Niemann–Pick type C disease: Novel NPC1 mutations and characterization of the concomitant acid sphingomyelinase deficiency

Tracking Sphingosine Metabolism and Transport in Sphingolipidoses: NPC1 Deficiency as a Test Case

“…We observed that NPC1-deficient cells exhibited defective degradation of SM, in agreement with earlier studies (17,19 Figure S3). To our knowledge, reduced SM synthesis has not been earlier reported in NPC1-deficient cells, and the basis for this is at present unknown.…”

“…This is probably because Sph may dissociate from the LDL particle and equilibrate between membranes at neutral pH (4,16 (Figure 2A). The cells were incubated with 50 μg/mL of [ 3 H]-SM/LDL for 4 h, after which lipids were extracted and analyzed by high performance thin layer chromatography (HPTLC) and (17)(18)(19). Indeed, we found that NPC1 silenced cells (with a ≥90% reduction in NPC1 protein levels, Figure 2A) exhibited elevated [ 3 H]-SM levels ( Figure 2B; see also Figure 3B, 4B).…”

Improvement in Lipid and Protein Trafficking in NPC1 Cells by Correction of a Secondary Enzyme Defect

“…It remains unclear whether the primary defect in NPC1 mutants is directly associated with cholesterol transport or whether the cholesterol accumulation is secondary to accumulation of other lipids, which associate with cholesterol in membranes (4,10). In this regard, it is noteworthy that cholesterol-enriched NPC cells and tissues from NPC1-mutant mice and humans have a secondary, post-translational defect in the activity of a lysosomal enzyme, acid sphingomyelinase (SMase) (14)(15)(16)(17)(18)(19). This alteration in acid SMase activity can be observed in wild-type (WT) cells with increased levels of late endosomal cholesterol resulting from incubation with low-density lipoprotein (LDL) and progesterone (16).…”

Niemann-Pick Disease Type C: Cataplexy and Hypocretin in Cerebrospinal Fluid

“…For filipin staining, fibroblast cells from the patient and normal control were stained with 300 μ g/ml of filipin complex (Sigma-Aldrich Corporation, St. Louis, MO, USA) after fixation with 2% paraformaldehyde. As a positive control, fibroblast cell from a patient with the severe phenotype of NPC due to a homozygous NPC1 gene mutation of c.3615delA was also stained with filipin (Tamura et al 2006). Stained cells were examined using a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Thornwood, NY, USA) equipped with UV laser.…”

“…The analysis was conducted following the method described before (Tamura et al 2006). A homozygous c.2974G > T mutation, which predicts a glycine (GGG) to tryptophan (TGG) change at codon 992 (designated as p.G 992 W), was identified in the sequence of the NPC1 gene from the patient (Fig.…”