VHL, NF-1, c-Ret, and Succinate Dehydrogenase Subunits B and D act on a developmental apoptotic pathway that is activated when nerve growth factor (NGF) becomes limiting for neuronal progenitor cells and requires the EglN3 prolyl hydroxylase as a downstream effector. Germline mutations of these genes cause familial pheochromocytoma and other neural crest-derived tumors. Using an unbiased shRNA screen we found that the kinesin KIF1B acts downstream from EglN3 and is both necessary and sufficient for neuronal apoptosis when NGF becomes limiting. KIF1B maps to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors including neuroblastomas. We identified inherited loss-of-function KIF1B missense mutations in neuroblastomas and pheochromocytomas and an acquired loss-of-function mutation in a medulloblastoma, arguing that KIF1B is a pathogenic target of these deletions.[Keywords: Apoptosis; kinesin; neuroblastoma; pheochromocytoma; prolyl hydroxylase] Supplemental material is available at http://www.genesdev.org.
The p75 neurotrophin receptor regulates neuronal survival, promoting it in some contexts yet activating apoptosis in others. The mechanism by which the receptor elicits these differential effects is poorly understood. Here, we demonstrate that p75 is cleaved by gamma-secretase in sympathetic neurons, specifically in response to proapoptotic ligands. This cleavage resulted in ubiquitination and subsequent nuclear translocation of NRIF, a DNA binding protein essential for p75-mediated apoptosis. Inhibition of gamma-secretase or expression of a mutant p75 resistant to this protease prevented receptor proteolysis, blocked NRIF nuclear entry, and prevented apoptosis. In contrast, overexpression of the p75 ICD resulted in NRIF nuclear accumulation and apoptosis. The receptor proteolysis and NRIF nuclear localization were also observed in vivo during naturally occurring cell death in the superior cervical ganglia. These results indicate that p75-mediated apoptosis requires gamma-secretase dependent release of its ICD, which facilitates nuclear translocation of NRIF.
SUMMARY Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Recent structural studies have shown that neurotrophins interact with dimers of the p75 neurotrophin receptor (p75NTR), but the actual mechanism of receptor activation has remained elusive. Here we show that p75NTR forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys257 in its transmembrane domain. Mutation of Cys257 abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75NTR/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75NTR. FRET experiments revealed a close association of p75NTR intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys257 did not alter the oligomeric state of p75NTR, the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75NTR by a novel mechanism involving rearrangement of disulphide-linked receptor subunits.
Seizure-induced damage elicits a loss of hippocampal neurons mediated to a great extent by the p75 neurotrophin receptor (NTR).Proneurotrophins, which are potent apoptosis-inducing ligands for p75 NTR , were increased in the hippocampus, particularly in astrocytes, by pilocarpine-induced seizures; and infusion of anti-pro-NGF dramatically attenuated neuronal loss after seizures. The p75 NTR is expressed in many different cell types in the nervous system, and can mediate a variety of different cellular functions by recruiting specific intracellular binding proteins to activate distinct signaling pathways. In this study, we demonstrate that neurotrophin receptorinteracting factor (NRIF) mediates apoptotic signaling via p75 NTR in hippocampal neurons in vitro and in vivo. After seizure-induced injury, NRIF ؊/؊ mice showed an increase in p75 NTR expression in the hippocampus; however, these neurons failed to undergo apoptosis in contrast to wild-type mice. Treatment of cultured hippocampal neurons with proneurotrophins induced association of NRIF with p75 NTR and subsequent translocation of NRIF to the nucleus, which was dependent on cleavage of the receptor. Neurons lacking NRIF were resistant to p75 NTR -mediated apoptosis in vitro and in vivo. In addition, we demonstrate some mechanistic differences in p75 NTR signaling in hippocampal neurons compared with other cell types. Overall, these studies demonstrate the requirement for NRIF to signal p75 NTR -mediated apoptosis of hippocampal neurons and that blocking pro-NGF can inhibit neuronal loss after seizures.
NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGEdeficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo. Cell Death and Differentiation (2008) 15, 1921-1929 doi:10.1038/cdd.2008; published online 5 September 2008The p75 neurotrophin receptor (p75NTR) is a TNF receptor superfamily member that is expressed at high levels during embryogenesis and functions as a pro-apoptotic receptor during the development of neuronal and non-neuronal tissues.
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 ␥-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-␣-converting enzyme (TACE)/ADAM17 mRNA and protein in wildtype, 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.
TRAF6 is an E3 ubiquitin ligase that mediates signaling from members of the tumor necrosis factor and Toll-like receptor superfamilies, including the p75 neurotrophin receptor. Recently, TRAF6 was shown to bind to another p75 cytoplasmic interactor, NRIF, and promote its nuclear localization. Here, we demonstrate that NRIF is a substrate for TRAF6-mediated K63 polyubiquitination and that this modification is necessary for its nuclear translocation. Activation of p75 resulted in NRIF polyubiquitination, association with TRAF6 and nuclear localization. NRIF was polyubiquitinated by TRAF6 in vitro and in cultured cells, and this was abrogated by mutation of K19 in the amino-terminus of NRIF. The K19R mutant NRIF displayed reduced TRAF6 association and neurotrophin-dependent nuclear localization. In neurons from traf6À/À mice, NRIF failed to enter the nucleus in response to p75 activation, and polyubiquitination and nuclear localization were attenuated in traf6À/À brain. Finally, unlike wild-type NRIF, the K19R NRIF failed to reconstitute p75-mediated apoptosis in nrifÀ/À neurons. These results reveal a unique mechanism of p75 signaling and a novel role for K63-linked ubiquitin chains.
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