Quantification of mRNAs encoding proteins of the glycosphingolipid catabolism in mouse models of GM2 gangliosidoses and sphingolipid activator protein precursor (prosaposin) deficiency.
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We have investigated the mRNA amounts of six lysosomal proteins (beta-hexosaminidase alpha- and beta-subunit, sphingolipid activator protein precursor, GM2 activator protein, lysosomal sialidase, beta-glucocerebrosidase) involved in the degradation of glycosphingolipids. We analyzed extracts from brain tissues of mouse models for lysosomal storage diseases, i.e., the GM2 gangliosidoses and the deficiency of the sphingolipid activator protein precursor (prosaposin). The mRNA levels were quantified by real-time reverse transcription-polymerase chain reaction. Although storage of the respective lysosomal proteins has been reported in human and mice, no increase of their mRNA amounts could be detected here. Our results indicate that there is no transcriptional upregulation of lysosomal proteins in the examined neuronal storage disorders. (+info)
Promoter characterization and expression of the gene coding for the human GM2 activator protein.
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Genomic clones of the human GM2 activator protein have been isolated and analyzed. The 5' region of the gene demonstrated promoter activity as ascertained by its ability to drive luciferase gene expression in transfected COS cells. This sequence contains GC rich region and several putative promoter elements were present, including Sp1, AP2, cAMP-responsive element, and B-cell-specific activating protein. Analysis of tissue distribution of the GM2 activator protein gene revealed tissue-specific variations in transcript levels. Placenta, bone marrow, mammary gland, bladder, lymph node, and spleen had the highest mRNA levels. (+info)
Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis.
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Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses. (+info)
Serum hexosaminidase and beta-glucuronidase activities in infants: effects of age and sex.
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We investigated the effect of age and sex on the serum activity of hexosaminidase (HEX) and -glucuronidase (BGLU) in 275 normal term infants aged 12 h to 12 months. Up to six weeks of life, HEX was significantly higher in boys (P<=0.023). During the age period of 1-26 weeks, BGLU was also higher in boys, but differences were significant only at 2-6 and 7-15 weeks (P<=0.016). The developmental pattern of HEX and BGLU was sex dependent. HEX activity increased in both sexes from 4-7 days of life, reaching a maximum of 1.4-fold the birth value at 2-6 weeks of age in boys (P<0.001) and a maximum of 1.6-fold at 7-15 weeks in girls (P<0.001). HEX activity gradually decreased thereafter, reaching significantly lower levels at 27-53 weeks than during the first three days of life in boys (P = 0.002) and the same level of this age interval in girls. BGLU increased in both sexes from 4-7 days of age, showing a maximum increase at 7-15 weeks (3.3-fold in boys and 2.9-fold in girls, both P<0.001). Then BGLU decreased in boys to a value similar to that observed at 4-7 days of age. In girls, BGLU remained elevated until the end of the first year of life. These results indicate a variation of HEX and BGLU activities during the first year of life and a sex influence on their developmental pattern. This observation should be considered in the diagnosis of GM2 gangliosidosis and mucopolysaccharidosis type VII. (+info)
Unusual presentation of GM2 gangliosidosis mimicking a brain stem tumor in a 3-year-old girl.
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We report a case of GM2 gangliosidosis revealed by MR imaging of an isolated brain stem abnormality in a 3-year-old girl referred for gait difficulties related to ataxia and pyramidal signs. Brain MR imaging displayed a brain stem lesion with high signal intensity on fluid-attenuated inversion recovery and T2-weighted images, suggesting either a tumor or an inflammatory process. Stereotactic biopsy findings showed the presence of swollen neurons with storage material in lysosomes. Enzyme study revealed deficiency of hexosaminidase A, variant B1. Gangliosidoses should be considered in the differential diagnosis of isolated infiltrating brain stem lesions in childhood. (+info)
Thermodynamic determination of plasma and leukocyte beta-hexosaminidase isoenzymes in homozygote and heterozygote carriers for the GM2 gangliosidosis B1 variant.
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In the GM2 gangliosidosis B1 variant, the mutated isoenzyme A of beta-hexosaminidase (Hex) is incapable of hydrolyzing ganglioside GM2 and negatively charged substrates. Biochemical characterization of this lysosomal disease is carried out using synthetic alpha-subunit-specific sulfated substrates, as heat-inactivation assays are not applicable. The apparent enzyme activation energy of Hex using the chromogenic substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide is related directly to the relative proportions of Hex A and Hex B isoenzymes. This thermodynamic variable was used for the study of Hex enzyme heterogeneity in 3 patients with the GM2 gangliosidosis B1 variant and 6 heterozygote carriers. Hex activity was determined at 25 degrees C, 30 degrees C, 35 degrees C, and 37 degrees C in a Cobas Bio analyzer (Roche Diagnostics, Basel, Switzerland), and Arrhenius plot slopes and apparent activation energies were calculated in plasma samples and mononuclear and polymorphonuclear leukocyte lysates. The determination of the Hex isoenzymes in plasma presented a high discrimination power for B1 variant patients but not for heterozygote carriers, in whom false-negative results may be obtained. However, thermodynamic evaluation of the isoenzyme composition of Hex in leukocyte lysates permits the biochemical identification of patients with the GM2 gangliosidosis B1 variant and of heterozygote carriers. (+info)
Possible role of autoantibodies in the pathophysiology of GM2 gangliosidoses.
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Mice containing a disruption of the Hexb gene have provided a useful model system for the study of the human lysosomal storage disorder known as Sandhoff disease (SD). Hexb(-/-) mice rapidly develop a progressive neurologic disease of ganglioside GM2 and GA2 storage. Our study revealed that the disease states in this model are associated with the appearance of antiganglioside autoantibodies. Both elevation of serum antiganglioside autoantibodies and IgG deposition to CNS neurons were found in the advanced stages of the disease in Hexb(-/-) mice; serum transfer from these mice showed IgG binding to neurons. To determine the role of these autoantibodies, the Fc receptor gamma gene (FcR gamma) was additionally disrupted in Hexb(-/-) mice, as it plays a key role in immune complex-mediated autoimmune diseases. Clinical symptoms were improved and life spans were extended in the Hexb(-/-)FcR gamma(-/-) mice; the number of apoptotic cells was also decreased. The level of ganglioside accumulation, however, did not change. IgG deposition was also confirmed in the brain of an autopsied SD patient. Taken together, these findings suggest that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in SD and therefore provides a target for novel therapies. (+info)
Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.
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In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained. (+info)