The ATP-binding cassette transporter-2 (ABCA2) regulates cholesterol homeostasis and low-density lipoprotein receptor metabolism in N2a neuroblastoma cells

The ATP-binding cassette transporter-2 (ABCA2) has been identified as a possible regulator of lipid metabolism. ABCA2 is most highly expressed in the brain but its effects on cholesterol homeostasis in neuronal-type cells have not been characterized. It is important to study the role of ABCA2 in regulating cholesterol homeostasis in neuronal-type cells because ABCA2 has been identified as a possible genetic risk factor for Alzheimer's disease. In this study, the effects of ABCA2 expression on cholesterol homeostasis were examined in mouse N2a neuroblastoma cells. ABCA2 reduced total, free- and esterified cholesterol levels as well as membrane cholesterol but did not perturb cholesterol distribution in organelle or lipid raft compartments. ABCA2 did not modulate de novo cholesterol biosynthesis from acetate. Cholesterol trafficking to the plasma membrane was not affected by ABCA2 but efflux to the physiological acceptor ApoE3 and mobilization of plasma membrane cholesterol to the endoplasmic reticulum for esterification were reduced by ABCA2. ABCA2 reduced esterification of serum and low-density lipoprotein-derived cholesterol but not 25-hydroxycholesterol. ABCA2 decreased low-density lipoprotein receptor (LDLR) mRNA and protein levels and increased its turnover rate. The surface expression of LDLR as well as the uptake of fluroresecent DiI-LDL was also reduced by ABCA2. Reduction of endogenous ABCA2 expression by RNAi treatment of N2a cells and rat primary cortical neurons produced the opposite effects of over-expression of ABCA2, increasing LDLR protein levels. This report identifies ABCA2 as a key regulator of cholesterol homeostasis and LDLR metabolism in neuronal cells.     

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.     

ABCA1. The gatekeeper for eliminating excess tissue cholesterol

It is widely believed that HDL functions to transport cholesterol from peripheral cells to the liver by reverse cholesterol transport, a pathway that may protect against atherosclerosis by clearing excess cholesterol from arterial cells. A cellular ATP-binding cassette transporter (ABC) called ABCA1 mediates the first step of reverse cholesterol transport: the transfer of cellular cholesterol and phospholipids to lipid-poor apolipoproteins. Mutations in ABCA1 cause Tangier disease (TD), a severe HDL deficiency syndrome characterized by accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. Studies of TD heterozygotes revealed that ABCA1 activity is a major determinant of plasma HDL levels and susceptibility to CVD. Drugs that induce ABCA1 in mice increase clearance of cholesterol from tissues and inhibit intestinal absorption of dietary cholesterol. Multiple factors related to lipid metabolism and other processes modulate expression and tissue distribution of ABCA1.Therefore, as the primary gatekeeper for eliminating tissue cholesterol, ABCA1 has a major impact on cellular and whole body cholesterol metabolism and is likely to play an important role in protecting against cardiovascular disease.     

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer’s disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT^−/− mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer’s-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.     

Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that affect protein structure and channel function. CFTR, localized in the apical membrane within cholesterol and sphingomyelin rich regions, is an ABC transporter that functions as a chloride channel. Here, we report that expression of defective CFTR (ΔF508CFTR or decreased CFTR) in human lung epithelial cell lines increases sphingolipid synthesis and mass of sphinganine, sphingosine, four long-chain saturated ceramide species, C16 dihydroceramide, C22, C24, C26-ceramide, and sphingomyelin, and decreases mass of C18 and unsaturated C18:1 ceramide species. Decreased expression of CFTR is associated with increased expression of long-chain base subunit 1 of serine-palmitoyl CoA, the rate-limiting enzyme of de novo sphingolipid synthesis and increased sphingolipid synthesis. Overexpression of ΔF508CFTR in bronchoalveolar cells that do not express CFTR increases sphingolipid synthesis and mass, whereas overexpression of wild-type CFTR, but not of an unrelated ABC transporter, ABCA7, decreases sphingolipid synthesis and mass. The data are consistent with a model in which CFTR functions within a feedback system that affects sphingolipid synthesis and in which increased sphingolipid synthesis could reflect a physiological response to sequestration of sphingolipids or altered membrane structure.     

