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Alzheimer's disease (AD) is characterized by the presence of amyloid β (Aβ) 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 Aβ 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 β- and γ-secretases to generate Aβ-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-, β-, and γ-secretase trafficking, suggesting that the ratio of Aβ 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 Aβ accumulation and enables the astrocyte regulation of Aβ accumulation by cholesterol signaling., Competing Interests: Competing interest statement: D.M.H. is an inventor on a patent licensed by Washington University to C2N Diagnostics on the therapeutic use of anti-tau antibodies. D.M.H. cofounded and is on the scientific advisory board of C2N Diagnostics. C2N Diagnostics has licensed certain anti-tau antibodies to AbbVie for therapeutic development. D.M.H. is on the scientific advisory board of Denali and consults for Genentech, Merck, Cajal Neuroscience, and Eli Lilly., (Copyright © 2021 the Author(s). Published by PNAS.)

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by the presence of amyloid β (Aβ) 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 Aβ 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 β- and γ-secretases to generate Aβ-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-, β-, and γ-secretase trafficking, suggesting that the ratio of Aβ 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 Aβ accumulation and enables the astrocyte regulation of Aβ accumulation by cholesterol signaling. [ABSTRACT FROM AUTHOR]

Targeting metabolism through bioactive key metabolites is an upcoming future therapeutic strategy. We questioned how modifying intracellular lipid metabolism could be a possible means for alleviating inflammation. Using a recently developed chemical probe (SH42), we inhibited distal cholesterol biosynthesis through selective inhibition of Δ24-dehydrocholesterol reductase (DHCR24). Inhibition of DHCR24 led to an antiinflammatory/proresolving phenotype in a murine peritonitis model. Subsequently, we investigated several omics layers in order to link our phenotypic observations with key metabolic alterations. Lipidomic analysis revealed a significant increase in endogenous polyunsaturated fatty acid (PUFA) biosynthesis. These data integrated with gene expression analysis, revealing increased expression of the desaturase Fads6 and the key proresolving enzyme Alox-12/15. Protein array analysis, as well as immune cell phenotype and functional analysis, substantiated these results confirming the antiinflammatory/proresolving phenotype. Ultimately, lipid mediator (LM) analysis revealed the increased production of bioactive lipids, channeling the observed metabolic alterations into a key class of metabolites known for their capacity to change the inflammatory phenotype. [ABSTRACT FROM AUTHOR]

The malignant progression of pancreatic ductal adenocarcinoma (PDAC) is accompanied by a profound desmoplasia, which forces proliferating tumor cells to metabolically adapt to this new microenvironment. We established the PDAC metabolic signature to highlight the main activated tumor metabolic pathways. Comparative transcriptomic analysis identified lipid-related metabolic pathways as being the most highly enriched in PDAC, compared with a normal pancreas. Our study revealed that lipoprotein metabolic processes, in particular cholesterol uptake, are drastically activated in the tumor. This process results in an increase in the amount of cholesterol and an overexpression of the low-density lipoprotein receptor (LDLR) in pancreatic tumor cells. These findings identify LDLR as a novel metabolic target to limit PDAC progression. Here, we demonstrate that shRNA silencing of LDLR, in pancreatic tumor cells, profoundly reduces uptake of cholesterol and alters its distribution, decreases tumor cell proliferation, and limits activation of ERK1/2 survival pathway. Moreover, blocking cholesterol uptake sensitizes cells to chemotherapeutic drugs and potentiates the effect of chemotherapy on PDAC regression. Clinically, high PDAC Ldlr expression is not restricted to a specific tumor stage but is correlated to a higher risk of disease recurrence. This study provides a precise overview of lipid metabolic pathways that are disturbed in PDAC. We also highlight the high dependence of pancreatic cancer cells upon cholesterol uptake, and identify LDLR as a promising metabolic target for combined therapy, to limit PDAC progression and disease patient relapse. [ABSTRACT FROM AUTHOR]

Epifluorescence microscopy is used to determine phase diagrams for lipid monolayers containing binary mixtures of cholesterol or dihydrocholesterol and dimyristoylphosphatidylserine, as well as ternary mixtures that also contain ganglioside [G.sub.M1]. The phase diagrams are unusual in that they show multiple critical points: two upper miscibility critical points and one lower miscibility critical point. These critical points all are associated with the formation of condensed complexes between these lipids and cholesterol (or dihydrocholesterol). The miscibility critical pressures depend on subphase pH and ionic strength. Changes in critical pressures due to changes in subphase composition are closely related to changes in membrane electrostatic pressure and surface ionization. phospholipids | sphingomyelin | phase diagrams | condensed complexes | liquid immiscibility

