Determination of 7-ketocholesterol in plasma by LC-MS for rapid diagnosis of acid SMase-deficient Niemann-Pick disease.

Acid sphingomyelinase (ASMase)-deficient Niemann-Pick disease (NPD) is caused by mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, resulting in accumulation of sphingomyelin in the lysosomes and secondary changes in cholesterol metabolism. We hypothesized that the oxidation product of cholesterol, 7-ketocholesterol (7-KC), might increase in the plasma of patients with ASMase-deficient NPD. In this study, a rapid and nonderivatized method of measurement of plasma 7-KC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Plasma samples from healthy subjects, patients with ASMase-deficient NPD, nonaffected ASMase-deficient NPD heterozygotes, Niemann-Pick type C (NPC) disease, glycogen storage disorder type II (GSDII), Gaucher disease (GD), mucopolysaccharidosis type II (MPSII), Krabbe disease (KD), and metachromatic leukodystrophy (MLD) were tested retrospectively. Markedly elevated 7-KC was found in patients with ASMase-deficient NPD and NPC disease that showed significant differences from ASMase-deficient NPD heterozygotes; patients with GSDII, GD, MPSII, KD, and MLD; and normal controls. The analysis of plasma 7-KC by LC-MS/MS offers the first simple, quantitative, and highly sensitive method for detection of ASMase-deficient NPD and could be useful in the diagnosis of both ASMase-deficient NPD and NPC disease.

Cathepsin B overexpression due to acid sphingomyelinase ablation promotes liver fibrosis in Niemann-Pick disease.

Niemann-Pick disease (NPD) is a lysosomal storage disease caused by the loss of acid sphingomyelinase (ASMase) that features neurodegeneration and liver disease. Because ASMase-knock-out mice models NPD and our previous findings revealed that ASMase activates cathepsins B/D (CtsB/D), our aim was to investigate the expression and processing of CtsB/D in hepatic stellate cells (HSCs) from ASMase-null mice and their role in liver fibrosis. Surprisingly, HSCs from ASMase-knock-out mice exhibit increased basal level and activity of CtsB as well as its in vitro processing in culture, paralleling the enhanced expression of fibrogenic markers α-smooth muscle actin (α-SMA), TGF-ß, and pro-collagen-α1(I) (Col1A1). Moreover, pharmacological inhibition of CtsB blunted the expression of α-SMA and Col1A1 and proliferation of HSCs from ASMase-knock-out mice. Consistent with the enhanced activation of CtsB in HSCs from ASMase-null mice, the in vivo liver fibrosis induced by chronic treatment with CCl(4) increased in ASMase-null compared with wild-type mice, an effect that was reduced upon CtsB inhibition. In addition to liver, the enhanced proteolytic processing of CtsB was also observed in brain and lung of ASMase-knock-out mice, suggesting that the overexpression of CtsB may underlie the phenotype of NPD. Thus, these findings reveal a functional relationship between ASMase and CtsB and that the ablation of ASMase leads to the enhanced processing and activation of CtsB. Therefore, targeting CtsB may be of relevance in the treatment of liver fibrosis in patients with NPD.

Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.

Types A and B Niemann-Pick disease (NPD) result from the deficient activity of acid sphingomyelinase (ASM). An animal model of NPD has been created by gene targeting. In affected animals, the disease followed a severe, neurodegenerative course and death occurred by eight months of age. Analysis of these animals showed their tissues had no detectable ASM activity, the blood cholesterol levels and sphingomyelin in the liver and brain were elevated, and atrophy of the cerebellum and marked deficiency of Purkinje cells was evident. Microscopic analysis revealed 'NPD cells' in reticuloendothelial organs and characteristic NPD lesions in the brain. Thus, the ASM deficient mice should be of great value for studying the pathogenesis and treatment of NPD, and for investigations into the role of ASM in signal transduction and apoptosis.

[Adult psychiatric aspects of Niemann-Pick disease]. / A Niemann-Pick betegség felnött pszichiátriai vonatkozásai.

