Cholesterol pathways affected by small molecules that decrease sterol levels in Niemann-Pick type C mutant cells.

BACKGROUND: Niemann-Pick type C (NPC) disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles. METHODOLOGY/PRINCIPAL FINDINGS: The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds. CONCLUSIONS/SIGNIFICANCE: Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165).

Investigation of N-aryl-3-alkylidenepyrrolinones as potential Niemann-Pick type C disease therapeutics.

A five-step synthesis of an array of N-aryl-3-alkylidenepyrrolinones, which are potential Niemann-Pick type C (NPC) disease therapeutics, is described. The synthetic route allows for the production of analogues, including photoaffinity and biotinylated derivatives. Compound 1a increased esterification by acyl-coenzyme A:cholesteryl acyltransferase in NPC1 mutant cells. It also decreased LDL uptake and increased cholesterol efflux in both NPC1-deficient and normal cells.

Chemical screen to reduce sterol accumulation in Niemann-Pick C disease cells identifies novel lysosomal acid lipase inhibitors.

Niemann-Pick C disease (NPC) is a lysosomal storage disorder causing abnormal accumulation of unesterified free cholesterol in lysosomal storage organelles. High content phenotypic microscopy chemical screens in both human and hamster NPC-deficient cells have identified several compounds that partially revert the NPC phenotype. Cell biological and biochemical studies show that several of these molecules inhibit lysosomal acid lipase, the enzyme that hydrolyzes LDL-derived triacylglycerol and cholesteryl esters. The effects of reduced lysosomal acid lipase activity in lowering cholesterol accumulation in NPC mutant cells were verified by RNAi-mediated knockdown of lysosomal acid lipase in NPC1-deficient human fibroblasts. This work demonstrates the utility of phenotypic cellular screens as a means to identify molecular targets for altering a complex process such as intracellular cholesterol trafficking and metabolism.

Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive genetic disorder manifested by abnormal accumulation of unesterified cholesterol and other lipids. We screened combinatorially synthesized chemical libraries to identify compounds that would partially revert cholesterol accumulation. Cultured CHO cells with NPC phenotypes (CT60 and CT43) were used for screening along with normal CHO cells as a control. We developed an automated microscopy assay based on imaging of filipin fluorescence for estimating cholesterol accumulation in lysosomal storage organelles. Our primary screen of 14,956 compounds identified 14 hit compounds that caused significant reduction in cellular cholesterol accumulation at 10 microM. We then screened a secondary library of 3,962 compounds selected based on chemical similarity to the initial hits and identified 7 compounds that demonstrated greater efficacy and lower toxicity than the original hits. These compounds are effective at concentrations of 123 nM to 3 microM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.

In situ MALDI-TOF MS regional analysis of neutral phospholipids in lens tissue.

Phospholipids (PLs) are important sources of lipid second messengers that participate in cell signaling pathways. Consequently, their analysis in biological tissues has received increased attention. Current approaches for PL analysis include an extraction step and subsequent identification of the main PL classes by either 31P NMR spectroscopy or chromatographic separation followed by mass spectrometric detection. Previous in vitro studies revealed regional changes in the PL composition of mammalian lenses at different growth stages. In this report, we demonstrate the feasibility of direct in situ analysis of two relevant PL classes, phosphatidylcholines (PCs) and sphingomyelins (SMs), in slices of fresh or fixed (2.5% formaldehyde) mammalian lenses. The chosen matrix was p-nitroaniline, as it generated superior sensitivity when compared to 2,5-dihydroxybenzoic acid, the compound most commonly used for in vitro analysis of PLs by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Regional differences in the relative amounts of PCs and SMs were in agreement with trends demonstrated by previous in vitro studies. Fresh and fixed tissue of the same lens gave comparable relative levels of PCs and SMs. In situ analysis of PLs by MALDI-TOF MS offers a rapid and sensitive tool for the mapping of PLs in biological tissues.

Glycero- versus sphingo-phospholipids: correlations with human and non-human mammalian lens growth.

