Hahn Nina, Knorr Debbra Y, Liebig Johannes, Wüstefeld Liane, Peters Karsten, Büscher Marita, Bucher Gregor, Ehrenreich Hannelore, Heinrich Ralf
Department of Cellular Neurobiology, Institute for Zoology, Georg-August-University GoettingenGoettingen, Germany.
Clinical Neuroscience, Max Planck Institute of Experimental MedicineGoettingen, Germany.
Front Mol Neurosci. 2017 Jul 14;10:223. doi: 10.3389/fnmol.2017.00223. eCollection 2017.
The cytokine erythropoietin (Epo) mediates various cell homeostatic responses to environmental challenges and pathological insults. While stimulation of vertebrate erythrocyte production is mediated by homodimeric "classical" Epo receptors, alternative receptors are involved in neuroprotection. However, their identity remains enigmatic due to complex cytokine ligand and receptor interactions and conflicting experimental results. Besides the classical Epo receptor, the family of type I cytokine receptors also includes the poorly characterized orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insect species. By making use of the more simple genetic makeup of insect model systems, we studied whether CRLF3 is a neuroprotective Epo receptor in animals. We identified a single ortholog of CRLF3 in the beetle , and established protocols for primary neuronal cell cultures from brains and efficient RNA interference. Recombinant human Epo as well as the non-erythropoietic Epo splice variant EV-3 increased the survival of serum-deprived brain neurons, confirming the previously described neuroprotective effect of Epo in insects. Moreover, Epo completely prevented hypoxia-induced apoptotic cell death of primary neuronal cultures. Knockdown of CRLF3 expression by RNA interference with two different double stranded RNA (dsRNA) fragments abolished the neuroprotective effect of Epo, indicating that CRLF3 is a crucial component of the insect Epo-responsive receptor. This suggests that a common urbilaterian ancestor of the orphan human and insect cytokine receptor CRLF3 served as a neuroprotective receptor for an Epo-like cytokine. Our work also suggests that vertebrate CRLF3, like its insect ortholog, might represent a tissue protection-mediating receptor.
细胞因子促红细胞生成素（Epo）介导各种细胞对环境挑战和病理损伤的稳态反应。虽然脊椎动物红细胞生成的刺激是由同二聚体“经典”Epo受体介导的，但其他受体参与神经保护作用。然而，由于复杂的细胞因子配体和受体相互作用以及相互矛盾的实验结果，它们的身份仍然成谜。除了经典的Epo受体外，I型细胞因子受体家族还包括在包括人类和各种昆虫物种在内的脊椎动物中特征不明确的孤儿细胞因子受体样因子3（CRLF3）。通过利用昆虫模型系统更简单的基因组成，我们研究了CRLF3是否是动物中的神经保护性Epo受体。我们在甲虫中鉴定出CRLF3的单个直系同源物，并建立了从大脑中进行原代神经元细胞培养和高效RNA干扰的方案。重组人Epo以及非促红细胞生成的Epo剪接变体EV-3提高了血清剥夺的脑神经元的存活率，证实了先前描述的Epo在昆虫中的神经保护作用。此外，Epo完全预防了原代神经元培养物中缺氧诱导的凋亡细胞死亡。用两种不同的双链RNA（dsRNA）片段通过RNA干扰敲低CRLF3表达消除了Epo的神经保护作用，表明CRLF3是昆虫Epo反应性受体的关键组成部分。这表明孤儿人类和昆虫细胞因子受体CRLF3的共同双侧对称祖先作为一种类Epo细胞因子的神经保护受体。我们的工作还表明，脊椎动物的CRLF3与其昆虫直系同源物一样，可能代表一种介导组织保护的受体。
