Abstract
Erythropoietin (Epo) plays a dual role as an erythropoiesis-stimulating hormone and a locally produced cytoprotectant in various vertebrate tissues. Splice variants and engineered derivatives of Epo that mediate neuroprotection but do not stimulate erythropoiesis suggest that alternative receptors, different from the classical' homodimeric receptor involved in haematopoiesis, mediate neuroprotective Epo functions. Previous studies on grasshoppers demonstrated neuroprotective and neuroregenerative effects of Epo that involved similar transduction pathways as in mammals. To advance the characterization of yet unidentified neuroprotective Epo receptors, we studied the neuroprotective potency of the human non-erythropoietic Epo splice variant EV-3 in primary cultured locust brain neurons. We demonstrate that EV-3, like Epo, protects locust neurons from hypoxia-induced apoptotic death through activation of the Janus kinase/signal transducer and activator of transcription transduction pathway. Using the fluorescent dye FM1-43 to quantify endocytotic activity we show that both Epo and EV-3 increase the number of fluorescently labelled endocytotic vesicles. This reveals that binding of Epo to its neuroprotective receptor induces endocytosis, as it has been described for the mammalian homodimeric Epo-receptor expressed by erythroid progenitors. Reduction in Epo-stimulated endocytotic activity following pre-exposure to EV-3 indicated that both Epo and its splice variant bind to the same receptor on locust neurons. The shared neuroprotective potency of Epo and EV-3 in insect and mammalian neurons, in the absence of erythropoietic effects of EV-3 in mammals, suggests a greater similarity of the unidentified nervous Epo receptors (or receptor complexes) across phyla than between mammalian haematopoietic and neuroprotective receptors. Insects may serve as suitable models to evaluate the specific protective mechanisms mediated by Epo and its variants in non-erythropoietic mammalian tissues.