fumonisin-b1 and conduritol-epoxide

We have recently demonstrated that incubation of cultured rat hippocampal neurons with conduritol beta-epoxide (CBE), an inhibitor of glucocerebrosidase, the enzyme defective in Gaucher disease, results in changes in intracellular morphology and in functional calcium stores. Changes in levels of functional calcium stores are directly related to neuronal cell death. We now show that neurons incubated with either CBE or a non-hydrolysable analogue of GlcCer (glucosylthioceramide), are more sensitive to the toxic effects of high concentrations of glutamate and of a variety of metabolic inhibitors. A linear relationship exists between level of accumulation of GlcCer and the extent of neuronal cell death. The deleterious effects of elevated GlcCer levels can be completely reversed by addition of human glucocerebrosidase (imiglucerase) to the culture medium. Imiglucerase is internalized to lysosomes, where it presumably degrades excess GlcCer. This suggests that the limited success of enzyme replacement therapy in neuronopathic forms of Gaucher disease is not due to lack of efficacy of glucocerebroside in degrading GlcCer in neurons of the central nervous system, and adds impetus to attempts to develop ways to efficiently deliver glucocerebrosidase to the brains of neurologically compromised Gaucher disease patients.

Topics: Animals; Antimetabolites; Carboxylic Acids; Cell Death; Cells, Cultured; Enzyme Inhibitors; Fumonisins; Gaucher Disease; Glucosylceramidase; Glucosylceramides; Hippocampus; Inositol; Neurons; Neurotoxins; Rats; Rats, Wistar; Recombinant Proteins

Sphingolipids, particularly gangliosides, are enriched in neuronal membranes where they have been implicated as mediators of various regulatory events. We recently provided evidence that sphingolipid synthesis is necessary to maintain neuronal growth by demonstrating that in hippocampal neurons, inhibition of ceramide synthesis by Fumonisin B1 (FB1) disrupted axonal outgrowth (Harel, R. and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481). We now analyze further the relationship between neuronal growth and sphingolipid metabolism by examining the effect of an inhibitor of glucosylceramide synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1- propanol (PDMP) and by examining the effects of both FB1 and PDMP at various stages of neuronal development. No effects of FB1 or PDMP were observed during the first 2 days in culture, but by day 3 axonal morphology was significantly altered, irrespective of the time of addition of the inhibitors to the cultures. Cells incubated with FB1 or PDMP had a shorter axon plexus and less axonal branches. FB1 appeared to cause a retraction of axonal branches between days 2 and 3, although long term incubation had no apparent effect on neuronal morphology or on the segregation of axonal or dendritic proteins. In contrast, incubation of neurons with conduritol B-epoxide, an inhibitor of glucosylceramide degradation, caused an increase in the number of axonal branches and a corresponding increase in the length of the axon plexus. A direct correlation was observed between the number of axonal branch points per cell and the extent of inhibition of either sphingolipid synthesis or degradation. These results suggest that sphingolipids play an important role in the formation or stabilization of axonal branches.

Topics: Animals; Axons; Cells, Cultured; Dendrites; Fumonisins; Hippocampus; In Vitro Techniques; Inositol; Morpholines; Mycotoxins; Neurons; Rats; Rats, Wistar; Sphingolipids