Neuroprotective functions of the orphan cytokine receptor CRLF3 – a study in human stem cell-derived neurons

细胞因子促红细胞生成素(EPO)通过激活与红系祖细胞上典型的同源二聚体EPO受体不同的特性不佳的受体来介导哺乳动物神经系统的保护和再生功能。已经在脊椎动物(从人到鱼)中发现了EPO基因，这表明EPO信号的进化早于脊椎动物的谱系。除了经典的促红细胞生成素受体，I型细胞因子受体家族还包括进化上保守的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。我们已经在甲虫和蝗虫身上证明了CRLF3是一种神经保护性的EPO受体。此外，几种保护哺乳动物神经元但不激活“经典”同源二聚体EPO受体的配体激活昆虫CRLF3，这表明昆虫CRLF3与未知的哺乳动物EPO神经保护受体结构相似。这项拟议的研究旨在证明人类CRLF3(与其昆虫同源基因一样)是一种具有神经保护作用的EPO受体，具有治疗退行性疾病的临床潜力。我们将使用CRISPR/Cas9系统在两个独立的人诱导多能干细胞(HiPSC)系中突变CRLF3。野生型和CRLF3突变的HiPSC将分化为神经元。我们将在EV-3存在和不存在的情况下诱导这些神经元的凋亡，EV-3是哺乳动物组织保护性EPO受体类型的特异性激活剂，也可以诱导CRLF3介导的昆虫神经元保护。通过比较EV-3对野生型和CRLF3敲除(KO)神经元的抗凋亡作用，我们将确定人CRLF3是否代表人类EPO的组织保护性受体。我们将使用带有潜在相关信号因子抑制剂的存活试验来识别CRLF3启动的细胞内转导通路。最有可能的是，Janus Kinase/STAT将参与其中，但PI3K、MAPK和NFB的作用也将得到测试，这些信号与Epo刺激的哺乳动物神经保护机制有关。利用EPO和EPO类似物的神经保护机制，在不激活CRLF3的情况下激活CRLF3，而不会激活不必要的造血或肿瘤副作用，将扩大神经退行性疾病和精神疾病的治疗选择。我们目前正在鉴定昆虫CRLF3的内源配体。如果成功，我们将搜索人类昆虫配体的同源物的数据库，并使用hiPSC衍生的神经元的存活实验来测试这些细胞因子是否也可以激活人类CRLF3。由于HiPSC主要可以分化为任何体细胞类型，所以CRLF3在人类其他组织和器官中的有益功能可以很容易地在体外条件下进行研究。

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 “classical” erythropoietic Epo receptor, the family of type I cytokine receptors also includes the phylogenetically conserved orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including humans and various insects. We have demonstrated in the beetle T. castaneum and the locust L. migratoria that CRLF3 is a neuroprotective Epo-receptor. In addition, several ligands that protect mammalian neurons but do not activate “classical” homodimeric Epo receptor activate insect CRLF3, indicating structural similarities between insect CRLF3 and unknown mammalian neuroprotective receptors for Epo. The proposed study aims to demonstrate that human CRLF3 (like its insect ortholog) is a neuroprotective Epo receptor with clinical potential for treatment of degenerative diseases. We will use the CRISPR/Cas9 system to mutate CRLF3 in two independent lines of human induced pluripotent stem cells (hiPSC). Wild type and CRLF3-mutated hiPSC will be differentiated into neurons. We will induce apoptosis in these neurons in the presence and absence of EV-3, a specific activator of mammalian tissue-protective Epo-receptor types that also induces CRLF3-mediated protection of insect neurons. By comparing anti-apoptotic effects of EV-3 on wild type and CRLF3-knock out (KO) neurons, we will determine whether human CRLF3 represents a tissue-protective receptor for Epo in humans. We will use the survival assay with inhibitors of potentially involved signaling factors to identify CRLF3-initiated intracellular transduction pathways. Most likely, janus kinase /STAT will be involved but contribution of Pi3K, MAPK and NFB, which have been implicated in Epo-stimulated neuroprotective mechanisms in mammals, will also be tested. Exploiting the neuroprotective mechanisms of Epo and Epo-mimetics by activation of CRLF3 without activation of unwanted hematopoietic or tumorigenic side effects, would expand treatment options for neurodegenerative and psychiatric diseases. We are currently in the process of identifying the endogenous ligand of insect CRLF3. If successful, we will search data bases for human orthologs of the insect ligand and use the survival assay with hiPSC-derived neurons to test whether these cytokines may also activate human CRLF3. Since hiPSC can principally be differentiated into any somatic cell type, beneficial functions of CRLF3 in other human tissues and organs can easily be studied under controlled in vitro conditions.