Presence and function of CRLF3 and erythropoietin-like ligands in invertebrate nervous systems

Erythropoietin (Epo) is a hematopoietic cytokine with multiple functions that are not exclusively related to vertebrate erythropoiesis. Epo is also expressed in the vertebrate nervous system where it serves important functions during neurodevelopment. Epo has been demonstrated to exert neuroprotective effects by interfering with apoptotic pathways and to promote the regeneration of damaged neurites in mammalian nervous systems. There is accumulating evidence that the functions of Epo/Epo-receptor in nervous tissues are independent from effects on the maturation of red blood cells. Particularly, the existence of endogenous splice variants and artificial derivatives of Epo that mediate neuroprotection but do not stimulate erythropoiesis, suggested different Epo-responsive receptors on erythroid progenitor cells and cells of other tissues including the nervous system. While the classical Epo-receptor EpoR is present in erythrocyte progenitors and other tissues, non-hematopoietic tissues express additional types of Epo-receptors with different ligand profiles.

Insects and other invertebrates lack genes for Epo and EpoR. Nevertheless, similar to its neuroprotective and neuroregenerative functions in mammals, recombinant human Epo initiates beneficial mechanisms in grasshopper and beetle nervous systems. In vitro, Epo increases survival of primary cultured brain neurons in normoxic and hypoxic conditions and promotes the regeneration of neurites. Epo interferes with apoptotic mechanisms by activating AG490-sensitive Januskinase and STAT signaling. In vivo, Epo accelerates and improves grasshopper axonal regeneration and reestablishment of sound source localization after crush injury of the tympanal nerve.




Locust and beetle brain neurons are also protected by the non-erythropoietic human Epo splice variant EV-3 and other non-erythropoietic Epo derivatives, suggesting that insect and mammalian neuroprotective Epo receptors share common structures that allow activation by the same non-erythropoietic agonists.

Insect species like Locusta migratoria and Tribolium castaneum, in which recombinant human Epo mediates neuroprotection, express a single orthologue of the human orphan cytokine receptor-like factor 3 (CRLF3). CRLF3 belongs to the cytokine type 1 receptor family, which also includes the hematopoietic cytokine receptors EpoR, thrombopoietin receptor and GCSF receptor. Of this family only CRLF3 has orthologs in invertebrates and it is highly conserved from cnidarians to mammals including humans. Primary cultured insect neurons have been demonstrated to lose Epo-mediated protection in hypoxia following RNAi-mediated downregulation of CRLF3, indicating that this receptor represents the neuroprotective Epo-receptor in insects. Its endogenous ligand (insects do not contain Epo) is contained in the circulating hemolymph since locust hemolymph extract protects both locust and beetle neurons from hypoxia-induced apoptosis through a CRLF3-dependentmechanism.




CRLF3 is highly conserved in eumetazoan species ranging from cnidarians to mammals. RNAi-mediated knockdown of CRLF3 expression abolishes Epo-mediated protection of hypoxia-challenged locust brain neuron in vitro. Locust hemolymph extracts protect both locust and beetle neurons against hypoxia-induced cell death.