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

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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.