Expression of the ACE2 virus entry protein in the nervus terminalis reveals the potential for an alternative route to brain infection in COVID-19

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Abstract

Previous studies suggested that the SARS-CoV-2 virus may gain access to the brain by using a route along the olfactory nerve. However, there is a general consensus that the obligatory virus entry receptor, angiotensin converting enzyme 2 (ACE2), is not expressed in olfactory receptor neurons, and the timing of arrival of the virus in brain targets is inconsistent with a neuronal transfer along olfactory projections. We determined whether nervus terminalis neurons and their peripheral and central projections should be considered as a potential alternative route from the nose to the brain. Nervus terminalis neurons in postnatal mice were double-labeled with antibodies against ACE2 and two nervus terminalis markers, gonadotropin-releasing hormone (GnRH) and choline acetyltransferase (CHAT). We show that a small fraction of CHAT-labeled nervus terminalis neurons, and the large majority of GnRH-labeled nervus terminalis neurons with cell bodies in the region between the olfactory epithelium and the olfactory bulb express ACE2 and cathepsins B and L. Nervus terminalis neurons therefore may provide a direct route for the virus from the nasal epithelium, possibly via innervation of Bowman’s glands, to brain targets, including the telencephalon and diencephalon. This possibility needs to be examined in suitable animal models and in human tissues.

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  1. SciScore for 10.1101/2021.04.11.439398: (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 approved by the local ethics committee for animal research at Bydgoszcz (Poland).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableTwo male homozygous ACE2 KO mice at age 3 weeks old were processed and immunolabeled as described below for wildtype mice.

    Table 2: Resources

    Antibodies
    SentencesResources
    ACE2 -/- knockout (ACE2 KO) control: To verify the specificity of the ACE2 antibody, an ACE2 knock-out (KO) mouse line was obtained from Taconic (strain #18180).
    ACE2
    suggested: None
    Next day sections were washed five times in PBST (PBS with 0.05% Triton X-100) and incubated with a mixture of secondary anti-rabbit-AF488 antibody and anti-goat-AF594 at 1/500 dilution for 60 min at room temperature.
    anti-rabbit-AF488
    suggested: None
    anti-goat-AF594
    suggested: None
    Alternatively, cryosections were stained with rabbit polyclonal anti-CHAT (choline acetyltransferase) instead of rabbit anti-GnRH antibody in the double staining primary antibody mixture.
    anti-CHAT
    suggested: None
    anti-GnRH
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals and tissue processing: A total of six wildtype C57BL/6J mice (Jackson Laboratory) at age 3-4 weeks old were used to obtain tissue material for experiments.
    C57BL/6J
    suggested: None
    ACE2 -/- knockout (ACE2 KO) control: To verify the specificity of the ACE2 antibody, an ACE2 knock-out (KO) mouse line was obtained from Taconic (strain #18180).
    ACE2 -/- knockout
    suggested: None
    Software and Algorithms
    SentencesResources
    The results were analyzed using GraphPad Prism 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 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.