Erythrocyte Plasma Membrane Lipid Composition Mirrors That of Neurons and Glial Cells in Murine Experimental In Vitro and In Vivo Inflammation.

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly.

High-content screen for modifiers of Niemann-Pick type C disease in patient cells.

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disease caused primarily by mutations in NPC1. NPC1 encodes the lysosomal cholesterol transport protein NPC1. The most common NPC1 mutation is a missense mutation (NPC1I1061T) that causes misfolding and rapid degradation of mutant protein in the endoplasmic reticulum. Cholesterol accumulates in enlarged lysosomes as a result of decreased levels of lysosomal NPC1I1061T protein in patient cells. There is currently no cure or FDA-approved treatment for patients. We sought to identify novel compounds that decrease lysosomal cholesterol storage in NPC1I1061T/I1061T patient fibroblasts using a high-content screen with the cholesterol dye, filipin and the lysosomal marker, LAMP1. A total of 3532 compounds were screened, including 2013 FDA-approved drugs, 327 kinase inhibitors and 760 serum metabolites. Twenty-three hits were identified that decreased both filipin and LAMP1 signals. The majority of hits (16/21) were histone deacetylase (HDAC) inhibitors, a previously described class of modifiers of NPC cholesterol storage. Of the remaining hits, the antimicrobial compound, alexidine dihydrochloride had the most potent lysosomal cholesterol-reducing activity. Subsequent analyses showed that alexidine specifically increased levels of NPC1 transcript and mature protein in both control and NPC patient cells. Although unsuitable for systemic therapy, alexidine represents a unique tool compound for further NPC studies and as a potent inducer of NPC1. Together, these findings confirm the utility of high-content image-based compound screens of NPC1 patient cells and support extending the approach into larger compound collections.

Molecular and biochemical biomarkers for diagnosis and therapy monitorization of Niemann-Pick type C patients.

BACKGROUND: Niemann-Pick type C (NP-C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP-C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP-C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP-C patients are arising as biomarkers for this disease screening: 7-ketocholesterol and cholestane-3ß,5α,6ß-triol, both oxidized cholesterol products. OBJECTIVE: In this work, we aimed to evaluate the performance of cholestane-3ß,5α,6ß-triol analysis for the screening and monitoring of NPC patients, correlating it with chitotriosidase levels, Filipin staining and molecular analysis. It was investigated 76 non-treated individuals with NP-C suspicion and also 7 patients with previous NP-C diagnosis under treatment with miglustat, in order to verify the cholestane-3ß,5α,6ß-triol value as a tool for therapy monitoring. RESULTS: Considering molecular assay as golden standard, it was verified that cholestane-3ß,5α,6ß-triol analysis presented 88% of sensitivity, 96.08% of specificity, a positive and negative predictive value calculated in 91.67% and 94.23%, respectively, for the diagnosis of NP-C. Chitotriosidase levels were increased in patients with positive molecular analysis for NP-C. For Filipin staining, it was found 1 false positive, 7 false negative and 24 inconclusive cases, showing that this assay has important limitations for NP-C diagnosis. Besides, we found a significant decrease in cholestane-3ß,5α,6ß-triol concentrations in NP-C patients under therapy with miglustat when compared to non-treated patients. CONCLUSION: Taken together, the present data show that cholestane-3ß,5α,6ß-triol analysis has a high potential to be an important NP-C screening assay, and also can be used for therapy monitorization with miglustat in NP-C patients.

Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-ß-cyclodextrin (MßC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MßC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MßC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution.

An automated fluorescence microscopy assay using a nontoxic cholesterol binding protein, toxin domain 4, (D4), was developed in order to identify chemical compounds modifying intracellular cholesterol metabolism and distribution. Using this method, we screened a library of 1,056 compounds and identified 35 compounds that decreased D4 binding to the cell surface. Among them, 8 compounds were already reported to alter the biosynthesis or the intracellular distribution of cholesterol. The remaining 27 hit compounds were further analyzed biochemically and histochemically. Cell staining with another fluorescent cholesterol probe, filipin, revealed that 17 compounds accumulated cholesterol in the late endosomes. Five compounds decreased cholesterol biosynthesis, and two compounds inhibited the binding of D4 to the membrane. This visual screening method, based on the cholesterol-specific probe D4 in combination with biochemical analyses, is a cell-based, sensitive technique for identifying new chemical compounds and modifying cholesterol distribution and metabolism. Furthermore, it is suitable for high-throughput analysis for drug discovery.

n-3 and n-6 Polyunsaturated fatty acids suppress sterol regulatory element binding protein activity and increase flow of non-esterified cholesterol in HepG2 cells.

The plasma lipid-lowering effect of PUFA, one of their main beneficial effects, is considered to be related to the regulation of lipid biosynthesis through transcription factors including sterol regulatory element binding proteins (SREBP). In the present study, we compared the effect of different PUFA on SREBP activity in HepG2 cells, using a sterol regulatory element-luciferase reporter construct as a probe. Supplementation with different fatty acids reduced SREBP activity in the order 20 : 5n-3 = 18 : 2n-6 = 20 : 4n-6 " 18 : 3n-3 = 22 : 6n-3 = 22 : 5n-6 " 18 : 1n-9. The suppression of SREBP activity greatly depended on the degree of incorporation of the supplemented PUFA into cellular lipids, and correlated positively with the unsaturation index (r 0.831; P < 0.01) of total cell lipids. Supplemented PUFA were also metabolised to longer and more unsaturated species. These processing activities were higher for n-3 than n-6 PUFA (P < 0.01). We studied the effect of PUFA on the intracellular distribution of non-esterified cholesterol, using filipin staining and fluorescence microscopy with or without the cholesterol traffic blocker U18666A. The data show that the incorporation of PUFA increases non-esterified cholesterol flow from the plasma membrane to intracellular membranes. We conclude that suppression of SREBP activity by PUFA depends on the degree of incorporation into cellular lipids, and is associated with increased flow of non-esterified cholesterol between the plasma membrane and intracellular membranes.

Effects of sterols on the development and aging of Caenorhabditis elegans.

Although Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterols as essential nutrients. Supplemental cholesterol undergoes extensive enzymatic modification in C. elegans to form certain sterols of unknown function. Since sterol metabolism in C. elegans differs from that in other species, such as mammals and yeast, it is important to examine how sterols regulate worm physiology. To examine the functions of sterols in C. elegans, a sterol-feeding experiment was carried out and several critical parameters, such as brood size, growth rate, and life span, were measured. In addition, the change in lipid distribution in C. elegans can be both qualitatively and quantitatively determined by various methods, including staining and chromatographic techniques. Taken together, the effects of sterols on C. elegans are very prominent and can be easily assessed using the techniques described here.