Metabolism of cerebroside sulphate and subcellular distribution of its metabolites in cultured skin fibroblasts derived from controls, metachromatic leukodystrophy, globoid cell leukodystrophy and farber disease

Abstract

The sphingolipidoses are generally understood to be lysosomal lipid storage diseases due to genetic enzymic defects of sphingolipid metabolism. However, the molecular mechanisms that lead to the clinical and pathological manifestations rernain largely obscure. Recent morphological and biochemical studies in neuronal storage diseases (Purpura and Suzuki, 1976) and animal models of gangliosidoses (Wood et al., 1985) suggested that altered membrane structure could be one of the causes of neuronal dysfunction. To understand the mechanism causing neurological dysfunctions in sphingolipidoses, clarification of the synthesis, translocation and insertion of sphingolipids in the different subcellular components in normal and pathological states is essential. This paper reports the metabolism of cerebroside sulphate (CS) and the subcellular distribution of its metabolite in cultured skin fibroblasts from normal control, metachromatic leukodystrophy (MLD; McKusick 25010), globoid cell leukodystrophy (GLD; McKusick 24520) and Farber disease (McKusick 22800). MATERIALS AND METHODS[1-14C]Stearic acid-labelled cerebroside sulphate (14C-CS) was prepared according to the methods of Dubois et al. (1980). Fibroblast cultures from controls, and patients with MLD, GLD and Farber disease, were grown and maintained as described previously (Inui et al., 1985). Feeding experiments of 14C-CS were undertaken as described by Kudoh and Wenger (1982) with minor modifications. After a 24h pulse, cells were harvested by trypsin (0.25%) and washed once with 1% bovine serum albumin in phosphate buffered saline (PBS) and twice with PBS. Cell pellets were suspended in 0.5 mL of 0.25 tool L -~ sucrose solution containing 1 mmol L -~ EDTA adjusted to pH7.0 with 1 molL -1 KOH and homogenized with 10 strokes in a Dual i homogenizer. Then the homogenate was centrifuged at 800g 293Journal of Inherited Metabolic Disease. ISSN 0141-8955.

Functional compartments of sulphatide metabolism in cultured living cells: evidence for the involvement of a novel sulphatide-degrading pathway

“…All these studies were performed on cultured skin fibroblasts and were mainly concerned with diagnosis or characterization of the genetic heterogeneity of leucodystrophies. On the basis of the degradation defect in MLD and on subcellular fractionation observations [16,18,19], these studies supported the conclusion that the hydrolysis of CS in cultured skin fibroblasts exclusively takes place in the lysosome. Up to now, there has been no investigation of in situ sulphatide metabolism in normal and mutant cultured cell types other than fibroblasts.…”

“…This sulphatide-loading test has also been employed to distinguish between MLD homozygotes and heterozygotes, patients carrying the pseudo-deficiency allele (ASA pseudodeficiency is a quite common condition observed among healthy individuals with in vitro ASA activities similar to those seen in MLD [1]), and patients with the sulphatide activator deficiency [4,[10][11][12][13][14][15][16]. Uptake and intracellular metabolism of the exogenous sulphatide has been followed by using a sulphatide radiolabelled in the sulphate [4,5,[8][9][10][11]13], fatty acid [12,[17][18][19] or sphingosine [15,16,20] moieties. Alternatively, various fluorescent derivatives of CS have been employed [21][22][23][24][25][26].…”

Sphingolipids—Their Metabolic Pathways and the Pathobiochemistry of Neurodegenerative Diseases

“…[408] The demonstration of substrate storage in cultured patient cells after loading with radiolabeled substrate or catabolic substrate precursors is an additional diagnostic confirmation. For example, the degradation of ceramide [410,411] or ceramide precursors such as sulfatide [410,412,413] and sphingomyelin [414] can be investigated for the diagnosis of Farbers disease. Such lipid loading studies show high degradation rates of endogenous ceramide despite existing Farbers disease.…”

A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease

“…Although aCDase is a very well known enzyme, existing methods for determining its activity and for FD diagnosis still exhibit many disadvantages. The methods that have been used for FD diagnostic purposes can be classifi ed into three groups: i ) aCDase enzymatic assays ( 4-16 ), classically performed with radiolabeled substrates ( 4-13 ), which are rather water-insoluble and require at least one detergent (see Table 1 ) ; ii ) loading tests, consisting of the addition of exogenous radiolabeled sphingolipids, e.g., ceramide ( 17, 18 ), sulfatide ( 19,20 ), or sphingomyelin ( 21 ), on cultured living cells and the study of their metabolism; and iii ) determination of accumulated ceramide, either by sophisticated chromatographic methods ( 22,23 ) or through the use of the diacylglycerol kinase assay in the presence of ␥ [ Farber disease (FD) is a rare inherited lipid storage disorder, also known as lipogranulomatosis, which is characterized by accumulation of ceramide in the cells and tissues of patients ( 1, 2 ). This accumulation is the consequence of a defi cient intracellular activity of aCDase, a lysosomal …”