title: Amino acid substitution in NPC1 that abolishes cholesterol binding reproduces phenotype of complete NPC1 deficiency in mice.
abstract: Substitution mutations in adjacent amino acids of the N-terminal domain of NPC1, a lysosomal membrane protein, abolish its cholesterol binding activity and impair its ability to export cholesterol from lysosomes of cultured cells lacking npc1 [Kwon HJ, et al. (2009) Cell 137:1213-1224]. Here, we show that the same two mutations (proline-202 and phenylalanine-203, both changed to alanine) reproduce the phenotype of complete NPC1 deficiency when knocked into the mouse npc1 gene by homologous recombination. Homozygous npc1(pf/pf) mice exhibited neurodegeneration beginning at day 49 and died at a median age of 84 d, as previously reported for mice that lack npc1. Liver and other organs of the npc1(pf/pf) mice accumulated excess cholesterol in lysosomes. In liver, mRNAs encoding several lysosomal proteins were elevated, including NPC1 and NPC2 and several digestive enzymes (acid lipase, β-glucuronidase, and cathepsins B and D). Weekly treatment with hydroxypropyl-β-cyclodextrin (HPCD) beginning at 7 wk reduced hepatic cholesterol accumulation and diminished the lysosomal mRNAs. We conclude that the cholesterol binding site in the N-terminal domain of NPC1 is essential for cholesterol export from lysosomes in living animals as it is in cultured cells. The HPCD-mediated reduction of excess lysosomal enzymes may contribute to the ability of this drug to delay the progression of NPC disease in mice.
authors: Departments of Molecular Genetics and Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.
authors: Xuefen
authors: X
authors: Xie
authors: Michael S
authors: MS
authors: Brown
authors: John M
authors: JM
authors: Shelton
authors: James A
authors: JA
authors: Richardson
authors: Joseph L
authors: JL
authors: Goldstein
authors: Guosheng
authors: G
authors: Liang
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D005260:Female; D005732:Gangliosides / Q000378:metabolism; D055879:Gene Knock-In Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D000077296:Niemann-Pick C1 Protein; D009928:Organ Specificity / Q000187:drug effects; D010641:Phenotype; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D016019:Survival Analysis; D014162:Transfection; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D002784:Cholesterol; D003505:Cyclodextrins; D005732:Gangliosides; D047908:Intracellular Signaling Peptides and Proteins; D051907:Lysosomal Membrane Proteins; D000077296:Niemann-Pick C1 Protein; D011506:Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C106831:Npc1 protein, mouse