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

Pipalia, Nina H.; Huang, Amy Y.; Ralph, Harold; Rujoi, Madalina; Maxfield, Frederick R.

doi:10.1194/jlr.m500388-jlr200

Cited by 79 publications

(134 citation statements)

References 62 publications

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“…MβCD conjugated to dextran fixed with paraformaldehyde, stained, and imaged as described previously (21). Cholesterol accumulation in LSOs was measured by quantifying filipin fluorescence as described previously (20). Data represent averages ± SEM of one representative experiment (n = 8, where n is total number of wells per condition used for quantification).…”

Section: Resultsmentioning

confidence: 99%

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Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells

Rosenbaum

Zhang

Warren

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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239

215

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Niemann-Pick type C disease (NPC) is a lysosomal storage disorder causing accumulation of unesterified cholesterol in lysosomal storage organelles. Recent studies have shown that hydroxypropyl-β-cyclodextrin injections in npc1 −/− mice are partially effective in treating this disease. Using cultured fibroblasts, we have investigated the cellular mechanisms responsible for reduction of cholesterol accumulation. We show that decreased levels of cholesterol accumulation are maintained for several days after removal of cyclodextrin from the culture medium. This suggests that endocytosed cyclodextrin can reduce the cholesterol storage by acting from inside endocytic organelles rather than by removing cholesterol from the plasma membrane. To test this further, we incubated both NPC1 and NPC2 mutant cells with cholesterol-loaded cyclodextrin for 1 h, followed by chase in serum-containing medium. Although the cholesterol content of the treated cells increased after the 1-h incubation, the cholesterol levels in the storage organelles were later reduced significantly. We covalently coupled cyclodextrin to fluorescent dextran polymers. These cyclodextrin-dextran conjugates were delivered to cholesterol-enriched lysosomal storage organelles and were effective at reducing the cholesterol accumulation. We demonstrate that methyl-β-cyclodextrin is more potent than hydroxypropyl-β-cyclodextrin in reducing both cholesterol and bis(monoacylglycerol) phosphate accumulation in NPC mutant fibroblasts. Brief treatment of cells with cyclodextrins causes an increase in cholesterol esterification by acyl CoA:cholesterol acyl transferase, indicating increased cholesterol delivery to the endoplasmic reticulum. These findings suggest that cyclodextrin-mediated enhanced cholesterol transport from the endocytic system can reduce cholesterol accumulation in cells with defects in either NPC1 or NPC2.acyl CoA:cholesterol acyl transferase | cholesterol accumulation | lysosomal storage organelles | bis(monoacylgycerol)phosphate, pinocytosis

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Section: Resultsmentioning

confidence: 99%

“…We used a previously described (20,21) quantitative assay to measure filipin labeling of the LSOs in NPC-defective cells. The CD effects on LSO cholesterol accumulation in NPC1 and NPC2 mutant cells were dose and time dependent (Fig.…”

Section: Resultsmentioning

confidence: 99%

Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells

Rosenbaum

Zhang

Warren

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

239

215

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“…One possible explanation for this could be the broad distribution of fi lipin signal intensities we observed, particularly in cultures from "classic" NPC patients: Within the same sample, cells with a high storage capacity for cholesterol coexisted with cells that seemed to be already saturated at lower amounts of storage material but in which cholesterol homeostatic responses nevertheless were likely to be maximally corrupted. The possibility that NPC fi broblast cultures are mosaics of cells that respond to an environmental stimulus such as LDL or also cyclodextrins, to a different extent may be of relevance for large-scale screening approaches that apply the fi lipin test to query for substances that may alleviate cholesterol storage in NPC disease ( 34,35 ). Indeed, assessing the distribution of fi lipin signal intensities within a population of individual cells rather than averaging parameters over the whole population proved a successful strategy in a recent fi lipin-based siRNA screen in NPC cells ( 36 ).…”

Section: Discussionmentioning

confidence: 99%

Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage

Tängemo

Weber

Theiß

et al. 2011

Journal of Lipid Research

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“…For example, the sterol dye filipin has been used to stain cells with lipid storage defects in order to screen for compounds that partially revert their cholesterol accumulation defect (Pipalia et al, 2006). In another example, a fluorescent analog of lactosylceramide (BODIPY-LacCer) was used to follow lipid accumulation in fibroblasts from patients with different lipidstorage diseases (Chen et al, 1999).…”

Section: Lipid Labelingmentioning

confidence: 99%

Getting the whole picture: combining throughput with content in microscopy

Rimon

Schuldiner

2011

Journal of Cell Science

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Commentary 3743Introduction Technological developments in the past years have led to the emergence of high-throughput methods in cell biology (Box 1), where multiple perturbations of a system are automatically repeated in a controlled and identical fashion. These capabilities allow the acquisition of vast amounts of data on a biological system. Evaluating all aspects of such data using computational and mathematical tools can then lead to an unbiased systematic representation of the process studied (Ahn et al., 2006). Technical breakthroughs, often driven by the pharmaceutical industry, have pushed this field forwards and have led to major advances in drug screening (reviewed by Zanella et al., 2010) and numerous discoveries in basic biology.The need for systematic and unbiased approaches has always been at the core of scientific efforts. However, the technology that is required for such efforts often displays an inverse relationship between throughput (speed) and content (biological information). Sydney Brenner referred to this phenomenon by saying that highthroughput experiments are in danger of creating "low-input, high-throughput, no-output biology" (Brenner, 2008). Therefore, it remains a major goal to develop high-throughput science that will give all the advantages of being systematic, accurate, fast and unbiased without giving up the requirement to provide profound and highly informative data.Among the variety of high-throughput approaches available to date (Box 1), one of the methods that holds the promise to bridge the gap between these expectations is high-throughput microscopy, often also referred to as high-content screening (HCS). This is mainly owing to the ability of microscopy methods to focus on single cells at a subcellular resolution, in a time-dependent manner and to measure a large number of parameters in each frame. In this Commentary, we focus on the current frontiers of HCS by presenting the wide range of tools that are utilized and the biological questions that can be tackled using such approaches. We also discuss possible ways to increase content while maintaining throughput at all levels of the screen -from sample design and preparation through to the acquisition and analysis capabilities of the high-content imager system. The power of high-throughput microscopyHigh-throughput microscopy can be employed to address a wide spectrum of biological questions. At the basis of this capability lies the ever-growing variety of labeling methods (discussed below) that allow visualization of cellular architecture and function, as well as developmental or behavioral processes (Fig. 1). In addition, the technological advance of biological tools and microscopic platforms now enables screens to be performed in an array of genetic backgrounds, under different growth conditions and at various time points, as well as allowing the comparison of multiple tissues, cell lines or organisms. Combining the richness of visualization approaches with various experimental strategies creates nearly endless options ...

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

Cited by 79 publications

References 62 publications

Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells

Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells

Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage

Getting the whole picture: combining throughput with content in microscopy

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