The partial erythropoietin receptor agonist ML1-R is a potent neuroprotective drug with a distinct signaling profile.

The partial erythropoietin receptor agonist ML1-R is a potent neuroprotective drug with a distinct signaling profile. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The partial erythropoietin receptor agonist ML1-R is a potent neuroprotective drug with a distinct signaling profile. Oh-Hoon Kwon, Jinsik Bae, Jun Chul Byun, Hyun-Joo Jeong, Jixing Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5779616/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Erythropoietin (EPO) is a glycoprotein that stimulates red blood cell production in the bone marrow and protects neurons from oxidative stress, making it a potential treatment for various neurological diseases. However, EPO analogs often lead to side effects like excessive erythropoiesis and tumor growth. In this study, we aimed to develop ML1-R, a peptide derived from the C-helix of EPO, to enhance neuroprotection while minimizing adverse effects. By modifying amino acids that interact with EPO receptors (EPORs), ML1-R activated EPORs differently from recombinant EPO (reEPO). ML1-R provided stronger neuroprotection than reEPO without promoting cell proliferation. In a murine stroke models and in-vitro neuron cultures, ML1-R reduced brain injury and prevented neuronal death caused by glutamate-induced excitotoxicity and hypoxia-reoxygenation. AlphaFold3 computational analysis showed distinct binding affinity and geometric structures between ML1-R–EPOR and EPO–EPOR complexes. ML1-R prolonged JAK2 activation and activated Akt/Erk signaling in distinct patterns, increasing EPORs on cell surface membranes. This reduced apoptosis and alleviated calcium overload, reactive oxygen species generation, and mitochondrial dysfunction induced by glutamate-induced excitotoxicity and hypoxia–reoxygenation. In conclusion, these findings highlight ML1-R as a promising candidate to treat ischemic stroke, reperfusion brain injury, and neurodegenerative diseases. Cellular & Molecular Neuroscience Erythropoietin Erythropoietin receptor Ischemic stroke Reperfusion brain injury Partial agonist Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Erythropoietin (EPO), a well-known hypoxia-inducible growth factor, promotes the proliferation, survival, and differentiation of erythroid progenitor cells into erythrocytes 1 . This hematopoietic function of EPO is widely recognized; however, in other organs, including the brain, kidney, heart, and muscle, it plays nonhematopoietic roles under various stress conditions 2 . Neurons and astrocytes also produce EPO, and the EPO receptor (EPOR) is expressed in brain tissue cells including neurons, astrocytes, microglia, and oligodendrocytes 3 , 4 . EPO is related to neuroprotection, neurogenesis, and regeneration. EPO gene expression is regulated by hypoxia-inducible factor-1 5, 6, 7 . EPO and EPOR are upregulated in the brain under pathological conditions in neurological diseases as a neuroprotective response to brain injury 8 , 9 , 10 . Because EPORs mediate anti-apoptotic and anti-oxidant effects, EPO has been proposed as a drug target for various neuronal diseases including ischemic stroke 11 , 12 . Stroke is a complex and dynamic brain disease that can lead to disability and mortality. The unique physiological properties of the brain make it extremely sensitive to blood supply changes; it is the organ with the highest energy demand in the body, making continuous oxygen and nutrient supply through blood flow crucial. Ischemic stroke, caused by occlusion of blood flow to the brain, is the most common type of stroke 13 , 14 . Such an occlusion disrupts the balance of oxygen and nutrients required for energy generation in the brain, resulting in the loss of physiological functions and cellular homeostasis 15 , 16 , 17 . Ischemic conditions rapidly deplete energy, inducing an imbalance in electrochemical gradients and leading to neuronal depolarization. This neuronal depolarization triggers intracellular calcium overload, reactive oxygen species (ROS) generation, mitochondrial dysfunction, and massive glutamate-induced excitotoxicity and neuronal death 18 , 19 . Although reperfusion to restore blood flow is very important, reperfusion also substantially upregulates oxidative stress and mitochondrial damage, resulting in potentially lethal reperfusion injury of the brain 20 , 21 . Thus, pharmacological alleviation of reperfusion injury is critical for the treatment of ischemic stroke. Although some drugs that inhibit glutamate release