Erythropoietin-mediated neuroprotection by cytokine receptor-like factor 3

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The cytokine erythropoietin (Epo) mediates protective and regenerative functions in mammalian nervous systems via activation of poorly characterized receptors that differ from “classic” homodimeric Epo receptors on erythroid progenitor cells. Epo genes have been identified in vertebrates (ranging from human to fish), suggesting that Epo signaling evolved earlier than the vertebrate lineage. Besides the classic erythropoietic Epo receptor, the family of type I cytokine receptors also includes the orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insects. We have demonstrated that CRLF3 is a neuroprotective Epo-receptor in the beetle T. castaneum. The proposed study aims to characterize the function of CRLF3 in insects and human cell lines by answering the following questions:1) Is CRLF3 a neuroprotective Epo-receptor in humans? We will study human cell lines with endogenous CRLF3 expression that derived from different tissues. We will overexpress and knock down CRLF3 in these cells. We will subject the cells to hypoxia-survival assays in the absence or presence of Epo or its neuroprotective (but non-erythropoietic) splice variant EV-3 and determine whether expression of CRLF3 correlates with suppression of apoptosis and cellular survival2) Can heterologous expression of CRLF3 confer Epo-sensitivity to Drosophila? D. melanogaster lacks a CRLF3 orthologue and beneficial effects of Epo on cellular survival are absent. In contrast, other components of Epo-stimulated anti-apoptotic pathways are present in Drosophila. We will heterologously express T. castaneum CRLF3 in Drosophila S2- and ML-DmBG2- cell lines to create Epo-responsive cells. In contrast to primary neuronal cultures, cell lines offer the possibility to use photometric assays (e.g. MTT) for the assessment of cell-culture vitality. We will also attempt to express human CRLF3 in Drosophila cell lines. If successful, CRLF3-expressing Drosophila cell lines will serve as a quick and reliable assay to screen potential Epo-mimetics for neuroprotective properties.3) Which insect tissues express CRLF3 and what is the function of this cytokine receptor? We will determine CRLF3 expression in embryos, larvae and pharate pupae of T. castaneum by in situ hybridisation. In addition, we will subject the same developmental stages to systemic RNAi to suppress CRLF3 expression. RNAi-treated beetles will be analysed for morphological alterations of outer structures and inner organs and for behavioural impairments.4) What is the endogenous “Epo-like” ligand in insects? We will generate and fractionate extracts of hypoxia-stimulated insect nervous systems and test these fractions for neuroprotective effects in hypoxia assays. We will crosslink potential ligands contained in these extracts with eGFP-tagged CRLF3. Extracts and crosslinked complexes will be separated from other proteins on gels and subjected to mass spectroscopy.