The Orphan Cytokine Receptor CRLF3 Emerged With the Origin of the Nervous System and Is a Neuroprotective Erythropoietin Receptor in Locusts

The orphan cytokine receptor-like factor 3 (CRLF3) was identified as a neuroprotective erythropoietin receptor in locust neurons and emerged with the evolution of the eumetazoan nervous system. Human CRLF3 belongs to class I helical cytokine receptors that mediate pleiotropic cellular reactions to injury and diverse physiological challenges. It is expressed in various tissues including the central nervous system but its ligand remains unidentified. A CRLF3 ortholog in the holometabolous beetle Tribolium castaneum was recently shown to induce anti-apoptotic mechanisms upon stimulation with human recombinant erythropoietin. To test the hypothesis that CRLF3 represents an ancient cell-protective receptor for erythropoietin-like cytokines, we investigated its presence across metazoan species. Furthermore, we examined CRLF3 expression and function in the hemimetabolous insect Locusta migratoria. Phylogenetic analysis of CRLF3 sequences indicated that CRLF3 is absent in Porifera, Placozoa and Ctenophora, all lacking the traditional nervous system. However, it is present in all major eumetazoan groups ranging from cnidarians over protostomians to mammals. The CRLF3 sequence is highly conserved and abundant amongst vertebrates. In contrast, relatively few invertebrates express CRLF3 and these sequences show greater variability, suggesting frequent loss due to low functional importance. In L. migratoria, we identified the transcript Lm-crlf3 by RACE-PCR and detected its expression in locust brain, skeletal muscle and hemocytes. These findings correspond to the ubiquitous expression of crlf3 in mammalian tissues. We demonstrate that the sole addition of double-stranded RNA to the culture medium (called soaking RNA interference) specifically interferes with protein expression in locust primary brain cell cultures. This technique was used to knock down Lm-crlf3 expression and to abolish its physiological function. We confirmed that recombinant human erythropoietin rescues locust brain neurons from hypoxia-induced apoptosis and showed that this neuroprotective effect is absent after knocking down Lm-crlf3. Our results affirm the erythropoietin-induced neuroprotective function of CRLF3 in a second insect species from a different taxonomic group. They suggest that the phylogenetically conserved CRLF3 receptor may function as a cell protective receptor for erythropoietin or a structurally related cytokine also in other animals including vertebrate and mammalian species.