or cell death pathways have been developed, most clinical trials failed because of unexpected adverse effects. Therefore, the development of neuroprotective drugs against ischemic stroke and reperfusion brain injury is still urgently needed. Protective EPO effects involve the activation of multiple downstream signaling pathways. When EPO binds to EPORs, their interaction induces the autophosphorylation of Janus kinase 2 (JAK2), which subsequently activates the signal transducer and activator of transcription 5 (STAT5), protein kinase B (Akt), and extracellular signal-regulated kinases 1/2 (Erks) pathways, which are involved in cell survival and proliferation 22 , 23 , 24 . These pathways induce anti-oxidative, anti-cytotoxic, anti-apoptotic, and anti-inflammatory effects 25 , 26 . Thus, EPO has been suggested as a neuroprotective drug in treating neurological diseases including stroke, epilepsy, Parkinson’s disease, and Alzheimer’s disease 27 , 28 . However, studies of using EPO for neuroprotection have shown its limitations including erythropoietic and tumorigenic adverse effects 24 , 29 . Therefore, EPO-based strategies that alleviate these adverse effects are required to treat ischemic stroke, reperfusion brain injury, and neurodegenerative diseases. Various EPO analogs have been developed to overcome these unwanted effects 30 . Structural biology studies have shown that EPO consists of four α-helices (A, B, C, and D) clustered through hydrophobic interactions 31 . A single EPO molecule binds to a dimeric EPOR consisting of the high-affinity EPOR1 and low-affinity EPOR2. Small peptides derived from EPO subregions involved in EPOR binding may selectively mediate non-hematopoietic protective effects. Distinct from the binding of the entire EPO molecule, their interactions with EPOR may differentially activate downstream pathways, potentially allowing targeted neuroprotection without unwanted proliferative and erythropoietic effects 32 , 33 . Our previous research suggests that novel peptides derived from the helix C of EPO, MK-X and ML1-h3, have neuroprotective effects without affecting cell proliferation via differential activation of EPOR-mediated signaling pathways 34 , 35 , 36 . Modification of the binding structure or EPOR affinity of EPO derivatives may provide important insights into the development of EPO-based neuroprotective strategies without unwanted erythropoietic and tumorigenic effects. In this study, we aimed to develop a new peptide, ML1-R, based on the C-helix of EPO and identify its molecular mechanisms in relation to EPOR neuroprotective effects. Our study suggests that ML1-R, a novel EPOR-binding partial agonist, is a therapeutic strategy for protection against ischemic stroke, reperfusion brain injury, and neurodegenerative disorders. Results ML1-R efficiently inhibits ischemia-reperfusion brain injury in a stroke model and apoptosis of primary cultured cortical neurons following hypoxia–reoxygenation. To investigate the potential of ML1-R as a novel drug with neuroprotective effects against ischemia-reperfusion brain injury, we induced transient ischemia via middle cerebral artery occlusion (MCAO) in mice for 1.5 h, followed by reperfusion and human recombinant EPO (reEPO) or ML1-R injection (Fig. 1 a). After 24 h, MCAO-reperfusion significantly induced severe unilateral infarction in the brain, as confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining (Fig. 1 b). The average TTC intensity on the MCAO-treated side was < 56% of that on the contralateral side in vehicle-injected mouse brains. Treatment with reEPO efficiently decreased the infarction area, and ML-1R administration strongly inhibited MCAO-reperfusion-induced brain injury (Fig. 1 c). To further investigate the in-vitro protective effects of ML1-R against apoptosis under hypoxia–reoxygenation conditions, we conducted a Hoechst staining assay with cultured primary cortical neurons. These neurons were maintained under hypoxic conditions for 18 h and then reoxygenated for 4 h (Fig. 1 d). The neurons were then kept with or without reEPO or ML1-R for 18 h, and the ratio of apoptotic cells to total cells was determined using the Hoechst staining assay (Fig. 1 e). Similar to the in-vivo results, treatment with ML1-R substantially reduced the number of apoptotic neurons compared to treatment with reEPO (Fig. 1 f). Taken together, in-vivo and in-vitro experiments indicated that ML1-R has better neuroprotective effects than reEPO. ML1-R prevents glutamate-induced neuronal cell death by suppressing calcium overload, oxidative stress, and