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Sandhoff disease is a lysosomal storage disease in which ganglioside GM2 accumulates because of a defective beta-subunit of beta-hexosaminidase. This disease is characterized by neurological manifestations, although the pathogenic mechanisms leading from GM2 accumulation to neuropathology are largely unknown. We now examine the viability, development and rates of neurite growth of embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease, the Hexb-/- mouse. GM2 was detected by metabolic labelling at low levels in wild type (Hexb+/+) neurones, and increased by approximately three-fold in Hexb-/- neurones. Hexb-/- hippocampal neurones were as viable as their wild type counterparts and, moreover, their developmental programme was unaltered because the formation of axons and of the minor processes which eventually become dendrites was similar in Hexb-/- and Hexb+/+ neurones. In contrast, once formed, a striking difference in the rate of axonal and minor process growth was observed, with changes becoming apparent after 3 days in culture and highly significant after 5 days in culture. Analysis of various parameters of axonal growth suggested that a key reason for the decreased rate of axonal growth was because of a decrease in the formation of collateral axonal branches, the major mechanism by which hippocampal axons elongate in culture. Thus, although the developmental programme with respect to axon and minor process formation and the viability of hippocampal neurones are unaltered, a significant decrease occurs in the rate of axonal and minor process growth in Hexb-/- neurones. These results appear to be in contrast to dorsal root ganglion neurones cultured from 1-month-old Sandhoff mice, in which cell survival is impaired but normal outgrowth of neurones occurs. The possible reasons for these differences are discussed.

This work describes the in vivo expression and distribution of glioma-associated gangliosides (GD3, GM2, 3′-isoLM1) in a novel human brain tumour nude rat xenograft model. In this model, the tumours, which are established directly from human glioblastoma biopsies, show extensive infiltrative growth within the rat brain. This model therefore provides an opportunity to study ganglioside expression not only within the macroscopic tumour, but also in brain areas with tumour cell infiltration. The ganglioside expression was studied by confocal microscopy of immunostained brain sections using antiganglioside monoclonal antibodies. Xenografts from four human glioblastoma multiformes were established in rats and the brains removed after 3–4 months. Ganglioside GD3 was expressed in the tumour parenchyma while ganglioside 3′-isoLM1 was more abundantly expressed in the periphery of the tumour associated with areas of tumour cell invasion. GM2 expression was only seen in one tumour, where it was located within the main tumour mass. Double staining with a pan antihuman monoclonal antibody (3B4) and the antiganglioside monoclonal antibodies confirmed that the ganglioside expression was associated with tumour cells. This work supports the concept of different biological roles for individual gangliosides and indicates that antibodies or ligands directed against GD3 and 3′-isoLM1 might be complementary when applied in the treatment of human glioblastomas.

Schwerer B., Lassman H. & Bernheimer H. (1982) Neuropathology and Applied Neurobiology 8, 217–226 Antisera against ganglioside GM2: immunochemical and immunohistological studies Antisera against ganglioside GM2 were raised in rabbits and tested by immunodiffusion, complement fixation and immunohistology. The presence of precipitating antibodies was demonstrated by immunodiffusion techniques (Ouchterlony and reversed Mancini). GM2-antibody titres were determined by a quantitative microcomplement fixation assay. The GM2-antibodies were shown to be specifically directed against GM2 and did not cross-react with the major brain gangliosides. Application of GM2-antiserum to brain sections from a case of GM2-gangliosidosis (Sandhoff s disease) in an immunofluorescence study produced a specific fluorescence of the granular GM2-storage material within the cytoplasm of cortical neurons. With immunoperoxidase staining by electron microscopy, labelling of the surface of the membraneous cytoplasmic bodies was obtained.