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.

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism.

Cytokine receptor-like factor 3 (CRLF3) is a highly conserved but largely uncharacterized orphan cytokine receptor that shares structural similarity with vertebrate classical erythropoietin receptor. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin and involves partly similar anti-apoptotic mechanisms as erythropoietin-mediated neuroprotection in mammals. To identify potential mechanisms of CRLF3-mediated neuroprotection we studied the expression and function of acetylcholinesterase which promotes apoptosis in different cell types, including mammalian neurons. We exposed primary brain neurons from Locusta migratoria and Tribolium castaneum to apoptogenic stimuli and/or dsRNA to interfere with acetylcholinesterase gene expression and compared survival and/or acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increases both apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in both normal and apoptogenic condictions. Pharmacological inhibition of both acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevent hypoxia-induced apoptosis of primary brain neurons. Activation of CRLF3 with protective concentrations of rhEpo prevents the increased expression of pro-apoptotic acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of rhEpo that commonly (and seemingly paradoxically) cause death of insect and mammalian neurons induced ace-1 expression and hence promoted apoptosis in insect neurons. Our study confirms the cell-intrinsic role of acetylcholinesterase as a major regulator of apoptotic death, that was previously described in mammalian neurons only. Moreover, we identify a mechanism (prevention of upregulation of pro-apoptotic acetylcholinesterase), by which CRLF3 activation mediates neuroprotection under apoptogenic conditions. Since both apoptosis and CRLF3 are conserved throughout the animal kingdom, the direct link between cytokine/CRLF3 activation and suppression of increased acetylcholinesterase expression underlying neuroprotection in insects may also be present in other cell types and other non-insect species.