Neuroprotection and endocytosis: erythropoietin receptors in insect nervous systems

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.

The Insect Ortholog of the Human Orphan Cytokine Receptor CRLF3 Is a Neuroprotective Erythropoietin Receptor

“…In both groups of animals neuroprotection is mediated via JAK and STAT activation, while mammalian neurons may additionally employ transduction pathways involving MAPK, PI3K and NF κ B (Digicaylioglu and Lipton, 2001; Sirén et al, 2001; Miljus et al, 2014). Various studies demonstrated optimum-type dose-response curves for different concentrations of experimentally applied Epo (this study; Sirén et al, 2001; Weishaupt et al, 2004; Ostrowski et al, 2011; Miller et al, 2015) and Epo-induced receptor endocytosis has been described for locust neurons (Miljus et al, 2017) and mammalian erythroid progenitors (Bulut et al, 2011). Importantly, the non-erythropoietic splice variant EV-3 increased the survival of serum-deprived primary cultured T. castaneum brain neurons (this study) and protected both hypoxia-exposed locust brain neurons (Miljus et al, 2017) and rat cortex neurons challenged with glucose and oxygen deprivation (Bonnas, 2012; Bonnas et al, 2017) from apoptotic cell death.…”

“…Various studies demonstrated optimum-type dose-response curves for different concentrations of experimentally applied Epo (this study; Sirén et al, 2001; Weishaupt et al, 2004; Ostrowski et al, 2011; Miller et al, 2015) and Epo-induced receptor endocytosis has been described for locust neurons (Miljus et al, 2017) and mammalian erythroid progenitors (Bulut et al, 2011). Importantly, the non-erythropoietic splice variant EV-3 increased the survival of serum-deprived primary cultured T. castaneum brain neurons (this study) and protected both hypoxia-exposed locust brain neurons (Miljus et al, 2017) and rat cortex neurons challenged with glucose and oxygen deprivation (Bonnas, 2012; Bonnas et al, 2017) from apoptotic cell death. This suggested a greater similarity of the ligand binding regions of locust and mammalian neuroprotective Epo receptors than between mammalian neuroprotective and classical erythropoietic EpoR.…”

“…In these studies recombinant human Epo rescued primary cultured brain neurons from hypoxia-induced apoptotic cell death by activating Janus kinase/signal transducers and activators of transcription (JAK/STAT) transduction pathways (Miljus et al, 2014). In addition, locust brain neurons were also protected by the non-erythropoietic human splice variant EV-3 (Miljus et al, 2017; characterization of EV-3 by Bonnas et al, 2017), suggesting that insect and mammalian neuroprotective Epo receptors share common structures that allow activation by both Epo and EV-3. These findings support the hypothesis of a pre-vertebrate evolution of Epo-like signaling in neuroprotection (Brines and Cerami, 2005).…”

EV-3, an endogenous human erythropoietin isoform with distinct functional relevance

“…EPO’s protective effects against hypoxia-induced cell death is well described in the literature 44, 63, 85 . We sought to investigate previously reported conservation of neuroprotective properties in the naturally occurring EPO-isoform EV-3 19 .…”

Erythropoietin as a Modulator of Pathology in a Toxicant Mouse Model of Parkinson’s Disease

“…In this regard, trophic factors can be internalized at distal sites together with their receptors, following which they are compartmentalized into signalling endosomes that engage in protein-based transport to the cell soma (Zahavi et al, 2017). Indeed, endocytosis of EPO and its receptor has been demonstrated in vitro (Bulut, Sulahian, Yao, & Huang, 2013) and more recently in the insect nervous system (Miljus et al, 2017), suggesting this may be one possible mechanism by which intra-striatal EPO can influence cell survival in the SNc.…”