Membrane lipids are essential for biological functions ranging from membrane trafficking to signal transduction. The composition of lipid membranes influences their organization and properties, so it ...is not surprising that disorders in lipid metabolism and transport have a role in human disease. Significant recent progress has enhanced our understanding of the molecular and cellular basis of lipid-associated disorders such as Tangier disease, Niemann-Pick disease type C and atherosclerosis. These insights have also led to improved understanding of normal physiology.
Despite its importance for mammalian cell biology and human health, there are many basic aspects of cholesterol homeostasis that are not well understood. Even for the well-characterized delivery of ...cholesterol to cells via lipoproteins, a novel regulatory mechanism has been discovered recently, involving a serum protein called PCSK9, which profoundly affects lipoproteins and their receptors. Cells can export cholesterol by processes that require the activity of ABC transporters, but the molecular mechanisms for cholesterol transport remain unclear. Cholesterol levels in different organelles vary by 5–10-fold, and the mechanisms for maintaining these differences are now partially understood. Several proteins have been proposed to play a role in the inter-organelle movement of cholesterol, but many aspects of the mechanisms for regulating intracellular transport and distribution of cholesterol remain to be worked out. The endoplasmic reticulum is the main organelle responsible for regulation of cholesterol synthesis, and careful measurements have shown that the proteins responsible for sterol sensing respond over a very narrow range of cholesterol concentrations to provide very precise, switch-like control over cholesterol synthesis.
In addition to their roles in normal cell physiology, endocytic processes play a key role in many diseases. In this review, three diseases are discussed as examples of the role of endocytic processes ...in disease. The uptake of cholesterol via LDL is central to our understanding of atherosclerosis, and the study of this disease led to many of the key breakthroughs in understanding receptor-mediated endocytosis. Alzheimer's disease is a growing burden as the population ages. Endosomes and lysosomes play important but only partially understood roles in both the formation and the degradation of the amyloid fibrils that are associated with Alzheimer's disease. Inherited lysosomal storage diseases are individually rare, but collectively they affect many individuals. Recent advances are leading to improved enzyme replacement therapy and are also leading to small-molecule drugs to treat some of these diseases.
J. Neurochem. (2011) 116, 789-795. ABSTRACT: Cholesterol is an important lipid of mammalian cells. Its unique physicochemical properties modulate membrane behavior and it serves as the precursor for ...steroid hormones, oxysterols and vitamin D. Cholesterol is effluxed from the late endosomes/lysosomes via the concerted action of at least two distinct proteins: Niemann-Pick C (NPC)1 and NPC2. Mutations in these two proteins manifest as NPC disease - a very rare, usually fatal, autosomal, recessive, neurovisceral, lysosomal storage disorder. In this review, we discuss the possible mechanisms of action for NPC1 and NPC2 in mediating cholesterol efflux, as well as the different therapeutic approaches being pursued for the treatment of this lipid storage disorder.
Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including ...atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr−/− mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo.
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•Continued efferocytosis requires Drp1-mediated mitochondrial fission•Mitochondrial fission allows release of ER Ca2+ into the cytoplasm•Ca2+-mediated vesicular trafficking enables phagocytosis of a second apoptotic cell•Mice lacking myeloid Drp1 have defective efferocytosis and its pathologic sequelae
How are dead cells in our bodies rapidly cleared by phagocytes in order to avoid inflammation and necrosis?
With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) ...or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.
The impact of apolipoprotein E ε4 (APOE4), the strongest genetic risk factor for Alzheimer's disease (AD), on human brain cellular function remains unclear. Here, we investigated the effects of APOE4 ...on brain cell types derived from population and isogenic human induced pluripotent stem cells, post-mortem brain, and APOE targeted replacement mice. Population and isogenic models demonstrate that APOE4 local haplotype, rather than a single risk allele, contributes to risk. Global transcriptomic analyses reveal human-specific, APOE4-driven lipid metabolic dysregulation in astrocytes and microglia. APOE4 enhances de novo cholesterol synthesis despite elevated intracellular cholesterol due to lysosomal cholesterol sequestration in astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co-cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain. Thus, APOE4 initiates glia-specific cell and non-cell autonomous dysregulation that may contribute to increased AD risk.
Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular ...transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly.
There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain ...has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain–containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.
The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. ...While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.
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