Defreitas et al p75ntr signaling promotes subplate

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DeFreitas et al. p75NTR Signaling Promotes Subplate Neuron Survival J. Neurosci., July 15, 2001, 21 (14):5121–5129 5123
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Signaling through Trk kinases is not required or sufficient for BDNF-dependent survival Because p75NTR binds all three neurotrophins, but NGF by itself is without effect, this data suggests that BDNF-dependent survival of subplate neurons might also require the function of ligand-specific Trk receptors in addition to p75NTR. This hy- pothesis is supported by the observation that subplate neurons express the Trk receptors for both BDNF and NT3 (TrkB and TrkC, respectively) but not the Trk receptor for NGF TrkA (Fig. 1). To assess the role of Trk tyrosine kinases in subplate neurons, K252a, which potently inhibits several tyrosine kinases including the Trks (Berg et al., 1992), was added to subplate neurons in the presence or absence of neurotrophin. Addition of K252a alone increases the background level of subplate neuron survival. A similar increase in survival by K252a has been shown in studies of cultured sensory, striatal, and basal forebrain neurons and likely results from inhibition of other kinases (Borasio, 1990; Glicksman et al., 1995). However, BDNF and NT3 are still able to further augment survival beyond that of K252a alone (Fig. 4 a ). To confirm that Trk activity is completely blocked by K252a, extracts of subplate neurons were probed with an antibody that recognizes all Trks (A, B, and C) phosphorylated at Y490 (Segal et al., 1996; Binder et al., 1999). Because Y490 is autophospho- rylated by Trk tyrosine kinases, its phosphorylation state is indic- ative of Trk activity (Segal et al., 1996). As shown in Figure 4 b , treatment of subplate neurons with BDNF results in the robust phosphorylation of Trk at Y490 compared with control. However, K252a completely inhibits the BDNF-dependent phosphorylation of Y490. It is, however, possible that undetectable Trk kinase activity is enough to activate downstream signaling pathways. The Trk tyrosine kinases activate several signaling cascades, including one leading to phosphorylation and activation of MAPK (Segal and Greenberg, 1996). In some types of neurons, including retinal ganglion cells and sympathetic neurons, Trks mediate survival via the activation of this downstream pathway Figure 2. Immunopurification of subplate neurons. a , p75NTR- immunopanned cells grown in culture 4 d, stained with calcein-AM, and visualized by fluorescence microscopy. b , Birth dating of immunopanned cells ( black bars ) and unbound cells ( white bars ). A BrdU pulse was given at E12 (birth date of subplate neurons) or E15 (birth date of neurons in layers V and VI), and the percentage of cells labeled with BrdU at E17 was determined. c , Percentage of panned cells ( black bars ) and unbound cells ( white bars ) immunostained for Tau, vimentin, nestin, or p75NTR. Results are mean 6 SEM. Figure 3. Neurotrophin-mediated survival of subplate neurons is depen- dent on p75NTR. a , Survival of subplate neurons in the absence or presence of 30 ng/ml exogenous BDNF. Anti-p75NTR FAbs ( black bars ) or normal rabbit FAbs ( white bars ) were added and compared with survival in the absence of FAbs ( shaded bars ). * p , 0.01 compared with BDNF with no FAbs.
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