Abstract
During the development of the sympathetic nervous system, the p75 neurotrophin receptor (p75NTR) has a dual function: promoting survival together with TrkA in response to NGF, but inducing cell death upon binding pro or mature brain-derived neurotrophic factor (BDNF). Apoptotic signaling through p75NTR requires activation of the stress kinase, JNK. However, the receptor also undergoes regulated proteolysis, first by a metalloprotease, and then by gamma-secretase, in response to pro-apoptotic ligands and this is necessary for receptor mediated neuronal death (Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L., and Carter, B. D. (2006) Neuron 50, 219-232). Hence, the relationship between JNK activation and receptor proteolysis remains to be defined. Here, we report that JNK3 activation is necessary for p75NTR cleavage; however, following release of the intracellular domain, there is a secondary activation of JNK3 that is cleavage dependent. Receptor proteolysis and apoptosis were prevented in sympathetic neurons from jnk3(-/-) mice, while activation of JNK by ectopic expression of MEKK1 induced p75NTR cleavage and cell death. Proteolysis of the receptor was not detected until 6 h after BDNF treatment, suggesting that JNK3 promotes cleavage through a transcriptional mechanism. In support of this hypothesis, BDNF up-regulated tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17 mRNA and protein in wild-type, but not jnk3(-/-) sympathetic neurons. Down-regulation of TACE by RNA interference blocked BDNF-induced p75NTR cleavage and apoptosis, indicating that this metalloprotease is responsible for the initial processing of the receptor. Together, these results demonstrate that p75NTR-mediated activation of JNK3 is required for up-regulation of TACE, which promotes receptor proteolysis, leading to prolonged activation of JNK3 and subsequent apoptosis in sympathetic neurons.
Highlights
In addition to its pro-survival effects in conjunction with TrkA, p75 neurotrophin receptor (p75NTR) can initiate independent signals that lead to axonal degeneration [10] and apoptotic cell death in sympathetic neurons [11]. p75NTR-mediated apoptosis contributes to the normal developmental elimination of these neurons in vivo, as indicated by the fact that p75ntrϪ/Ϫ mice exhibit excess neurons in the superior cervical ganglia (SCG) during the apoptotic period [11, 12]
Activation of Jun N-terminal kinase (JNK) Is Necessary and Sufficient to Induce p75NTR Proteolysis in Sympathetic Neurons—Recent studies from our laboratory revealed that p75NTR-mediated apoptosis in sympathetic neurons requires regulated proteolysis of the receptor [18]
Because previous studies demonstrated that the activation of the stress kinase JNK was necessary for p75NTR to induce cell death [21,22,23,24], we investigated whether these signals are in a common pathway
Summary
P75NTR Activation and Western Blotting—Sympathetic neurons from P4 rats, wild-type, jnk3Ϫ/Ϫ, and traf6Ϫ/Ϫ P4 mice were cultured and maintained in NGF for 48 h, NGF was removed, cells were rinsed, and switched to medium containing anti-NGF (0.1 g/ml, Chemicon, Temecula, CA) together with 12.5 mM KCl, to promote survival, with or without the addition of 200 ng/ml BDNF (generously provided by Regeneron Inc.) for p75NTR activation for various times, as indicated. The cells were harvested, lysed in Nonidet P-40 lysis buffer, and subjected to p75NTR ICD, TACE/ADAM-17, and tubulin Western blotting. In some of the experiments the neurons were treated with 200 ng/ml BDNF as described above, lysed in Nonidet P-40 lysis buffer and subjected to phospho-JNK and total-JNK Western blotting. JNK Assay—Cultured sympathetic neurons from wild-type P4 rats were treated with or without 200 ng/ml BDNF, as described above, and after the indicated time harvested and lysed in Nonidet P-40 lysis buffer. The neurons were kept in medium lacking NGF in the presence of the pan-Caspase inhibitor boc-aspartyl-(OMe)-fluoromethyl ketone (40 M, MP Biomedicals) lysed and subjected to Western blot analysis, as indicated
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