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Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases

Nature Cell Biology volume 1, pages 386–388 (1999)Cite this article

Sphingolipid-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 lysosomes2,3,4, 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.

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. We found increased Filipin staining and altered NPC1 distribution in all of the SLSD cell types that accumulate BODIPY-LacCer (see Fig. 1b), except that Filipin staining of GM2 gangliosidosis cells (Tay–Sachs variant) was similar to that of control fibroblasts.

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Figure 1: Altered sphingolipid trafficking, Filipin staining and NPC1 protein distribution in SLSD fibroblasts.
Figure 2: Effect of cholesterol on sphingolipid targeting in normal and SLSD fibroblasts.

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Acknowledgements

This work was supported by grants from the Ara Parseghian Medical Research Foundation and US PHS Grant GM-22942 (to R.E.P.) and a Kendall-Mayo Fellowship (to M.D.). We thank G. Bach and K. Sandhoff for providing fibroblast samples and S. Patel and P. Pentchev for an antibody to the NPC1 protein.

Correspondence and requests for materials should be addressed to R.E.P.

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

    Vishwajeet Puri, Rikio Watanabe, Michel Dominguez, Xiaofeng Sun, Christine L. Wheatley, David L. Marks & Richard E. Pagano

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Correspondence to Richard E. Pagano.

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Puri, V., Watanabe, R., Dominguez, M. et al. Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases. Nat Cell Biol 1, 386–388 (1999). https://doi.org/10.1038/14084

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