Niemann-Pick disease (NPD) is a group of distinct rare disorders (i.e. NPD-A; NPD-B; NPD-C) - with autosomal recessive inheritance pattern - within the class of the inborn disorders of the sphingolipid metabolism (called sphingolipidoses). Since patients with NPD-A do not survive into adulthood and most patients with NPD-B are free from neuropsychiatric symptoms we discuss only briefly type-A and -B NPD and mainly constrict our review discussing the neuropsychiatric symptoms along with the pathomechanism and the treatment of NPD-C. NPD-C is clinically heterogeneous, with notable variations in age at onset, course and symptoms. Along with systemic signs, neurologic and psychiatric symptoms are quite frequent in NPD-C and in its adult form sometimes psychiatric symptoms are the first ones appearing. Unfortunately, the majority of clinicans (including adult psychiatrists and neurologists) are not aware of the symptom group characteristic to NPD-C so patients with this disorder are frequently misdiagnosed in the clinical practice. Since neuropsychiatric manifestations of NPD-C may be treated with a substrate reduction agent (miglustat) with greater awareness of the identification of neuropsychiatric symptoms in due course is the prerequisite of proper and early diagnosis and treatment.

Pulmonary delivery of recombinant acid sphingomyelinase improves clearance of lysosomal sphingomyelin from the lungs of a murine model of Niemann-Pick disease.

Systemic administration of recombinant acid sphingomyelinase (rhASM) into ASM deficient mice (ASMKO) results in hydrolysis of the abnormal storage of sphingomyelin in lysosomes of the liver, spleen and lung. However, the efficiency with which the substrate is cleared from the lung, particularly the alveolar macrophages, appears to be lower than from the other visceral tissues. To determine if delivery of rhASM into the air spaces of the lung could enhance clearance of pulmonary sphingomyelin, enzyme was administered to ASMKO mice by intranasal instillation. Treatment resulted in a significant and dose-dependent reduction in sphingomyelin levels in the lung. Concomitant with this reduction in substrate levels was a decrease in the amounts of the pro-inflammatory cytokine, MIP-1alpha, in the bronchoalveolar lavage fluids and an improvement in lung pathology. Maximal reduction of lung sphingomyelin levels was observed at 7 days post-treatment. However, reaccumulation of the substrate was noted starting at day 14 suggesting that repeated treatments will be necessary to effect a sustained reduction in sphingomyelin levels. In addition to reducing the storage abnormality in the lung, intranasal delivery of rhASM also resulted in clearance of the substrate from the liver and spleen. Hence, pulmonary administration of rhASM may represent an alternative route of delivery to address the visceral pathology associated with ASM deficiency.

The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases.

Acid sphingomyelinase (ASM; E.C. 3.1.4.12) is best known for its involvement in the lysosomal storage disorder Niemann-Pick disease (NPD). Through studies that began by investigating this rare disease, recent findings have uncovered the important role of this enzyme in the initiation of ceramide-mediated signal transduction. This unique function involves translocation of the enzyme from intracellular compartments to the outer leaflet of the cell membrane, where hydrolysis of sphingomyelin into ceramide initiates membrane reorganization and facilitates the formation and coalescence of lipid microdomains. These microdomains are sites of protein-protein interactions that lead to downstream signaling, and perturbation of microdomain formation influences the pathophysiology of many common diseases. The initial observations implicating ASM in this process have come from studies using cells from patients with NPD or from ASM knockout (ASMKO) mice, where the genetic deficiency of this enzymatic activity has been shown to protect these cells and animals from stress-induced and developmental apoptosis. This review will discuss the complex biology of this enzyme in the context of these new findings and its recently reported importance in common human diseases, including cancer, sepsis, cardiovascular, pulmonary, liver, and neurological diseases as well as the potential for using ASM (or ASM inhibitors) as therapeutic agents.

Niemann-Pick disease: a genetic model in Siamese cats.

Three Siamese cats were found to have a progressive neurological disease that became obvious when they were 4 to 5 months of age. Their brains contained an excess of GM2 and GM3 gangliosides, and their livers a nine- to tenfold excess of sphingomyelin and cholesterol. A total deficiency of lysosomal (pH 5.0) sphingomyelinase was found in the leukocytes, liver, and brain of the cats, although the activity of the microsomal (pH 7.4, magnesium-dependent) sphingomyelinase was normal in brain. These cats appear to have a genetic disease identical to Niemann-Pick disease type A.

