sphingosine-phosphorylcholine and Niemann-Pick-Diseases

The molecular basis of Niemann-Pick disease, type A and B, has been confirmed by detection of mutations causing deficiency of the acid sphingomyelinase activity in the patients. It has been shown that mutations, which cause no activity of acid sphingomyelinase, are responsible for the type A and mutations which cause residual activities of the enzyme are responsible for the type B. Acid sphingomyelinase deficient mice have been established and confirmed to show a similar abnormality observed in human disease type A. These knock-out mice should serves as a useful model for somatic gene therapy. The mechanism of neuronal cell dysfunctions in the type A patients has not been well-characterized. Progress suggest that ceramide, which is produced by sphingomyelinase from sphingomyelin, is an important factor for signal transduction of cell differentiation. In addition, a lysosphingolipid (sphingosylphocholine), which accumulates in the tissues of the patients, has been reported to act as a strong mitogen in several types of cells through activating a transcription factor. AP-1. It is possible that abnormal phospholipid signaling is involved in the pathogenesis of neuronal cell dysfunction of Niemann-Pick disease type A.

Topics: Animals; Cell Differentiation; Ceramides; Glycoproteins; Humans; Lysosomes; Mice; Mutation; Niemann-Pick Diseases; Phosphorylcholine; Saposins; Signal Transduction; Sphingolipid Activator Proteins; Sphingomyelin Phosphodiesterase; Sphingosine

Acid sphingomyelinase deficiency (ASMD) is a lysosomal disorder caused by deficiency of acid sphingomyelinase (ASM) leading to the accumulation of sphingomyelin (SM) in a variety of cell types. Lysosphingomyelin (LysoSM) is the de-acetylated form of SM and it has been shown as a biomarker for ASMD in tissues, plasma, and dried blood spots (DBS) and lysosphingomyelin-509 (LysoSM509) is the carboxylated analogue of LysoSM. High levels of Lysosphingomyelin 509 (LysoSM509) have also been shown in ASMD patients. In this study, we report the utility of the quantification of LysoSM and LysoSM509 in DBS of patients from Latin America with ASMD by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).. DBS samples from 14 ASMD patients were compared with 15 controls, and 44 general newborns. All patients had their diagnosis confirmed by the quantification of ASM and the measurement of the activity of chitotriosidase. All patients had significantly higher levels of lysoSM and lysoSM509 compared to controls and general newborns.. The quantification of lysosphingolipids in DBS is a valuable tool for the diagnosis of ASMD patients and lysoSM can be useful in the differential diagnosis with NPC. This method is also valuable in the ASMD newborn screening process.

Topics: Chromatography, Liquid; Humans; Infant, Newborn; Niemann-Pick Disease, Type A; Niemann-Pick Diseases; Sphingomyelin Phosphodiesterase; Tandem Mass Spectrometry

Topics: Chromatography, Liquid; Humans; Niemann-Pick Diseases; Phosphatidylcholines; Phosphorylcholine; Sphingosine; Tandem Mass Spectrometry

Niemann-Pick disease types A and C, and Gaucher disease are glycolipid storage disorders characterized by the systemic deposition of glycosphingolipids, i.e., sphingomyelin in Niemann-Pick disease types A and C tissues and glucosylceramide in Gaucher disease ones, respectively. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we analyzed the sphingolipids in liver and spleen specimens from patients with Niemann-Pick disease types A and C, and Gaucher disease. Crude lipids were extracted from tissue containing 5mg protein with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by MALDI-TOF/MS. The results were as follows: (a) ion peaks with m/z values corresponding to different sphingomyelin and ceramide monohexoside (CMH) species were clearly detected. (b) With sphingosylphosphorylcholine as the internal standard for quantification of sphingomyelin and CMH, the relative peak heights of sphingomyelin and CMH were calculated and plotted versus their contents. The relative peak heights of sphingomyelin and CMH showed linearity between 50 and 1500 ng sphingomyelin content, and between 5 and 150 ng CMH content, respectively. (c) Quantitative analysis revealed the accumulation of sphingomyelin in the liver and spleen specimens from the patients with Niemann-Pick disease types A and C. Striking accumulation of CMH was also detected in the liver and spleen specimens from the patients with Gaucher disease. This investigation indicated that accumulated sphingomyelin and CMH in small amounts of tissues from sphingolipidosis patients can be detected quantatively with the MALDI-TOF/MS method. This method will be useful not only for the diagnosis but also for biochemical pathophysiology evaluation of patients with various sphingolipidosis.

