Mild respiratory SARS-CoV-2 infection can cause multi-lineage cellular dysregulation and myelin loss in the brain

  1. Anthony Fernández-Castañeda
  2. Peiwen Lu
  3. Anna C. Geraghty
  4. Eric Song
  5. Myoung-Hwa Lee
  6. Jamie Wood
  7. Belgin Yalçın
  8. Kathryn R. Taylor
  9. Selena Dutton
  10. Lehi Acosta-Alvarez
  11. Lijun Ni
  12. Daniel Contreras-Esquivel
  13. Jeff R. Gehlhausen
  14. Jon Klein
  15. Carolina Lucas
  16. Tianyang Mao
  17. Julio Silva
  18. Mario A. Peña-Hernández
  19. Alexandra Tabachnikova
  20. Takehiro Takahashi
  21. Laura Tabacof
  22. Jenna Tosto-Mancuso
  23. Erica Breyman
  24. Amy Kontorovich
  25. Dayna McCarthy
  26. Martha Quezado
  27. Marco Hefti
  28. Daniel Perl
  29. Rebecca Folkerth
  30. David Putrino
  31. Avi Nath
  32. Akiko Iwasaki
  33. Michelle Monje

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Abstract

Survivors of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection frequently experience lingering neurological symptoms, including impairment in attention, concentration, speed of information processing and memory. This long-COVID cognitive syndrome shares many features with the syndrome of cancer therapy-related cognitive impairment (CRCI). Neuroinflammation, particularly microglial reactivity and consequent dysregulation of hippocampal neurogenesis and oligodendrocyte lineage cells, is central to CRCI. We hypothesized that similar cellular mechanisms may contribute to the persistent neurological symptoms associated with even mild SARS-CoV-2 respiratory infection. Here, we explored neuroinflammation caused by mild respiratory SARS-CoV-2 infection – without neuroinvasion - and effects on hippocampal neurogenesis and the oligodendroglial lineage. Using a mouse model of mild respiratory SARS-CoV-2 infection induced by intranasal SARS-CoV-2 delivery, we found white matter-selective microglial reactivity, a pattern observed in CRCI. Human brain tissue from 9 individuals with COVID-19 or SARS-CoV-2 infection exhibits the same pattern of prominent white matter-selective microglial reactivity. In mice, pro-inflammatory CSF cytokines/chemokines were elevated for at least 7-weeks post-infection; among the chemokines demonstrating persistent elevation is CCL11, which is associated with impairments in neurogenesis and cognitive function. Humans experiencing long-COVID with cognitive symptoms (48 subjects) similarly demonstrate elevated CCL11 levels compared to those with long-COVID who lack cognitive symptoms (15 subjects). Impaired hippocampal neurogenesis, decreased oligodendrocytes and myelin loss in subcortical white matter were evident at 1 week, and persisted until at least 7 weeks, following mild respiratory SARS-CoV-2 infection in mice. Taken together, the findings presented here illustrate striking similarities between neuropathophysiology after cancer therapy and after SARS-CoV-2 infection, and elucidate cellular deficits that may contribute to lasting neurological symptoms following even mild SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2022.01.07.475453: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All procedures used in this study (sex and age matched) complied with federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee.
    Euthanasia Agents: Briefly, animals were anaesthetized using a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg), injected i.p. The rostral neck was shaved and disinfected.
    Consent: Isolation of patient plasma and cytokine/chemokine measurements: Patient plasma was collected with informed consent and IRB approval as previously described (Lucas et al., 2020).
    Sex as a biological variableMouse models: 6- to 12-week-old female CD1 and BALB/c mice were purchased from Charles River and Jackson Laboratory, respectively, and housed at Yale University.
    Randomizationnot detected.
    BlindingConfocal imaging and quantification: All cell counting was performed by experimenters blinded to experimental conditions on a Zeiss LSM700 or LSM800 scanning confocal microscope (Zeiss).
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Rabbit anti-SARS-CoV-2 nucleocapsid (1:250, GeneTex GTX135357), rabbit anti-IBA1 (1:1000, Wako Chemicals 019-19741), rat anti-CD68 (1:200, Abcam ab53444), rabbit anti-DCX (1:500, Abcam ab18723), rabbit anti-ASPA (1:250, EMD Millipore ABN1698), goat anti-PDGFRa (1:500; R&D Systems AF1062), mouse anti-CC1 (1:100; EMD Millipore OP80) were diluted in 1% normal donkey serum in 0.3% Triton X-100 in TBS and incubated overnight at 4°C (CC1 was incubated for 7 days at 4°C).
    anti-DCX
    suggested: (Abcam Cat# ab18723, RRID:AB_732011)
    Sections were then rinsed three times in 1X TBS and incubated in secondary antibody solution (Alexa 647 donkey anti-rabbit IgG, 1:500 (Jackson Immunoresearch); Alexa 488 donkey anti rat IgG, 1:500 (Jackson Immunoresearch); Alexa 594 donkey anti-rabbit IgG, 1:500 (Jackson Immunoresearch); Alexa 488 donkey anti goat IgG, 1:500 (Jackson Immunoresearch); Alexa 647 donkey anti-mouse IgG, 1:500 (Jackson Immunoresearch) in 1% blocking solution at room temperature protected from light for two hours.
    anti rat IgG
    suggested: None
    anti-rabbit IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 711-545-152, RRID:AB_2313584)
    anti goat IgG
    suggested: None
    anti-mouse IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 015-500-003, RRID:AB_2337222)
    Antibodies included IBA1 (Wako 019-19741) and CD68 (Thermo Fisher Scientific 14-0688-82).
    IBA1
    suggested: (FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504)
    CD68
    suggested: (Thermo Fisher Scientific Cat# 14-0688-82, RRID:AB_11151139)
    Antibody binding was visualized with 3,3′- diaminobenzidine (DAB; Vector Laboratories).
    DAB
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Generation of SARS-CoV-2 stocks: To generate SARS-CoV-2 viral stocks, Huh7.5 cells were inoculated with SARS-CoV-2 isolate USA-WA1/2020 (NR-52281; BEI Resources) to generate a P1 stock.
    Huh7.5
    suggested: RRID:CVCL_7927)
    Virus titer was determined by plaque assay using Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse models: 6- to 12-week-old female CD1 and BALB/c mice were purchased from Charles River and Jackson Laboratory, respectively, and housed at Yale University.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Software and Algorithms
    SentencesResources
    Images were taken at 20X magnification and analyzed using FIJI software.
    FIJI
    suggested: (Fiji, RRID:SCR_002285)
    The cingulum of the corpus callosum in each hemisphere was imaged with one 319.5 × 319.5 μm field and cells were quantified using the “Spots” function in Imaris image analysis software (Oxford Instruments).
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    g-ratios were measured by dividing the axonal diameter by the diameter of the entire fiber (diameter of axon/diameter of axon + myelin sheath) using ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    All statistics were performed using Prism Software (Graphpad).
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)

    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.

    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.

  2. Version published to 10.1101/2022.01.07.475453 on bioRxiv