The Sterol-sensing Domain of the Niemann-Pick C1 (NPC1) Protein Regulates Trafficking of Low Density Lipoprotein Cholesterol

Millard, Elizabeth E.; Gale, Sarah E.; Dudley, Nicole; Zhang, Jessie; Schaffer, Jean E.; Ory, Daniel S.

doi:10.1074/jbc.m414024200

Cited by 96 publications

(99 citation statements)

References 38 publications

(62 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To address this possibility, we performed both cholesterol esterification and cholesterol efflux assays using the same cells evaluated in this study to see if we could observe phenotypic differences in cells with mutations in either NPC1 or NPC2. Results from these evaluations were virtually identical to previously established trends (16). Specifically, both NPC1-and NPC2-deficient cells have shown marked impairments in FIGURE 7.…”

Section: Discussionsupporting

confidence: 75%

See 1 more Smart Citation

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

Goldman

Krise

2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [ 3 H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

show abstract

Section: Discussionsupporting

confidence: 75%

“…Cholesterol Esterification-Esterification assays were based on a previously published report (16). Cells were plated on 6-well plates at 100,000 cells per well and allowed to adhere overnight.…”

Section: Methodsmentioning

confidence: 99%

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

Goldman

Krise

2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…NPC2 is a soluble glycoprotein in the lumen of the late endosomes/ lysosomes (LE/LYS) (9). Both NPC1 and NPC2 bind to cholesterol (10)(11)(12)(13)(14)(15) and may cooperate in endosomal cholesterol transport (16). Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not yet been demonstrated.…”

Section: Transport Of Ldl-derived Cholesterol From the Npc1 Compartmementioning

confidence: 99%

Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex

Urano

Watanabe

Murphy

et al. 2008

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

105

131

View full text Add to dashboard Cite

Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The hydrolyzed cholesterol (LDL-CHOL) enters the Niemann–Pick type C1 (NPC1)-containing endosomal compartment en route to various destinations. Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not been demonstrated. Using subcellular fractionation and immunoadsorption to enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary (CHO) cells are briefly exposed to 3 H-cholesteryl linoleate (CL) labeled-LDL, newly liberated 3 H-LDL-CHOL appears in membranes rich in trans-Golgi network (TGN) long before it becomes available for re-esterification at the endoplasmic reticulum (ER) or for efflux at the plasma membrane. In mutant cells lacking NPC1, the appearance of newly liberated 3 H-LDL-CHOL in the TGN-rich fractions is much reduced. We next report a reconstituted transport system that recapitulates the transport of LDL-CHOL to the TGN and to the ER. The transport system requires ATP and cytosolic factors and depends on functionality of NPC1. We demonstrate that knockdown by RNAi of 3 TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) reduces ≥50% of the LDL-CHOL transport in intact cells and in vitro. These results show that vesicular trafficking is involved in transporting a significant portion of LDL-CHOL from the NPC1-containing endosomal compartment to the TGN before its arrival at the ER.

show abstract

“…RNA isolation, cDNA synthesis, and real-time quantitative RT-PCR using SYBR Green Master Mix or TaqMan Fast Universal PCR Master Mix with template-specific primers (Table 1, which is published as supporting information on the PNAS web site) were performed as described (30). Fold changes in gene expression for a particular target were determined as previously described and normalized to 36B4 expression.…”

Section: Methodsmentioning

confidence: 99%

Pregnane X receptor (PXR) activation: A mechanism for neuroprotection in a mouse model of Niemann–Pick C disease

Langmade

Gale

Frolov

et al. 2006

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

Self Cite

152

145

View full text Add to dashboard Cite

Niemann-Pick type C1 (NPC1) disease is a fatal neurodegenerative disease characterized by neuronal lipid storage and progressive Purkinje cell loss in the cerebellum. We investigated whether therapeutic approaches to bypass the cholesterol trafficking defect in NPC1 disease might delay disease progression in the npc1 ؊/؊ mouse model. We show that the neurosteroid allopregnanolone (ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset of neurological symptoms and prolong the lifespan of npc1 ؊/؊ mice. ALLO and T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of microglial-associated genes and inflammatory mediators, and reduced infiltration of activated microglia in the cerebellar tissue. To establish whether the mechanism of neuroprotection in npc1 ؊/؊ mice involves GABAA receptor activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer that has identical physical properties of natural ALLO but is not a GABA A receptor agonist. ent-ALLO provided identical functional and survival benefits as natural ALLO in npc1 ؊/؊ mice, strongly supporting a GABAA receptor-independent mechanism for ALLO action. On the other hand, the efficacy of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these compounds to activate pregnane X receptor-dependent pathways in vivo. These findings suggest that treatment with pregnane X receptor ligands may be useful clinically in delaying the progressive neurodegeneration in human NPC disease.cholesterol ͉ neurosteroid ͉ allopregnanolone ͉ neurodegeneration N iemann-Pick type C (NPC) disease is an autosomal recessive neurodegenerative disorder characterized by accumulation of cholesterol and other lipids in the viscera and central nervous system and patterned Purkinje cell death in the cerebellum (1). Mutations in the NPC1 gene are responsible for Ϸ95% of human NPC disease. NPC1 loss-of-function mutants exhibit marked impairment of low-density lipoprotein (LDL) cholesterol esterification and mobilization of newly hydrolyzed LDL cholesterol to the plasma membrane (2-4), resulting in lysosomal sequestration of LDL cholesterol, delayed down-regulation of the LDL receptor and de novo cholesterol biosynthesis, and impaired ABCA1-mediated cholesterol efflux (5-7). Despite recent progress in characterizing the biochemical and genetic defects in NPC disease, the mechanisms underlying the neurodegenerative phenotype are not well understood. Moreover, at present there are no effective therapies that delay progression of human NPC disease.Many of the prominent neuropathological features of human NPC disease [e.g., neuronal lipid storage and progressive loss of Purkinje neurons (1)] are recapitulated in the BALB͞c NPC nih (npc1 Ϫ/Ϫ ) mouse, a naturally occurring murine model that harbors a retroposon insertion in the Npc1 gene (8, 9). In NPC1 mice, accumulation of unesterified cholesterol and gangliosides occurs in morphologically normal neurons as early as postnatal day 9 (P9) and precedes neuronal injury and c...

show abstract

The Sterol-sensing Domain of the Niemann-Pick C1 (NPC1) Protein Regulates Trafficking of Low Density Lipoprotein Cholesterol

Cited by 96 publications

References 38 publications

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex

Pregnane X receptor (PXR) activation: A mechanism for neuroprotection in a mouse model of Niemann–Pick C disease

Contact Info

Product

Resources

About