p75 neurotrophin receptor and its novel interaction partner, NIX, are involved in neuronal apoptosis after intracerebral hemorrhage

Recently, NIX, a pro-apoptotic BH3-only protein, was found to be a novel p75 neurotrophin receptor (p75NTR) binding protein by screening a human fetal brain two-hybrid library in our laboratory. We further study the interaction of these two proteins and the possible roles of p75NTR and NIX in intracerebral hemorrhage (ICH)-induced neuronal death. Using the split-ubiquitin yeast two-hybrid system, we found that the “Copper” domain in p75NTR and the TM region in NIX were sufficient for the interaction of these two proteins. Co-immunoprecipitation and in vitro binding assays demonstrated the direct interaction between p75NTR and NIX. NIX protein was stabilized by p75NTR at post-translational levels. Moreover, p75NTR was able to work together with NIX to promote apoptosis and affected the NIX-induced JNK-p53-Bax pathway in neuronal PC12 cells. Previous work has indicated that p75NTR and NIX are induced in neurons in human ICH and the rat ICH model, respectively. We confirm that both p75NTR and NIX levels were up-regulated in glutamate-treated primary cortical neurons (a cellular in vitro model for ICH) and in the rat ICH model. Glutamate exposure increased the association between p75NTR and NIX and elevated the activation of the JNK-p53-Bax pathway and neuronal apoptosis; all of these observations were similar in the rat ICH model. Importantly, p75NTR and NIX appeared to be involved in cortical neuronal apoptosis, because knockdown of p75NTR or NIX not only inhibited the JNK pathway but also impaired neuronal apoptosis. Thus, p75NTR and NIX may play critical roles in ICH-induced neuronal apoptosis in vitro and in vivo.