How mechanical forces are sensed remains largely mysterious. The forces that gate prokaryotic and several eukaryotic channels were found to come from the lipid membrane. Our survey of animal cells found that membrane force foci all have cholesterol-gathering proteins and are reinforced with cholesterol. This result is evident in overt force sensors at the tips of stereocilia for vertebrate hearing and the touch receptor of Caenorhabditis elegans and mammalian neurons. For less specialized cells, cadherins sustain the force between neighboring cells and integrins between cells and matrix. These tension bearers also pass through and bind to a cholesterol-enriched platform before anchoring to cytoskeleton through other proteins. Cholesterol, in alliance with sphingomyelin and specialized proteins, enforces a more ordered structure in the bilayer. Such a stiffened platform can suppress mechanical noise, redirect, rescale, and confine force. We speculate that such platforms may be dynamic. The applied force may allow disordered-phase lipids to enter the platform-staging channel opening in the thinner mobile neighborhood. The platform may also contain specialized protein/lipid subdomains enclosing mechanosensitive channels to open with localized tension. Such a dynamic stage can mechanically operate structurally disparate channels or enzymes without having to tie them directly to cadherin, integrin, or other protein tethers. [ABSTRACT FROM AUTHOR]

According to the lipid raft hypothesis, biological lipid membranes are laterally heterogeneous and filled with nanoscale ordered "raft" domains, which are believed to play an important role for the organization of proteins in membranes. However, the mechanisms stabilizing such small rafts are not clear, and even their existence is sometimes questioned. Here, we report the observation of raft-like structures in a coarse-grained molecular model for multicomponent lipid bilayers. On small scales, our membranes demix into a liquid ordered (lo) phase and a liquid disordered (ld) phase. On large scales, phase separation is suppressed and gives way to a microemulsion-type state that contains nanometer-sized lo domains in an ld environment. Furthermore, we introduce a mechanism that generates rafts of finite size by a coupling between monolayer curvature and local composition. We show that mismatch between the spontaneous curvatures of monolayers in the lo and ld phases induces elastic interactions, which reduce the line tension between the lo and ld phases and can stabilize raft domains with a characteristic size of the order of a few nanometers. Our findings suggest that rafts in multicomponent bilayers might be closely related to the modulated ripple phase in one-component bilayers. [ABSTRACT FROM AUTHOR]

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165-3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective. [ABSTRACT FROM AUTHOR]

Cholesterol plays an important role in regulating the properties of phospholipid membranes. To obtain a detailed understanding of the lipid-cholesterol interactions, we have developed a mesoscopic water-lipid-cholesterol model. In this model, we take into account the hydrophobic-hydrophilic interactions and the structure of the molecules. We compute the phase diagram of dimyristoylphosphati- dylcholine-cholesterol by using dissipative particle dynamics and show that our model predicts many of the different phases that have been observed experimentally. In quantitative agreement with experimental data our model also shows the condensation effect; upon the addition of cholesterol, the area per lipid decreases more than one would expect from ideal mixing. Our calculations show that this effect is maximal close to the main-phase transition temperature, the lowest temperature for which the membrane is in the liquid phase, and is directly related to the increase of this main-phase transition temperature upon addition of cholesterol. We demonstrate that no condensation is observed if we slightly change the structure of the cholesterol molecule by adding an extra hydrophilic head group or if we decrease the size of the hydrophobic part of cholesterol. [ABSTRACT FROM AUTHOR]

Plasma membranes of cells are asymmetric in both lipid and protein composition. The mechanism by which proteins on both sides of the membrane colocalize during signaling events is unknown but may be due to the induction of inner leaflet domains by the outer leaflet. Here we show that liquid domains form in asymmetric Montal-Mueller planar bilayers in which one leaflet's composition would phase-separate in a symmetric bilayer and the other's would not. Equally important, by tuning the lipid composition of the second leaflet, we are able to suppress domains in the first leaflet. When domains are present in asymmetric membranes, each leaflet contains regions of three distinct lipid compositions, implying strong interleaflet interactions. Our results show that mechanisms of domain induction between the outer and inner leaflets of cell plasma membranes do not necessarily require the participation of membrane proteins. Based on these findings, we suggest mechanisms by which cells could actively regulate protein function by modulating local lipid composition or interleaflet interactions. [ABSTRACT FROM AUTHOR]

