Erythropoietin-mediated neuroprotection in insects
von Natasa Miljus
Datum der mündl. Prüfung:2016-05-18
Betreuer:Prof. Dr. Ralf Heinrich
Gutachter:Prof. Dr. Ralf Heinrich
Gutachter:Prof. Dr. Dr. Hannelore Ehrenreich
EnglischThe cytokine erythropoietin (Epo) initiates adaptive cellular responses to a variety of mechanical and physiological insults in various non-hematopoietic mammalian tissues including the nervous system. Previous studies on insects demonstrated neuroprotective and regenerative effects of recombinant human Epo (rhEpo) in acridid grasshoppers in vitro and in vivo, similar to those in mammalian nervous system. This suggested that Epo-like signaling involved in tissue protection could represent an ancient cell-protective system shared by vertebrates and invertebrates that was later adopted for erythropoiesis in the vertebrate lineage. This PhD thesis provides further evidence for a pre-vertebrate evolution of a tissue-protective Epo-like signaling system. In the first part of the thesis I studied intracellular transduction pathways involved in Epo-mediated protection of locust brain neurons. I demonstrate that rhEpo effectively rescues primary cultured locust brain neurons from apoptotic cell death induced by hypoxia or the chemical compound H-7. The protective effects of rhEpo on locust brain neurons were abolished by the Janus kinase (JAK) inhibitor AG-490 and signal transducer and activator of transcription (STAT) inhibitor sc-355797. In contrast, the phosphoinositol-3-kinase (PI3K) inhibitor LY294002 and an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) PDTC did not prevent rhEpo-mediated neuroprotection. The results indicate that rhEpo mediates the protection of locust brain neurons through interference with apoptotic pathways by the activation of a JAK-associated receptor and STAT transcription factors. In the second part of the thesis I characterized some functional properties of the locust Epo-binding receptor. Using the fluorescent dye FM1-43 to quantify endocytotic activity I demonstrated that binding of rhEpo to a surface receptor initiates endocytotic internalization of the ligand-receptor complex. The results were confirmed by the detection of fluorescently labeled rhEpo in endocytotic vesicles. Epo-stimulated endocytosis has been demonstrated in vertebrate erythroid progenitor cells but so far not in any other tissue outside the erythropoietic system. In another series of experiments I could show that the neuroprotective but non-erythropoietic human Epo splice variant EV3 protected locust neurons from hypoxia-induced apoptosis with equal potency as rhEpo. The shared neuroprotective potency of EV3 in mammals and insects in the absence of erythropoietic effects suggests a greater similarity of the unidentified nervous erythropoietin-binding receptors across phyla than between mammalian hematopoietic and neuroprotective receptors. In the third part of the thesis I studied the potential role of rhEpo in the regulation of insect neurogenesis using three different preparations including a permanent cell line from a moth, a brain region with known neurogenic activity in a beetle and cells from a developing brain region with proliferative activity in the locust. With the tools and conditions applied, I found no evidence for an involvement of Epo in the regulation of neurogenesis neither at the level of progenitor cells proliferation nor at the level of neuronal differentiation. In summary, the present study demonstrated three important characteristics of Epo–like neuroprotective signaling in locust brain neurons that underline the similarity of mechanisms involved in insect and mammalian Epo-mediated neuroprotection. The involvement of similar transduction pathways in Epo-mediated neuroprotection, the endocytosis of Epo following its binding to a receptor and the capacity of EV3 to stimulate neuroprotection in mammals and insects, indicate that an Epo/Epo receptor-like signaling system with high structural and functional similarity exists in both groups of animals and may have originally evolved to provide tissue protection against various types of stressors.
Keywords: Erythropoietin; Neuroprotection; Primary brain cell culture; Insect; Locusta migratoria; Apoptosis; Signal transduction; Erythropoietin-binding receptor; Endocytosis; Non-erythropoietic splice variant of human erythropoietin; Neurogenesis