Sterol transfer between cyclodextrin and membranes: similar but not identical mechanism to NPC2-mediated cholesterol transfer.

Cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of the autosomal recessive disease Niemann–Pick C. In healthy cells, LDL-derived cholesterol is efficiently cleared and moves primarily to the plasma membrane and the endoplasmic reticulum (ER). In cells lacking wild type expression of either Niemann–Pick C 1 (NPC1) or Niemann–Pick C2 (NPC2) protein, the post-LE/LY transport and metabolism of cholesterol is blocked. The resulting physiological consequences, almost always including neurodegeneration, are thought to arise secondary to the specific absence of normal postlysosomal cholesterol metabolism and the effects of general lysosomal dysfunction arising from the buildup of cholesterol and other lipids in the LE/LY. Effective therapeutics that can restore cholesterol egress are under active investigation. Cyclodextrins (CD) are cyclic oligosaccharides shaped like hollow truncated cones. The exterior of the cone is hydrophilic and the interior hydrophobic, imparting the ability to bind small hydrophobic molecules such as cholesterol in the interior, thereby solubilizing them in aqueous media.1-3 Cyclodextrins can have over 15 glucopyranose units per ring; derivatives of β-cyclodextrin, containing seven units, are most widely used in pharmaceuticals because of their high affinity for hydrophobic compounds, low toxicity, and price.4,5 β-Cyclodextrin (BCD), methyl-β-cyclodextrin (MBCD), and 2-hydroxypropyl-β-cyclodextrin (HPCD) are the most commonly used β-cyclodextrin derivatives, with relative cholesterol affinities as MBCD > HPCD > BCD.6,7 HPCD was shown to have lower toxicity compared with MBCD and to have greater specificity for cholesterol and triacylglycerol.5,8 In mouse models of NPC disease, CD was used as a vehicle to deliver potential therapeutic compounds to the animals, and it was noted that vehicle alone appeared to have substantial benefit.9,10 Indeed, Liu et al. demonstrated that the administration of a single dose of HPCD at 7 days of age to npc1−/− mice resulted in the rapid release of cholesterol accumulation from the LE/LY compartment, as monitored by cholesterol esterification in the ER and the restoration of steroldependent regulation of SREBP2 and LXR-mediated target gene expression.10 Remarkably, the CD-treated mice also showed diminished neuropathology and >40% extension of lifespan over untreated controls.11 Similar benefits were found in studies using chronic administration of HPCD.12 Interestingly, two other lipid storage diseases, characterized by primary accumulation of gangliosides (GM1 gangliosidosis) or mucopolysaccharides (MPS IIIA) as well as secondary cholesterol accumulation, were not ameliorated by CD treatment.12 The molecular mechanisms by which CD leads to the rapid restoration of normal post-LE/LY cholesterol transport are beginning to be understood. Rosenbaum et al. recently showed that CDs were functioning within the LE/LY compartment following fluid phase pinocytosis, rather than acting at the plasma membrane level.13 On the basis of the effectiveness of CD in treatment of NPC1-deficient mice, it was suggested that the CD may be functioning by delivering acid lipase-derived cholesterol to NPC2, thereby substituting for the defective NPC1.11 While specific interactions between CD and NPC2 have not been reported, interactions of CD with a number of other proteins have been observed,14 making NPC2–CD interactions a plausible hypothesis. We have shown that NPC2 catalyzes the rapid transfer of cholesterol to and from phospholipid membranes and that the mechanism of transfer involves direct protein–membrane interactions, with sterol transfer rates highest when membranes contain the LE/LY-specific lipid lysobisphosphatidic acid (LBPA, also known as bis-monoacylglycerol phosphate, BMP).15,16 Since the LDL-derived free cholesterol that accumulates in NPC disease is likely to be present largely in the internal membrane network of the LE/LY compartment, we considered that CD could also be acting by extracting membrane-bound cholesterol in the process of cellular rescue as well as, perhaps, delivering cholesterol to the limiting lysosomal membrane. While CD has long been used to manipulate plasma membrane cholesterol levels in cultured cells,6,17 its mechanism of action is not fully understood.14 Thus, to explore the underlying mechanism by which CD decreases cholesterol accumulation in NPC1 deficiency, and the potential role of NPC2 in the CD-mediated amelioration of cholesterol accumulation in NPC1 cells, the present studies used kinetic approaches and spectroscopy to examine the mechanism of sterol transfer between CD and NPC2, the rates and mechanism of sterol transfer between phospholipid membranes and CD, and the effects of CD on intermembrane sterol transfer. The dose- and time-dependent effects of HPCD on cholesterol accumulation in npc2−/− fibroblasts were also examined. The results do not provide evidence for a specific interaction of CD with NPC2, implying that the beneficial effects of CD in NPC1 disease are independent of NPC2. Nevertheless, CD functions to accelerate the rate of cholesterol transport from membranes, potentially behaving as a mimic for NPC2. At high concentrations, CD can generate sterol transfer rates that are on the order of those obtained with lower levels of NPC2. Similar to NPC2, the mechanism of CD action in cholesterol transfer between membranes appears to be collisional, involving direct interactions of CD with membranes. However, the rates of sterol transfer between CD and membranes are independent of membrane LBPA, in contrast to the marked effects of this lipid on increasing sterol transfer rates between NPC2 and membranes. Finally, the results also show that CD can rescue the cholesterol accumulation phenotype of npc2−/− cells, indicating that the actions of CD are independent of NPC2, in agreement with the kinetic studies.