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

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Abstract

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.

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Acknowledgments

We thank Prof. Aiguo Shen for his advice and invaluable help with this paper.

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    Authors and Affiliations

    1. Department of Neurology, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, People’s Republic of China

      Jiabing Shen & Kaifu Ke

    2. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, 2005 Songhu Rd., Shanghai, 200438, People’s Republic of China

      Xiaomei Chen, Hongmei Li, Yang Wang & Keke Huo

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    1. Jiabing Shen
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    2. Xiaomei Chen
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    Correspondence to Keke Huo or Kaifu Ke.

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    Additional information

    Jiabing Shen and Xiaomei Chen contributed equally to this work.

    This work is supported by the National Key Basic Research Program of China (2103CB531603) to Keke Huo and by the National Natural Science Foundation of China (nos. 81371299, 81671135) to Kaifu Ke.

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    sFig. 1

    Split-ubiquitin yeast two-hybrid system used to screen potential interacting partners of p75NTR. a Yeast two-hybrid screening system control. For detection of the interaction between p75NTR and NIX, yeast NMY32 were transformed with plasmids as indicated in b–e and then the transformants were screened on SD-Trp-Leu (SD-2) and SD-Trp-Leu-His-Ade+5 mM 3-AT (SD-4) medium containing X-gal. (GIF 144 kb)

    High resolution image (TIF 12790 kb)

    sFig. 2

    a, b Overexpression of p75NTR (a) or NIX (b) induces an increase in the p-JNK level in vivo. HEK293T cells were transiently transfected with increasing amounts of FLAG-tagged p75NTR or MYC-tagged NIX expressing plasmid (1 μg, 2 μg, and 4 μg) in 60-mm dishes. After 30 h transfection, cells were harvested and the protein lysates were analyzed by Western blot for FLAG-tagged p75NTR and p-JNK (a) or MYC-tagged NIX and p-JNK (b). c Neuronal apoptosis-associated markers are increased in primary cortical neurons after glutamate exposure. Samples were prepared from cortical cultures exposed to 150 μM glutamate for various times and Western blot was performed to test the levels of PCNA and Cl caspase 3. NeuN was used as a loading control. The relative protein levels for PCNA and Cl caspase 3 (c’) were calculated by densitometric analysis, and the readings were normalized to that of the corresponding NeuN. Each value is the mean ± SD from three independent experiments. *,# P < 0.05 indicates significant differences. (GIF 50 kb)

    High resolution image (TIF 6885 kb)

    sFig. 3

    Assessments and scores of behavioral tests on rats after ICH. The neurological function damage of rats used in this study was assessed by forelimb placing tests (a) and corner turn tests (b). Compared with the sham group, the ICH groups showed remarkable functional deficits. *P < 0.05. (GIF 15 kb)

    High resolution image (TIF 3015 kb)

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    Shen, J., Chen, X., Li, H. et al. p75 neurotrophin receptor and its novel interaction partner, NIX, are involved in neuronal apoptosis after intracerebral hemorrhage. Cell Tissue Res 368, 13–27 (2017). https://doi.org/10.1007/s00441-016-2510-y

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