Robert Ehehalt

Amyloidogenic processing of the Alzheimer β-amyloid precursor protein depends on lipid rafts

Formation of senile plaques containing the β-amyloid peptide (Aβ) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimers disease (AD). APP is cleaved either by β-secretase or by α-secretase to initiate amyloidogenic (release of Aβ) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating Aβ generation. Reducing cholesterol levels in N2a cells decreased Aβ production. APP and the β-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of Aβ in a cholesterol-dependent manner. Aβ generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by β-secretase, APP outside rafts undergoes cleavage by α-secretase. Thus, access of α- and β-secretase to APP, and therefore Aβ generation, may be determined by dynamic interactions of APP with lipid rafts.

Cholesterol, lipid rafts, and disease

Lipid rafts are dynamic assemblies of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes but can also cluster to form larger, ordered platforms. Rafts are receiving increasing attention as devices that regulate membrane function in eukaryotic cells. In this Perspective, we briefly summarize the structure and regulation of lipid rafts before turning to their evident medical importance. Here, we will give some examples of how rafts contribute to our understanding of the pathogenesis of different diseases. For more information on rafts, the interested reader is referred to recent reviews (1, 2). Composition of lipid rafts Lipid rafts have changed our view of membrane organization. Rafts are small platforms, composed of sphingolipids and cholesterol in the outer exoplasmic leaflet, connected to phospholipids and cholesterol in the inner cytoplasmic leaflet of the lipid bilayer. These assemblies are fluid but more ordered and tightly packed than the surrounding bilayer. The difference in packing is due to the saturation of the hydrocarbon chains in raft sphingolipids and phospholipids as compared with the unsaturated state of fatty acids of phospholipids in the liquid-disordered phase (3). Thus, the presence of liquid-ordered microdomains in cells transforms the classical membrane fluid mosaic model of Singer and Nicholson into a more complex system, where proteins and lipid rafts diffuse laterally within a two-dimensional liquid. Membrane proteins are assigned to three categories: those that are mainly found in the rafts, those that are present in the liquid-disordered phase, and those that represent an intermediate state, moving in and out of rafts. Constitutive raft residents include glycophosphatidylinositol-anchored (GPI-anchored) proteins; doubly acylated proteins, such as tyrosine kinases of the Src family, Gα subunits of heterotrimeric G proteins, and endothelial nitric oxide synthase (eNOS); cholesterol-linked and palmitate-anchored proteins like Hedgehog (see Jeong and McMahon, this Perspective series, ref. 4); and transmembrane proteins, particularly palmitoylated proteins such as influenza virus hemagglutinin and β-secretase (BACE) (1). Some membrane proteins are regulated raft residents and have a weak affinity for rafts in the unliganded state. After binding to a ligand, they undergo a conformational change and/or become oligomerized. When proteins oligomerize, they increase their raft affinity (5). A peripheral membrane protein, such as a nonreceptor tyrosine kinase, can be reversibly palmitoylated and can lose its raft association after depalmitoylation (6). By these means, the partitioning of proteins in and out of rafts can be tightly regulated.

Lipids as Modulators of Proteolytic Activity of BACE INVOLVEMENT OF CHOLESTEROL, GLYCOSPHINGOLIPIDS, AND ANIONIC PHOSPHOLIPIDS IN VITRO

The β-secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic β-amyloid peptide (Aβ). Previous results have suggested that the regulation of β-secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10–150 μm) unilamellar vesicles, and found ∼20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large (∼100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol).

Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4.

Long-chain fatty acids are important metabolites for the generation of energy and the biosynthesis of lipids. The molecular mechanism of their cellular uptake has remained controversial. The fatty acid transport protein (FATP) family has been named according to its proposed function in mediating this process at the plasma membrane. Here, we show that FATP4 is in fact localized to the endoplasmic reticulum and not the plasma membrane as reported previously. Quantitative analysis confirms the positive correlation between expression of FATP4 and uptake of fatty acids. However, this is dependent on the enzymatic activity of FATP4, catalyzing the esterification of fatty acids with CoA. Monitoring fatty acid uptake at the single-cell level demonstrates that the ER localization of FATP4 is sufficient to drive transport of fatty acids. Expression of a mitochondrial acyl-CoA synthetase also enhances fatty acid uptake, suggesting a general relevance for this mechanism. Our results imply that cellular uptake of fatty acids can be regulated by intracellular acyl-CoA synthetases. We propose that the enzyme FATP4 drives fatty acid uptake indirectly by esterification. It is not a transporter protein involved in fatty acid translocation at the plasma membrane.

