Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient mice were used to study the toxicity of this potent sphingolipid metabolite in terminally differentiated postmitotic neurons. Based on earlier findings with the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate, we hypothesized that accumulation of S1P above a certain threshold induces neuronal apoptosis. The present studies confirmed this conclusion and further revealed that for S1P to induce apoptosis in lyase-deficient neurons it must also be produced by sphingosine-kinase2 (SK2). These conclusions are based on the finding that incubation of lyase-deficient neurons with either sphingosine or S1P results in a similar elevation in cellular S1P; however, only S1P addition to the culture medium induces apoptosis. This was not due to S1P acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was phosphorylated by the cells, as described before for cis-4-methylsphingosine. Although the cells produced S1P from both exogenously added sphingosine as well as sphingosine derived from exogenous S1P, the S1P from these two sources were not equivalent, because the former was primarily produced by SK1, whereas the latter was mainly formed by SK2 (as also was cis-4-methylsphingosine phosphate), based on studies in neurons lacking SK1 or SK2 activity. Thus, these investigations show that, due to the existence of at least two functionally distinct intracellular origins for S1P, exogenous S1P can be neurotoxic. In this model, S1P accumulated due to a defective lyase, however, this cause of toxicity might also be important in other cases, as illustrated by the neurotoxicity of cis-4-methylsphingosine phosphate.Sphingosine 1-phosphate (S1P) 2 is a potent lipid mediator that has been shown to regulate a wide range of physiological processes, including proliferation, differentiation, motility, cytoskeleton rearrangements, and calcium homeostasis (1, 2). There is convincing experimental evidence that this bioactive sphingolipid can act both extracellularly, as a ligand for a family of five specific G protein-coupled receptors, and inside the cells, as a second messenger (3, 4). In most cell types described so far, S1P and its metabolic precursor ceramide exert antagonistic effects on cell survival with S1P being generally regarded as a survival signal, whereas ceramide and sphingosine are generally toxic (5, 6). Interestingly, generation of sphingosine and S1P is generally thought to be dependent on the availability of ceramide (7), however, relatively high amounts of S1P are also present in blood, lymph, and cerebrospinal fluid (8, 9) and may serve as additional sources for some cells.More than a decade ago, we introduced the synthetic sphingosine analog cis-4-methylsphingosine as a tool for studies of sphingoid base metabolism and function (10). When added to the culture medium, this analog is taken up and mainly phosphorylated to the respective cis-4-methylsphingosine phosphate, which accumulates intracellularly, because it ...

We have recently reported that the bioactive lipid sphingosine-1-phosphate (S1P), usually signaling proliferation and anti-apoptosis induces neuronal death when generated by sphingosine-kinase2 and when accumulation due to S1P-lyase deficiency occurs. In the present study, we identify the signaling cascade involved in the neurotoxic effect of sphingoid-base phosphates. We demonstrate that the calcium-dependent cysteine protease calpain mediates neurotoxicity by induction of the endoplasmic reticulum stress-specific caspase cascade and activation of cyclin-dependent kinase5 (CDK5). The latter is involved in an abortive reactivation of the cell cycle and also enhances tau phosphorylation. Neuroanatomical studies in the cerebellum document for the first time that indeed neurons with abundant S1P-lyase expression are those, which degenerate first in S1P-lyase-deficient mice. We therefore propose that an impaired metabolism of glycosphingolipids, which are prevalent in the central nervous system, might be linked via S1P, their common catabolic intermediate, to neuronal death.

As shown before in three different cell types, cis -4-methylsphingosine is a synthetic, membrane permeable, pro-drug, that is taken up by cells and phosphorylated to a metabolically stable cis -4-methylsphingosine-phosphate. The synthetic compound mimicked the mitogenic effect of sphingosine-1-phosphate (S1P) in Swiss 3T3 fibroblasts, but induced apoptosis in B104 neuroblastoma cells. We now investigated its effect in differentiated primary cultured neurons. In contrast to S1P, which had no effect on growth of these postmitotic cells, cis -4-methylsphingosine-phosphate induced apoptosis. Interestingly, both compounds stimulated extracellular regulated kinase (ERK) and also p38 mitogen-activated protein kinase (MAPK). Additionally, both compounds induced an increased expression of cyclin D1 but not of cyclin E. Our results document that the different physiological effects, apoptosis in the case of the accumulating metabolically stable synthetic compound vs. no apoptosis in the case of the short-living S1P, rely only on nuances of impact. In other words both sphingoid phosphates affect similar pathways albeit in a sustained and more pronounced manner in case of the metabolically stable synthetic compound. Experiments with several pharmacological inhibitors indicate that cis -4-methylsphingosine-phosphate-induced neuronal apoptosis is mediated on the one hand by a caspase dependent and p38 MAPK forwarded pathway and on the other hand by an abortive reactivation of the cell cycle, a caspase independent process.