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Neural stem cells depend on insulin-like growth factor I (IGF-I) for differentiation. We analysed how activation and inhibition of the PI 3-kinase-Akt signalling affects the number and differentiation of mouse olfactory bulb stem cells (OBSCs). Stimulation of the pathway with insulin and/or IGF-I, led to an increase in Akt phosphorylated on residues Ser473 and Thr308 (P-Akt(Ser473) and P-Akt(Thr308), respectively) in proliferating OBSCs, and in differentiating cells. Conversely, P-Akt(Ser473) levels decreased by 50% in the OB of embryonic day 16.5-18.5 IGF-I knockout mouse embryos. Overexpression of PTEN, a negative regulator of the PI 3-kinase pathway, caused a reduction in the basal levels of P-Akt(Ser473) and P-Akt(Thr308) and a minor reduction in IGF-I-stimulated P-Akt(Ser473). Although PTEN overexpression decreased the proportion of neurons and astrocytes in the absence of insulin/IGF-I, it did not alter the proliferation or survival of OBSCs. Accordingly, overexpression of a catalytically inactive PTEN mutant promoted OBSCs differentiation. Inhibition of PI 3-kinase by LY294002 produced strong and moderate reductions in IGF-I-stimulated P-Akt(Ser473) and P-Akt(Thr308), respectively. Consequently, LY294002 reduced the proliferation of OBSCs and the number of neurons and astrocytes, and also augmented cell death. These findings indicate that OBSC differentiation is more sensitive to lower basal levels of P-Akt than proliferation or death. By regulating P-Akt levels in opposite ways, IGF-I and PTEN contribute to the fine control of neurogenesis in the olfactory bulb.

Nerve growth factor induces cell death in organotypic cultures of otic vesicle explants. This cell death has a restricted pattern that reproduces the in vivo pattern of apoptosis occurring during inner ear development. In this study, we show that binding of nerve growth factor to its low affinity p75 neurotrophin receptor is essential to achieve the apoptotic response. Blockage of binding to p75 receptor neutralized nerve-growth-factor-induced cell death, as measured by immunoassays detecting the presence of cytosolic oligonucleosomes and by TUNEL assay to visualize DNA fragmentation. Nerve growth factor also induced a number of cell-death-related intracellular events including ceramide generation, caspase activation and poly-(ADP ribose) polymerase cleavage. Again, p75 receptor blockade completely abolished all of these effects. Concerning the intracellular pathway, ceramide increase depended on initiator caspases, whereas its actions depended on both initiator and effector caspases, as shown by using site-specific caspase inhibitors. Conversely, insulin-like growth factor I, which promotes cell growth and survival in the inner ear, abolished apoptosis induced by nerve growth factor. Insulin-like growth factor cytoprotective actions were accomplished, at least in part, by decreasing endogenous ceramide levels and activating Akt. Taken together, these results strongly suggest that regulation of nerve-growth-factor-induced apoptosis in the otocysts occurs via p75 receptor binding and is strictly controlled by the interaction with survival signalling pathways.

Regulation of normal development involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. We have investigated the signalling mechanisms involved in regulation of the balance between cell proliferation and apoptotic cell death in the otic vesicle. The sphingomyelin pathway signals apoptosis for nerve growth factor upon binding to p75 receptors. It is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide. In the present study, we show that nerve growth factor stimulates sphingomyelin hydrolysis and the concomitant ceramide release in organotypic cultures of otic vesicles. Both nerve growth factor and ceramide induce apoptotic responses to a different extent. Ceramide-induced apoptosis was suppressed by insulin-like growth factor-I which is a strong promoter of cell growth and morphogenesis for the developing inner ear. In contrast, ceramide-1-phosphate protected the explants from apoptosis induced by serum withdrawal but did not antagonise ceramide-induced cell death. This study suggests that sphingomyelin-derived second messengers might be key modulators of programmed cell death during development.