Retarded release phosphatidylcholine benefits patients with chronic active ulcerative colitis

Background and aims: We examined the hypothesis of an anti-inflammatory effect of phosphatidylcholine in ulcerative colitis. Methods: A phase IIA, double blind, randomised, placebo controlled study was performed in 60 patients with chronic active, non steroid dependent, ulcerative colitis, with a clinical activity index (CAI) of ⩾4. Retarded release phosphatidylcholine rich phospholipids and placebo were administered at a dose of 6 g daily over three months. The primary end point was a change in CAI towards clinical remission (CAI ⩽3) or CAI improvement by ⩾50%. Secondary end points included ⩾50% changes in endoscopic activity index (EAI), histology, and quality of life scores. Results: Induction of clinical remission (CAI ⩽3) as the primary outcome variable was attained by 16 (53%) patients in the phosphatidylcholine treated group compared with three (10%) in the placebo group (p<0.00001). The rate of clinical remission and CAI improvement was 90% in the phosphatidylcholine group and only 10% in the placebo group. A median drop of seven points in the CAI score (70% improvement) was recorded in the phosphatidylcholine group compared with no change in the placebo group. Secondary end point analysis revealed concomitant drops in EAI and histology scores (p = 0.00016 and p = 0.0067 compared with placebo, respectively). Improvement in quality of life was reported by 16 of 29 evaluated patients in the phosphatidylcholine group compared with two of 30 in the placebo group (p = 0.00005). Conclusion: Retarded release oral phosphatidylcholine is effective in alleviating inflammatory activity caused by ulcerative colitis.

Anti-inflammatory effects of phosphatidylcholine

We recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release preparations is beneficial. The positive role of PC in this disease is still elusive. Here we tested the hypothesis that exogenous application of PC has anti-inflammatory properties using three model systems. First, human Caco-2 cells were treated with tumor necrosis factor-α (TNF-α) to induce a pro-inflammatory response via activation of NF-κB. Second, latex bead phagosomes were analyzed for their ability to assemble actin in vitro, a process linked to pro-inflammatory signaling and correlating with the growth versus killing of mycobacteria in macrophages. The third system used was the rapid assembly of plasma membrane actin in macrophages in response to sphingosine 1-phosphate. TNF-α induced a pro-inflammatory response in Caco-2 cells, including 1) assembly of plasma membrane actin; 2) activation of both MAPKs ERK and p38; 3) transport of NF-κB subunits to the nucleus; and 4) subsequent up-regulation of the synthesis of pro-inflammatory gene products. Exogenous addition of most PCs tested significantly inhibited these processes. Other phospholipids like sphingomyelin or phosphatidylethanolamine showed no effects in these assays. PC also inhibited latex bead phagosome actin assembly, the killing of Mycobacterium tuberculosis in macrophages, and the sphingosine 1-phosphate-induced actin assembly in macrophages. TNF-α induces the activation of signaling molecules and the reorganization of the actin cytoskeleton in human intestinal cells. Exogenous application of PC blocks pro-inflammatory signaling in Caco-2 cells, in phagosomes in vitro and facilitates intracellular survival of mycobacteria. We provide further evidence that actin assembly by membranes is part of the pro-inflammatory response. Collectively, these results provide a molecular foundation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis.

Localization of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) in Lipid Rafts/Caveolae and Modulation of Its Activity by Cholesterol in Vitro

Breast cancer resistance protein (BCRP/ABCG2) is an active efflux pump that belongs to the ATP-binding cassette (ABC) transporter family. It is located in various tissues involved in drug absorption, distribution, and elimination and plays an important role in multidrug resistance. For P-glycoprotein, another member of the ABC transporter family, it is well established that it is at least partly located in cholesterol and sphingolipid-enriched domains of the plasma membrane called “lipid rafts” and that the composition of the membrane lipids may modulate its efflux activity. This study addressed the compartmentalization of BCRP in the plasma membrane and the influence of membrane cholesterol on the efflux activity of BCRP. As a cell model, we used the canine kidney epithelial cell line MDCKII-BCRP transfected with the cDNA encoding human BCRP and the corresponding parental cell line MDCKII. Cholesterol depletion with methyl-β-cyclodextrin (MβCD) provoked a 40% decrease in BCRP activity (p < 0.01) assessed with flow cytometry (pheophorbide A efflux assay). Cholesterol repletion with MβCD/cholesterol-inclusion complexes restored BCRP function, and cholesterol saturation of native cells did not further enhance BCRP activity. Coimmunoprecipitation experiments indicated a physical interaction between BCRP and caveolin-1, and Western blot analysis after density gradient ultracentrifugation demonstrated that BCRP is located in detergent-resistant membranes that also contain caveolin-1. In conclusion, our results demonstrate for the first time that BCRP is located in membrane rafts and that cholesterol has impact on its efflux activity.

