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Improvement in lipid and protein trafficking in Niemann-Pick C1 cells by correction of a secondary enzyme defect.

ABSTRACT: Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann-Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation of cholesterol and other lipids, leading to NPC disease. NPC1 cells also develop a secondary defect in acid sphingomyelinase (SMase) activity despite a normal acid SMase gene (SMPD1). When acid SMase activity was restored to normal levels in NPC1-deficient CHO cells through SMPD1 transfection, there was a dramatic reduction in lysosomal cholesterol. Two other defects, excess lysosomal bis-(monoacylglycerol) phosphate (BMP) and defective transferrin receptor (TfR) recycling, were also markedly improved. To test its relevance in human cells, the acid SMase activity defect in fibroblasts from NPC1 patients was corrected by SMPD1 transfection or acid SMase enzyme replacement. Both treatments resulted in a dramatic reduction in lysosomal cholesterol. These data show that correcting one aspect of a complex lysosomal lipid storage disease can reduce the cellular consequences even if the primary genetic defect is not corrected.

SUBMITTER: Devlin C

PROVIDER: S-EPMC3000737 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Improvement in lipid and protein trafficking in Niemann-Pick C1 cells by correction of a secondary enzyme defect.

Devlin Cecilia C Pipalia Nina H NH Liao Xianghai X Schuchman Edward H EH Maxfield Frederick R FR Tabas Ira I

Traffic (Copenhagen, Denmark) 20100222 5

Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann-Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation of cholesterol and other lipids, leading to NPC disease. NPC1 cells also develop a seconda ...[more]

PMID: 20412078

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Project description:Niemann-Pick Type C (NPC) is an autosomal recessive lysosomal storage disease leading to progressive neurodegeneration. Mutations in the NPC1 gene, which accounts for 95% of the cases, lead to a defect in intra-lysosomal trafficking of cholesterol and an accumulation of storage material including cholesterol and sphingolipids in the endo-lysosomal system. Symptoms are progressive neurological and visceral deterioration, with variable onset and severity of the disease. This study investigates the influence of two different NPC1 mutations on the biochemical phenotype in fibroblasts isolated from NPC patients in comparison to healthy, wild type (WT) cells. Skin derived fibroblasts were cultured from one patient compound-heterozygous for D874V/D948Y mutations, which presented wild-type like intracellular trafficking of NPC1, and a second patient compound- heterozygous for I1061T/P887L mutations, which exhibited a more severe biochemical phenotype as revealed in the delayed trafficking of NPC1. Biochemical analysis using HPLC and TLC indicated that lipid accumulations were mutation-dependent and correlated with the trafficking pattern of NPC1: higher levels of cholesterol and glycolipids were associated with the mutations that exhibited delayed intracellular trafficking, as compared to their WT-like trafficked or wild type (WT) counterparts. Furthermore, variations in membrane structure was confirmed in these cell lines based on alteration in lipid rafts composition as revealed by the shift in flotillin-2 (FLOT2) distribution, a typical lipid rafts marker, which again showed marked alterations only in the NPC1 mutant showing major trafficking delay. Finally, treatment with N-butyldeoxynojirimycin (NB-DNJ, Miglustat) led to a reduction of stored lipids in cells from both patients to various extents, however, no normalisation in lipid raft structure was achieved. The data presented in this study help in understanding the varying biochemical phenotypes observed in patients harbouring different mutations, which explain why the effectiveness of NB-DNJ treatment is patient specific.

Project description:OBJECTIVES:The aim of this study was to comprehensively evaluate the auditory phenotype in Niemann-Pick disease, type C1 (NPC1), to understand better the natural history of this complex, heterogeneous disorder, and to define further the baseline auditory deficits associated with NPC1 so that use of potentially ototoxic interventions (e.g., 2-hydroxypropyl-ß-cyclodextrin) may be more appropriately monitored and understood. DESIGN:Fifty patients with NPC1 ranging in age from 4 months to 21 years (mean = 9.3 years) enrolled in a natural history/observational study at the National Institutes of Health. The auditory test battery included, when possible, immittance audiometry, pure-tone and speech audiometry, otoacoustic emission testing, and a neurotologic auditory brainstem response study. Longitudinal data were collected on a subset of patients. RESULTS:Over half of the cohort exhibited hearing loss involving the high frequencies ranging from a slight to moderate degree, and 74% of patients presented with clinically significant hearing loss involving the frequencies most important to speech understanding (0.5, 1, 2, 4 kHz). Despite the heterogeneity of the sample, results among patients were sufficiently consistent to implicate retrocochlear dysfunction in the majority (66%) of individuals, with (22%) or without (44%) accompanying cochlear involvement. Some patients (10%) presented with a profile for auditory neuropathy spectrum disorder. The combination of cross-sectional and longitudinal data indicates these patients are at risk for a progressive decline in auditory function. CONCLUSIONS:This is the largest cohort of patients with NPC1 evaluated comprehensively for auditory dysfunction, and results implicate the pathological processes of NPC1 in the manifestation of hearing loss. Patients with NPC1 should be monitored audiologically throughout their lives, beginning at the time of diagnosis. Clinicians and researchers should be aware of this historically overlooked aspect of the phenotype.

Dataset Information

Improvement in lipid and protein trafficking in Niemann-Pick C1 cells by correction of a secondary enzyme defect.

Publications

Improvement in lipid and protein trafficking in Niemann-Pick C1 cells by correction of a secondary enzyme defect.

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