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Accumulation of ordered ceramide-cholesterol domains in farber disease fibroblasts.

ABSTRACT: Farber disease is an inherited metabolic disorder caused by mutations in the acid ceramidase gene, which leads to ceramide accumulation in lysosomes. Farber disease patients display a wide variety of symptoms with most patients eventually displaying signs of nervous system dysfunction. We now present a novel tool that could potentially be used to distinguish between the milder and more severe forms of the disease, namely, an antibody that recognizes a mixed monolayer or bilayer of cholesterol:C16-ceramide, but does not recognize either ceramide or cholesterol by themselves. This antibody has previously been used to detect cholesterol:C16-ceramide domains in a variety of cultured cells. We demonstrate that levels of cholesterol:C16-ceramide domains are significantly elevated in fibroblasts from types 4 and 7 Farber disease patients, and that levels of the domains can be modulated by either reducing ceramide or cholesterol levels. Moreover, these domains are located in membranes of the endomembrane system, and also in two unexpected locations, namely, the mitochondria and the plasma membrane. This study suggests that the ceramide that accumulates in severe forms of Farber disease cells is sequestered to distinct membrane subdomains, which may explain some of the cellular pathology observed in this devastating lysosomal storage disease.

SUBMITTER: Ferreira NS

PROVIDER: S-EPMC3897794 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Accumulation of ordered ceramide-cholesterol domains in farber disease fibroblasts.

Ferreira Natalia Santos NS Goldschmidt-Arzi Michal M Sabanay Helena H Storch Judith J Levade Thierry T Ribeiro Maria Gil MG Addadi Lia L Futerman Anthony H AH

JIMD reports 20130712

Farber disease is an inherited metabolic disorder caused by mutations in the acid ceramidase gene, which leads to ceramide accumulation in lysosomes. Farber disease patients display a wide variety of symptoms with most patients eventually displaying signs of nervous system dysfunction. We now present a novel tool that could potentially be used to distinguish between the milder and more severe forms of the disease, namely, an antibody that recognizes a mixed monolayer or bilayer of cholesterol:C1 ...[more]

PMID: 23846911

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Project description:Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can't achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients.

Project description:The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C(12:0)), N-myristoyl- (C(14:0)) and N-palmitoyl- (C(16:0)) sphingosine (C(18:1)) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C(18:0)) substrate. The activity towards N-linolenoylsphingosine (C(18:3)/C(18:1)), N-linoleoylsphingosine (C(18:2)/C(18:1)) and N-oleoylsphingosine (C(18:1)/C(18:1)) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C(18:0)/C(18:1)). The activity towards N-stearoyldihydrosphingosine (C(18:0)/C(18:0)) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C(6:0)/C(18:1)), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2-12)-fold and 7 (4-17)-fold higher K(m) values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.

Project description:A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.

Dataset Information

Accumulation of ordered ceramide-cholesterol domains in farber disease fibroblasts.

Publications

Accumulation of ordered ceramide-cholesterol domains in farber disease fibroblasts.

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