Erythropoietin (Epo) is the primary humoral mediator of hypoxic induction of vertebrate erythropoiesis (Bunn 2013). Apart from regulating erythropoiesis, evidence emerging over the last decade underlined the importance of Epo in mediating adaptive cellular responses triggered by diverse harmful stimuli in various non-hematopoietic mammalian tissues including the nervous system.
Acting in a paracrine manner (Ruscher et al. 2002), Epo induces a wide range of cellular responses in the nervous system to protect and repair physiologically challenged or injured tissue. Beneficial functions include protection of neurons from apoptosis (Sirén et al. 2001a; Wen et al. 2002), glutamate excitotoxicity (Morishita et al. 1996) and oxidative damage (Chong et al. 2003; Kumral et al.
2005; Wu et al. 2007a), prevention of inflammatory responses (Agnello et al.
2002; Villa et al. 2003; Sättler et al. 2004; Chen et al. 2007a), promotion of angiogenesis (Wang et al. 2004) and neurogenesis (Shingo et al. 2001). Thus, by targeting not only injured mature neurons directly, but also neural progenitors, astrocytes, oligodendrocytes, microglia and endothelial cells (Byts & Sirén 2009), Epo coordinates and orchestrates differential cell type-specific responses to promote healing and repair of injured nervous tissues.
As in the kidney, Epo production in the mammalian brain was observed to be hypoxia-inducible (Marti et al. 1996) and regulated via hypoxia-inducible factor (HIF) (Semenza & Wang 1992). In addition to hypoxia other potentially harmful stimuli, such as hypoglycemia, insulin release and reactive oxygen species activate HIF and lead to increased expression of Epo (Masuda et al. 1997;
Chandel et al. 1998). Epo exerts its physiological role by binding to a cell surface receptor. Epo receptor (EpoR) expressed on the surface of erythroid progenitor
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cells is a homodimeric cytokine class I receptor (Youssoufian et al. 1993). Signal transduction in erythropoiesis involves binding of one Epo molecule to an EpoR homodimer which leads to the activation of intracellular signaling cascades (Constantinescu et al. 1999). In contrast, tissue-protective functions of Epo are suggested to be mediated by binding to a heteromeric receptor, consisting of one hematopoietic EpoR monomer and another, probably cell-specific, receptor subunit. Potential heteromeric partners in non-hematopoietic tissues include one or more units of the beta common receptor chain (Brines et al. 2004) and the ephrine B4 receptor (Jackson et al. 2012; Debeljak et al. 2014).
The signaling pathways involved in neuroprotection overlap partially with those engaged in erythropoiesis. Both, neuroprotection and erythropoiesis are initiated by trans-phosphorylation and activation of receptor-associated tyrosine kinases of the Janus kinase 2 (JAK2) type as a result of conformational change in the receptor induced upon Epo binding (Witthuhn et al. 1993). Three principal downstream signaling pathways are subsequently activated. These include signal transducer and activator of transcription 5 (STAT5), phosphoinositol-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) (Sirén et al. 2001a). STAT5 contributes to an universal antiapoptotic pathway activated in erythroid precursors (Silva et al. 1999;
Socolovsky et al. 2001), neuronal cells (Sirén et al. 2001a) and other cell types.
The PI3K/Akt pathway has been demonstrated to be involved in neural progenitor cell migration to the area of injury (Wang et al. 2006a) and regulation of endothelial responses (Chong et al. 2002). The MAPK pathway has been implicated in proliferation (Sui et al. 1998; Lawson et al. 2000) and differentiation of erythroid precursors (Klingmüller et al. 1997), as well as in reduction of inflammation (Brines 2014). All three pathways contribute to antiapoptotic effects of Epo through interference with apoptotic processes, accomplished either directly (e.g. via Akt that regulates the activity of caspases) (Digicaylioglu et al. 2004; Wu et al. 2007) or via activation of transcription factors that either suppress transcription of pro-apoptotic genes (e.g. bad) (Ruscher et al. 2002) or activate transcription of anti-apoptotic genes (e.g. bcl-XL)
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(Wen et al. 2002). Epo-mediated neuroprotection in mammals additionally involves signaling pathways different from those involved in erythropoiesis, such as the NF-kB pathway (Digicaylioglu & Lipton 2001; Liu et al. 2005).
The fact that Epo initiates similar transduction pathways to stimulate erythropoiesis and tissue protection suggests a common evolutionary origin of Epo signaling for both systems in vertebrates. Epo signaling seems to be common to all vertebrates since homologues of the human epo gene have been identified in various mammalian (Wen et al., 1993), amphibian (Nogawa-Kosaka et al. 2010) and fish species (Chou et al. 2004; Chu et al. 2007; Chu et al.
2008; Ostrowski et al. 2011). In contrast, orthologues of epo and epor genes could so far not be identified in any invertebrate species, while downstream components of mammalian Epo signaling pathways, namely JAK, STAT, PI3K, Akt, NF-κB are present in invertebrates, including various insect species (Ghosh et al. 1998; Scanga et al. 2000; Arbouzova & Zeidler 2006). The significance of these signaling networks in insect cells is reflected by their requirement for normal developmental processes (Bina & Zeidler 2009) and for innate immune responses against invading pathogens (Agaisse et al. 2003). Despite the absence of Epo and EpoR orthologues in insects, a previous study on grasshoppers found neuroprotective and neuroregenerative effects of rhEpo in vitro and in vivo (Ostrowski et al. 2011). Hence, neuroprotection or even general tissue protection might have been the original function of an ancient Epo/EpoR-like signaling system that evolved in a common ancestor of vertebrates and insects as a part of innate immunity. With the evolution of specialized oxygen carrying erythrocytes at the basis of the vertebrate lineage the Epo/EpoR system was subsequently adapted for its role in vertebrate erythropoiesis (Svoboda &
Bartunek 2015).
Although orthologues of mammalian components of Epo-stimulated intracellular transduction pathways have been described in insects (most completely in Drosophila melanogaster), their requirement for Epo-mediated protection of insect neurons have not been studied so far. Therefore, the aim of
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the first part of this thesis was to examine whether signaling pathways involved in Epo-mediated protection of insect (locust) neurons are similar to those required for neuroprotection in mammals. Similarities in signaling cascades leading to Epo-mediated neuroprotection between mammals and insects would thus provide further support for the hypothesis of a pre-vertebrate evolution of Epo/EpoR-like signaling with an original function in cell and tissue protection.
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