Cholesterol trapping in Niemann-Pick disease type B fibroblasts can be relieved by expressing the phosphotyrosine binding domain of GULP.

Background: Impairment of acid sphingomyelinase (SMase) results in accumulation of sphingomyelin (SM) and cholesterol in late endosomes, the hallmarks of a lysosomal storage disease.

Objective: We describe cellular lipid metabolism in fibroblasts from two patients with novel compound heterozygote mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene manifesting as Niemann-Pick disease type B (NPB) and demonstrate mechanisms to overcome the storage defect.

Methods: Using biochemical assays and confocal microscopy, we provide evidence that accumulated lysosomal SM and cholesterol can be released by different treatments.

Results: Defective SMase activity in these fibroblasts results in a 2.5-fold increased cellular mass of SM and cholesterol, increased de novo endogenous cholesterol synthesis, and decreased cholesterol esterification, demonstrating impaired intracellular cholesterol homeostasis. Depletion of exogenous addition of cholesterol for 24 hours or addition of the cholesterol acceptor apolipoprotein A-I are sufficient to restore normal homeostatic responses. In an effort to correct the lysosomal storage phenotype of NPB, we infected the fibroblasts with a lentivirus expressing the phosphotyrosine binding domain of the adapter protein GULP (PTB-GULP). We have previously shown that expression of PTB-GULP in Chinese hamster ovary cells promotes intracellular cholesterol trafficking and ABCA1-mediated cholesterol efflux. We find that expression of PTB-GULP in NPB fibroblasts results in increased ABCA1 expression, increased cellular cholesterol efflux and lysosomal cholesterol redistribution, independent of the impaired SMase and cholesterol presence.

Conclusion: We provide extensive functional characterization of a novel compound heterozygote mutation and provide a novel functional mechanism to overcome lysosomal storage disease defects.