g(m1)-ganglioside and Niemann-Pick-Diseases

Topics: alpha-Galactosidase; Arylsulfatases; beta-Galactosidase; Cystine; Fabry Disease; G(M1) Ganglioside; G(M2) Ganglioside; Galactosylceramidase; Gangliosidoses; Genetic Carrier Screening; Glycoproteins; Heparitin Sulfate; Humans; Hydrolases; Isoelectric Focusing; Isoenzymes; Kinetics; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lipid Metabolism, Inborn Errors; Lysosomes; Metabolism, Inborn Errors; Molecular Weight; Mucolipidoses; Niemann-Pick Diseases; Sphingolipidoses; Sphingomyelin Phosphodiesterase

Niemann-Pick disease type C (NPC) is a progressive neurodegenerative disorder with characteristic storage of glycolipids in the brain. This study investigated cellular origin and temporal changes of monosialoganglioside storage in the Balb/c npc(nih) mouse brain by immunohistochemistry. Anti-GM1 gave positive staining of the hippocampus, thalamus, cerebellar molecular and Purkinje cell layers in the 3-week old NPC mouse brain and in general, the staining progressively diminished in an age-dependent manner. Anti-GM2 gave positive staining of the hippocampus, thalamus, cerebellar granule cell layer and brainstem nuclei in the 3-week old NPC mouse brain. In contrast to GM1, GM2 staining in these regions, except for the hippocampus, progressively augmented in an age-dependent manner. Double labeling experiments with antibodies against glial fibrillary acidic protein and lysozyme showed localization of GM1 and GM2 in reactive astrocytes and macrophages, respectively. Thus in the NPC mouse brain, GM1 accumulated primarily in neurons and astrocytes whereas GM2 accumulated primarily in neurons and macrophages. Temporal profiles of storage were different from each other and depended on the cell type, presumably reflecting both developmental changes and progression of the disease process. We also investigated subcellular sites of storage in primary-cultured Purkinje cells from the neonatal NPC mouse by immunocytochemistry. In NPC Purkinje cells, GM1 accumulated both in the cytoplasm and dendrites whereas GM2 showed punctuate accumulation in perinuclear vesicles. Thus, subcellular sites of storage were also different between GM1 and GM2 in NPC neurons.

Topics: Aging; Animals; Animals, Newborn; Brain; Brain Chemistry; Cells, Cultured; Cerebellum; Fluorescent Antibody Technique; G(M1) Ganglioside; G(M2) Ganglioside; Glial Fibrillary Acidic Protein; Immunohistochemistry; Mice; Mice, Inbred BALB C; Niemann-Pick Diseases; Purkinje Cells

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.

Topics: Animals; Carrier Proteins; CHO Cells; Cholera Toxin; Cricetinae; Endosomes; G(M1) Ganglioside; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Niemann-Pick C1 Protein; Niemann-Pick Diseases

An improved method, which combined a number of published techniques, is described for the polyethylene-glycol-induced fusion of mononuclear human skin fibroblasts in the presence of phytohaemagglutinin-P and for the subsequent isolation of polynuclear cells by Ficoll gradient sedimentation. Enriched cultures contain between 60 and 75% multinucleated cells and may be maintained in culture without fetal calf serum for up to 14 days without significant overgrowth by the few contaminating mononuclear parental cells. Complementation appears not to occur between GM1 gangliosidosis and mucopolysaccharidosis, type VI B (Morquio) cell strains; this experimental observation provides support for the earlier hypothesis that the mutations for these conditions are allelic. Earlier observations that complementation does not occur between selected phenotypic variants (viz., neuronopathic forms and those without neurological involvement) of sphingomyelin storage (Niemann-Pick) disease or Gaucher's disease are confirmed.

