Repeated ethanol exposure and withdrawal alters angiotensin‐converting enzyme 2 expression in discrete brain regions: Implications for SARS‐CoV ‐2 neuroinvasion
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Abstract
Background
People with alcohol use disorder (AUD) may be at higher risk for COVID‐19. Angiotensin‐converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are required for cellular entry by SARS‐CoV‐2, but information on their expression in specific brain regions after alcohol exposure is limited. We sought to clarify how chronic alcohol exposure affects ACE2 expression in monoaminergic brainstem circuits and other putative SARS‐CoV‐2 entry points.
Methods
Brains were examined for ACE2 using immunofluorescence after 4 weeks of chronic intermittent ethanol (CIE) vapor inhalation. We also examined TMPRSS2, Cathepsin L, and ADAM17 by Western blot and RAS pathway mediators and pro‐inflammatory markers via RT‐qPCR.
Results
ACE2 was increased in most brain regions following CIE including the olfactory bulb (OB), hypothalamus (HT), raphe magnus (RMG), raphe obscurus (ROB), locus coeruleus (LC), and periaqueductal gray (PAG). We also observed increased colocalization of ACE2 with monoaminergic neurons in brainstem nuclei. Moreover, soluble ACE2 (sACE2) was elevated in OB, HT, and LC. The increase in sACE2 in OB and HT was accompanied by upregulation of ADAM17, an ACE2 sheddase, while TMPRSS2 increased in HT and LC. Cathepsin L, an endosomal receptor involved in viral entry, was also increased in OB. Alcohol can increase Angiotensin II, which triggers a pro‐inflammatory response that may upregulate ACE2 via activation of RAS pathway receptors AT1R/AT2R. ACE2 then metabolizes Angiotensin II to Angiotensin (1‐7) and provokes an anti‐inflammatory response via MAS1. Accordingly, we report that AT1R/AT2R mRNA decreased in OB and increased in the LC, while MAS1 mRNA increased in both OB and LC. Other mRNAs for pro‐inflammatory markers were also dysregulated in OB, HT, raphe, and LC.
Conclusions
Our results suggest that alcohol triggers a compensatory upregulation of ACE2 in the brain due to disturbed RAS and may increase the risk or severity of SARS‐CoV‐2 infection.
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SciScore for 10.1101/2022.03.29.486282: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animals: All experiments were performed on adult male C57BL/6J mice (3-6 months) following the ethical guidelines of NIH guidelines for animal research and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Iowa.
Euthanasia Agents: (RT-qPCR): A separate group of mice was decapitated under an isoflurane chamber to collect brains for RT-qPCR experiments.Sex as a biological variable Animals: All experiments were performed on adult male C57BL/6J mice (3-6 months) following the ethical guidelines of NIH guidelines for animal research and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Iowa. Randomization not detected. SciScore for 10.1101/2022.03.29.486282: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animals: All experiments were performed on adult male C57BL/6J mice (3-6 months) following the ethical guidelines of NIH guidelines for animal research and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Iowa.
Euthanasia Agents: (RT-qPCR): A separate group of mice was decapitated under an isoflurane chamber to collect brains for RT-qPCR experiments.Sex as a biological variable Animals: All experiments were performed on adult male C57BL/6J mice (3-6 months) following the ethical guidelines of NIH guidelines for animal research and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Iowa. Randomization not detected. Blinding Images were analyzed by trained researchers blind to experimental conditions for expression parameters in ImageJ (Fiji) software as described previously (Sagarkar et al., 2021). Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources Further, the membranes were incubated overnight with antibodies specific to ACE2, TMPRSS2, and β-actin (Table 2) at 4°C. ACE2suggested: NoneTMPRSS2suggested: Noneβ-actin ( Table 2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Animals: All experiments were performed on adult male C57BL/6J mice (3-6 months) following the ethical guidelines of NIH guidelines for animal research and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Iowa. C57BL/6Jsuggested: NoneSoftware and Algorithms Sentences Resources Immuno-colocalization was analyzed by measuring the percentage of co-localization area pixels by total immunoreactive area pixels after setting color thresholding parameters in ImageJ (Fiji) software (Sagarkar et al., 2021). ImageJsuggested: (ImageJ, RRID:SCR_003070)Fijisuggested: (Fiji, RRID:SCR_002285)Data and statistical analysis: The statistical analysis was performed using GraphPad Prism v.9 (CA, USA). GraphPad Prismsuggested: (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.
Results from scite Reference Check: We found no unreliable references.
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