Translocation of long chain fatty acids across the plasma membrane - lipid rafts and fatty acid transport proteins

Translocation of long chain fatty acids across the plasma membrane is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but transport can also be accelerated by certain membrane proteins as well as lipid rafts. Lipid rafts are dynamic assemblies of proteins and lipids, that float freely within the two dimensional matrix of the membrane bilayer. They are receiving increasing attention as devices that regulate membrane function in vivo and play an important role in membrane trafficking and signal transduction. In this review we will discuss how lipid rafts might be involved in the uptake process and how the candidate proteins for fatty acid uptake FAT/CD36 and the FATP proteins interact with these domains. We will also discuss the functional role of FATPs in general. To our understanding FATPs are indirectly involved in the translocation process across the plasma membrane by providing long chain fatty acid synthetase activity.

Alterations of phospholipid concentration and species composition of the intestinal mucus barrier in ulcerative colitis: a clue to pathogenesis.

Background: Phospholipids are essential for the normal function of the intestinal mucus barrier. The objective of this study was to systematically investigate phospholipids in the intestinal mucus of humans suffering from inflammatory bowel diseases, where a barrier defect is strongly supposed to be pathogenetic. Methods: Optimal mucus recovery was first validated in healthy mice and the method was then transferred to the endoscopic acquisition of ileal and colonic mucus from 21 patients with ulcerative colitis (UC), 10 patients with Crohns disease (CD), and 29 healthy controls. Nano‐electrospray ionization tandem mass spectrometry (ESI‐MS/MS) was used to determine phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in lipid extracts of mucus specimens. Results: Human and rodent mucus contained very similar phospholipid species. In the ileal and colonic mucus from patients suffering from UC, the concentration of PC was highly significantly lower (607 ± 147 pmol/100 &mgr;g protein and 745 ± 148 pmol/100 &mgr;g protein) compared to that of patients with CD (3223 ± 1519 pmol/100 &mgr;g protein and 2450 ± 431 pmol/100 &mgr;g protein) and to controls (3870 ± 760 pmol/100 &mgr;g protein and 2790 ± 354 pmol/100 &mgr;g protein); overall, P = 0.0002 for ileal specimens and P < 0.0001 for colonic specimens. Independent of disease activity, patients suffering from UC showed an increased saturation grade of PC fatty acid residues and a higher LPC‐to‐PC ratio. Conclusions: The intestinal mucus barrier of patients with UC is significantly altered concerning its phospholipid concentration and species composition. These alterations may be very important for the pathogenesis of this disease and underline new therapeutic strategies. Inflamm Bowel Dis 2009

Phosphatidylcholine and lysophosphatidylcholine in intestinal mucus of ulcerative colitis patients. A quantitative approach by nanoelectrospray‐tandem mass spectrometry

Background: A defective mucus composition represents a key pathogenetic factor for intestinal injury. Phosphatidylcholine (PC) is an essential component contributing to formation of a hydrophobic mucus layer. For evaluation of PC in the pathogenesis of inflammatory bowel disease, the concentration and composition of PC in the rectal mucus of patients with ulcerative colitis was determined. Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) allows quantification of PC species and enables analysis of crude extracts. Methods: Lipid extracts of material obtained by light scrapings of the intestinal lumen were analysed quantitatively by nanoESI MS/MS with synthetic internal PC and lysophosphatidylcholine (LPC) standards. PC and LPC species from rectoscopically acquired mucus aliquots of patients with ulcerative colitis were compared to Crohn disease and control subjects. Results: Patients with inactive ulcerative colitis showed significantly less PC and LPC (median 346 [IQR: 230–405] pmol total PC/mg dry weight) in rectal mucus compared to Crohn disease (median 1126 [IQR: 465–1941] pmol total PC/mg dry weight) and control subjects (median 1285 [IQR: 850–1639] pmol total PC/mg dry weight) (P < 0.05). The molecular species of PC and LPC were not significantly different between the groups. The most abundant species were PC 16:0/18:1; PC 16:0/18:2; PC 18:0/18:1; PC 18:0/18:2; LPC 16:0; and LPC 18:0. Conclusion: NanoESI MS/MS is a suitable tool for analysing and quantifying small amounts of PC in human mucus. Patients with ulcerative colitis have significant less PC in their intestinal mucus despite a comparable PC molecular species composition pattern. This suggests that a low amount of protective mucus PC is a characteristic feature in ulcerative colitis and explains an increased susceptibility to luminal contents.