Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Abstract

This article is available online at http://www.jlr.org Nearly all cells in the body, including neurons of the central nervous system (CNS), take up cholesteryl ester (CE) and/or unesterifi ed cholesterol (UC) carried in various lipoproteins from the surrounding pericellular fl uid by receptor-mediated and bulk-phase endocytosis ( 1, 2 ). The sterol in these particles is processed in the late endosomal/lysosomal (E/L) compartment of cells by the sequential action of at least three proteins, lysosomal acid lipase (LAL) ( 3 ), Niemann-Pick C2 (NPC2) ( 4 ), and Niemann-Pick C1 (NPC1) ( 5 ), before being exported into the cytosolic compartment. There it joins other UC, newly synthesized from acetyl-CoA, to provide a metabolically active pool of sterol critical for normal turnover of plasma membrane cholesterol. Because UC is a hydrophobic amphipath and potentially toxic to cells, the size of this metabolically active pool is tightly monitored by two systems, the sterol regulatory element binding proteins (SREBPs) ( 6 ) and the liver X receptors (LXRs) ( 7 ), which, in turn, regulate the expression of genes controlling the uptake, synthesis, degradation, and export of UC. In this manner, the size of the metabolically active pool is kept relatively small and constant even though rates of lipoprotein uptake and sterol synthesis may vary widely.Mutations in any one of these three proteins cause accumulation of CE (LAL, Wolman disease) or UC (NPC2 or NPC1, Niemann-Pick C disease) in every tissue, which leads to cell dysfunction and death, and to a variety of clinical syndromes including disease of the liver, lungs, and Abstract Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterifi ed cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED 50 values for this reversal varied from ‫ف‬ 40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2 Ϫ / Ϫ and npc1 Ϫ / Ϫ mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment. Press, February 2, 2011 DOI 10.1194 Abbreviations: ALT, alanine aminotransferase ; AST, aspartate aminotransferase ; bw, body weight; CD, cyclodextrin; CE, c...

Cyclodextrin mediates rapid changes in lipid balance in Npc1 mice without carrying cholesterol through the bloodstream

“…In addition, the similar behaviors of HP-␤ -CD and SBE-␤ -CD to elicit changes in intracellular sterol revealed by altered gene expression suggest that only the "shuttle" capacity of CD is required. This scenario is consistent with proposals that CD is promoting the translocation of unesterfi ed cholesterol from internal membranes of the LE/L across the limiting lysosomal membrane ( 16,17 ) for egress into the cytosolic compartment of Npc1 Ϫ / Ϫ cells.…”

“…Ϫ / Ϫ double-knockout mice ( 17 ). Studies performed within a variety of tissues harvested 24 h after a single HP-␤ -CD injection to 7-or 49-day-old Npc1 Ϫ / Ϫ mice have shown dramatic changes in sterol dynamics, including a decrease in cholesterol synthesis rates, an increase in the ratio of esterifi ed/unesterifi ed cholesterol, downregulation of the sterol regulatory element-binding protein 2 (SREBP2) and its target genes, and increased expression of liver X receptor (LXR) target genes ( 5,18 ).…”

Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

“…In vitro studies have shown that CYCLO markedly accelerates cholesterol transfer between membranes via a collisional mechanism involving direct interaction between CYCLO and membranes ( 177 ). The mechanism by which CYCLO releases cholesterol from LEs/Ls was further investigated using two types of ␤ -cyclodextrin ( 167,176 ). Previous studies had shown that CYCLO, at concentrations of >1 mM, removes cholesterol from membranes into the aqueous medium by formation of a 2:1 CYCLO-cholesterol molar complex.…”

“…At lower concentrations (<1 mM), however, both CYCLO and sulfobutylether-␤ -cyclodextrin can shuttle cholesterol between membranes ( 171 ). To determine whether CYCLO removed cholesterol from LEs/Ls of NPC-defi cient cells by solubilization or by a shuttle mechanism, Npc1 Ϫ / Ϫ mice were injected with CYCLO or sulfobutylether-␤ -cyclodextrin ( 167,176 ). With both types of cyclodextrin, the mobilized cholesterol did not rapidly and prolongs life are very encouraging.…”

“…When CYCLO (4,000 mg/kg) was subcutaneously injected into Npc1 Ϫ / Ϫ mice on alternate days, starting at either day 7 or day 21 after birth, the neuronal storage of cholesterol and gangliosides was reduced, neurodegeneration was delayed, and life span was prolonged ( 99 ). Importantly, injection of CYCLO into NPC2-defi cient mice was similarly beneficial ( 99,167 ). In careful "balance" studies on quantifi cation of the cholesterol excreted into bile by Npc1 Ϫ / Ϫ mice, CYCLO reduced cholesterol storage in almost all tissues and promoted the excretion of cholesterol-derived bile acids ( 100 ).…”