mitochondrial dysfunction. Following ischemia, energy depletion occurs within minutes, leading to the failure of energy-dependent cellular homeostasis and maintenance of brain function. Severe energy depletion under ischemic conditions disrupts the electrochemical gradient of the plasma membrane (PM), leading to depolarization and excessive glutamate release. This causes calcium influx through glutamate receptors, resulting in glutamate-induced excitotoxicity 15 , 16 , 17 , 18 , 19 . To understand the neuroprotective effects of ML1-R, we examined in cultured neurons with or without reEPO or ML1-R treatment various processes that are critical for cellular homeostasis under glutamate-induced excitotoxicity (Fig. 2 a). Intracellular calcium levels increased after 3 min of glutamate treatment. Both reEPO and ML1-R similarly inhibited calcium influx (Fig. 2 b). Moreover, reEPO and ML1-R decreased glutamate-induced ROS levels; ML1-R more strongly inhibited ROS generation (Fig. 2 c). As calcium overload and oxidative stress cause mitochondrial dysfunction in glutamate-induced excitotoxicity, we also measured the mitochondrial membrane potential. Both reEPO and ML1-R suppressed the glutamate-induced reduction in mitochondrial membrane potential, but the effect of ML1-R was more pronounced than that of reEPO (Fig. 2 d). ML1-R also substantially decreased the loss of cell viability compared to reEPO (Fig. 2 e). These data indicate that ML1-R has neuroprotective effects by regulating calcium overload and ROS production, thereby mitigating mitochondrial dysfunction. Mitochondrial damage can activate caspase-dependent and caspase-independent cell death pathways 18 , 19 . Glutamate treatment increased the expression of Bax, cleaved caspase 3 (Casp3), and cleaved Parp1, known apoptosis marker proteins, whereas ML1-R efficiently suppressed their expression and increased Bcl-2 levels which inhibits Bax activity (Fig. 2 f-j). Overall, except for cleaved Parp1 (Fig. 2 j), ML1-R reversed the upregulation of pro-apoptotic markers more strongly than reEPO and effectively restored Bcl-2 expression (Fig. 2 h). These results indicated that ML1-R effectively prevents cellular dyshomeostasis and the activation of cell death pathways following glutamate-induced excitotoxicity. ML1-R protects neurons against hypoxia–reoxygenation effects by inhibiting oxidative stress, mitochondrial dysfunction, and apoptosis. Prompt restoration of cerebral blood flow is crucial in ischemic stroke, but the restoration of blood flow can induce reperfusion injury 20 , 21 . To understand the protective effects of ML1-R against reperfusion brain injury in vitro, we assessed cellular processes under hypoxia–reoxygenation conditions in the presence or absence of reEPO or ML1-R in primary cultured cortical neurons (Fig. 3 a). ML1-R inhibited ROS generation (Fig. 3 b), mitochondrial dysfunction (Fig. 3 c), and the loss of neuronal viability (Fig. 3 d) more strongly than reEPO. Previous studies reported that hypoxia–reoxygenation induces caspase-dependent and caspase-independent cell death 35 . Immunoblotting showed that hypoxia–reoxygenation upregulated Bax, cleaved Casp3, and Parp1 levels and down-regulated Bcl-2 levels (Fig. 3 e-i). ML1-R reversed the expression of apoptosis markers more effectively than reEPO under hypoxia–reoxygenation conditions, significantly increasing Bcl-2 (Fig. 3 g) and reducing cleaved Casp3 (Fig. 3 h) and cleaved Parp1 levels to control levels (Fig. 3 i), except for Bax (Fig. 3 f). We also assessed the cell proliferative effects of high reEPO and ML1-R concentrations because proliferative EPO functions may induce oncogenicity. We found that ML1-R did not induce the proliferation of PC12 and SH-SY5Y cells, whereas high doses of reEPO significantly increased cell proliferation (Supplementary Fig. 1a, b). Taken together, these results indicate that ML1-R suppresses hypoxia–reoxygenation-induced cell death by alleviating ROS production and mitochondrial dysfunction, resulting in neuroprotection without proliferative effects. As ML1-R exhibited a stronger neuroprotective effect than reEPO and significantly influenced cellular homeostasis and the expression of apoptosis-related proteins, gene expression in EPOR signaling pathways might be differentially activated by ML1-R and reEPO. RNA-seq analysis revealed distinct gene expression profiles for reEPO- and ML1-R-treated primary cultured cortical neurons (Supplementary Fig. 2a). ML1-R was found to influence EPOR-mediated signaling pathways, with a particular impact on gene expression related to the