Endothelial Autonomous Mechanisms of Exacerbated Outcomes in Mice with Autoimmune Demyelinating Disease and SARS-CoV-2

Background: Neurological signs and neuropathology are reported in up to 84% of COVID-19 patients and up to 100% of autopsied brains of individuals who died from COVID-19 in the United States. Hypoxia and cerebral hypoperfusion are ubiquitous. Even patients who experience "mild" COVID-19 report persistent brain fog, headache, and extreme fatigue for months. Thus, neuroinflammation and neurovascular dysfunction are central features of COVID-19. Neurological problems may be especially pronounced in patients with pre-existing neuroinflammation and neurovascular pathology, as in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have shown that neurovascular leakage in MS/EAE enables inflammatory solutes and leukocytes to access the central nervous system (CNS) with complex outcomes. For example, interferon-gamma producing Th1 cells promote the antiviral immune response, but also autoimmune tissue destruction. Other respiratory infections are known to exacerbate MS/EAE. In COVID-19, cellular targets of infection are debated, and we therefore cannot answer the question of whether COVID-19 neurological sequelae are due to CNS infection, vascular infection, or are secondary to systemic inflammation. CNS endothelial infection with SARS-CoV-2 could lead to neurovascular leakage and demyelination. Neurovascular inflammation in MS/EAE could enhance CNS infection, decrease viral suppression, and induce/exacerbate long-term autoimmune neurodegenerative damage. We have identified VEGF-A as a potential common driver of neurovascular leakage in COVID-19 and MS. In MS/EAE, interleukin-1b drives VEGF production. In hypoxia, HIF1a drives VEGF production. We have shown that VEGF-A is a critical node in pro-angiogenic transcriptional signatures (Kitajewski lab, unpublished). We predict that in COVID-19 and MS these pathways will converge on elevated VEGF endothelial plasticity, angiogenesis, and neurovascular leakage. MSRP Focus Area: We will develop a novel mouse model for SARS-CoV-2 demyelination and autoimmunity and use it to establish therapeutic targets to enhance Central Nervous System Regenerative Potential in Demyelinating Conditions. Hypothesis/Objective: We hypothesize that central nervous system endothelial cell (EC) infection with SARS-CoV-2 causes demyelination and blood brain barrier (BBB) leakage. We test whether this exacerbates EAE neuropathology. Our objective is to develop a novel mouse model to test whether aberrant VEGF signaling represents an actionable common feature of neurovascular pathology in SARS-CoV-2 infection with autoimmune demyelination. Specific Aim: (1) Assess neurovascular inflammation and demyelination in mice exposed to SARS-CoV-2 and EAE. (1A) Test whether BBB EC dysfunction due to EAE promotes CNS infection by SARS-CoV-2. (1B) Determine if SARS-CoV-2 causes or exacerbates BBB leakage and demyelination. (1C) Evaluate neurovascular VEGF signaling in EAE and SARS-CoV-2. Study Design: *To model multiple sclerosis neurovascular pathology, we utilize the acute myelin oligodendrocyte glycoprotein (MOG35-55) EAE model in male C57Bl/6 mice. In our hands, MOG35-55 EAE in males results in peak clinical disease with BBB permeability at 14 days post immunization, followed by a near-complete remission. *Mice are not naturally susceptible to SARS-CoV-2, so we will introduce the obligate receptor human ACE2 in CNS endothelial cells by intravenous delivery of hACE2 with CNS EC-trophic AAV2-BR1 serotype. In preliminary data we successfully transduced >95% of brain ECs with AAV2-BR1. *During the peak of acute EAE clinical signs, we will intravenously inoculate live SARS-CoV-2 into AAV2-BR1:hACE2+ mice in our BSL3 facility. *We will examine four treatment group cohorts: SARS-CoV-2 + EAE, SARS-CoV-2 alone, EAE alone, and vector control. *Neuroinvasion and viral replication will be measured by plaque assay, QPCR for viral RNA, and staining viral protein. *Blood brain barrier leakage, T cell infiltration, demyelination, and neuronal death will be measured by immunostaining. *VEGF signaling will be assessed by nuclear localized HIF1a; VEGF isoforms, and VEGF receptor levels/phosphorylation by immunostaining and Western blot. Innovation: The impact of COVID-19 on MS disease pathogenesis and progression is unexplored. We will develop a novel mouse model to assess demyelination and neurovascular pathology in SARS-CoV-2. Impact: These studies are designed to resolve the debate regarding whether EC infection by SARS-CoV-2 causes and/or contributes to neurovascular pathology and demyelination. We will determine if VEGF therapy is an actionable biological link between the two disease states that accounts for severe outcomes. Less