SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier

  1. Laura Pellegrini
  2. Anna Albecka
  3. Donna L. Mallery
  4. Max J. Kellner
  5. David Paul
  6. Andrew P. Carter
  7. Leo C. James
  8. Madeline A. Lancaster

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Abstract

Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.20.259937: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: All cells were regularly tested and were mycoplasma free.

    Table 2: Resources

    Antibodies
    SentencesResources
    After blocking and permeabilisation with 0.25% Triton and 1% donkey serum buffer, sections were incubated overnight with the following primary antibodies according to their optimized instruction: sheep anti-TTR (1:500, Abcam, ab9015), rabbit anti-ACE2 (1:100, Abcam, ab108209), goat anti-ACE2 (1:100, R&D Systems, AF933), rabbit anti-HTR2C (1:500, Abcam, ab133570), mouse anti-TMPRSS2 (with antigen retrieval, 1:50, SantaCruz, Sc515727), rabbit anti-claudin 5 (1:200, ThermoFisher, 34-1600), rabbit anti-Sox2 (1:300, Abcam, ab97959), rabbit anti-APOA1 (1:100, ThermoFisher, PA5-88109), sheep anti-Tbr2 (1:200, R&D Systems, AF6166), rat anti-CTIP2 (1:300, Abcam, ab18465), mouse anti-HuC/D (1:500, LifeTech, A21271), rabbit anti-DLK1 (1:200, Abcam, ab21682), rabbit anti-GFAP antibody (1:200, Abcam, ab7260), chicken anti-MAP2 (1:1000, Abcam, ab5392),_chicken anti-GFP (1:500, Abcam, ab13970), goat anti-MSX1 (1:200, R&D, AF5045), rabbit anti-ND2 (1:500, Abcam, ab104430), mouse anti-SMI312 (1:500, BioLegend, 837934), mouse anti-SARS-CoV-2 spike glycoprotein (1:200, GeneTex, GTX632604), rabbit anti-SARS-CoV-2 spike glycoprotein C-terminal (1:200, Abcam, ab252690).
    anti-TTR
    suggested: (Abcam Cat# ab9015, RRID:AB_306943)
    anti-ACE2
    suggested: (Abcam Cat# ab108209, RRID:AB_10862654)
    anti-HTR2C
    suggested: (Antibodies.com Cat# A84098, RRID:AB_2124559)
    anti-TMPRSS2
    suggested: None
    anti-claudin 5
    suggested: (Thermo Fisher Scientific Cat# 34-1600, RRID:AB_2533157)
    anti-Sox2
    suggested: (Abcam Cat# ab97959, RRID:AB_2341193)
    anti-APOA1
    suggested: (Thermo Fisher Scientific Cat# PA5-88109, RRID:AB_2804657)
    anti-Tbr2
    suggested: (Millipore Cat# AB2283, RRID:AB_10806889)
    anti-CTIP2
    suggested: (Abcam Cat# ab18465, RRID:AB_2064130)
    anti-HuC/D
    suggested: (V.A. Lennon, Mayo Clinic College of Medicine Cat# HuC/D_Lennon, RRID:AB_2813895)
    anti-DLK1
    suggested: (Abcam Cat# ab21682, RRID:AB_731965)
    anti-GFAP
    suggested: (Abcam Cat# ab7260, RRID:AB_305808)
    anti-MAP2
    suggested: (Abcam Cat# ab5392, RRID:AB_2138153)
    anti-GFP
    suggested: (Abcam Cat# ab13970, RRID:AB_300798)
    anti-MSX1
    suggested: (R and D Systems Cat# AF5045, RRID:AB_2148804)
    anti-ND2
    suggested: (Abcam Cat# ab104430, RRID:AB_10975628)
    anti-SMI312
    suggested: None
    anti-SARS-CoV-2 spike glycoprotein
    suggested: None
    anti-SARS-CoV-2 spike glycoprotein C-terminal
    suggested: None
    Incubation with HCS LipidTox (1:1000 in PBS, Thermo Fisher, H34477) was carrier for 20min after secondary antibody to visualize lipid droplets.
    H34477
    suggested: None
    Membranes were blocked in 5% milk in PBS-T and incubated overnight at 4°C with primary antibodies (rabbit anti-ACE2 and mouse anti-β-actin).
    anti-β-actin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and plasmids: HEK 293T CRL-3216 cells were purchased from ATCC and authenticated by the supplier.
    CRL-3216
    suggested: None
    Vero-hACE2 were generated by transducing Vero cells with lentiviral particles expressing hACE2 ORF and cultured in DMEM 10% FCS with 5 μg/ml blasticidin.
    Vero
    suggested: None
    Lenti-viral particles were generated by cotransfection of HEK 293T cells with pLenti6-Dest_Puro_ACE2-2A-Bla, pCMVR8.74 (Addgene plasmid 22036) and pMD2.G (Addgene plasmid 12259) using PEI.
    HEK 293T
    suggested: None
    For titer determination of pseudotyped viruses, 293T ACE2 cells were plated into 96 well plates at a density of 7.5×103 cells per well and allowed to attach overnight.
    293T ACE2
    suggested: RRID:CVCL_YZ65)
    Virus titers were assessed by plaque assays in Vero ACE2/TMPRSS2 cells.
    Vero ACE2/TMPRSS2
    suggested: None
    For plaque assays, Vero hAce2-TMPRSS2 cells were seeded on 12-well dishes day prior infection.
    Vero hAce2-TMPRSS2
    suggested: None
    Organoid infection experiments: Organoid infections were performed by addition of virus to the culture medium at an expected MOI of 0.5 for each virus based on viral titre determined in 293T ACE2 cells (in the case of pseudotyped virus) or plaque assay (in the case of live SARS-CoV-2).
    ACE2
    suggested: RRID:CVCL_DR94)
    Software and Algorithms
    SentencesResources
    Images were acquired using a Zeiss LSM 780 confocal microscope (Carl Zeiss) and prepared using Fiji (NIH).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Single cell sequencing data were previously published and accessible on NCBI GEO, accession number GSE150903, and through the UCSC cell browser.
    UCSC cell browser
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    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.

  3. Version published to 10.1101/2020.08.20.259937v1 on bioRxiv