* Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, Minnesota 55905-0001, USA

† e-mail: pagano. richard@ mayo. edu phingolipid-storage diseases (SLSDs) generally result from a defective lysosomal hydrolase or activator-protein cofactors, which lead to accumulation of endogenous lipids in lysosomes1. In addition, lipid accumulation in Niemann–Pick type C (NPC) and mucolipidosis type IV (ML-IV) diseases arises from defects in transport to or from lysosomes2–4, rather than from defects in degradation of substances within lysosomes. A fluorescent analogue of the glycosphingolipid lactosylceramide (BODIPY-LacCer) is internalized from the plasma membrane to the Golgi complex in normal human skin fibroblasts, but is targeted predominantly to endosomes and lysosomes in fibroblasts from ten different SLSDs, suggesting a common mechanism of cellular dysfunction in these biochemically distinct disorders5. Here we show that multiple SLSD cell types have an altered distribution of intracellular cholesterol and/or NPC1 (a cholesterol-sensing protein that is defective in NPC disease) 6, 7. Furthermore, depletion of cholesterol from SLSD cells restores normal BODIPY-LacCer targeting to the Golgi, whereas overloading normal cells with cholesterol redirects BODIPY-LacCer to the endosomal/lysosomal compartment. These results indicate that sphingolipid traffic from the plasma membrane is regulated by cellular cholesterol and that cholesterol homeostasis is perturbed in multiple SLSDs secondary to sphingolipid accumulation. When normal human skin fibroblasts were pulse-labelled with BODIPY-LacCer, we observed marked labelling of the Golgi complex (Fig. 1a), as shown by co-localization studies using a marker of the trans-Golgi network (TGN38; data not shown). In contrast, most of the LacCer fluorescence was distributed in punctate structures throughout the cytoplasm in GM2 gangliosidosis cells (Fig. 1a) and eight other SLSD cell types4, 5. Qualitatively similar results to those seen with BODIPY-LacCer were obtained using BODIPY-analogues of sphingomyelin, galactosylceramide or GM1 ganglioside (data not shown). The lysosomal accumulation of BODIPY-LacCer, as measured by ratio imaging4, 8, 9 of BODIPY-LacCer, varied in nine different SLSD cell types from 4 to 14 times that found in normal cells (Fig. 1b). Little or no lysosomal accumulation was observed in Gaucher type II and Farber disease fibroblasts. The results shown in Fig. 1a, b indicated that a common feature of SLSD cells may have been responsible for the altered trafficking of BODIPY-LacCer compared with control cells. One possibility was that the distribution and/or amount of intracellular cholesterol was altered in most SLSD cells as a result of the lysosomal accumulation of various sphingolipids. To evaluate this possibility, we examined cells labelled with the cholesterol-binding antibiotic Filipin. When we used Niemann–Pick type A (NPA) or GM1 gangliosidosis cells we observed intense labelling of cytoplasmic vesicles, whereas in normal control fibroblasts relatively little cytoplasmic fluorescence was seen (Fig. 1c). We also studied the intracellular distribution of NPC1, which normally resides in a new set of endocytic vesicles and redistributes to lysosomes upon accumulation of cholesterol in this compartment6, 7. This protein was distributed in a fine punctate pattern throughout the cytoplasm of normal fibroblasts, whereas in NPA and GM1 gangliosidosis cells the intensity of NPC1 staining was much greater and the protein was associated with larger punctate structures …