Niemann-Pick disease experimental model: sphingomyelinase reduction induced by AY-9944.

Organs of rats treated with the drug A Y-9944 for 5 days showed a significant reduction in sphingomyelinase activity. Evidence is presented which suggests that the reduction is due to impaired enzyme synthesis.

Characterization of common SMPD1 mutations causing types A and B Niemann-Pick disease and generation of mutation-specific mouse models.

Herein we describe detailed characterization of four common mutations (L302P, H421Y, R496L and DeltaR608) within the acid sphingomyelinase (ASM) gene causing types A and B Niemann-Pick disease (NPD). In vitro and in situ enzyme assays revealed marked deficiencies of ASM activity in NPD cell lines homoallelic for each mutation, although Western blotting and fluorescent microscopy showed that the mutant ASM polypeptides were expressed at normal levels and trafficked to lysosomes. Co-immunoprecipitation of the polypeptides with the ER chaperone, BiP, confirmed these findings, as did in vitro expression of the mutant cDNAs in reticulocyte lysates. We further developed a computer assisted, three-dimensional model of human ASM based on homologies to known proteins, and used this model to map each NPD mutation in relation to putative substrate binding, hydrolysis and zinc-binding domains. Lastly, we generated transgenic mice expressing the R496L and DeltaR608 mutations on the complete ASM knock-out background (ASMKO), and established breeding colonies for the future evaluation of enzyme enhancement therapies. Analysis of these mice demonstrated that the mutant ASM transgenes were expressed at high levels in the brain, and in the case of the DeltaR608 mutation, produced residual ASM activity that was significantly above the ASMKO background.

Niemann-Pick type B disease. Identification of a single codon deletion in the acid sphingomyelinase gene and genotype/phenotype correlations in type A and B patients.

Types A and B Niemann-Pick disease both result from the deficient activity of the lysosomal hydrolase, acid sphingomyelinase (E.C. 3.1.4.12). Type A Niemann-Pick disease is a severe neurodegenerative disorder of infancy which leads to death by three years of age, whereas Type B disease has a later age at onset, little or no neurologic involvement, and most patients survive into adulthood. To investigate the molecular basis for the remarkable phenotypic heterogeneity, the nature of the mutations causing Type B Niemann-Pick disease in Ashkenazi Jewish patients was determined. The entire acid sphingomyelinase coding region from an Ashkenazi Jewish Type B patient was polymerase chain reaction-amplified, subcloned, and completely sequenced. A three-base deletion was identified of nucleotides 1821-1823 in the cDNA which predicted the removal of an arginine residue from position 608 of the acid sphingomyelinase polypeptide (delta R608). The other cDNA clones from this patient had the R496L mutation previously identified in Type A Niemann-Pick disease patients. Both Ashkenazi Jewish Type B patients were heteroallelic for the delta R608 mutation, whereas this allele was not present in 15 unrelated non-Jewish Type B patients, with the notable exception of one mildly affected patient of Arabic descent who was homoallelic for the delta R608 mutation. These results indicate that the delta R608 mutation predicts the Type B Niemann-Pick disease phenotype, even in the presence of the R496L Type A allele, thereby providing the first genotype/phenotype correlation for this lysosomal storage disease. Although only two patients have been studied, it appears that the delta R608 mutation occurs frequently in Type B Niemann-Pick disease patients of Ashkenazi Jewish descent.

Enzyme deficiency in cholesteryl ester storage idisease.

Cholesteryl ester storage disease has been shown to involve severe deficiency of acid cholesteryl ester hydrolase and triglyceride lipase activity in liver, spleen, and lymph node. The cholesteryl ester hydrolase was also deficient in aorta. Tissue storage of both cholesteryl esters and triglycerides is generalized. Both the lipid and enzymatic changes are very similar to those in Wolman's disease.

Acid sphingomyelinase-deficient Niemann-Pick disease: novel findings in a Greek child.

