SARS-CoV-2 spike proteins uptake mediated by lipid raft ganglioside GM1 in human cerebrovascular cells

While there is clinical evidence of neurological manifestation in coronavirus disease-19, its unclear whether this is due to differential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake from blood by cells of the cerebrovasculature. SARS-CoV-2 and its spike protein (SP) interact with the endothelium but the roles of extracellular peptidase domain on angiotensin converting enzyme 2 receptors (ACE2) and ACE2 independent pathways (such as glycans) are not fully elucidated. In addition, for SARS-CoV-2 to enter the brain parenchyma from blood it has to cross several cell types, including the endothelium, pericytes and vascular smooth muscle. Since SARS-CoV-2 interacts with host cells via it SP at the entry point of it life cycle, we used fluorescently labelled SP (SP-555) (wild type and mutants) to model viral behaviour, in vitro, for these cell types (endothelial, pericytes and vascular smooth muscle) to explore pathways of viral entry into brain from blood. There was differential SP uptake by these cell types. The endothelial cells had the least uptake, which may limit SP uptake into brain from blood. Uptake was mediated by ACE2, but it was dependent on SP interaction with ganglioside GM1 in the lipid raft. Mutation sites, N501Yand E484K and D614G, as seen in variants of interest, were differentially taken up by these cell types. There was greater uptake but neutralization with anti-ACE2 and anti-GM1antibodies was less effective. Our data suggested that GM1/lipid raft is an important entry point of SARS-CoV-2 into these cells since inhibition of SP uptake with both anti-ACE2 and anti-GM1 together was similar to that with only anti-GM1, and both ACE2 and GM1 are within the lipid raft region of plasma membrane. Thus, GM1 is a potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.