Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

Sugii, Shigeki; Reid, Patrick; Ohgami, Nobutaka; Shimada, Yukiko; Maue, Robert A.; Ninomiya, Haruaki; Ohno‐Iwashita, Yoshiko; Chang, Ta−Yuan

doi:10.1194/jlr.d200036-jlr200

Cited by 44 publications

(40 citation statements)

References 50 publications

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“…The cell type-dependent defect observed in the current work may also be explained in part by the following scenario: the cells used in our current study were maintained in FBS-containing medium, then incubated in lipid-free medium for 2 days before the assay began. By using a very sensitive intracellular cholesterol stain, BC-, we recently showed that mouse NPC1 MEFs grown under this condition still contained a significant amount of cholesterol accumulated in the late endosome/lysosome (63). Similar results were obtained in the NPC1 MPMs, hepatocytes, and glial cells (P. C. Reid, S. Sugii, and T-Y.…”

Section: Discussionsupporting

confidence: 68%

Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice

Reid

Sugii

Chang

2003

Journal of Lipid Research

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Niemann-Pick type C1 disease (NPC1) is an inherited neurovisceral lipid storage disorder, hallmarked by the intracellular accumulation of unesterified cholesterol and glycolipids in endocytic organelles. Cells acquire cholesterol through exogenous uptake and endogenous biosynthesis. NPC1 participation in the trafficking of LDL-derived cholesterol has been well studied; however, its role in the trafficking of endogenously synthesized cholesterol (endo-CHOL) has received much less attention. Previously, using mutant Chinese hamster ovary cells, we showed that endo-CHOL moves from the endoplasmic reticulum (ER) to the plasma membrane (PM) independent of NPC1. After arriving at the PM, it moves between the PM and internal compartments. The movement of endoCHOL from internal membranes back to the PM and the ER for esterification was shown to be defective in NPC1 cells. To test the generality of these findings, we have examined the trafficking of endoCHOL in four different physiologically relevant cell types isolated from wild-type, heterozygous, and homozygous BALB/c NPC1 NIH mice. The results show that all NPC1 homozygous cell types (embryonic fibroblasts, peritoneal macrophages, hepatocytes, and cerebellar glial cells) exhibit partial trafficking defects, with macrophages and glial cells most prominently affected. Our findings suggest that endoCHOL may contribute significantly to the overall cholesterol accumulation observed in selective tissues affected by Niemann-Pick type C disease.

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Section: Discussionsupporting

confidence: 68%

Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice

Reid

Sugii

Chang

2003

Journal of Lipid Research

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“…Cholesterol was visualized by incubating coverslips in 0.05 mg/ml filipin (Sigma) for 1 h prior to antibody staining. Alternatively, following permeabilization, cholesterol was visualized by incubating coverslips with 10 g/ml of biotinylated -toxin (BC) then Alexa 488-conjugated streptavidin (Invitrogen) as described (32). BC was kindly supplied by Dr. Yoshiko Ohno-Iwashita, Tokyo Metropolitan Institute of Gerontology.…”

Section: Methodsmentioning

confidence: 99%

Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells

Ganley

Pfeffer

2006

Journal of Biological Chemistry

106

127

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Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompanied by stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent mannose 6-phosphate receptors were missorted to the lysosome for degradation, a process that was reversed by overexpression of GFPtagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking. Niemann-Pick type C (NPC)2 is an autosomal recessive, neurodegenerative disorder. A hallmark of NPC is the massive accumulation of cholesterol and glycosphingolipids within late endosomes and lysosomes (reviewed in Refs. 1-3). In normal cells, endocytosed low density lipoproteins are delivered to endosomes, where they are hydrolyzed and free cholesterol is released. This cholesterol is transported rapidly out of endosomes to the plasma membrane and endoplasmic reticulum (4, 5). In NPC cells, the cholesterol does not exit the endocytic pathway and it accumulates within lysosomes.Approximately 95% of NPC patients harbor mutations in the NPC1 gene that encodes a large, late endosomal protein with 13 transmembrane domains (6 -8). Although NPC1 binds cholesterol weakly (9), the precise function of NPC1 is unknown; it may be involved in cholesterol export from late endosomes (10). The remainder of NPC patients carry mutations in the NPC2 gene that encodes a small, soluble protein present in the lumen of late endosomes and lysosomes (11). Unlike NPC1, NPC2 binds cholesterol with high affinity (12), but like NPC1, its precise role is unclear.Late endosomes act as sorting stations to deliver endocytosed molecules to lysosomes for degradation, while at the same time, retrieving other classes of proteins and lipids for transport back to non-degradative compartments. Mannose 6-phos...

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“…For GM1 and GM2 staining, sections were permeabilized with 0.5% Triton X-100 as described previously (28). Filipin (125 g/ml) staining was performed under light-protected conditions for 2 h at room temperature, as previously described (25). After incubation with primary reagents, slides were washed with 0.5 M Tris containing no FBS and counterstained with Neurotrace TM …”

Section: Animal and Tissue Preparationsmentioning

confidence: 99%

“…Concurrently, the cells become leaky to various macromolecules, allowing BC to enter the cell interior. At the cell interior, BC was shown to stain mainly cholesterol-rich domains; the complexes formed can be visualized under fluorescence microscopy in a manner far superior to that of filipin (25). In the current study, we employ this new method to monitor cholesterol accumulation in the brains of NPC1 mice from the first signs of neurodegeneration at postnatal day 9 to a stage of low levels of neuronal cell loss at postnatal day 22.…”

mentioning

confidence: 99%

“…By employing avidinconjugated fluorescent dyes, BC bound to cellular cholesterol can be visualized under fluorescence microscopy (23). In unfixed cells or cells fixed with 1% or 2% paraformaldehyde, BC cannot enter the cell interior and binds mainly to cholesterol-rich domain(s) at the cell surface (24,25). In contrast, when cells are fixed with 4% paraformaldehyde, a significant portion of the cholesterol binding sites at the plasma membrane (PM) are inactivated, presumably because of extensive cross-linking of membrane proteins (26).…”

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confidence: 99%

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A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain

Reid

Sakashita

Sugii

et al. 2004

Journal of Lipid Research

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Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

Cited by 44 publications

References 50 publications

Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice

Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice

Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells

A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain

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