Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan China in 2019. The disease in notably severe in elderly and those with underlying chronic conditions. The molecular mechanism as to why the elderly are vulnerable and why children are resistant is largely unknown. Understanding these differences is critical for safeguarding the vulnerable and guiding effective policy and treatments. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the endocytic entry of pseudotyped SARS-CoV-2. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol showed markedly increased apparent diameter (~10% to 100 nm) and almost twice the total number of viral entry points. The cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the viral entry site where SARS-CoV-2 docks to properly exploit entry into the cell. Furthermore, we show cholesterol enhances binding of SARS-CoV-2 to the cell surface which increases association with the endocytic pathway. Decreasing cellular cholesterol has the opposite effect. Based on these findings and known loading of cholesterol into peripheral tissue during aging and inflammation, we build a cholesterol dependent model for COVID19 lethality in elderly and the chronically ill. As cholesterol increases with age and inflammation (e.g. smoking and diabetes), the cell surface is coated with viral entry points and optimally assembled viral entry proteins. Importantly our model suggests high levels of cholesterol is most alarming in the tissue, not the blood. In fact, rapidly dropping cholesterol in the blood may indicate severe loading of cholesterol in peripheral tissue and a dangerous situation for escalated SARS-CoV-2 infectivity. Polyunsaturated fatty acids (PUFAs) oppose the effects of cholesterol and provide a molecular basis for eating healthy diets to avoid severe cases of COVID19.

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K+channels (TREK-1) and reversibly induce loss of consciousness. For 100 y, anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of phospholipase D2 (PLD2) localization to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in whole-cell patch-clamp recordings. Localization of PLD2 renders the TRAAK channel sensitive, a channel that is otherwise anesthetic insensitive. General anesthetics, such as chloroform, isoflurane, diethyl ether, xenon, and propofol, disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnullflies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.

Alzheimer's Disease (AD), a common and burdensome neurodegenerative disorder, is characterized by the presence of β-Amyloid (Aβ) plaques, inflammation, and loss of cognitive function. A cholesteroldependent process sorts Aβ-producing enzymes into nanoscale lipid compartments (also called lipid rafts). Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic marker for sporadic AD. Evidence suggests apoE links to Aβ production through lipid rafts, but so far there has been little scientific validation of this link in vivo. Here we use super-resolution imaging to show apoE utilizes astrocyte-derived cholesterol to specifically traffic amyloid precursor protein (APP) into lipid rafts where it interacts with β-and g-secretases to generate Aβ-peptide. We find that targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid burden in a mouse model of AD. Treatment with cholesterol-free apoE or knockdown of cholesterol synthesis in astrocytes decreases cholesterol levels in cultured neurons and causes APP to traffic out of lipid rafts where it interacts with αsecretase and gives rise to soluble APPα (sAPPα), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, β-, and g-secretase trafficking, suggesting the ratio of Aβ to sAPPα is regulated by the trafficking of the substrate, not the enzymes. Treatment of astrocytes with inflammatory cytokines IL-1β, IL-6 and TNF-α upregulates the synthesis of cholesterol in the astrocytes. We conclude that cholesterol is a signaling molecule kept low in neurons to inhibit raft function, decrease Aβ formation, and enable astrocyte regulation of APP by cholesterol control of substrate presentation. Highlights:ApoE regulates amyloid precursor protein localization to rafts and its exposure to α-vs. β-secretase. α-, β-, and g-Secretases are activated by substrate presentation. ApoE specifically transports astrocyte cholesterol to neurons. Astrocyte cholesterol synthesis disruption prevents Alzheimer's-associated amyloid pathology in mice.

No abstract

Background: SARS-coronavirus 2 (SARS-CoV-2) is currently causing a worldwide pandemic. Potential drugs identified for the treatment of SARS-CoV-2 infection include chloroquine (CQ), its derivative hydroxychloroquine (HCQ), and the anesthetic propofol. Their mechanism of action in SARS-CoV-2 infection is poorly understood. Recently, anesthetics, both general and local, were shown to disrupt ordered lipid domains. These same lipid domains recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to an endocytic entry point and their disruption by cholesterol depletion decreases ACE2 recruitment and viral entry. Methods: Viral entry was determined using a SARS-CoV-2 pseudovirus (SARS2-PV) and a luciferase reporter gene expressed by the virus after treatment of the cells with 50 micromolar propofol, tetracaine, HCQ, and erythromycin. HCQ disruption of monosialotetrahexosylganglioside1 (GM1) lipid rafts, phosphatidylinositol 4,5-bisphosphate (PIP2) domains, and ACE2 receptor at nanoscale distances was monitored by direct stochastic reconstruction microscopy (dSTORM). Cells were fixed, permeabilized, and then labeled with either fluorescent cholera toxin B (CTxB) or antibody and then fixed again prior to imaging. Cluster analysis of dSTORM images was used to determine size and number and cross pair correlation was used to determine trafficking of endogenously expressed ACE2 in and out of lipid domains. Results: Propofol, tetracaine, and HCQ inhibit SARS2-PV viral entry. HCQ directly perturbs both GM1 lipid rafts and PIP2 domains. GM1 rafts increased in size and number similar to anesthetic disruption of lipid rafts; PIP2 domains decreased in size and number. HCQ blocked both GM1 and PIP2 domains ability to attract and cluster ACE2. Conclusions: We conclude HCQ is an anesthetic-like compound that disrupts GM1 lipid rafts similar to propofol and other local or general anesthetics. Furthermore, we conclude disruption of GM1 raft function, and not the concentration of GM1 raft molecules, governs the antiviral properties of HCQ. HCQ disruption of the membrane appears to also disrupt the production of host defense peptide, hence an antimicrobial such as erythromycin could be an important combined treatment. Nonetheless erythromycin has anti-SARS-CoV-2 activity and may combine with HCQ to reduce infection.