To verify that bdnf dependent survival is not limited

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To verify that BDNF-dependent survival is not limited by the concentration of sphingomyelin, subplate neurons were grown in fumonisin with increasing concentrations of exogenous sphingo- myelin. As shown in Figure 5 c , sphingomyelin does not affect the survival of subplate neurons at concentrations as high as 3 m M but at 10 m M becomes toxic. In this same concentration range, sphin- gomyelin rescue of BDNF-dependent survival is maximal at 0.3 and 1 m M (Fig. 5 c ). Survival at these concentrations is equivalent to the level of BDNF-dependent survival in the absence of fumonisin or exogenous sphingomyelin ( dotted line ). Thus, the sphingomyelin concentration is not limiting for BDNF- dependent survival. In addition, increased sphingomyelin levels do not directly promote subplate neuron survival but are a nec- essary substrate for BDNF-mediated survival, likely through the regulation of sphingomyelinase. These results provide additional evidence that a p75NTR- specific signaling pathway is necessary for BDNF-dependent sur- vival and provide an unusual example in which p75NTR signaling through the sphingolipid pathway supports neuronal survival, not death. DISCUSSION The results of this study show that ligand binding to p75NTR can promote survival, not death, of a neocortical neuronal population via a sphingolipid signaling pathway. This is surprising in the context of well known examples of neuronal death triggered by ligand-dependent p75NTR activation of ceramide signaling. Research into the role of p75NTR in neuronal survival has provided contradictory results depending on the type of neurons examined. Studies of mice carrying a gene deletion of p75NTR reveal that it is necessary for survival of sensory and basal forebrain cholinergic neurons, although, conversely, it is neces- sary for naturally occurring cell death in sympathetic, motor, and retinal neurons (Lee et al., 1992; Bamji et al., 1998; Brennan et al., 1999; Frade and Barde, 1999; Peterson et al., 1999). However, because both neuronal and non-neuronal cells express p75NTR, it is unclear from these studies, as well as studies of unpurified neurons in vitro , whether or not p75NTR functions in a cell- autonomous manner for all of these neuronal populations. Our study is thus novel in that we used a highly purified population of CNS neurons cultured at low density, allowing assessment of the direct role of p75NTR and associated signaling pathways on neuronal survival. p75NTR signaling is required for BDNF-dependent survival Although p75NTR-mediated cell death has been linked to the ceramide signaling pathway (Dobrowsky et al., 1994; Casaccia- Bonnefil et al., 1996), the mechanism of p75NTR-mediated sur- vival is unclear. One model is that p75NTR indirectly promotes survival by facilitating neurotrophin binding to the Trk receptors (Davies et al., 1993; Lee et al., 1994; Chao et al., 1998). The Trk receptor tyrosine kinases, and not p75NTR, then transduce the survival signal. Thus, high concentrations of neurotrophin can support survival even in the absence of p75NTR (Davies et al., 1993; Lee et al., 1994). This mechanism is not consistent with our results in subplate neurons. First, inhibition of Trk phosphoryla-
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  • Spring '10
  • Bier
  • tyrosine kinase, subplate neurons, coactivated Trk, K252a

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