Accumulation of Glycosphingolipids in Niemann-Pick C Disease Disrupts Endosomal Transport

Abstract

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1 ؊/؊ mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.Increasing evidence suggests that the membranes of eukaryotic cells are not homogeneously fluid. They contain membrane microdomains, often referred to as lipid rafts and/or caveolae, enriched in glycosphingolipids (GSLs), 1 specific proteins, and cholesterol (1-3). Being operationally defined, i.e. by detergent insolubility and low buoyant density, the natural (in situ) existence of such membrane microdomains has been and continues to be controversial (4). However, they have recently been visualized by using electron microscopy as small structures covering as much as 35% of the cell surface (5). Although membrane microdomains on the cell surface tend to be transient (6), relatively stable microdomains have been visualized in the endocytic pathway (7) where they are proposed to play a role in protein and lipid sorting (8). Many GSL storage diseases, including Niemann-Pick type C (NPC), are often characterized by enlarged liver and spleen as well as severe neurodegeneration. In these diseases, cholesterol and GSLs accumulate in the endocytic pathway because of reduced breakdown. Hence, they may be a useful tool for determining the relative importance of GSLs in endocytic transport. Fibroblasts from patients suffering f...

Alkaline methanolysis of lipid extracts extends shotgun lipidomics analyses to the low-abundance regime of cellular sphingolipids

“…The contents of sphingosine (and sphinganine) in the determined mouse brain and plasma samples are compiled in Table 2. These results are within the range of the values published previously [40,41].…”

“…The contents of sphingosine (and sphinganine) in the determined mouse brain and plasma samples are compiled in Table 2. These results are within the range of the values published previously [40,41].Similar to other choline-containing phospholipids, product ion analysis of protonated lysoSM shows a predominant fragment at m/z 184.1 (Fig. 4A).…”

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

“…N-Butyl deoxynojirimycin (NB-DNJ) is an inhibitor of the enzyme glucosylceramide synthetase, a key enzyme involved in the biosynthesis of gangliosides in animal cells. In NPC1 cells, some of the endosomal malfunction can be corrected by treating cells with NB-DNJ (35); however, the drug has little effect on reversing the cholesterol trafficking defect (22,35). Thus, it is unlikely that the cholesterol trafficking defects observed in NPC1 cells are due to secondary consequence of glycosphingolipid accumulation.…”

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

“…Niemann Pick type C (NPC) disease (NPC1, MIM 257220; NPC2, MIM 607625) is an autosomal recessive neurovisceral disorder with an estimated incidence of 1:150,000 live birth and characterized by the accumulation of a broad spectrum of lipids including unesterified cholesterol, glycosphingolipids (GSLs), sphingosine and sphingomyelin within the lysosomes/late endosomes (LE) due to a defect in the intracellular lipid trafficking (Patterson et al 2001;te Vruchte et al 2004;Lloyd-Evans et al 2008).…”