Linda M. Hoffman

GM2* ganglioside in fetal Tay-Sachs disease brain cultures: A model system for the disease

Cultured cells derived from Tay-Sachs disease (TSD) fetal cerebellum were shown to accumulate GM2 ganglioside when compared with control cultures. In contrast, fibroblasts derived from TSD fetal lung do not contain GM2. GM2 was identified by thin-layer chromatography (TLC) and confirmed by gas-liquid chromatography (GLC). Unlike fetal TSD brain, the cell cultures established from fetal TSD brain have high concentrations of globoside and GD3 and little or no asialo GM2(GA2). The GM2 and related glycosphingolipids in these cultured cells contain a high percentage of C24:0 and C24:1 fatty acids. In spite of these differences, this TSD cell strain is unique in that it accumulates GM2, and can therefore serve as a useful in vitro model for the study of TSD.

Enzyme replacement treatment for Tay-Sachs disease brain cells in culture utilizing Concanavalin A-mediated hexosaminidase A uptake: Biochemical and morphological evidence of GM2 mobilization

SummaryWhen Concanavalin A (Con A) is bound to the cell membrane, it functions as an artificial enzyme receptor, mediating the binding and intracellular incorporation of significant amounts of exogenous hexosaminidase A (Hex A) into Tay-Sachs disease (TSD) glial cells. The treated cells retained almost 50% of incorporated Hex A activity after 3 days incubation in Hex A free medium. Hex A was released from Con A within the cell and was available as free enzyme. Biochemical analysis of gangliosides in Con A and Hex A treated cells depicted a greater than 50% reduction in stored GM2 ganglioside and a fourfold reduction in GM2label (14C) when compared to controls. Ultrastructural evidence of GM2 breakdown is presented which supports the biochemical and labeling studies.

Ceramide hexosides in niemann-pick disease brain

ZusammenfassungIn normalem Gehirngewebe und in Gehirngewebe einer Niemann-Pickschen Erkrankung vom Typus A wurden Glykosphingolipide unter Verwendung der Dünnschichtchromatographie und der Flüssig-Gaschromatographie bestimmt. Bei der Niemann-Pickschen Erkrankung fand sich eine Zunahme der Ceramid-Trihexoside, die durch Digalactosylglucosylceramide bedingt war. Auch das Glucosylcerebrosid war vermehrt.

Human fetal brain cells in culture. Increase in GM2 ganglioside after SV-40 transformation

Abstract The composition of gangliosides in a cultured human fetal brain cell line was examined before and after SV-40 transformation. In contrast to most reports on other cell lines, an increase was observed in the complex ganglioside G M2 in the SV-40-ttransformed brain cell line.

Fetal tay-sachs disease brain cells in culture: lack of turnover in [14C]glucosamine-labeled GM2

Abstract The glycosphingolipids in a cultured cell strain derived from Tay-Sachs disease fetal cerebellum were pulse-labeled with radioactive [ 14 C]glucosamine. The turnover of individual gangliosides in the cells were followed during a 10 day period. All of the gangliosides except G M2 had a buildup of counts which peaked between 24 and 96 h, then decreased. In contrast, the counts incorporated into G M2 did not decrease during the entire course of the experiment. The lack of turnover in G M2 ganglioside provides support for the use of these cultured brain cells as a model for Tay-Sachs disease.

Gangliosides containing glucosamine and galactosamine in transformed Tay-Sachs disease and normal human brain cell lines

Human SV-40 transformed brain cell lines derived from Tay-Sachs disease (TSD) and normal fetal cerebra were grown in culture and analyzed for their ganglioside content. Both the TSD and normal cells contained GM3, GM2, and a novel triheoxyl N-acetylglucosamine-containing ganglioside. In order to increase tumorigenicity, the cells were cloned on soft agar. The cloned cells still contained GM3, GM2, and the N-acetylglucosamine-containing ganglioside. The per cent distribution of gangliosides in the TSD and normal SV-40 transformed cell lines was surprisingly similar despite the fact that the TSD transformed cells still lacked hexosaminidase A, the isoenzyme which is required to break down GM2.

Gangliosides in SV-40-transformed cells derived from Tay-Sachs disease fetal brain

A human glial brain cell line derived from a Tay-Sachs disease fetal cerebellum was transformed with SV-40 virus in order to obtain a transformed brain cell line which reflected the characteristics of the disease. It was shown that the transformed TSD cell line maintained an elevated level of GM2 which was similar to that shown by the nontransformed precursor. In addition, the TSD transformed line lacked hexosaminidase A.

Glycolipids in Cultured Fetal Tay-Sachs Disease Cerebellar Cells

Tay-Sachs disease (TSD) is a fatal, genetically determined disorder of sphingoglycolipid metabolism, associated with the absence of the lysosomal enzyme B-D-N-acetylhexosaminidase A (Hex A) (1). There is a massive accumulation of GM2 ganglioside, and its asialo derivative GA2 in cells of the central nervous system. A cell culture which reproduces these biochemical parameters would permit one to measure the effect of enzyme replacement therapy under controlled conditions that are not easily attainable in vivo. Although skin fibroblasts cultured from TSD patients lack Hex A, these cultured cells do not accumulate GM2 ganglioside (2). Since TSD is a neuronal lipid storage disease, and since fetal TSD brain has the characteristic glycolipid patterns found in infant TSD brain, a cell strain from fetal TSD cerebellum was established and the glycolipid patterns were evaluated by TLC and GLC (3). The cells were labelled with 14C-glucosamine in order to compare the metabolic activity of the gangliosides over an extended time period. The cells were also transformed with the oncogenic DNA SV-40 virus (4) since we wished to establish a permanent cell line for the study of this disease.

Enzyme-replacement therapy in cultured tay-sachs disease brain cells

The clinical results of in vivo enzyme-replacement therapy for sphingolipidoses have been uniformly disappointing, especially when the central nervous system is the target organ, as in Tay-Sachs Disease. In vitro enzyme-replacement studies using cultured fibroblasts have serious limitations when applied to Tay-Sachs disease (TSD, McKusick 27280). Whereas TSD fibroblasts lack N-acetyl-13-D-hexosaminidase A (HexA), they do not demonstrate the increased concentration of GM2 ganglioside and the membranous cytoplasmic bodies that are characteristic of the TSD brain. Thus, they are not ideal models for the disease. In contrast, cells cultured from TSD fetal cerebellum contain increased concentrations of GM2 and membranous cytoplasmic bodies. The increased concentrations of GMa are due to lack of degradation (Hoffman et al., 1978). The uptake of lysosomal hydrolases is a receptormediated phenomenon, with the low uptake variety predominating in certain purified preparations (Neufeld and Hickman, 1972). Liposomes have been employed as carriers to increase enzyme uptake, but intracellular uptake may be difficult to achieve. Therefore, for these studies, the plant lectin concanavalin A (Con A) has been used as a ligand to link Hex A to the target-cell surface and to facilitate endocytosis of the enzyme by the cells,