Elsevier

Neuroscience

Volume 94, Issue 2, September 1999, Pages 405-415
Neuroscience

Sphingosine-1-phosphate induces apoptosis of cultured hippocampal neurons that requires protein phosphatases and activator protein-1 complexes

https://doi.org/10.1016/S0306-4522(99)00288-2Get rights and content

Abstract

In this study, we report that mobilization of internal Ca2+ by sphingosine-1-phosphate, a metabolite of ceramide, induces apoptosis in cultured hippocampal neurons. This sphingosine-1-phosphate-induced apoptosis is dependent upon the activation of protein phosphatases, possibly calcineurin and phosphatase 2A (or a related phosphatase). In addition, pretreatment of neurons with double-stranded oligonucleotides containing the metallothionein phorbol-12-myristate-13-acetate response element sequence as transcription factor decoys suppressed apoptosis. In contrast, double-stranded oligonucleotides containing either the c-jun or SV40 phorbol-12-myristate-13-acetate response element sequences were ineffective. Electrophoretic mobility shift assays and supershift assays revealed that c-Fos-containing activator protein-1 complexes preferentially bound the metallothionein phorbol-12-myristate-13-acetate response element sequence-containing oligonucleotides. Furthermore, antisense oligonucleotides to c-fos and c-jun were also protective.

The apoptotic death of hippocampal neurons has been hypothesized to contribute to the cognitive impairments observed following insults to the brain. While increases in intracellular calcium are thought to be key mediators of neuronal apoptosis, the biochemical cascade(s) activated as a result of increased Ca2+ which mediates apoptosis of hippocampal neurons is (are) not well understood. The findings presented in this study suggest that mobilization of internal calcium via prolonged exposure of sphingosine-1-phosphate induces apoptosis of hippocampal neurons in culture. Sustained increases in intracellular calcium activate a phosphatase cascade that includes calcineurin and a phosphatase 2A-like phosphatase, and leads to the expression of genes containing metallothionein phorbol-12-myristate-13-acetate response element (TGAGTCA)-type enhancer sequences. The expression of genes containing TGAGTCA-type enhancer sequences appears to be essential for sphingosine-1-phosphate-induced apoptosis of hippocampal neurons.

Section snippets

Materials

Cell culture media were purchased from Gibco-BRL (Grand Island, NY). SPP, microcysteine-LR, okadaic acid, H7, KN62, staurosporin, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetate acetoxymethyl ester (BAPTA-AM), genestein and PD098059 were purchased from Biomol (Plymouth Meeting, PA). Calpain inhibitors I (N-acetyl-Leu-Leu-norleucinal) and II (N-acetyl-Leu-Leu-methioninal) were purchased from Boehringer Mannheim (Indianapolis, IN). The caspase inhibitor (N-acetyl-YVAD-CMK) was obtained

Exposure of cultured hippocampal neurons to sphingosine-1-phosphate induces apoptosis

SPP is generated by the enzymatic breakdown of the lipid second messenger ceramide (Fig. 1), and has been shown to stimulate the release of Ca2+ from IP3-sensitive stores.18., 31. While the precursor ceramide has been implicated in various forms of apoptosis, SPP is thought to be protective.12., 27., 41. Prolonged exposure of neuronal cultures to 2 μM SPP for 15 h did not cause any obvious morphological changes (Fig. 2a). This is consistent with previous studies using cell lines, which indicated

Discussion

Hippocampal neurons have been shown to be selectively vulnerable to many insults to the CNS. Studies have shown that traumatic brain injury and ischemic insults cause apoptosis (programmed cell death) of hippocampal neurons, possibly via sustained increases in intracellular Ca2+ levels.8., 10., 49., 61. In this report, we have employed a hippocampal culture system to investigate the mechanism of Ca2+-mediated apoptosis. Our results suggest that prolonged exposure of hippocampal neurons to 10 μM

Acknowledgements

We would like to thank Dr Neal Waxham for helpful comments, and Rama Grenda for photographic and artistic expertise. This work was supported by the National Institutes of Health (NS35457, MH49662 to P.K.D.) and the Austrian Science Foundation (FNFP12287MED to A.W.K.).

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