Nanocrystal Quantum Dot Biomimetics of SARS-CoV-2 to Interrogate Neutrophil-Mediated Neuroinflammation at the Blood-Brain Barrier

PROJECT SUMMARY/ABSTRACT Public/health/relevance: Chronic, or recurring, neurological deficits in 60% of recovered COVID-19 patients are now an unmet medical need to treat the aftermath of SARS-CoV-2 infection of the central nervous system (CNS). A recent study from Germany suggests that these symptoms persist beyond a year, similarly to patients suffering from chronic symptoms due to SARS-CoV-1 infection. Thus, there is clear need for interventions against chronic neurologic symptoms after COVID. Elucidating the mechanism for SARS-CoV-2 impact on the CNS is essential to inform the design of such interventions. Objective: This proposal aims to identify a pathway for SARS-CoV-2's effects on the CNS through a dysregulated blood-brain barrier (BBB) mediated by a neutrophil-dependent "storm" of bradykinin (BK). We hypothesize that this storm induces neuroinflammation that ultimately disrupts normal neuronal signaling, providing the substrate for enduring neurological symptoms. Research Plan: Recent studies have reported altered integrity of the BBB in response to the spike (S) protein of SARS-CoV-2, thereby suggesting a neuroinvasive pathway for SARS-CoV-2 or inflammatory immune cells through the BBB. In line with these observations, this proposal will investigate how pro-inflammatory mediators associated with COVID infection activate neutrophil-mediated upregulation of BK; this leads to an increased permeability through paracellular gaps across the BBB due to dysregulated tight junctions (TJs). Such a model aligns with the upregulated levels of BK observed in bronchoalveolar fluid taken from COVID-19 patients coupled with the ability of neutrophils to engage the kinin system to remodel endothelial barriers in acute inflammation. As a proxy for native SARS-CoV-2, we will construct S protein coated quantum dots as high fidelity biomimetics of SARS-CoV-2 to investigate the size and structural constraints regulating SARS-CoV-2 permeability across the BBB. These constructs will be used to bias neutrophils to a pro-inflammatory state in the presence of relevant kallikrein-kinin factors to increase the permeability of cultured bEnd.3 monolayers, a high-fidelity in vitro model system for murine BBB. A leakier BBB will be indicated by increased permeability of our fluorescent SARS-CoV-2 biomimetic and corroborated with complementary measurements of global barrier health, as measured by transendothelial electrical resistance (TEER). Lastly, we will construct a correlated scanning ion conductance and confocal microscope system to examine the heterogeneity of dysregulated barrier function and the specific nanoscale changes in TJ expression and localization that regulate it.