Nerve Growth Factor-induced p75-mediated Death of Cultured Hippocampal Neurons Is Age-dependent and Transduced through Ceramide Generated by Neutral Sphingomyelinase

Abstract

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75 exonIII؊/؊ mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase ؊/؊ mice were as susceptible to NGF-induced death as wild type neurons. The p75 neurotrophin receptor (p75) 1 is the shared receptor for all four mammalian neurotrophins (1) as well as other unrelated ligands (2-6). It is expressed in a wide range of neuronal and non-neuronal cells (7,8), with a corresponding diversity of roles attributed throughout development and in the adult (9 -11). In addition to enhancing responsiveness of cells co-expressing p75 and Trk receptors (12), p75 has in recent years been established as a signaling receptor in its own right (13,14). Independent signaling of p75 has been reported to modulate many aspects of neuronal physiology including sensory functions (15), axon outgrowth (16 -18), and survival or apoptotic effects of neurotrophins (19 -24). Typically, the diverse effects observed after p75 activation are explained by "cell context," which may involve the differential activation of a number of intracellular signaling pathways, including NFB translocation (25), Jun kinase phosphorylation (26), and ceramide generation (27).Ceramide is a lipid second messenger implicated in diverse intracellular pathways, most prominently those regulating cell death in assorted cell types (28 -30). A plethora of studies have looked at the effects of exogenously added ceramide analogues on cultured cells, and in cultured neurons both outgrowth and survival/death effects have been reported (31-33). However, less attention has been paid to endogenous ceramide generation in neurons. Endogenous ceramide can be generated by hydrolysis of sphingomyelin (SM) or by de novo synthesis, processes that occur at different intracellular locations, and by different modes of regulation (29). Ceramide generated by SM hydrolysis can be produced by either neutral or acid sphingomyelinases (N-SMase or A-SMase, respectively) (30), which may be differentially distributed between the cell body and the axon (34).A prominent example of an endogenous signaling system that generates ceramide in the nervous system is p75 (27, 35). Although an early report (26) suggested a role for ceramide in p75-mediated cell death of oligodendrocytes, subsequent s...

Mitochondrial Superoxide Production and Nuclear Factor Erythroid 2-Related Factor 2 Activation in p75 Neurotrophin Receptor-Induced Motor Neuron Apoptosis

“…As observed for other apoptotic stimuli, including trophic factor deprivation and Fas (Estévez et al 1998;Raoul et al, 2002), the mechanism of p75 NTR -induced apoptosis in motor neurons involves downstream production of nitric oxide and peroxynitrite formation (Pehar et al, 2004). In the present study, we found that the p75 NTR apoptotic pathway in motor neurons involved increased ceramide production and cytochrome c release into the cytosol, as observed in other cell types (Casaccia-Bonnefil et al, 1996;Kuner and Hertel, 1998;Brann et al, 2002;Bhakar et al, 2003). p75…”

Nerve Growth Factor-induced Glutamate Release Is via p75 Receptor, Ceramide, and Ca2+ from Ryanodine Receptor in Developing Cerebellar Neurons

“…To investigate this, we examined the involvement of ceramide, because the generation of ceramide following the activation of p75 regulates many aspects of neuronal response, including cell survival and apoptotic cell death by neurotrophins (24,28). Blochl and Sirrenberg (33) suggested the possibility that NGF affected dopamine release through the sphingomyelin pathway.…”

“…For example, ceramide, which is produced by sphingomyelinase, is involved in NGF-mediated neuronal cell death (24,28). Therefore, we attempted to clarify whether ceramide production was involved in the NGF-induced glutamate release.…”

“…Endogenous ceramide can be generated by the hydrolysis of sphingomyelin stimulated by neurotrophins via activation of p75 (20) and is known to be a second messenger that works in diverse intracellular pathways, with effects on neuronal outgrowth/survival and death (27,28). Several reports (29 -31) indicated that the NGF-selective binding of p75 resulted in the promotion of survival and protection from apoptosis.…”

Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

“…High levels of p75 NTR expression can induce apoptosis when there are low levels of Trk or when Trk is absent (10,11). Apoptosis occurs through increased ceramide production (12), activation of c-Jun N-terminal kinase (JNK1), and p53 (10,13). p75 NTR requires a co-receptor called sortilin to induce cell death (14).…”