Protection of insect neurons by erythropoietin/CRLF3-mediated regulation of pro-apoptotic acetylcholinesterase

Abstract

Cytokine receptor-like factor 3 (CRLF3) is a highly conserved but largely uncharacterized orphan cytokine receptor that shares structural similarity with vertebrate classical erythropoietin receptor. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin and involves partly similar anti-apoptotic mechanisms as erythropoietin-mediated neuroprotection in mammals. To identify potential mechanisms of CRLF3-mediated neuroprotection we studied the expression and function of acetylcholinesterase which promotes apoptosis in different cell types, including mammalian neurons. We exposed primary brain neurons from Locusta migratoria and Tribolium castaneum to apoptogenic stimuli and/or dsRNA to interfere with acetylcholinesterase gene expression and compared survival and/or acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increases both apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in both normal and apoptogenic condictions. Pharmacological inhibition of both acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevent hypoxia-induced apoptosis of primary brain neurons. Activation of CRLF3 with protective concentrations of rhEpo prevents the increased expression of pro-apoptotic acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of rhEpo that commonly (and seemingly paradoxically) cause death of insect and mammalian neurons induced ace-1 expression and hence promoted apoptosis in insect neurons. Our study confirms the cell-intrinsic role of acetylcholinesterase as a major regulator of apoptotic death, that was previously described in mammalian neurons only. Moreover, we identify a mechanism (prevention of upregulation of pro-apoptotic acetylcholinesterase), by which CRLF3 activation mediates neuroprotection under apoptogenic conditions. Since both apoptosis and CRLF3 are conserved throughout the animal kingdom, the direct link between cytokine/CRLF3 activation and suppression of increased acetylcholinesterase expression underlying neuroprotection in insects may also be present in other cell types and other non-insect species.