GABA and astrocytic cholesterol determine the lipid environment of GABAAR in cultured cortical neurons.

The γ-aminobutyric acid (GABA) type A receptor (GABAAR), a GABA activated pentameric chloride channel, mediates fast inhibitory neurotransmission in the brain. The lipid environment is critical for GABAAR function. How lipids regulate the channel in the cell membrane is not fully understood. Here we employed super resolution imaging of lipids to demonstrate that the agonist GABA induces a rapid and reversible membrane translocation of GABAAR to phosphatidylinositol 4,5-bisphosphate (PIP2) clusters in mouse primary cortical neurons. This translocation relies on nanoscopic separation of PIP2 clusters and lipid rafts (cholesterol-dependent ganglioside clusters). In a resting state, the GABAAR associates with lipid rafts and this colocalization is enhanced by uptake of astrocytic secretions. These astrocytic secretions enhance endocytosis and delay desensitization. Our findings suggest intercellular signaling from astrocytes regulates GABAAR location based on lipid uptake in neurons. The findings have implications for treating mood disorders associated with altered neural excitability.

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

"Ouch!": you have just stabbed your little toe on the sharp corner of a coffee table. That painful sensation stems from nerve cells converting information about external forces into electric signals the brain can interpret. Increasingly, new evidence is suggesting that this process may be starting at fat-based structures within the membrane of these cells. The cell membrane is formed of two interconnected, flexible sheets of lipids in which embedded structures or molecules are free to move. This organisation allows the membrane to physically respond to external forces and, in turn, to set in motion chains of molecular events that help fine-tune how cells relay such information to the brain. For instance, an enzyme known as PLD2 is bound to lipid rafts ­ precisely arranged, rigid fatty 'clumps' in the membrane that are partly formed of cholesterol. PLD2 has also been shown to physically interact with and then activate the ion channel TREK-1, a membrane-based protein that helps to prevent nerve cells from relaying pain signals. However, the exact mechanism underpinning these interactions is difficult to study due to the nature and size of the molecules involved. To address this question, Petersen et al. combined a technology called super-resolution imaging with a new approach that allowed them to observe how membrane lipids respond to pressure and fluid shear. The experiments showed that mechanical forces disrupt the careful arrangement of lipid rafts, causing PLD2 and TREK-1 to be released. They can then move through the surrounding membrane where they reach a switch that turns on TREK-1. Further work revealed that the levels of cholesterol available to mouse cells directly influenced how the clumps could form and bind to PLD2, and in turn, dialled up and down the protective signal mediated by TREK-1. Overall, the study by Petersen et al. shows that the membrane of nerve cells can contain cholesterol-based 'fat sensors' that help to detect external forces and participate in pain regulation. By dissecting these processes, it may be possible to better understand and treat conditions such as diabetes and lupus, which are associated with both pain sensitivity and elevated levels of cholesterol in tissues.

Super-resolution imaging of potassium channels with genetically encoded EGFP.

The plasma membrane is a well-organized structure of lipids and proteins, segmented into lipid compartments under 200 nm in size. This specific spatial patterning is crucial for the function of proteins and necessitates super-resolution imaging for its elucidation. Here, we establish that the genetically encoded enhanced green fluorescent protein (EGFP), when combined with direct optical reconstruction microscopy (dSTORM), tracks shear- and cholesterol-induced nanoscopic patterning of potassium channels overexpressed in HEK293T cells. Leveraging EGFP in dSTORM (EGFP-STORM), our findings indicate that cholesterol directs the C-terminus of TWIK-related potassium channel (TREK-1) to ceramide-enriched lipid ganglioside (GM1) clusters. In the absence of the C-terminus, the channel associates with phosphatidylinositol 4,5-bisphosphate (PIP2) cluster. Similarly, cholesterol derived from astrocytes repositions EGFP-tagged inward-rectifying potassium (Kir) channels into GM1 clusters. Without cholesterol, the channel aligns with PIP2 lipids. We deduce that cholesterol's interaction with Kir sequesters the channel, separating it from its activating lipid PIP2. Fundamentally, a genetically encoded EGFP tag should make any protein amenable to dSTORM analysis.

Cholesterol's Function and Origin in the Alzheimer's Disease Brain.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with neuroinflammation and altered lipids in the brain. Cholesterol is a key component of inflammatory lipids. However, the role of cholesterol in AD, specifically in sporadic or late-onset AD, has remained poorly understood due to the belief that most brain cholesterol is separate from circulating blood cholesterol. A new theory suggests that the permeation of circulating cholesterol into the brain is a causal event critical for the onset of AD. As research in this area continues, new hypotheses and insights into AD are expected to emerge.

The shared role of cholesterol in neuronal and peripheral inflammation.