The human lens differs from other mammalian lenses in its very slow growth and unusual phospholipid composition of its cell membranes. Dihydrosphingomyelins (DHSMs) make up about half of all phospholipids in adult human fiber membranes. In all other membranes, sphingomyelins(SMs) with a trans double bond in their backbone, are prevalent. In our quest to understand the biological implications of such elevated DHSM levels, we analyzed membranes from various regions of human, elephant, giraffe, polar bear, pig and cow lenses. The levels of DHSMs were minor in non-human lens membranes. A strong correlation was observed between growth rate and relative contents of phosphatidylcholines(PCs) in epithelia and outer cortical fibers. Sphingomyelins became increasingly predominant in differentiated fibers and this increase was age dependent. Indeed, nuclear fiber membranes of aged non-human mammals were composed, almost exclusively, of (SMs). Although human lens membranes followed comparable compositional trends, the magnitude of the changes was much smaller. We postulate that the high relative contents of DHSMs provide a biochemically inert matrix in which only small amounts of PCs and SMs and their metabolites, known to promote and arrest growth, respectively, are present. This compositional difference is proposed to contribute to the slow multiplication and elongation of human lens cells.

Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers.

PURPOSE: Human lens membranes contain unusually high levels of cholesterol and sphingolipids, lipids known to segregate into liquid-ordered domains. The current study was conducted to pursue the determination and characterization of these domains in membranes of clear and cataractous human lenses. METHODS: Cortical and nuclear regions of aged clear and cataractous lenses were obtained. After lysis with Triton X-100 at 4 degrees C and sucrose linear-density centrifugation, sedimenting and nonsedimenting fractions (when present) were collected. Phospholipids were analyzed by (31)P-nuclear magnetic resonance (NMR) and mass spectrometry. Caveolae and raft markers were tested by Western blot analysis. RESULTS: Only samples from clear lenses exhibited a nonsedimenting band. Phospholipid contents were comparable for sedimenting fractions of clear and cataractous membranes. Cholesterol to phospholipid molar ratios in light-density bands were nearly 7, three times greater than in sedimenting fractions. The portion of total cholesterol present in nonsedimenting fractions increased from 5.5% in the cortex to 14% in the nucleus. Two lysophospholipids comprising approximately 10% of all phospholipids in total membranes were undetectable in nonsedimenting fractions. Caveolin-1 was enriched in these fractions. CONCLUSIONS: Phospholipid compositional differences between lighter and heavier fractions from clear lenses were relatively minor and could not, alone, account for the substantial enrichment of cholesterol in the lighter fractions. Specific proteins, such as caveolin-1, must recruit cholesterol and induce clustering. Undetectable amounts of light-density domains in cataractous membranes suggest either disruption of these aggregates and thus the function of proteins within them, possibly relevant to lens transparency, and/or greater density of these clusters due to stronger binding of insoluble crystallins to membranes.

Interactions of Ca(2+) with sphingomyelin and dihydrosphingomyelin.

The changes induced by Ca(2+) on human lens sphingolipids, sphingomyelin (SM), and dihydrosphingomyelin were investigated by infrared spectroscopy. Ca(2+)-concentration-dependent studies of the head group region revealed that, for both sphingolipids, Ca(2+) partially dehydrates some of the phosphate groups and binds to others. Ca(2+) affects the interface of each sphingolipid differently. In SM, Ca(2+) shifts the amide I' band to frequencies lower than those in dehydrated samples of SM alone. This could be attributed to the direct binding of Ca(2+) to carbonyl groups and/or strong tightening of interlipid H-bonds to levels beyond those in dehydrated samples of SM only. In contrast, Ca(2+) induces relatively minor dehydration around the amide groups of dihydrosphingomyelin and a slight enhancement of direct lipid-lipid interactions. Temperature-dependent studies reveal that 0.2 M Ca(2+) increases the transition temperature T(m) from 31.6 +/- 1.0 degrees C to 35.7 +/- 1.1 degrees C for SM and from 45.5 +/- 1.1 degrees C to 48.2 +/- 1.0 degrees C for dihydrosphingomyelin. Binding of Ca(2+) to some phosphate groups remains above T(m). The strength of the interaction is, however, weaker. This allows for the partial rehydration of these moieties. Similarly, above T(m), Ca(2+)-lipid and/or direct inter-lipid interactions are weakened and lead to the rehydration of amide groups.