Multiple Mammalian Cytokines and Erythropoietin‐Mimetic Peptides Protect Insect Neurons via Phylogenetically Conserved Cytokine Receptor‐Like Factor 3 (CRLF3)

Erythropoietin (Epo) and its non-erythropoietic splice variant EV-3 have demonstrated potent neuroprotective effects across species, although the respective mechanisms are variable and incompletely understood. Unlike vertebrates, insects lack both Epo and the classical Epo receptor but express Cytokine Receptor-Like Factor 3 (CRLF3), a conserved type I receptor that serves as a neuroprotective receptor for Epo and EV-3 in insects and human iPSC-derived neuron-like cells. Insects, which express CRLF3 but lack all other group 1 type I cytokine receptors, represent a suitable model to study the function of CRLF3 in neuroprotection. Previous experiments with primary brain neurons from the locust Locusta migratoria demonstrated that human Epo and EV-3 prevented hypoxia-induced apoptosis via activation of CRLF3 and downstream signaling mediated by JAK/STAT activity. Here we demonstrate that locust neurons are also protected by three small peptides (≤ 20 amino acids) with previously established erythropoietic and/or neuroprotective capacities in mammals. These peptides included non-erythropoietic HBSP and P16, as well as erythropoietic EMP1. To further characterize the ligand spectrum of locust CRLF3, we tested specific ligands of vertebrate group 1 type I cytokine receptors for their neuroprotective potential. While human thrombopoietin protected locust neurons via activation of CRLF3, human prolactin and growth hormone showed no anti-apoptotic effects. Our findings highlight CRLF3 as a versatile neuroprotective receptor that can be activated by the three natural human cytokines Epo, EV-3, and thrombopoietin, and three artificial short peptides with and without erythropoietic activity. In contrast to vertebrate classical Epo receptor, which is only activated by Epo and EMP1, CRLF3 has an extended and more generalist ligand spectrum to mediate neuroprotection. This wide ligand spectrum may support the hypothesis of CRLF3 being the evolutionary precursor of various vertebrate-specific cytokine receptors and may qualify this more general cell protective receptor as a target for pharmacological intervention in neurodegenerative processes.