Origins of intestinal ABCA1-mediated HDL-cholesterol

The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. Influx of apical membrane cholesterol increases ABCA1 mRNA and mass, resulting in enhanced efflux of HDL-cholesterol. Luminal (micellar) cholesterol and newly synthesized cholesterol are not transported directly to ABCA1 but reach the ABCA1 pool after incorporation into the apical membrane. Depleting the apical or the basolateral membrane of cholesterol by cyclodextrin attenuates the amount of cholesterol transported by ABCA1 without altering ABCA1 expression. Filipin added to the apical side but not the basal side attenuates ABCA1-mediated cholesterol efflux, suggesting that apical membrane "microdomains," or rafts, supply cholesterol for HDL. Preventing cholesterol esterification increases the amount of cholesterol available for HDL. Ezetimibe, a Niemann-Pick C1-like 1 protein inhibitor, does not alter ABCA1-mediated cholesterol efflux. U18666A and imipramine, agents that mimic cholesterol trafficking defects of Neimann-Pick type C disease, attenuate cholesterol efflux without altering ABCA1 expression; thus, intestinal NPC1 may facilitate cholesterol movement to ABCA1. ABCA1-mediated cholesterol efflux is independent of cholesterol synthesis. The results suggest that following incorporation into plasma membrane and rafts of the apical membrane, dietary/biliary and newly synthesized cholesterol contribute to the ABCA1 pool and HDL-cholesterol. NPC1 may have a role in this process.     

Peroxisome proliferator-activated receptor alpha controls cellular cholesterol trafficking in macrophages

The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant that governs its availability for efflux to extracellular acceptors. NPC1 and NPC2 are proteins localized in the late endosome and control cholesterol transport from the lysosome to the plasma membrane. Here, we report that NPC1 and NPC2 gene expression is induced by oxidized LDL (OxLDL) in human macrophages. Because OxLDLs contain natural activators of peroxisome proliferator-activated receptor alpha (PPARalpha), a fatty acid-activated nuclear receptor, the regulation of NPC1 and NPC2 by PPARalpha and the consequences on cholesterol trafficking were further studied. NPC1 and NPC2 expression is induced by synthetic PPARalpha ligands in human macrophages. Furthermore, PPARalpha activation leads to an enrichment of cholesterol in the plasma membrane. By contrast, incubation with progesterone, which blocks postlysosomal cholesterol trafficking, as well as NPC1 and NPC2 mRNA depletion using small interfering RNA, abolished ABCA1-dependent cholesterol efflux induced by PPARalpha activators. These observations identify a novel regulatory role for PPARalpha in the control of cholesterol availability for efflux that, associated with its ability to inhibit cholesterol esterification and to stimulate ABCA1 and scavenger receptor class B type I expression, may contribute to the stimulation of reverse cholesterol transport.     

Thematic Review Series: High Density Lipoprotein Structure,Function, and Metabolism: Regulation of signal transduction by HDL

High density lipoprotein (HDL) cholesterol has direct effects on numerous cell types that influence cardiovascular and metabolic health. These include endothelial cells, vascular smooth-muscle cells, leukocytes, platelets, adipocytes, skeletal muscle myocytes, and pancreatic β cells. The effects of HDL or apoA-I, its major apolipoprotein, occur through the modulation of intracellular calcium, oxygen-derived free-radical production, numerous kinases, and enzymes, including endothelial nitric-oxide synthase (eNOS). ApoA-I and HDL also influence gene expression, particularly genes encoding mediators of inflammation in vascular cells. In many paradigms, the change in intracellular signaling occurs as a result of cholesterol efflux, with the cholesterol acceptor methyl-β-cyclodextrin often invoking responses identical to HDL or apoA-I. The ABC transporters ABCA1 and ABCG1 and scavenger receptor class B, type I (SR-BI) frequently participate in the cellular responses. Structure-function relationships are emerging for signal initiation by ABCA1 and SR-BI, with plasma membrane cholesterol binding by the C-terminal transmembrane domain of SR-BI uniquely enabling it to serve as a sensor of changes in membrane cholesterol. Further investigation of the processes underlying HDL and apoA-I modulation of intracellular signaling will potentially reveal new prophylactic and therapeutic strategies to optimize both cardiovascular and metabolic health.     

