Abstract 1725: Cholesterol Trapping in the Endosomal Pathway Does not Impair Cholesterol Efflux in Niemann-Pick Disease Type B Fibroblasts
Subjects with the genetic disorder Niemann-Pick disease type I (subtypes A and B; NPA, NPB) due to mutation in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, which codes for acid sphingomyelinase (SMase), are characterized by low plasma HDL-C level. SMase hydrolyzes sphingomyelin (SM), a key regulator of cellular cholesterol homeostasis. The role of SMase in cellular cholesterol trafficking pathways is poorly understood. In this study, we report that defective SMase activity in fibroblasts from NPB results in a 2.5-fold increase in cellular SM mass, but not phosphatidylcholine (PC). Furthermore, we observed a specific increase in SM and cellular cholesterol content in NPB cells compared to controls. To determine if cholesterol trapping in the late endocytic compartment is rate-limiting in the ABCA1-mediated efflux pathway, we examined cholesterol trafficking in NPB fibroblasts. Using confocal microscopy to examine the sub-cellular compartmentalization of SM and cholesterol, we provide evidence that SM and cholesterol are retained and co-localized as endocytic punctates in NPB fibroblasts. Despite this sequestration, the expression of ABCA1 transporter and the binding to its ligand, apo AI, in NPB cells are comparable to that of controls. In addition, the sterol regulatory element binding protein-2 (SREBP-2) hydrolysis is regulated normally upon cholesterol deprivation and repletion. Finally, LDL-derived [3H]-cholesterol is normally removed by apo AI in both NPA and NPB fibroblasts. This observation stands in sharp contrast to that observed in Niemann-Pick disease type C (NPC1) and Tangier disease (ABCA1) fibroblasts where the removal of LDL-derived cholesterol is markedly decreased. Altogether, these data suggest that defective SMase activity leads to SM accumulation in the lysosomal pathway, which causes the retention of cholesterol within endocytic compartments. Nevertheless, this sequestered pool of lipids is dynamic and exchangeable with those of ER regulatory sites and plasma membrane. Thus, despite the molecular trapping of cholesterol in the late endocytic pathway in NPB fibroblasts, apo AI-mediated cholesterol efflux via the ABCA1 transporter is not impaired.