Niemann-Pick Disease (NPD) is a heterogeneous group of autosomal recessive disorders characterized by progressive accumulation of sphingomyelin and cholesterol in lysosomes. Six types of NPD have been described based on clinical presentation and involved organs. The primary defect in NPD types A and B is a deficiency of lysosomal acid sphingomyelinase (ASM). We present a case of a 5-year-old boy with type B NPD who had severe clinical manifestations, including heart involvement. He was first admitted to the hospital at 2 months because of vomiting, refusal to feed, lethargy, hepatomegaly and mild transaminasaemia. Liver biopsy at 12 months showed lipid accumulation and fibrosis. Investigations for lysosomal storage disorders revealed increased plasma chitotriosidase (549 nmol/h per ml, normal value 0-150). At 18 months, no detectable ASM activity was observed in cultured fibroblasts (normal range 23-226 nmol/h per mg protein) confirming NPD B. Pulmonary involvement was detected with high-resolution computerized tomography which revealed reticulonodular infiltrations and thickening of the interlobular septa. At 2 years growth retardation and kyphosis were noted. At 2.5 years he manifested neurodevelopment regression, indicating CNS involvement. Cardiac involvement (grade III mitral valve insufficiency) developed at 4 years and heart failure at 5 years. Genetic analysis revealed two mutations: a H421Y mutation that is common in Saudi Arabian and Turkish patients, and a W32X mutation, which has been found in other Mediterranean patients.

Neutral sphingomyelinase 1 deficiency in the mouse causes no lipid storage disease.

Sphingomyelin is a major lipid in the bilayer of subcellular membranes of eukaryotic cells. Different sphingomyelinases catalyze the initial step in the catabolism of sphingomyelin, the hydrolysis to phosphocholine and ceramide. Sphingomyelinases have been postulated to generate ceramide as a lipophilic second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. To elucidate the function of the first cloned Mg(2+)-dependent, neutral sphingomyelinase (nSMase 1) in sphingomyelin catabolism and its potential role in signaling processes in a genetic and molecular approach, we have generated an nSMase 1-null mutant mouse line by gene targeting. The nSMase 1-deficient mice show an inconspicuous phenotype and no accumulation or changed metabolism of sphingomyelin or other lipids, despite grossly reduced nSMase activity in all organs except brain. We also addressed the recent proposal that nSMase 1 possesses lysophospholipase C activity. The unaltered metabolism of lysophosphatidylcholine or lyso-platelet-activating factor excludes the proposed role of nSMase 1 as a lysophospholipase C.

Clinical evaluation of chitotriosidase enzyme activity in Gaucher and Niemann Pick A/B diseases: A retrospective study from India.

Plasma chitotriosidase originates from activated macrophages and is reported to be elevated in many Lysosomal Storage Disorders. Measurement of this enzyme activity has been an available tool for monitoring therapy of Gaucher disease. The degree of elevation of chitotriosidase is useful for differential diagnosis of Gaucher disease and Niemann Pick A/B. However the potential utility of this chitotriosidase assay depends on the frequency of deficient chitotriosidase activity in a particular population. We therefore aim to study the clinical utility of this assay Gaucher and Niemann Pick A/B diseases in the backdrop of chitotriosidase deficiency in our population. The study comprises 173 patients with clinical suspicion of either Gaucher disease (n=108) or Niemann Pick A/B (n=65) and 92 healthy controls. The plasma samples of controls, Gaucher disease, and Niemann Pick A/B showed chitotriosidase deficiency of 12%, 25% and 27% respectively. The degree of elevation of chitotriosidase in Gaucher disease and Niemann Pick A/B patients is 40-326 (11,325.7±6395.4nmol/h/ml) and 7-22 folds (1192.5±463.0nmol/h/ml) respectively. In view of these findings of distinguishable fold elevation of chitotriosidase in Gaucher disease or Niemann Pick A/B, it can be a potential surrogate differential diagnostic marker for these groups of diseases, except in the patients in whom this enzyme is deficient.

Regulation of sphingomyelin metabolism.