Critical fluctuations are investigated in lipid membranes near miscibility critical points in bilayers composed of dioleoylphos- phatidylcholine, chain perdeuterated dipalmitoylphosphatidylcholine, and cholesterol. Phase boundaries are mapped over the temperature range from 10°C to 60°C by deuterium NMR. Tie-lines and three-phase triangles are evaluated across two-phase and three-phase regions, respectively. In addition, a line of miscibility critical points is identified. NMR resonances are broadened in the vicinity of critical points, and broadening is attributed to increased transverse relaxation rates arising from modulation of chain order with correlation times on a microsecond time scale. We conclude that spectral broadening arises from composition fluctuations in the membrane plane with dimensions of <50 nm and speculate that similar fluctuations are commonly found in cholesterol-containing membranes. [ABSTRACT FROM AUTHOR]

Rhodopsin, the G protein-coupled receptor primarily responsible for sensing light, is found in an environment rich in polyunsaturated lipid chains and cholesterol. Biophysical experiments have shown that lipid unsaturation and cholesterol both have significant effects on rhodopsin's stability and function; ω-3 polyunsaturated chains, such as docosahexaenoic acid (DHA), destabilize rhodopsin and enhance the kinetics of the photocycle, whereas cholesterol has the opposite effect. Here, we use molecular dynamics simulations to investigate the possibility that polyunsaturated chains modulate rhodopsin stability and kinetics via specific direct interactions. By analyzing the results of 26 independent 100-ns simulations of dark-adapted rhodopsin, we found that DHA routinely forms tight associations with the protein in a small number of specific locations qualitatively different from the nonspecific interactions made by saturated chains and cholesterol. Furthermore, the presence of tightly packed DHA molecules tends to weaken the interhelical packing. These results are consistent with recent NMR work, which proposes that rhodopsin binds DHA, and they suggest a molecular rationale for DHA's effects on rhodopsin stability and kinetics. [ABSTRACT FROM AUTHOR]

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [³H]7,7-azocholestanol ([³H]AC). After immunoprecipitation, ³H-labeled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [³H]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin A1, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [³H]AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1. [ABSTRACT FROM AUTHOR]

Niemann-Pick C (NPC) disease is a fatal neurodegenerative disorder characterized by a lysosomal accumulation of cholesterol and other lipids within the cells of patients. Clinically identical forms of NPC disease are caused by defects in either of two different proteins: NPC1, a lysosomal-endosomal transmembrane protein and NPCZ, a soluble lysosomal protein with cholesterol binding properties. Although it is clear that NPC1 and NPC2 are required for the egress of lipids from the lysosome, the precise roles of these proteins in this process is unknown. To gain insight into the normal function of NPC2 and to investigate its interactions, if any, with NPC1, we have generated a murine NPC2 hypomorph that expresses 0-4% residual protein in different tissues and have examined its phenotype in the presence and absence of NPC1. The phenotypes of NPC1 and NPC2 single mutants and an NPC1;NPC2 double mutant are similar or identical in terms of disease onset and progression, pathology, neuronal storage, and biochemistry of lipid accumulation. These findings provide genetic evidence that the NPC1 and NPC2 proteins function in concert to facilitate the intracellular transport of lipids from the lysosome to other cellular sites. [ABSTRACT FROM AUTHOR]

Beauveriolides I and Ill, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and Ill strongly inhibited the cholesteryl ester (CE) synthesis with IC[sub50] values of 0.78 and 0.41 μM, respectively, without showing significant effects on the tri-acylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyl-transferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and Ill with lC[sub50] values of 6.0 and 5.5 μM. respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and Ill exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveniolides I and Ill are the first microbial cyclodepsipeptides having an in viva antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents. [ABSTRACT FROM AUTHOR]

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macro phages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10-15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (< 1 μm[SUP2]), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis. [ABSTRACT FROM AUTHOR]

HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host. [ABSTRACT FROM AUTHOR]

Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative highresolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergentresistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts. [ABSTRACT FROM AUTHOR]