensitivity is limited and thus negative results should be interpreted with care. SARS-CoV-2 shows systemic effects affecting multiple organ systems including the brain, liver, kidney, heart, and others, and increasing evidence suggests that blood vessel endothelial cells exert crucial roles in the underlying pathogenesis30,49,81,82. Whereas cellular entry of SARS-CoV-1 exclusively depends on CTSL and TMPRSS150,53, cellular entry of SARS-CoV-2 can occur either via TMPRSS2 or CTSB/CTSL53,59 (Extended Data Figure 16). Thus, the widespread expression of CTSB in the brain (vasculature) and the expression of CTSL in multiple other SARS-CoV-2 affected organs/tissues suggest that CTSB and CTSL might be involved in alternative entry mechanisms and transmission routes that could be responsible for the neurovascular- and vascular phenotypes observed in COVID-19 patients30,32. Furthermore, our findings that CTSB is – in addition to the brain endothelium – also highly expressed in microglia (and that CTSL shows high expression in peripheral endothelial cells and macrophages) suggests a potential mechanism invo...

    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.

    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 10.1101/2020.10.10.334664v1 on bioRxiv