The cytokine receptor CRLF3 is a human neuroprotective EV-3 (Epo) receptor.

The evolutionary conserved orphan cytokine receptor-like factor 3 (CRLF3) has been implicated in human disease, vertebrate hematopoiesis and insect neuroprotection. While its specific functions are elusive, experimental evidence points toward a general role in cell homeostasis. Erythropoietin (Epo) is a major regulator of vertebrate hematopoiesis and a general cytoprotective cytokine. Erythropoietic functions mediated by classical Epo receptor are understood in great detail whereas Epo-mediated cytoprotective mechanisms are more complex due to involvement of additional Epo receptors and a non-erythropoietic splice variant with selectivity for certain receptors. In the present study, we show that the human CRLF3 mediates neuroprotection upon activation with the natural Epo splice variant EV-3. We generated CRLF3 knock-out iPSC lines and differentiated them toward the neuronal lineage. While apoptotic death of rotenone-challenged wild type iPSC-derived neurons was prevented by EV-3, EV-3-mediated neuroprotection was absent in CRLF3 knock-out neurons. Rotenone-induced apoptosis and EV-3-mediated neuroprotection were associated with differential expression of pro-and anti-apoptotic genes. Our data characterize human CRLF3 as a receptor involved in Epo-mediated neuroprotection and identify CRLF3 as the first known receptor for EV-3.

Locust Hemolymph Conveys Erythropoietin-Like Cytoprotection via Activation of the Cytokine Receptor CRLF3.

The cytokine receptor-like factor 3 (CRLF3) is an evolutionary conserved class 1 cytokine receptor present in all major eumetazoan groups. Endogenous CRLF3 ligands have not been identified and the physiological responses mediated by mammalian CRLF3 are poorly characterized. Insect CRLF3 is activated by erythropoietin (Epo) and several related molecules that protect mammalian neurons from stress-induced apoptosis. However, insects neither express Epo nor "classical" Epo receptor. Cell-protective effects of insect hemolymph have been described for several species. In this study, we explored the possibility that the endogenous CRLF3 ligand is contained in locust hemolymph. PCR analyses confirmed expression of crfl3-transcripts in neurons and hemocytes of Locusta migratoria and Tribolium castaneum. Survival of locust hemocytes in primary cultures was significantly increased by supplementation of culture medium with locust hemolymph serum. Locust primary neuron cultures were also protected by locust hemolymph, though preceding exposure to fetal bovine serum changed the hemolymph dose-dependency of neuroprotection. Direct comparison of 10% hemolymph serum with recombinant human Epo in its optimal neuroprotective concentration revealed equivalent anti-apoptotic effects on hypoxia-exposed locust neurons. The same concentration of locust hemolymph serum also protected hypoxia-exposed T. castaneum neurons. This indicates that the neuroprotective factor in locust hemolymph is sufficiently conserved in insects to allow activation of neuroprotective receptors in different species. Locust hemolymph-induced neuroprotection in both L. migratoria and T. castaneum was abolished after RNAi-mediated suppression of crlf3-expression. In summary, we report the presence of a conserved endogenous cytokine in locust hemolymph that activates CRLF3 and connected anti-apoptotic processes in hemocytes and neurons. Identification and characterization of the CRLF3 ligand will promote knowledge about cytokine evolution and may unravel cell-protective agents with potential clinical application.