Characterization of NRAGE, a p75 neurotrophin receptor interacting protein and a mediator of p75 initiated apoptosis

The neurotrophins are a family of growth factors involved in the survival, development, and apoptosis of specific populations of neurons and non-neuronal cells. The pro-apoptotic action of neurotrophins is, at least partially, carried out by the p75 neurotrophin Receptor (p75NTR). However the mechanism employed by p75NTR to mediate apoptosis is poorly defined.We used a yeast two-hybrid screen to find proteins involved in p75NTR apoptotic signaling. This screen led to the identification NRAGE (for neurotrophin receptor-interacting M AGE homologue), as a p75NTR interacting molecule. NRAGE is a novel member of the MAGE family of proteins, a group of molecules with ill-defined function, sharing a ∼200 amino-acid region of homology. We found that NRAGE binds p75NTR in vitro and in vivo, and that the ectopic expression of NRAGE promotes p75NTR-dependent death in cells normally insensitive to p75NTR signaling. Furthermore, physical and functional assays indicated that the interaction of p75NTR with NRAGE, or that of p75NTR with its co-receptor TrkA, are mutually exclusive.To further dissect the mechanism of NRAGE mediated apoptosis, we developed an inducible recombinant NRAGE adenovirus. Induced NRAGE expression resulted in robust activation of the JNK pathway, cytosolic accumulation of Cytochrome c, activation of Caspases-3, -7 and -9 and caspase-dependent cell death. Accordingly, inhibition of the JNK pathway suppressed NRAGE-mediated caspase activation and NRAGE-induced cell death.The well-characterized activation of JNK-dependent cell death by NRAGE or p75NTR was then used as a model system to determine the mechanism-of-action of a novel anti-apoptotic pharmaceutical, AEG3482. Our studies revealed that AEG3482 almost completely blocks JNK-dependent apoptosis induced by p75NTR or NRAGE. The apoptotic inhibitory activity of AEG3482 was attributed to its ability to induce the expression of Heat-Shock Protein-70 (HSP70), which acts as a direct inhibitor of JNK activity. Therefore AEG3482 blocks cell death through an indirect inhbition of JNK. The identical sensitivity of p75NTR- and NRAGE-initiated signaling to AEG3482 or its analogues, provides further support for NRAGE as a mediator of p75NTR-induced apoptotic signaling.In summary, our results show that NRAGE contributes to p75NTR-dependent cell death via JNK-dependent activation of the mitochondrial death pathway