Sphingolipids (SFs) represent a large class of lipids playing diverse functions in a vast number of physiological and pathological processes. Sphingomyelin (SM) is the most abundant SF in the cell, with ubiquitous distribution within mammalian tissues, and particularly high levels in the Central Nervous System (CNS). SM is an essential element of plasma membrane (PM) and its levels are crucial for the cell function. SM content in a cell is strictly regulated by the enzymes of SM metabolic pathways, which activities create a balance between SM synthesis and degradation. The de novo synthesis via SM synthases (SMSs) in the last step of the multi-stage process is the most important pathway of SM formation in a cell. The SM hydrolysis by sphingomyelinases (SMases) increases the concentration of ceramide (Cer), a bioactive molecule, which is involved in cellular proliferation, growth and apoptosis. By controlling the levels of SM and Cer, SMSs and SMases maintain cellular homeostasis. Enzymes of SM cycle exhibit unique properties and diverse tissue distribution. Disturbances in their activities were observed in many CNS pathologies. This review characterizes the physiological roles of SM and enzymes controlling SM levels as well as their involvement in selected pathologies of the Central Nervous System, such as ischemia/hypoxia, Alzheimer disease (AD), Parkinson disease (PD), depression, schizophrenia and Niemann Pick disease (NPD).

Human acid sphingomyelinase structures provide insight to molecular basis of Niemann-Pick disease.

Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann-Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.

The structure and catalytic mechanism of human sphingomyelin phosphodiesterase like 3a--an acid sphingomyelinase homologue with a novel nucleotide hydrolase activity.

UNLABELLED: Human sphingomyelinase phosphodiesterase like 3a (SMPDL3a) is a secreted enzyme that shares a conserved catalytic domain with human acid sphingomyelinase (aSMase), the enzyme carrying mutations causative of Niemann-Pick disease. We have solved the structure of SMPDL3a revealing a calcineurin-like fold. A dimetal site, glycosylation pattern and a disulfide bond network are likely to be conserved also in human aSMase. We show that the binuclear site of SMPDL3a is occupied by two Zn(2+) ions and that excess Zn(2+) leads to inhibition of enzyme activity through binding to additional sites. As an extension of recent biochemical work we uncovered that SMPDL3a catalyses the hydrolysis of several modified nucleotides that include cytidine 5'-diphosphocholine, cytidine diphosphate ethanolamine and ADP-ribose, but not the aSMase substrate, sphingomyelin. We subsequently determined the structure of SMPDL3a in complex with the product 5'-cytidine monophosphate (CMP), a structure that is consistent with several distinct coordination modes of the substrate/product in the active site during the reaction cycle. Based on the structure of CMP complexes, we propose a phosphoryl transfer mechanism for SMPDL3a. Finally, a homology model of human aSMase was constructed to allow for the mapping of selected Niemann-Pick disease mutations on a three-dimensional framework to guide further characterization of their effects on aSMase function. DATABASE: Structural data are available in the PDB database under the accession numbers 5EBB and 5EBE.

Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology.

Niemann-Pick disease is caused by a genetic deficiency in acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. In the present study, we evaluated the effects of direct intracerebral transplantation of neural progenitor cells (NPCs) on the brain storage pathology in the ASM knock-out (ASMKO) mouse model of Type A Niemann-Pick disease. NPCs derived from adult mouse brain were genetically modified to express human ASM (hASM) and were transplanted into multiple regions of the ASMKO mouse brain. Transplanted NPCs survived, migrated, and showed region-specific differentiation in the host brain up to 10 weeks after transplantation (the longest time point examined). In vitro, gene-modified NPCs expressed up to 10 times more and released five times more ASM activity into the culture media compared with nontransduced NPCs. In vivo, transplanted cells expressed hASM at levels that were barely detectable by immunostaining but were sufficient for uptake and cross-correction of host cells, leading to reversal of distended lysosomal pathology and regional clearance of sphingomyelin and cholesterol storage. Within the host brain, the area of correction closely overlapped with the distribution of the hASM-modified NPCs. No correction of pathology occurred in brain regions that received transplants of nontransduced NPCs. These results indicate that the presence of transduced NPCs releasing low levels of hASM within the ASMKO mouse brain is necessary and sufficient to reverse lysosomal storage pathology. Potentially, NPCs may serve as a useful gene transfer vehicle for the treatment of CNS pathology in other lysosomal storage diseases and neurodegenerative disorders.