First inhibition of trk phosphoryla tion with k252a

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results in subplate neurons. First, inhibition of Trk phosphoryla- tion with K252a does not inhibit BDNF-dependent survival. Also, the concentration of BDNF used in conjunction with p75NTR FAbs was 30-fold higher than that required to stimulate Trk-dependent survival maximally in the absence of p75NTR (Davies et al., 1993; Lee et al., 1994). Finally, excess NGF, which competes for binding to p75NTR but not TrkB, blocks BDNF- dependent survival but not activation of the Trk-dependent MAPK signaling cascade. Thus, our data strongly support an alternative model that p75NTR directly signals survival in sub- plate neurons. A role for p75NTR signaling is further supported by the requirement for sphingolipid synthesis in BDNF-dependent sub- plate survival. Ligand binding to p75NTR, but not Trk receptors, activates sphingomyelinase, which converts sphingomyelin to cer- amide (Dobrowsky et al., 1995). Ceramide in turn acts as a second Figure 5. Sphingomyelin synthesis is necessary for p75NTR-dependent survival. a , Subplate neurons were cultured with BDNF ( black bars ) in the presence of fumonisin B1 ( white bars ) or myriocin ( hatched bars ). Survival is normalized to that in the absence of additives. * p , 0.01 compared with BDNF alone. b , Survival of subplate neurons grown in the presence of BDNF ( B ), fumonisin B1 ( F ), or sphingomyelin ( SM ) in combinations indicated. * p , 0.01 compared with control survival; t test. c , Dose– response curve for sphingomyelin rescue of BDNF-dependent survival. Subplate neurons were grown in the presence of fumonisin and increasing concentrations of sphingomyelin with BDNF ( black squares ) or without BDNF ( white squares ). Dashed line represents survival in BDNF alone. 5126 J. Neurosci., July 15, 2001, 21 (14):5121–5129 DeFreitas et al. p75NTR Signaling Promotes Subplate Neuron Survival
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messenger that activates apoptotic pathways in some cell types (Casaccia-Bonnefil et al., 1996; Hannun, 1996). In cells that express both p75NTR and a Trk, coactivation of the high-affinity Trk receptors has been found to suppress p75NTR activation of the ceramide pathway and apoptosis (Dobrowsky et al., 1995; Bamji et al., 1998; Davey and Davies, 1998; Yoon et al., 1998). By this model, the p75NTR apoptotic pathway is only activated by neurotrophins when the Trk receptor tyrosine kinase is not coactivated. This model, derived from studies of sensory and sympathetic neurons as well as non-neuronal cells, contrasts with our results in subplate neurons in several important respects. First, inhibition of ceramide synthesis prevents BDNF-dependent survival rather than increasing survival, as would be predicted if ceramide me- diates cell death. Second, BDNF-dependent survival is rescued by sphingomyelin, the precursor to ceramide. This indicates that p75NTR regulation of sphingomyelinase is a necessary compo- nent of BDNF-dependent survival signaling pathway in subplate neurons. This data also suggest that p75NTR promotes survival, not death, by increasing production of ceramide. Finally, because BDNF activates signaling through a Trk kinase (likely TrkB) in subplate neurons (Fig. 4), p75NTR regulation of the ceramide pathway does not appear to be suppressed by a coactivated Trk.
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