Ceramide Induces Apoptosis in Cultured Mesencephalic Neurons

Abstract

The death of dopaminergic and other neurons in primary cultures of the mesencephalon could be induced by treatment with ceramide, as in lymphocytes where it mediates activation by the cytokines tumor necrosis factor-a and interleukin-1 /3 of a novel sphingomyelin-dependent signaling pathway leading to apoptosis. The morphological hallmarks of this form of cell deathbleb formation, cell body shrinkage, nuclear chromatin condensation, and fragmentation-were observed in degenerating neurons. Internucleosomal DNA degradation could also be evidenced by gel electrophoresis. The 02 and 06 analogues as well as native ceramide, administered in a dodecane suspension, had a similar effect, whereas the closely related C 2-dihydroceramide, which lacks the 4-5 trans double bond in the sphingosine chain, failed to induce apoptosis. Neuronal death could be delayed by serum factors, dibutyryl cyclic AMP, and the protein synthesis inhibitor cycloheximide. Key Words: Programmed cell death-Sphingomyelin signal transduction-Parkinson's disease.

C-jun N-terminal kinases/c-Jun and p38 pathways cooperate in ceramide-induced neuronal apoptosis

“…The specificity of c 2 -ceramide was evaluated by comparing its effects with those of c 2 -dihydro-ceramide (Biomol Research Laboratory, Plymouth Meeting, PA, USA), an analog lacking the 4 -5 trans double bond in the sphingosine moiety of c 2 -ceramide, which has been shown to be incapable of activating the sphingomyelin pathway in lymphocytes (Bielawska et al, 1993;Obeid et al, 1993) and neurons (Brugg et al, 1996).…”

“…The cortex was dissected, mechanically dissociated and plated on polyethyleneimine (1 mg/ml) coated culture wells, in Eagle's basal medium (Eurobio, Les Ulis, France), supplemented with 5% horse serum (HS; Eurobio) and 2.5% fetal calf serum (FCS; Eurobio), at a density of 7.10 5 cells/cm 2 . After 2 days in culture the media was replaced with N5 medium (Kawamoto and Barrett, 1986) with 180mg/l glucose and changed daily; FCS content was reduced to 1%; Ara-C (cytosine arabinoside, 3 M, Sigma, Saint Louis, MO, USA) was added to prevent astrocyte proliferation (our cultures were at least 95% neuronal after this treatment), and MK-801 (1 M, Research Biochemicals International, Natick, MA, USA) to prevent excitotoxicity (Knusel et al, 1990;Brugg et al, 1996). All experiments conformed to French and international guidelines on the ethical use of animals.…”

“…That is why we reduced serum content to 1% HS, 24 h before any ceramide treatment (Brugg et al, 1996). Thus, the effects seen were not due to partial serum deprivation but rather to specific treatment, since control cells were in the same serum conditions during the same time as the treated cells.…”

“…Secondly, increased ceramide levels are observed during apoptosis induced by growth factor withdrawal in PC12 cells (Lambeng et al, 1999) and after lethal ischemia in the gerbil hippocampus (Nakane et al, 2000). Furthermore, apoptosis can be induced by exogenous ceramide treatment of PC12 cells (France-Lanord et al, 1997;Hartfield et al, 1997), mesencephalic (Brugg et al, 1996), hippocampal (Mitoma et al, 1998) or cortical neurons (Willaime et al, 2001).…”

IGF-I protects cortical neurons against ceramide-induced apoptosis via activation of the PI-3K/Akt and ERK pathways; is this protection independent of CREB and Bcl-2?

“…Increased ceramide levels are observed during apoptosis induced by growth factor-withdrawal in neuronally differentiated PC12 cells [32] or cortical neurons [62] and after ischemia in the gerbil hippocampus [41]. Furthermore, apoptosis can be induced by exogenous ceramide treatment of PC12 cells [20,24], mesencephalic [7], hippocampal [40] or cortical neurons [60]. Recently, we have shown that ceramide regulates pro-apoptotic MAP kinase pathways and that JNK and p38 activation is crucial in ceramide-induced neuronal apoptosis in primary cortical neurons [60,61].…”