Erythropoietin-mediated neuroprotection by cytokine receptor-like factor 3

细胞因子促红细胞生成素（Epo）在哺乳动物神经系统中通过激活特征不明显的受体介导保护和再生功能，这些受体不同于红祖细胞上的“经典”同型二聚体Epo受体。在脊椎动物（从人类到鱼类）中发现了促生成素基因，这表明促生成素信号的进化早于脊椎动物谱系。除了经典的促红细胞生成素受体外，I型细胞因子受体家族还包括孤儿细胞因子受体样因子3 (CRLF3)，存在于包括人类和各种昆虫在内的脊椎动物中。我们已经证明了CRLF3是甲虫中具有神经保护作用的epo受体。本研究旨在通过回答以下问题来表征CRLF3在昆虫和人类细胞系中的功能：1)CRLF3是否是人类的神经保护epo受体？我们将研究来自不同组织的内源性表达CRLF3的人类细胞系。我们将在这些细胞中过表达和敲低CRLF3。我们将在缺乏或存在Epo或其神经保护（但非促红细胞生成）剪接变体EV-3的情况下对细胞进行缺氧存活试验，并确定CRLF3的表达是否与抑制凋亡和细胞存活相关。D. melanogaster缺乏CRLF3同源基因，Epo对细胞存活的有益作用也不存在。相反，epo刺激的抗凋亡通路的其他成分存在于果蝇中。我们将在果蝇S2-和ML-DmBG2-细胞系中异源表达T. castaneum CRLF3，以产生epo反应细胞。与原代神经元培养相反，细胞系提供了使用光度测定法（例如MTT）来评估细胞培养活力的可能性。我们也将尝试在果蝇细胞系中表达人类CRLF3。如果成功的话，表达crlf3的果蝇细胞系将作为一种快速可靠的方法来筛选潜在的epo模拟物的神经保护特性。3)哪些昆虫组织表达CRLF3，这种细胞因子受体的功能是什么？我们将采用原位杂交的方法测定甘蔗胚、幼虫和舌蛹中CRLF3的表达。此外，我们将在相同的发育阶段采用系统性RNAi来抑制CRLF3的表达。经过rnai处理的甲虫将被分析其外部结构和内部器官的形态学改变以及行为障碍。4)昆虫内源性“类epo”配体是什么？我们将生成并提取缺氧刺激昆虫神经系统的提取物，并在缺氧试验中测试这些提取物的神经保护作用。我们将这些提取物中含有的潜在配体与egfp标记的CRLF3交联。萃取物和交联配合物将与凝胶上的其他蛋白质分离，并进行质谱分析。

The cytokine erythropoietin (Epo) mediates protective and regenerative functions in mammalian nervous systems via activation of poorly characterized receptors that differ from “classic” homodimeric Epo receptors on erythroid progenitor cells. Epo genes have been identified in vertebrates (ranging from human to fish), suggesting that Epo signaling evolved earlier than the vertebrate lineage. Besides the classic erythropoietic Epo receptor, the family of type I cytokine receptors also includes the orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insects. We have demonstrated that CRLF3 is a neuroprotective Epo-receptor in the beetle T. castaneum. The proposed study aims to characterize the function of CRLF3 in insects and human cell lines by answering the following questions:1) Is CRLF3 a neuroprotective Epo-receptor in humans? We will study human cell lines with endogenous CRLF3 expression that derived from different tissues. We will overexpress and knock down CRLF3 in these cells. We will subject the cells to hypoxia-survival assays in the absence or presence of Epo or its neuroprotective (but non-erythropoietic) splice variant EV-3 and determine whether expression of CRLF3 correlates with suppression of apoptosis and cellular survival2) Can heterologous expression of CRLF3 confer Epo-sensitivity to Drosophila? D. melanogaster lacks a CRLF3 orthologue and beneficial effects of Epo on cellular survival are absent. In contrast, other components of Epo-stimulated anti-apoptotic pathways are present in Drosophila. We will heterologously express T. castaneum CRLF3 in Drosophila S2- and ML-DmBG2- cell lines to create Epo-responsive cells. In contrast to primary neuronal cultures, cell lines offer the possibility to use photometric assays (e.g. MTT) for the assessment of cell-culture vitality. We will also attempt to express human CRLF3 in Drosophila cell lines. If successful, CRLF3-expressing Drosophila cell lines will serve as a quick and reliable assay to screen potential Epo-mimetics for neuroprotective properties.3) Which insect tissues express CRLF3 and what is the function of this cytokine receptor? We will determine CRLF3 expression in embryos, larvae and pharate pupae of T. castaneum by in situ hybridisation. In addition, we will subject the same developmental stages to systemic RNAi to suppress CRLF3 expression. RNAi-treated beetles will be analysed for morphological alterations of outer structures and inner organs and for behavioural impairments.4) What is the endogenous “Epo-like” ligand in insects? We will generate and fractionate extracts of hypoxia-stimulated insect nervous systems and test these fractions for neuroprotective effects in hypoxia assays. We will crosslink potential ligands contained in these extracts with eGFP-tagged CRLF3. Extracts and crosslinked complexes will be separated from other proteins on gels and subjected to mass spectroscopy.