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  • br ABCA mRNA expression is elevated in Niemann

    2024-07-10


    ABCA2 mRNA expression is elevated in Niemann-Pick type C and familial hypercholesterolemia fibroblasts Niemann-Pick Type C disease is a neurological disorder of adolescents due to autosomal-recessive mutations in either one or both Niemann-Pick-2 (NPC2) and NPC1 genes, which, like ABCA2, are localized to the LE/LY compartment [52]. Cholesterol exit from the LE/LY requires the concerted action of both NPC2 and NPC1 to deliver free cholesterol from the lumen to the limiting membrane of the LE/LY, where it traffics to other intracellular compartments, e.g., Golgi apparatus, endoplasmic reticulum, by vesicular and non-vesicular mechanisms [53], [54], [55]. Mutant NPC1 fibroblasts have a defect in the delivery of LDL-derived free cholesterol from the LE/LY leading to cholesterol accumulation within this compartment [56], [57]. In addition to cholesterol accumulation, other lipids including sphingomyelin, glycosphingolipids and sphingosine also accumulate in the LE/LY [58], [59]. The expression of the LDLR and the cholesterol synthetic genes are also elevated in mutant NPC1 fibroblasts. Fibroblasts from Familial Hypercholesterolemia (FHC) patients lack functional LDL receptors that are required for LDL uptake from the plasma [60].
    Role of ABCA2 in intracellular cholesterol transport We discovered that ABCA2 overexpression functioned to sequester cholesterol in the LE/LY compartment. In one set of experiments, we employed a CHO cell line that stably overexpressed ABCA2 [46]. Cells were cultured in lipoprotein-deficient serum (LPDS) to up-regulate LDL receptors and then cultured in increasing concentrations of LDL. Radioactive [14C]oleate fatty WZ3146 was added and the esterification of free cholesterol to [14C]cholesteryl ester by ACAT in the ER was determined following extraction of lipids, thin layer chromatography (TLC) and autoradiography. In control CHO cells cholesteryl ester formation increased in a dose-dependent fashion while overexpression of ABCA2 reduced the magnitude of cholesteryl ester formation. Measurement of in vitro ACAT activity confirmed that the decrease in cholesteryl ester was not due to defects in ACAT activity in ABCA2 overexpressing cells. Filipin, a polyene antibiotic that binds to free cholesterol, decorated the abundant cholesterol in the plasma membrane in control cells, as revealed by ultraviolet fluorescence microscopy. In contrast, in cells treated with U18666a or in CHO cells overexpressing ABCA2 there was reduced plasma membrane staining and increased punctate staining of free cholesterol in the cytosol (filipin positive droplets) that was indicative of the LE/LY. Since ABCA2 is most highly expressed in the brain, we extended our studies to determine the effects of ABCA2 overexpression on cholesterol metabolism in mouse N2a neuroblastoma cells. In the first set of experiments to measure the effects of ABCA2 on uptake and trafficking of LDL-derived cholesterol back to the plasma membrane, we replaced the cholesteryl ester in the LDL particle with radioactive [3H]cholesteryl linoleate ([3H]CL-LDL). We were able to measure the recycling of cholesterol to the plasma membrane by its efflux to the cholesterol-binding molecule 2-hydroxypropyl-β-cyclodextrin [61]. ABCA2 did not significantly alter cholesterol trafficking to the plasma membrane. These results suggested that following endocytosis of LDL/LDLR by receptor-mediated endocytosis into early endosomes, cholesterol recycling back to the plasma membrane occurred mainly between early endosomes and the endocytic-recycling compartment (ERC). Under these experimental conditions (i.e., short duration culture with [3H]CL-LDL), cholesterol recycling to the plasma membrane was not sensitive to ABCA2 activity because the free cholesterol did not reach the more mature acidic endosomes of the LE/LY, where it would be sequestered. As an alternative approach to determine the effects of ABCA2 overexpression on LDL-derived cholesterol recycling to the plasma membrane, we measured the efflux of lipoprotein-derived cholesterol from the plasma membrane to the physiological cholesterol acceptor apolipoprotein E (ApoE) that was added exogenously to the cell culture medium. When control N2a cells and N2a cells overexpressing ABCA2 were cultured for longer periods with [3H]CL-LDL, we did observe a modest reduction in efflux of free cholesterol from the plasma membrane to ApoE in ABCA2 overexpressing cells. We interpreted from these results that over extended periods of time of cells in culture a greater fraction of cholesterol had trafficked from the ERC to more mature endosomal compartments (LE/LY), where ABCA2 functions in cholesterol sequestration, thereby reducing cholesterol trafficking back to the plasma membrane for efflux to ApoE.