Neurodegeneration and its loss of cognitive function is associated with inflammation and an accumulation of lipids. In the periphery, cholesterol's uptake drives a major component of chronic inflammation. In this perspective, we describe the cellular and molecular roles of cholesterol in neuroinflammation and contrast them with those in the periphery. Incorporating shared mechanisms from the periphery, cholesterol emerges as a central signal originating in astrocytes and connecting inflammatory escalation in neurons and microglia. A cholesterol uptake pathway is proposed for neuroinflammation, and we speculate on the binding of cholesterol transport protein apolipoprotein E (apoE), including the Christchurch mutant (R136S), to cell surface receptors as a potential protective modality against uptake of astrocyte cholesterol and escalated neuroinflammation. Lastly, we discuss the molecular basis of cholesterol signaling through nanoscopic clustering and peripheral sources of cholesterol after opening of the blood brain barrier.

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity.

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.

The role of high cholesterol in SARS-CoV-2 infectivity.

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200 nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.

Getting in on the action: New tools to see SARS-CoV-2 infect a cell.

In this issue of Cell Chemical Biology, Miao et al. develop probes for live cell tracking of SARS-CoV-2. The probes reveal the endocytic pathway for viral entry. Unexpectedly, the antiviral compound BafA1 traps the virus on the cell surface, highlighting the power of super-resolution imaging in live cells.

Cholesterol Regulation of Membrane Proteins Revealed by Two-Color Super-Resolution Imaging.

Cholesterol and phosphatidyl inositol 4,5-bisphosphate (PIP2) are hydrophobic molecules that regulate protein function in the plasma membrane of all cells. In this review, we discuss how changes in cholesterol concentration cause nanoscopic (<200 nm) movements of membrane proteins to regulate their function. Cholesterol is known to cluster many membrane proteins (often palmitoylated proteins) with long-chain saturated lipids. Although PIP2 is better known for gating ion channels, in this review, we will discuss a second independent function as a regulator of nanoscopic protein movement that opposes cholesterol clustering. The understanding of the movement of proteins between nanoscopic lipid domains emerged largely through the recent advent of super-resolution imaging and the establishment of two-color techniques to label lipids separate from proteins. We discuss the labeling techniques for imaging, their strengths and weakness, and how they are used to reveal novel mechanisms for an ion channel, transporter, and enzyme function. Among the mechanisms, we describe substrate and ligand presentation and their ability to activate enzymes, gate channels, and transporters rapidly and potently. Finally, we define cholesterol-regulated proteins (CRP) and discuss the role of PIP2 in opposing the regulation of cholesterol, as seen through super-resolution imaging.

Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture.

Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP2) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP2 clusters, and HCQ moves ACE2 away from PIP2 clusters-erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.

Regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol.

Alzheimer's disease (AD) is characterized by the presence of amyloid ß (Aß) plaques, tau tangles, inflammation, and loss of cognitive function. Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic risk factor for sporadic AD. In vitro evidence suggests that apoE links to Aß production through nanoscale lipid compartments (lipid clusters), but its regulation in vivo is unclear. Here, we use superresolution imaging in the mouse brain to show that apoE utilizes astrocyte-derived cholesterol to specifically traffic neuronal amyloid precursor protein (APP) in and out of lipid clusters, where it interacts with ß- and γ-secretases to generate Aß-peptide. We find that the targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid and tau 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 clusters, where it interacts with α-secretase and gives rise to soluble APP-α (sAPP-α), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, ß-, and γ-secretase trafficking, suggesting that the ratio of Aß to sAPP-α is regulated by the trafficking of the substrate, not the enzymes. We conclude that cholesterol is kept low in neurons, which inhibits Aß accumulation and enables the astrocyte regulation of Aß accumulation by cholesterol signaling.

The role of high cholesterol in age-related COVID19 lethality.

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 is notably severe in elderly and those with underlying chronic conditions. A molecular mechanism that explains why the elderly are vulnerable and why children are resistant is largely unknown. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the entry of pseudotyped SARS-CoV-2 and the infectivity of the virion. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol shows almost twice the total number of endocytic entry points. Cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the endocytic entry site where SARS-CoV-2 presumably docks to efficiently exploit entry into the cell. Furthermore, in cells producing virus, cholesterol optimally positions furin for priming SARS-CoV-2, producing a more infectious virion with improved binding to the ACE2 receptor. In vivo, age and high fat diet induces cholesterol loading by up to 40% and trafficking of ACE2 to endocytic entry sites in lung tissue from mice. We propose a component of COVID19 severity based on tissue cholesterol level and the sensitivity of ACE2 and furin to cholesterol. Molecules that reduce cholesterol or disrupt ACE2 localization with viral entry points or furin localization in the producer cells, may reduce the severity of COVID19 in obese patients.

Hydroxychloroquine: mechanism of action inhibiting SARS-CoV2 entry.