Topics: Cell Fusion; Cells, Cultured; Enzymes; Fibroblasts; G(M1) Ganglioside; Gangliosidoses; Gaucher Disease; Genetic Complementation Test; Humans; Lysosomes; Mucopolysaccharidosis IV; Niemann-Pick Diseases

The results of the diagnostic application of first trimester trophoblast sampling in 100 pregnancies are reported in detail. Further improvement of the method for routine, direct chromosome analysis resulted in a technique which proved to be fast, simple, and efficient. We found that short-term incubation of villi permits the application of many experimental methods, such as visualization of sister chromatid exchanges and bromodeoxyuridine (BrdU) incorporation. Fetal karyotyping was successful in each of the 96 pregnancies in which fetal material was obtained from a total of 98 fetuses. There were 42 males and 56 females, and an abnormal chromosome constitution was found in 12 cases. Two trisomic fetuses were found among the eight pregnancies at risk for Duchenne muscular dystrophy, and this indicates that fetal sexing (which is achieved with our method in two hours) should not be performed without chromosome visualization. The results indicate a risk of 8% of an abnormal fetus for mothers aged 35 years or more, while the risk of failure of sampling and of spontaneous abortion after villi sampling were 4 and 6%, respectively. Enzyme determinations were performed in three pregnancies at risk for gangliosidosis GM1, Niemann-Pick disease, and Hurler syndrome. In this last case inconsistency between the results of the assay of iduronidase on chorionic villi and amniotic fluid cells was found. This unexplained error indicates the need for extensive characterisation in chorionic villi of the series of enzymes involved in metabolic diseases.

Topics: Adult; Chorionic Villi; Chromosome Aberrations; Chromosomes, Human, 13-15; Chromosomes, Human, 16-18; Clinical Enzyme Tests; Female; G(M1) Ganglioside; Gangliosidoses; Humans; Karyotyping; Male; Maternal Age; Niemann-Pick Diseases; Placenta; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Diagnosis; Trophoblasts

Sphingomyelinase activity of cultivated skin fibroblast extracts from normal individuals was resolved by chromatofocusing in the pH range 8-5 into three major components with pI's of 7.3, 6.3 and 5.9, respectively. Chromatofocusing proved a more efficient and reproducible separation technique than preparative flat-bed isoelectric focusing and it gave a constant profile even when detergent concentration varied. In skin fibroblasts from five patients with Niemann-Pick disease type C, a varying degree of reduction in the proportion of the 7.3 peak was observed. In a patient with clinical features of Niemann-Pick disease type C, the finding of such a profile would thus be a good argument for the diagnosis, but it is not pathognomonic as we found similar changes in two cases with GM1-gangliosidosis, while some cases of Niemann-Pick disease type C have borderline normal profiles. These results challenge the concept of a specific sphingomyelinase isoenzyme deficiency as the basic defect in Niemann-Pick disease type C.

Topics: Fibroblasts; G(M1) Ganglioside; Gangliosidoses; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Isoelectric Point; Niemann-Pick Diseases; Phosphoric Diester Hydrolases; Skin; Sphingomyelin Phosphodiesterase

Rectal mucosa biopsy specimens from patients with neuronal storage diseases were examined by electron microscopy. The diseases were Tay-Sachs disease, Sandhoff's disease, Niemann-Pick disease types B and C, late infantile metachromatic leukodystrophy, GM1 gangliosidosis type 1, beta-galactosidase-neuraminidase deficiency, I-cell disease, and mucopolysaccharidoses (Hunter's syndrome and Sanfilippo's syndrome type A). Unmyelinated nerve fibers, endothelial cells, fibroblasts, plasma cells, and histiocytes were seen in the specimens. Except for plasma cells, the results thus obtained for various cells were similar to those obtained from skin and conjunctival biopsy specimens, which have been already reported. There has been no report so far on ultrastructure of the plasma cell in these diseases. Storage materials, eg, dense bodies and membrane-bound vacuoles, were observed in the plasma cells in various storage diseases, with the exception of late infantile metachromatic leukodystrophy. Thus, electron microscopy of rectal mucosa is useful in making diagnoses and examining plasma cells in some neuronal storage diseases.

Topics: Adolescent; Axons; Biopsy; Brain Diseases, Metabolic; Child, Preschool; G(M1) Ganglioside; Humans; Infant; Intestinal Mucosa; Leukodystrophy, Metachromatic; Lymphocytes; Microscopy, Electron; Niemann-Pick Diseases; Plasma Cells; Rectum; Sandhoff Disease; Tay-Sachs Disease