Neuroprotective functions of the orphan cytokine receptor CRLF3 – a study in human stem cell-derived neurons

• Applicant: Professor Ralf Heinrich, Ph.D.
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells ; Experimental Models for the Understanding of Nervous System Diseases
• Term: from 2022 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 499371712

• Final Report Year: 2023

Final Report Abstract

Erythropoietin (Epo) mediates protective and regenerative functions in mammalian nervous systems via activation of “classic” homodimeric EpoR and additional poorly characterized receptors. Insect CRLF3 (a phylogenetically conserved type I cytokine receptor) is activated by human Epo and several ligands (including the non-erythropoietic Epo splice variant EV-3) that do not activate homodimeric EpoR. No endogenous ligand for CRLF3 and no receptor for EV- 3 have so far been identified. We generated CRLF3-knock out lines of human induced pluripotent stem cells (hiPSC) and differentiated them into neurons. Rotenone-induced apoptosis was prevented by both Epo and EV-3 in wild type hiPSC-derived neurons. In contrast, CRLF3-KO cell lines were not protected by these cytokines. Upon stimulation of hiPSC-derived neurons with labelled EV-3, only wild type but not CRLF3-KO neurons accumulated EV-3 associated fluorescence in endocytotic vesicles. EV-3 prevented rotenone-induced elevation of proapoptotic BAX and Caspase-3 and elevated anti-apoptotic BCL-2 transcript levels in wild type neurons. In contrast, BAX and Caspase-3 expression was not reduced and BCL-2 not elevated in CRLF3-KO cells. The results indicate that human CRLF3 functions as a cell membrane-located receptor for Epo and EV-3 that initiates anti-apoptotic mechanisms upon physio-/pathological challenge. In order to connect CRLF3 with intracellular anti-apoptotic mechanisms we explored its connection to the expression and pro-apoptotic function of acetylcholinesterase (AChE). Expression of both AChE-coding genes (ace-1 and ace-2) in Tribolium castaneum is elevated under apoptogenic (hypoxia) conditions and pharmacological inhibition of AChE prevents hypoxia-induced cell death. Epo-/CRLF3-signaling prevents hypoxia-induced upregulation of ace-1 and protects beetle neurons from apoptotic death. Thus, Epo-/CRLF3-mediated neuroprotection in beetles (also shown in locusts) relies on the suppression of elevated levels of pro-apoptotic AChE. A common (and yet not understood) feature of Epo- (and EV-3-) mediated effects on both mammalian and insect neurons is its optimum-type dose response, reaching optimal neuroprotection at a certain concentration, decreasing protection with further increasing concentrations and even cytotoxic effects at very high concentrations. In locust and beetle neurons, protective concentrations of Epo generally reduce ace expression whereas higher and toxic concentrations of Epo stimulate pro-apoptotic ace-1 expression. This suggests regulation of ace-1 as a mechanism underlying the dose dependence of Epo in neuroprotection / neurotoxicity. Whether protective and toxic Epo signaling includes AChE also in vertebrate/mammalian cells has not been studied.

Publications

• Anti-apoptotic and neuroprotective erythropoietin/CRLF3-signalling in insects and humans. University Goettingen Repository.
  Knorr, Debbra Yasemin
  
• Meeting of the Society for Neuroscience in San Diego; Poster “The human cytokine receptor CRLF3 is a neuroprotective EV-3 (erythropoietin) receptor”
  Knorr, Rodriguez Polo, Pies, Schwedhelm-Domeyer, Pauls, Behr & Heinrich
  
• Protection of insect neurons by erythropoietin/CRLF3-mediated regulation of pro-apoptotic acetylcholinesterase. Scientific Reports, 12(1).
  Knorr, Debbra Y.; Schneider, Kristin; Büschgens, Luca; Förster, Jan; Georges, Nadine S.; Geurten, Bart R. H. & Heinrich, Ralf
  
• 15th Göttingen meeting of the German Neuroscience Society; Poster “The human cytokine receptor CRLF3 is a neuroprotective EV-3 (erythropoietin) receptor”
  Knorr, Twellsieck, Rodriguez Polo, Pies, Schwedhelm-Domeyer, Pauls, Behr & Heinrich
  
• Multifaceted promotion of apoptosis by acetylcholinesterase. Frontiers in Cell Death, 2.
  Knorr, Debbra Y.; Demirbas, Defne & Heinrich, Ralf
  
• The cytokine receptor CRLF3 is a human neuroprotective EV-3 (Epo) receptor. Frontiers in Molecular Neuroscience, 16.
  Knorr, Debbra Y.; Rodriguez, Polo Ignacio; Pies, Hanna S.; Schwedhelm-Domeyer, Nicola; Pauls, Stephanie; Behr, Rüdiger & Heinrich, Ralf