Hydroxychloroquine (HCQ) has been proposed in the treatment of SARS-coronavirus 2 (SARS-CoV-2) infection, albeit with much controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed to resolve these conflicts. Recently, anesthetics were shown to disrupt ordered monosialotetrahexosylganglioside1 (GM1) lipid rafts. These same lipid rafts recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to an endocytic entry point, away from phosphatidylinositol 4,5 bisphosphate (PIP2) domains. Here we employed super resolution imaging of cultured mammalian cells to show HCQ directly perturbs GM1 lipid rafts and inhibits the ability of ACE2 receptor to associate with the endocytic pathway. HCQ also disrupts PIP2 domains and their ability to cluster and sequester ACE2. Similarly, the antibiotic erythromycin inhibits viral entry and both HCQ and erythromycin decrease the antimicrobial host defense peptide amyloid beta in cultured cells. We conclude HCQ is an anesthetic-like compound that disrupts GM1 lipid rafts similar to anesthetics. The disruption likely decreases viral clustering at both endocytic and putative PIP2 entry points.

Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization.

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.

Studies on the mechanism of general anesthesia.

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 PLDnull flies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.

Disruption of palmitate-mediated localization; a shared pathway of force and anesthetic activation of TREK-1 channels.

TWIK related K+ channel (TREK-1) is a mechano- and anesthetic sensitive channel that when activated attenuates pain and causes anesthesia. Recently the enzyme phospholipase D2 (PLD2) was shown to bind to the channel and generate a local high concentration of phosphatidic acid (PA), an anionic signaling lipid that gates TREK-1. In a biological membrane, the cell harnesses lipid heterogeneity (lipid compartments) to control gating of TREK-1 using palmitate-mediated localization of PLD2. Here we discuss the ability of mechanical force and anesthetics to disrupt palmitate-mediated localization of PLD2 giving rise to TREK-1's mechano- and anesthetic-sensitive properties. The likely consequences of this indirect lipid-based mechanism of activation are discussed in terms of a putative model for excitatory and inhibitory mechano-effectors and anesthetic sensitive ion channels in a biological context. Lastly, we discuss the ability of locally generated PA to reach mM concentrations near TREK-1 and the biophysics of localized signaling. Palmitate-mediated localization of PLD2 emerges as a central control mechanism of TREK-1 responding to mechanical force and anesthetic action. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Tools for Understanding Nanoscale Lipid Regulation of Ion Channels.

Anionic phospholipids are minor but prominent components of the plasma membrane that are necessary for ion channel function. Their persistence in bulk membranes, in particular phosphatidylinositol 4,5-bisphosphate (PIP2), initially suggested they act as channel cofactors. However, recent technologies have established an emerging system of nanoscale signaling to ion channels based on lipid compartmentalization (clustering), direct lipid binding, and local lipid dynamics that allow cells to harness lipid heterogeneity to gate ion channels. The new tools to study lipid binding are set to transform our view of the membrane and answer important questions surrounding ion channel-delimited processes such as mechanosensation.

Polymodal Mechanism for TWIK-Related K+ Channel Inhibition by Local Anesthetic.

BACKGROUND: Local anesthetics cause reversible block of pain and robustly inhibit TWIK-related K channel (TREK-1) currents. Before local anesthesia onset, injection of local anesthetics can cause unwanted transient pain. TREK-1 is an anesthetic-sensitive potassium channel that when inhibited produces pain. A disordered C-terminal loop of TREK-1 is thought to contribute to anesthetic sensitivity, but the molecular basis for TREK-1 inhibition by local anesthetics is unknown. Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA) required for TREK-1 activation and also binds to the channel's C terminus. METHODS: Here, we use biophysical and cellular techniques to characterize direct and indirect lipid-mediated mechanism for TREK-1 inhibition (respectively). We characterized direct binding of local anesthetic to TREK-1 by reconstituting the purified channel into artificial membranes and measuring ion flux. We characterized indirect PA-mediated inhibition of TREK-1 by monitoring lipid production in live whole cells using a fluorescent PLD2 product release assay and ion channel current using live whole-cell patch-clamp electrophysiology. We monitored anesthetic-induced nanoscale translocation of PLD2 to TREK-1 channels with super-resolution direct stochastic reconstruction microscopy (dSTORM). RESULTS: We find local anesthetics tetracaine, lidocaine, and bupivacaine directly bind to and inhibit PLD2 enzymatic activity. The lack of PLD2 activity indirectly inhibited TREK-1 currents. Select local anesthetics also partially blocked the open pore of TREK-1 through direct binding. The amount of pore block was variable with tetracaine greater than bupivacaine and lidocaine exhibiting a minor effect. Local anesthetics also disrupt lipid rafts, a mechanism that would normally activate PLD2 were it not for their direct inhibition of enzyme catalysis. CONCLUSIONS: We propose a mechanism of TREK-1 inhibition comprised of (1) primarily indirect PLD2-dependent inhibition of lipid catalysis and (2) limited direct inhibition for select local anesthetics through partial open pore block. The inhibition through PLD2 explains how the C terminus can regulate the channel despite being devoid of structure and putative binding sites for local anesthetics.