转运蛋白ABCG1/4 和ABCA1对脑胆固醇的调节作用

脑是富含胆固醇的器官,机体大约有25%的胆固醇集中在脑组织中.ATP结合盒超家族转运蛋白对脑组织中胆固醇的膜外转运和动态平衡起着重要的调节作用.研究发现,ATP结合盒超家族转运蛋白亚体ABCG1、ABCG4和ABCA1在成体脑组织中存在不同程度的表达,一种或多种亚体的缺失可以导致神经退行性病变.然而,ATP结合盒超家族转运蛋白亚体对脑发育过程中脑胆固醇动态变化的调节缺乏相关性的报道.在本研究中,从低胆固醇饮食喂养的C57BL/6J小鼠中获取出生后不同发育时期的脑组织,对ABCG1、ABCG4和ABCA1的mRNA与蛋白质表达水平进行测定,并对脑组织和血清中ATP结合盒超家族转运蛋白的表达水平与胆固醇水平的相关性进行研究.同时,使用ABCG1、ABCG4单一基因敲除鼠和ABCG1、ABCG4双基因敲除鼠,研究ATP结合盒超家族转运蛋白对与胆固醇合成的相关基因表达的影响以及对脑组织胆固醇代谢的调节作用.结果发现,ABCG1、ABCG4和ABCA1在机体多个器官中均有表达,但ABCG1和ABCG4在小鼠脑组织中表达量最高.在脑组织发育过程中,ABCG1和ABCG4mRNA水平呈现明显的表达时效性,小鼠于出生后42天达到峰值,而ABCA1 mRNA的表达水平无明显变化.血清和脑组织中中酯化型胆固醇水平呈双高峰分布,也于出生后42天达到最高.基因敲除鼠模型显示,单一敲除ABCG1或者ABCG4基因对脑组织胆固醇水平无明显影响,而ABCG1和ABCG4基因的同时缺失导致脑胆固醇水平显著升高,并明显降低胆固醇合成相关基因的表达水平.本研究表明,在脑发育成熟过程中,ATP结合盒超家族转运蛋白亚体ABCG1和ABCG4,而非ABCA1,以调节脑胆固醇的膜外转运;ABCG1和ABCG4互补调控脑胆固醇的动态平衡.     

ABCA1 overexpression in the liver of LDLr-KO mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of (125)I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7alpha1, CYP27alpha1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions.     

Intestinal cholesterol absorption is substantially reduced in mice deficient in both ABCA1 and ACAT2

The process of cholesterol absorption has yet to be completely defined at the molecular level. Because of its ability to esterify cholesterol for packaging into nascent chylomicrons, ACAT2 plays an important role in cholesterol absorption. However, it has been found that cholesterol absorption is not completely inhibited in ACAT2-deficient (ACAT2 KO) mice. Because ABCA1 mRNA expression was increased 3-fold in the small intestine of ACAT2 KO mice, we hypothesized that ABCA1-dependent cholesterol efflux sustains cholesterol absorption in the absence of ACAT2. To test this hypothesis, cholesterol absorption was measured in mice deficient in both ABCA1 and ACAT2 (DKO). Compared with wild-type, ABCA1 KO, or ACAT2 KO mice, DKO mice displayed the lowest level of cholesterol absorption. The concentrations of hepatic free and esterified cholesterol and gallbladder bile cholesterol were significantly reduced in DKO compared with wild-type and ABCA1 KO mice, although these measures of hepatic cholesterol metabolism were very similar in DKO and ACAT2 KO mice. We conclude that ABCA1, especially in the absence of ACAT2, can have a significant effect on cholesterol absorption, although ACAT2 has a more substantial role in this process than ABCA1.     

Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells

The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann-Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.     

Phospholipase A2-Modified Low-Density Lipoprotein Activates Liver X Receptor in Human Macrophages

Macrophages respond to cholesterol accumulation by increasing cholesterol efflux, which is mediated by activation of the nuclear liver X receptor (LXR) and ATP binding cassette (ABC) transporters. In the present study, we investigated whether foam cell formation induced by phospholipase A(2)-modified low-density lipoprotein (PLA-LDL) influences LXR activity and cholesterol efflux in primary human monocyte-derived macrophages (MDMs). Macrophages were treated with PLA-LDL and expression of the LXR target genes ABCA1 and ABCG1 was analyzed by quantitative PCR and western blot. PLA-LDL time-dependently up-regulated ABCA1 and ABCG1 mRNA and protein. Removal of non-esterified fatty acids from PLA-LDL particles did not influence the induction of ABC transporters. A role of LXR in PLA-LDL-stimulated ABCG1 expression was verified by LXR-knockdown and luciferase reporter assays using a construct containing a LXR response element from the ABCG1 gene. Functionally, cholesterol efflux to apolipoprotein A-I and high-density lipoprotein was higher in PLA-LDL treated cells compared to controls. Together, these results demonstrate that in primary human MDMs PLA-LDL induces ABC transporter expression via LXR activation. A concomitantly increased cholesterol efflux may prevent excessive cholesterol accumulation and thus, attenuate foam cell formation.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1(-/-) fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1(-/-) fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2(-/-) cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1(-/-) and NPC2(-/-) fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1(-/-) fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2(-/-) fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1(-/-) cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Inhibition of cholesterol absorption by SCH 58053 in the mouse is not mediated via changes in the expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte

Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL-C) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053. We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1^−/− fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1^−/− fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2^−/− cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1^−/− and NPC2^−/− fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1^−/− fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2^−/− fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1^−/− cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).