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

Abstract

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism.

Multifaceted promotion of apoptosis by acetylcholinesterase

“…Current knowledge suggests that complex regulation of apoptosis including mitochondrial cytochrome c release and apoptosomemediated activation of caspases evolved in early metazoans (Zmasek and Godzik, 2013;Chai and Shi, 2014;Green and Fitzgerald, 2016). Elevated levels of AChE expression induced by apoptogenic stressors and apoptosis inhibition by experimental reduction of AChE presence and/or catalytical activity have also been detected in insects (Knorr et al, 2020;Knorr et al, 2022) paralleling previous reports from studies with vertebrate cells. The molecular mechanisms underlying AChE's pro-apoptotic functions have not been revealed in any invertebrate species.…”

“…Elevated AChE levels may also result from reduced degradation, as has been demonstrated in amyloid beta-stimulated murine neuroblastoma cells (Hu et al, 2003). While presence of AChE per se does not initiate apoptosis, elevated levels sensitize the induction of apoptosis under pathogenic or physiologically challenging conditions (Jin et al, 2004;Knorr et al, 2022). Some studies indicated that only particular splice variants of mammalian AChE induce apoptosis in the investigated cell types, such as N-AChE-S in cholinergic cortex neurons and glioblastoma cells (Toiber et al, 2008).…”

“…The molecular mechanisms underlying AChE's pro-apoptotic functions have not been revealed in any invertebrate species. Pro-apoptotic elevation of AChE expression in locust and beetle neurons is suppressed by activation of CRLF3 (cytokine receptor-like factor 3) stimulation with various cytokine ligands (Knorr et al, 2022). CRLF3 represents a phylogenetically (from cnidarian to mammals) conserved cytokine receptor that initiates anti-apoptotic processes via activation of intracellular JAK/STAT signaling (Hahn et al 2017;Hahn et al 2019).…”