Topics: Child, Preschool; Gaucher Disease; Glucosylceramides; Humans; Liver; Niemann-Pick Diseases; Phosphorylcholine; Reference Standards; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingomyelins; Sphingosine; Spleen

6-Hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMUPC) was shown to be a specific substrate for the determination of acid (lysosomal) sphingomyelinase (ASM; gene SMPD1). Fibroblasts (n = 27) and leukocytes (n = 8) from both the A and B types of Niemann-Pick disease showed < 6% and < 10% of mean normal ASM activity, respectively. Niemann-Pick A or B patients bearing the Q292K mutation had apparently normal ASM activity with our new artificial substrate. These patients with false-normal sphingomyelinase activity, however, could readily be detected by determining the extent of inhibition of enzymatic hydrolysis of the artificial substrate HMU-PC by an unlabelled natural substrate, in particular lysosphingomyelin. This approach is generally applicable. Our novel assay for ASM combines the ease of a rapid and robust enzyme assay using a fluorogenic substrate with the specificity of an ASM assay using a natural substrate. Such assays are obviously more convenient to the diagnostic laboratory, since radiolabelled substrates are not required.

Topics: Blood Chemical Analysis; Ceramides; Chemistry, Clinical; Clinical Enzyme Tests; Diagnosis, Differential; Dose-Response Relationship, Drug; Fibroblasts; Fluorometry; Gene Expression Regulation, Enzymologic; Hexosaminidases; Humans; Hydrolysis; Leukocytes; Mutation; Niemann-Pick Diseases; Phospholipid Ethers; Phosphorylcholine; Protein Binding; Reproducibility of Results; Skin; Sphingomyelin Phosphodiesterase; Sphingomyelins; Sphingosine; Substrate Specificity; Time Factors

A study of brain lipids in patients with the sphingomyelinase-deficient types of Niemann-Pick disease demonstrated that abnormal accumulation of sphingomyelin occurs only in the brain of neuronopathic type A patients but not in the non-neuronopathic type B. Additional lipid abnormalities were present in the type A brain. In contrast, the brain lipid profile was normal in type B patients. Since lysosphingolipids have been implicated in the biochemical pathogenesis of other genetic lysosomal sphingolipidoses, the occurrence of sphingosylphosphorylcholine (lysosphingomyelin) was specifically investigated in brain and extraneural tissues, using an HPLC method with fluorescent detection of orthophtalaldehyde derivatives. Levels close to or below the limit of detection (10 pmol/mg tissue protein) were observed in normal and pathological controls. A striking accumulation was observed in brain of two Niemann-Pick type A patients (830 and 430 pmol/mg protein in 27-and 16-month-old children with severe and milder neurological course, respectively), which was not present at the fetal stage of the disease. No significant increase was found in brain tissue from a 3.5 year-old type B patient. In liver and spleen, abnormally high sphingosylphosphorylcholine levels were observed in both types of the disease, with indication of a progressive increase during development. This study establishes the integrity of brain tissue in Niemann-Pick disease type B and suggests that the lysocompound sphingosylphosphorylcholine could play a role in the pathophysiology of brain dysfunction in the neuronopathic type A.

Topics: Brain; Child, Preschool; Chromatography, High Pressure Liquid; Humans; Infant; Liver; Niemann-Pick Diseases; Phosphorylcholine; Sensitivity and Specificity; Sphingosine; Spleen

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

Topics: 3T3 Cells; Animals; Base Sequence; Binding Sites; Cell Division; Cell Nucleus; DNA; DNA-Binding Proteins; Humans; Male; Mice; Molecular Sequence Data; Niemann-Pick Diseases; Oligodeoxyribonucleotides; Phosphorylcholine; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Recombinant Proteins; Salmon; Signal Transduction; Spermatozoa; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transfection

Psychosine, sphingosylphosphorylcholine (52-104 microM), and other glycosphingolipids stimulate mitochondrial respiration (up to 500%) and inhibit oxidative phosphorylation to varying degrees. Above 104 microM these functions as well as uptake of Ca2+ are prevented. At 104 microM sphingosylphosphorylcholine inhibits the mitochondrial ATPase reaction in submitochondrial particles by 48%. Both sphingosylphosphorylcholine and psychosine enhance the active phosphate-dependent swelling of mitochondria. Passive swelling occurs in the presence of rotenone (when swelling does not normally occur) and under hypotonic conditions. A direct interaction of sphingosylphosphorylcholine with membranes is demonstrated by a discharge of the proton gradient across mitochondrial membranes, hemolysis of red blood cells, and binding to inner and outer mitochondrial membranes. Thus lysosphingolipids bind strongly to mitochondrial membranes and markedly alter mitochondrial function. This alteration would affect the ATP levels, thereby altering a wide range of ATP-dependent cellular functions. These results offer a partial explanation for the pathogenesis of representative lysosomal storage diseases.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Calcium; Choline; Erythrocytes; Hemolysis; Humans; Intracellular Membranes; Male; Mitochondria, Liver; Mitochondrial Swelling; Niemann-Pick Diseases; Oxygen Consumption; Phosphorylcholine; Rabbits; Rats; Rats, Inbred Strains; Sphingosine