MEMBRANE TRAFFIC IN SPHIGNOLIPID STORAGE DISEASES

Sphingolipid storage diseases (SLSD) generally result from a defective lysosomal hydrolase or activator protein, which leads to accumulation of endogenous lipids in the lysosomes of many different cell types in the body. Figure 1 shows a diagram of the catabolic pathways of the sphingolipids (SLs) and the diseases associated with defective breakdown of these lipids. In addition, cells from patients with Niemann Pick Type C (NP-C) and Mucolipidosis Type IV (ML-IV) diseases accumulate lipids in the lysosomes, but this accumulation is not the result of a defect in hydrolysis. Rather, in both cases it appears that accumulation results from defects in transport to or from the lysosomes

We are studying defective lipid transport and sorting along the endosome/lysosome and membrane recycling pathways in SLSD fibroblasts.

MAJOR FINDINGS

• BODIPY-LacCer and other SL analogs are internalized from the plasma membrane (PM) and transported predominantly to the Golgi (G) complex of control cells, while in ten different SLSD cell types, these lipids accumulated in endosomes and lysosomes (see Figure 2).
• This perturbation in endocytic trafficking in SLSD cells is due to elevated cholesterol. Cholesterol depletion of SLSD cell types induces lactosylceramide (LacCer) targeting to the Golgi (G) (see Figure 3), while excess cholesterol in normal fibroblasts induces LacCer accumulation in endosomal structures.
• Rapid recycling of LacCer and transferrin from early endosomes to the plasma membrane (PM) is inhibited in Niemann Pick Type A or Type C fibroblasts (see Figure 4). This inhibition is due to elevated cholesterol and can be reversed by cholesterol depletion.
• Detailed studies of the endocytic itineraries in normal vs. SLSD fibroblasts demonstrated that the function of selected rab GTPases (rab4, rab7 and rab9) is inhibited by elevated cholesterol (see Fig. 5).
• Over-expression of rab4, rab7 and rab9 in Niemann Pick Type C and GM1 gangliosidosis fibroblasts restores normal sphingolipid traffic and dramatically lowers cellular cholesterol (see Fig. 6).

OPEN QUESTIONS FOR FUTURE STUDY

1. How does cholesterol modulate the function of selected rab proteins, but not others?
2. How does over-expression of these rab proteins lower stored lipids and cholesterol (e.g., by stimulating cholesterol esterification and/or cholesterol secretion)?
3. Can modulation of membrane traffic be used as a therapeutic approach for treatment of SLSDs?

Selected References

• Chen CS, Bach G, Pagano RE. Abnormal transport along the lysosomal pathway in mucolipidosis, type IV disease. Proc Natl Acad Sci USA. 1998 May 26;95(11):6373-8.
• Chen CS, Patterson MC, Wheatley CL, O'Brien JF, Pagano RE. Broad screening test for sphingolipid-storage diseases. Lancet. 1999 Sep 11;354(9182):901-5.
• Puri V, Watanabe R, Dominguez M, Sun X, Wheatley CL, Marks DL, Pagano RE. Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases. Nat Cell Biol. 1999 Oct;1(6):386-8.
• Sun X, Marks DL, Park WD, Wheatley CL, Puri V, O'Brien JF, Kraft DL, Lundquist PA, Patterson MC, Pagano RE, Snow K. Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1. Am J Hum Genet. 2001 Jun;68(6):1361-72.
• Choudhury A, Dominguez M, Puri V, Sharma DK, Narita K, Wheatley CL, Marks DL, Pagano RE. Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J Clin Invest. 2002 Jun;109(12):1541-50.
• Marks, D.L. and Pagano, R.E. Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease (REVIEW). Trends Cell Biol. 2002 12: 605-613.
• Pagano RE. Endocytic trafficking of glycosphingolipids in sphingolipid storage diseases. Philos Trans R Soc Lond B Biol Sci. 2003 May 29; 358(1433):885-91.
• Puri V Jefferson JR Singh RD Wheatley CL Marks DL Pagano RE. Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage. J Biol Chem. 2003 Jun 6; 278(23):20961-70.
• Choudhury A, Sharma DK, Marks DL, Pagano RE. Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane recycling. Mol Biol Cell. 2004 Oct;15(10):4500-11.