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的“经典”同型二聚体Epo受体不同的特征不明确的受体来介导哺乳动物神经系统中的保护和再生功能。Epo基因已经在脊椎动物（从人类到鱼类）中被鉴定，表明Epo信号转导比脊椎动物谱系进化得更早。除了“经典的”红细胞生成Epo受体之外，I型细胞因子受体家族还包括存在于脊椎动物（包括人类和各种昆虫）中的遗传学上保守的孤儿细胞因子受体样因子3（CRLF 3）。我们已经在甲虫T. castaneum和刺槐L. CRLF 3是一种神经保护性Epo受体。此外，几种保护哺乳动物神经元但不激活“经典”同型二聚体Epo受体的配体激活昆虫CRLF 3，表明昆虫CRLF 3与未知的哺乳动物Epo神经保护受体之间的结构相似性。该研究旨在证明人CRLF 3（如其昆虫直系同源物）是一种神经保护性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类诱导多能干细胞（hiPSC）系中突变CRLF 3。野生型和CRLF 3突变的hiPSC将分化成神经元。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，也诱导CRLF 3介导的昆虫神经元保护。通过比较EV-3对野生型和CRLF 3敲除（KO）神经元的抗凋亡作用，我们将确定人CRLF 3是否代表人类Epo的组织保护性受体。我们将使用生存试验与抑制剂的潜在参与的信号传导因子，以确定CRLF 3启动的细胞内转导途径。最有可能的是，janus激酶/STAT将参与，但也将测试与哺乳动物中Epo刺激的神经保护机制有关的Pi 3 K、MAPK和NFB的贡献。通过激活CRLF 3而不激活不需要的造血或致瘤副作用来利用Epo和Epo模拟物的神经保护机制，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CRLF 3的内源性配体。如果成功，我们将搜索昆虫配体的人类直系同源物的数据库，并使用hiPSC衍生的神经元的存活测定来测试这些细胞因子是否也可以激活人CRLF 3。由于hiPSC原则上可以分化成任何体细胞类型，因此可以在受控的体外条件下容易地研究CRLF 3在其他人体组织和器官中的有益功能。

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.