The biological activity of synthetic ceramide analogues, having modified sphingoid and N-acyl chains, as well as fluorine substituents in the allylic position, was investigated in hippocampal neurons. Their influence on axonal growth was compared to that of C(6)-N-acyl analogues of natural ceramides. D-erythro-Ceramides with a phenyl group in the sphingoid moiety and a short N-acyl chain were able to reverse the inhibitory effect of fumonisin B(1) (FB(1)), but not of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), on accelerated axonal growth in hippocampal neurons. Moreover, we demonstrated that a ceramide analogue with an aromatic ring in the sphingoid moiety is recognized as a substrate by glucosylceramide synthase, which suggests that the observed biological effects are mediated by activation of the ceramide analogue via glucosylation. Introduction of a methyl, pentyl, fluoro, or methoxy substituent in the para position of the phenyl ring in the sphingoid moiety yielded partly active compounds. Likewise, substitution of the benzene ring for a thienyl group did not abolish the ability to reverse the inhibition of accelerated axonal growth by FB(1). Both D-erythro- and L-threo-ceramide analogues, having an allylic fluorine substituent, partly reversed the FB(1) inhibition.

Changes in the levels and types of gangliosides occur during neuronal differentiation and development, but no studies have correlated these changes with defined events in neuronal morphogenesis. Here, we have analyzed the relationship between ganglioside synthesis and the development of axons and dendrites in polarized neurons, using hippocampal neurons cultured in such a way that axons and dendrites are generated by a defined sequence of events and in which there is virtually no contamination by glial cells. Neurons were labeled with [4,5-3H]dihydrosphingosine, which was rapidly incorporated into cells and metabolized to 3H-labeled glycosphingolipids. The rate of 3H-labeled glycosphingolipid synthesis was directly proportional to the initial rate of [4,5-3H]dihydrosphingosine uptake and was linear versus time for up to 9 h of incubation. The major changes in 3H-labeled ganglioside synthesis occurred during the period of axonogenesis and rapid axon growth. During axonogenesis, there was a significant increase in the synthesis of complex gangliosides (i.e. GM1, GD1a, GD1b, and GT1b) with a corresponding reduction in the synthesis of glucosylceramide and ganglioside GD3. During the stage of rapid axon growth, the ratio of a- to b-series gangliosides increased significantly. However, during dendritogenesis, dendrite growth, and synaptogenesis, there was little change in ganglioside synthesis, with a small and gradual increase in the ratio of a- to b-series gangliosides and an increase in the synthesis of gangliosides GD1a and GT1b. These results indicate that despite major changes in neuronal morphology and functionality as neurons mature, changes in ganglioside synthesis are restricted to early stages of neuronal development, namely axonogenesis and rapid axon elongation.

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Axons of the central nervous system in adult mammals do not regenerate spontaneously after injury, partly because of the presence of oligodendrocytes that inhibit axonal growth. This is not the case in lower vertebrates (e.g., in fish), where regeneration of the optic nerve does occur spontaneously and has been correlated with the presence of factors cytotoxic to oligodendrocytes. The present study provides evidence that the substance originating from the fish optic nerves, which is cytotoxic to oligodendrocytes, is an interleukin 2-like substance.Axons of the central nervous system (CNS) in lower vertebrates regenerate after injury (1-4). Considerable research interest has recently been focused on the oligodendrocytes. It was shown that mature oligodendrocytes create an environment that is nonpermissive for growth (9). Monoclonal antibodies directed against the inhibitory molecules were shown to neutralize the growthinhibitory effect (10). In line with these observations is our finding that the high regenerative capacity of the fish optic nerve is correlated with the presence of a factor or factors cytotoxic to oligodendrocytes (12)(13)(14). This factor(s) was detected among the soluble substances derived from regenerating fish optic nerves, which were collectively termed conditioned medium (CM) and, when applied in vivo, helped to create conditions conducive to axonal growth in other species-e.g., in injured optic nerves of rabbit (15, 16). Recent studies in our laboratory suggest that the cytotoxic factor(s) in the fish might be associated with the postinjury inflammatory reaction-i.e., with the early invasion of injured nerves by blood-derived cells (14). In the present study, the cytotoxic factor was identified as an interleukin 2 (IL-2)-like molecule. MATERIALS AND METHODSPreparation of Soluble Substances Derived from Regenerating Fish Optic Nerves. Carp (Cyprinus carpio, 800-1200 g; Tnuva, Israel) were anesthetized with 0.05% 3-aminobenzoic acid ethyl ester (Sigma), and their optic nerves were crushed with forceps (for 30 sec). Eight days later, the nerves were removed by dissection and incubated in serum-free medium for 1.5 hr (four nerve segments per 300 pLI of medium) at 250C (15). The resulting medium, defined as CM, was then collected, and its protein content was determined by the Bradford assay (17).Preparation of Medium Conditioned by Fish Lymphocytes. Fish were anesthetized with 3-aminobenzoic acid ethyl ester (Sigma). Blood was withdrawn from the venous plexus of the eye orbit and was collected (usually 30 ml from two or three fish) from the socket into a tube containing heparin sulfate (100 units/ml; BDH Chemicals). The blood was diluted with an equal volume of phosphate-buffered saline (PBS) and allowed to stand for 5 min before being layered on top of the Percoll solution [30 ml of diluted blood was carefully layered on top of the solution in a 50-ml sterilized Corex (Corning) centrifuge tube by using a small pipet]. The tubes were capped and centrifuged in swinging buckets at 800 x g ...