Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

  1. Madeline G. Andrews
  2. Tanzila Mukhtar
  3. Ugomma C. Eze
  4. Camille R. Simoneau
  5. Yonatan Perez
  6. Mohammed A. Mostajo-Radji
  7. Shaohui Wang
  8. Dmitry Velmeshev
  9. Jahan Salma
  10. G. Renuka Kumar
  11. Alex A. Pollen
  12. Elizabeth E. Crouch
  13. Melanie Ott
  14. Arnold R. Kriegstein

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

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  1. ScreenIT

    SciScore for 10.1101/2021.01.17.427024: (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 cell lines tested negative for mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061-2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419-1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735-1-AP, 1:100), Aqp4 (Proteintech, 16473-1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940-1-AP, 1:50), CD147 (Invitrogen, 34-5600, 1:500), Arcn1 (Proteintech, 23843-1-AP, 1:50), Rat: Gfap (Thermofisher, 13-0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R&D, AF933, 1:200), Ace2 (Thermofisher, MA5-32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R&D, AF385, 1:100), Sheep: Eomes (R&D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R&D, AF7227, 1:200).
    MABE1134
    suggested: (Millipore Cat# MABE1134, RRID:AB_2819101)
    Sox2
    suggested: (Santa Cruz Biotechnology Cat# sc-365823, RRID:AB_10842165)
    S100B
    suggested: None
    S2532
    suggested: (Sigma-Aldrich Cat# S2532, RRID:AB_477499)
    Ki67
    suggested: (Abcam Cat# ab156956, RRID:AB_2732028)
    CD31
    suggested: None
    GA61061-2
    suggested: None
    Olig2
    suggested: (Millipore Cat# MABN50, RRID:AB_10807410)
    MABN50
    suggested: (Millipore Cat# MABN50, RRID:AB_10807410)
    Pax6
    suggested: (BioLegend Cat# 901301, RRID:AB_2565003)
    Cleaved Caspase-3 (Cell Signaling,
    suggested: (Cell Signaling Technology Cat# 9661, RRID:AB_2341188)
    20735-1-AP
    suggested: (Proteintech Cat# 20735-1-AP, RRID:AB_11042328)
    Aqp4
    suggested: (Novus Cat# NB600-716, RRID:AB_10001192)
    16473-1-AP
    suggested: (Proteintech Cat# 16473-1-AP, RRID:AB_2827426)
    Egfr (Abcam, ab32077
    suggested: (Abcam Cat# ab32077, RRID:AB_732101)
    Dpp4
    suggested: None
    CD147
    suggested: (Innovative Research Cat# 34-5600, RRID:AB_87290)
    Arcn1
    suggested: (Proteintech Cat# 23843-1-AP, RRID:AB_2879337)
    23843-1-AP
    suggested: (Proteintech Cat# 23843-1-AP, RRID:AB_2879337)
    Laminin
    suggested: (Abcam Cat# ab80580, RRID:AB_1604099)
    Nrp1
    suggested: (Abcam Cat# ab81321, RRID:AB_1640739)
    Map2
    suggested: (Abcam Cat# ab5392, RRID:AB_2138153)
    Ace2
    suggested: (Thermo Fisher Scientific Cat# MA5-32307, RRID:AB_2809589)
    Iba1
    suggested: (Abcam Cat# ab48004, RRID:AB_870576)
    AF385
    suggested: (R and D Systems Cat# AF385, RRID:AB_355339)
    AF6166
    suggested: (R and D Systems Cat# AF6166, RRID:AB_10569705)
    NeuN
    suggested: (Millipore Cat# ABN90, RRID:AB_11205592)
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 stocks were passaged in Vero cells (ATCC) and titer was determined via plaque assay on Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Sections were washed with PBS plus 0.5% Triton X-100 and then incubated in AlexaFluor secondary antibodies (Thermo Fisher and Jackson Labs) diluted in blocking buffer at 4 °C overnight.
    Thermo Fisher and Jackson Labs
    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:
    While cerebral organoid models have limitations in terms of the diversity and fidelity of neural cell types (36), our studies indicate that astrocytes are preferentially infected in both primary human cortical tissue and cortical organoid models. Moreover, organoid-derived and primary astrocytes had similar proportions of receptor abundance and reactivity phenotypes following infection, further emphasizing the utility of organoid models to study viral tropism and cellular responses. However, future studies exploring post-mortem patient neural tissues will be required to understand if mature astrocytes are as vulnerable as immature astrocytes, and to further define the receptors that may mediate infectivity. The results of our qPCR analysis indeed suggest moderate expression of coronavirus receptors ACE2, DPP4, CD147, and NRP1 in the adult cortex. The range of COVID-19 associated neurological symptoms including dizziness, seizures, and cognitive difficulties, may reflect the involvement of astrocytes that are vital to global brain homeostasis and function. Further studies elucidating potential routes of CNS infection as well as strategies to block viral entry may aid in controlling transmission and help alleviate disease symptoms.

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 7, 9, 11, 12 and 14. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

  2. Version published to 10.1101/2021.01.17.427024v1 on bioRxiv