Functional characterization and molecular identification of neuroprotective receptors for erythropoietin-like ligands

by Nina Hahn
Cumulative thesis
Date of Examination:2019-12-13
Date of issue:2019-12-19
Advisor:Prof. Dr. Ralf Heinrich
Referee:Prof. Dr. Ralf Heinrich
Referee:Prof. Dr. Gregor Bucher
Referee:Dr. Sven Bradler
Referee:Dr. Nico Posnien
Referee:Prof. Dr. Manuela Schmidt
Referee:Dr. Hauke Werner
Persistent Address: http://dx.doi.org/10.53846/goediss-7774

 

 

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Abstract

English

The cytokine erythropoietin (Epo) regulates erythropoiesis but also protects mammalian neurons from apoptosis. However, receptors and transduction pathways involved in neuroprotection are only partially characterized. Although insects lack Epo and classical Epo receptor genes, human recombinant Epo induces protection and promotes neuroregeneration of insect neurons. Thus, insect neurons are a suitable model system to study neuroprotective signaling pathways independent from erythropoietic pathways. My studies on insect primary brain neurons demonstrated that recombinant human Epo protects neurons against hypoxia-induced apoptosis by activating orthologs of cytokine receptor like factor 3 (CRLF3), a predicted cytokine receptor with unknown ligand and function in mammals. To this end, I established soaking RNA interference to knock down orthologs of CRLF3 in primary brain cell cultures of the beetle Tribolium castaneum and the locust Locusta migratoria. Furthermore, I tested various Epo-like molecules which have been described in vertebrates to be neuroprotective but not erythropoietic. All tested Epo-like molecules were also neuroprotective in locust primary brain neurons. These shared neuroprotective functions propose that the ligand-binding structures of neuroprotective receptors in insects and vertebrates are more similar than those of the neuroprotective and erythropoietic receptors within vertebrates. Moreover, I provided evidence that neuroprotective effects of the human splice variant EV-3 and an Epo-mimetic peptide are mediated via CRLF3. Phylogenetic analysis of 293 CRLF3 sequences revealed that CRLF3 emerged with the origin of the eumetazoan nervous system. This coincidence suggests that CRLF3 might have originally evolved for a function within the nervous system. However, I have demonstrated CRLF3 expression also in other tissues apart from the nervous system in human cell lines, Tribolium castaneum and Locusta migratoria. In both human neuroblastoma (SH-SY5Y) cells and Tribolium tissue CRLF3 expression is regulated by hypoxic stress. Moreover, I showed that CRLF3 expression is present at all developmental stages of Tribolium but to a variable extent. In this study, I tested the hypothesis that CRLF3 might be a neuroprotective Epo receptor. The results presented herein show that CRLF3 is crucial to mediate the neuroprotective effect of human Epo and Epo-like cell protective molecules in insect primary brain cells. Whether this function of CRLF3 is also present in human brain cells has to be proven in future experiments for which I have already established pivotal tools. This study introducing CRLF3 as a neuroprotective Epo receptor might support the development of safe, neuroprotective therapies that selectively stimulate CRLF3.
Keywords: Neuroprotection; Erythropoietin; CRLF3; Cytokine receptor-like factor 3; Orphan cytokine receptor; Neurodegenerativ diseases; Tribolium castaneum; Soaking RNAi; Locusta migratoria; Epo; Nervous system; Evolution
 
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