Pathophysiology

NPC Defects in Lipid Trafficking

The central biochemical defect in NPC is severely impaired intracellular trafficking of lipids,1 and is very distinct from acid lysosomal sphingomyelinase deficiencies that characterise NPA and NPB.^2,5 At the cellular level, the unique cholesterol trafficking defect in NPC occurs when endocytosed LDL-cholesterol becomes sequestered in lysosomes around the cell nucleus.¹ Transport from these perinuclear lysosomes to the cell membrane and endoplasmic reticulum is retarded in some, as yet, unknown way. As a result, unesterified cholesterol accumulates in great excess, which can be visualised by filipin staining of cultured NPC fibroblast cells (Figure 1).^1,6,7It has been suggested that, in turn, this may lead to a deficiency in cell membrane cholesterol with subsequent membrane dysfunction and/or a triggering of cell death (apoptosis).^5,6,8

Figure 1. Fluorescent filipin staining of (A) normal and (B) NPC fibroblasts showing characteristic peri-nuclear accumulation of LDL-derived cholesterol (images provided by J. Imrie)

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Cells are first cultured in conditions of cholesterol deprivation to create a baseline level of cholesterol and maximal expression of the NPC phenotype. Cells are then incubated in fresh medium with LDL, followed by washing, fixing and staining for cholesterol with filipin.⁹

The definitive biochemical diagnosis of NPC is currently based on the demonstration of abnormal intracellular cholesterol homeostasis in cultures of fibroblasts taken from the patient; cholesterol esterification assays (Figure 2) and filipin staining (Figure 1) are employed in tandem^14. The large majority (approximately 85%) of NPC1 mutations that have been identified have been associated with the ‘classic biochemical phenotype’ of severely impaired cellular cholesterol trafficking.¹⁰ However, some patients display relatively mild alterations of intracellular cholesterol transport that do not show any strict correlation with their clinical NPC symptomatology. Such patients are termed as having the ‘variant biochemical phenotype’.³

Figure 2. Cholesterol trafficking defect in cultured NPC fibroblasts (with permission from Pentchev et al.¹¹)

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Fibroblasts were cultured in lipoprotein-deficient serum to activate the LDL receptor pathway, and then LDL was added to measure their ability to respond to the endocytic uptake and accumulation of lipoprotein-derived cholesterol.¹¹

NPC results in a complex pattern of intracellular lipid storage, with the profile of abnormal lipid levels varying between tissues. In the liver and spleen of NPC patients, unesterified cholesterol, sphingomyelin, phospholipids and glycosphingolipids are stored in excess, with no particular lipid moiety predominating.¹Lipids accumulate to a greater extent in the spleen than in the liver, where alteration can be mild.⁹ However, it has been shown that glucosylceramide accumulates in the foetal NPC liver to an even greater level than that seen in Gaucher disease.¹ It is well established that there is no overt increase in cellular cholesterol or sphingomyelin levels in the brain in NPC.¹ However, studies have demonstrated a many-fold increase in levels of glucosylceramide (again, up to levels seen in Gaucher disease) and lactosylceramide.^12,13 GM2 and GM3 gangliosides are also significantly elevated in NPC brain.^9,14

At the histopathologic level, all clinical forms of NPC are associated with two key pathologic features: foamy storage cells in the visceral organs and accumulation of storage materials in neurones and glial cells. Foamy cells (lipid-laden macrophages) and sea-blue histiocytes can be seen in the spleen, liver, lung, lymph nodes and bone marrow preparations.¹ Only subtle pathologic involvement of skin, skeletal muscle and the eyes is seen during routine histopathologic assessment.

References:
1. Patterson MC, Vanier MT, Suzuki K et al. Niemann–Pick disease, type C: a lipid traffi cking disorder. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B (eds) The Metabolic and Molecular Bases of Inherited Disease, 8th ed, 2001. New York: McGraw-Hill, Ch 145, pp 3611–33.
2. Elleder M, Jirásek A. Niemann–Pick Disease. Report on a symposium held in Hlava‘s Institute of Pathology, Charles University, Prague 2nd-3rd September, 1982. Acta Univ Carol [Med] (Praha) 1983;29:259–67.
3. Vanier MT. Prenatal diagnosis of Niemann–Pick diseases types A, B and C. Prenat Diagn 2002;22:630–2.
4. Pentchev PG, Comly ME, Kruth HS et al. A defect in cholesterol esterifi cation in Niemann–Pick disease (type C) patients. Proc Natl Acad Sci USA 1985;82:8247–51.
5. Mukherjee S, Maxfield FR. Lipid and cholesterol trafficking in NPC. Biochim Biophys Acta 2004;1685:28–37.
6. Sokol J, Blanchette-Mackie J, Kruth HS et al. Type C Niemann–Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol. J Biol Chem 1988;263:3411–7.
7. Shamburek RD, Pentchev PG, Zech LA et al. Intracellular trafficking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann–Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation. J Lipid Res 1997;38:2422–35.
8. Beltroy EP, Richardson JA, Horton JD et al. Cholesterol accumulation and liver cell death in mice with Niemann–Pick type C disease. Hepatology 2005;42:886–93.
9. Vanier MT, Wenger DA, Comly ME et al. Niemann–Pick disease group C: clinical variability and diagnosis based on defective cholesterol esterifi cation. A collaborative study on 70 patients. Clin Genet 1988;33:331–48.
10. Vanier MT, Rodriguez-Lafrasse C, Rousson R et al. Type C Niemann–Pick disease: biochemical aspects and phenotypic heterogeneity. Dev Neurosci 1991;13:307–14.
11. Pentchev PG, Comly ME, Kruth HS et al. Group C Niemann–Pick disease: faulty regulation of low-density lipoprotein uptake and cholesterol storage in cultured fibroblasts. FASEB J 1987;1:40–5.
12. Dawson G, Matalon R, Stein AO. Lactosylceramidosis: lactosylceramide galactosyl hydrolase deficiency and accumulation of lactosylceramide in cultured skin fibroblasts. J Pediatr 1971;79:423–9.
13. Dawson G. Glycosphingolipid levels in an unusual neurovisceral storage disease characterized by lactosylceramide galactosyl hydrolase deficiency: lactosylceramidosis. J Lipid Res 1972;13:207–19.
14. Zervas M, Dobrenis K, Walkley SU. Neurons in Niemann–Pick disease type C accumulate gangliosides as well as unesterifi ed cholesterol and undergo dendritic and axonal alterations. J Neuropathol Exp Neurol 2001a;60:49–64.

Niemann-Pick Knowledge Centre
(Type C)