PI3K-Akt pathway rather than the JAK-STAT pathway. Specifically, genes associated with the PI3K-Akt pathway showed greater up-regulation or down-regulation (Supplementary Fig. 2b, c). This differential modulation of EPOR-mediated pathways likely explains the superior neuroprotective effects of ML1-R, promoting cell survival without inducing cell proliferation. ML1-R differentially activates EPOR-mediated JAK2 signaling by changing the geometric structure and binding interaction of EPOR. Previously, we developed other peptides derived from the C-helix of EPO that retained the neuroprotective effects of EPO while preventing cell proliferation 34 , 35 . Among these, ML1-R was identified as a promising candidate because of its ability to activate EPOR-mediated signaling without inducing proliferation. To confirm whether ML1-R directly activates EPOR signaling, we treated HAP1 EPOR wild-type (WT) and knockout (KO) cells with either reEPO or ML1-R and assessed JAK2 phosphorylation (Fig. 4 a). Immunoblotting showed that ML1-R promoted JAK2 activation more than reEPO in HAP1 EPOR-WT cells after 15 min (Fig. 4 b). However, in HAP1 EPOR-KO cells, JAK2 phosphorylation was not observed following reEPO or ML1-R treatment. Thus, the neuroprotective effects of ML1-R are induced by activating the JAK2 pathway downstream of EPORs. To investigate the structural differences contributing to JAK2 activation, we performed in-silico modeling and molecular dynamics (MD) simulations to compare the predicted structures of the EPO–EPOR and ML1-R–EPOR complexes. To investigate the structural changes in EPOR, we used AlphaFold3 to model these complexes during signaling (Fig. 4 c-g). The predicted binding sites matched those in the known EPO–EPOR crystal structure (PDB ID: 1EER) 37 , thus validating the accuracy of the models (Fig. 4 h). Dimerization is mainly mediated by the transmembrane domain of the EPOR and its membrane-spanning leucine zipper 38 . To investigate the structural changes in ligand–EPOR complexes, we targeted L251 located in the upper region, L263 in the middle region, and L272 in the lower region of the EPOR transmembrane domain and measured the distances between these residues. In the EPO-bound model, distances were 9.1, 8.1, and 11.9 Å. They increased in the ML1-R-bound model to 11.0, 11.7, and 12.2 Å (Fig. 4 i, j). These findings suggest that ML1-R binding results in a greater separation of transmembrane helices, indicating a weaker binding affinity. During a 1,000 ns simulation, EPO and ML1-R induced opposing distance patterns, suggesting distinct structural effects (Fig. 4 k). These results indicate that the weaker binding affinity of ML1-R may contribute to structural changes in EPOR, potentially influencing differential signaling and neuroprotective effects. Further MD simulations over 1,000 ns showed that the ML1-R–EPOR complex exhibited greater structural variability with higher root-mean-square deviation (RMSD) values than the EPO–EPOR complex. Structural changes were analyzed in 41 residues from transmembrane and juxtamembrane EPOR domains, and simulations were conducted to observe structural changes in the receptor (Fig. 5 a). Over the 1,000 ns simulation, the RMSD values of the overall structure of the ML1-R complex was more than twice as high as that of the EPO complex (Fig. 5 b). Moreover, the RMSD values of the key residues involved in JAK2 activation (L278, I282, and W283) in the ML1-R complex were higher than those in the EPO complex (Fig. 5 c-e). These results indicate that the key residues involved in JAK2 activation are structurally more unstable in the ML1-R–EPOR complex than in the EPO–EPOR complex. Root-mean-square fluctuation (RMSF) analysis of the BOX1 motif, which is the JAK2 binding site, showed distinct fluctuation patterns for ML1-R and EPO. RMSF analysis revealed that one side exhibited similar behavior for ML1-R and EPO (Fig. 5 f), whereas the other side exhibited reduced fluctuations in the ML1-R complex (Fig. 5 g). The MD simulation results confirmed that when ML1-R binds to EPOR, it may partially bind and induce conformational changes, resulting in differential JAK2 activation. In the activated EPOR, the switch tryptophan residue W283 shows a distance of 45 Å when forming a dimer 39 . To confirm the distance change, we measured simulated distances between the Cα carbons of G285 located in the central BOX1 motif sequence (Fig. 4 d) when EPO and ML1-R are bound. EMP1, an EPO-agonistic peptide, has been confirmed experimentally 40 . Therefore, we predicted ligand–EPOR complexes and conducted 100 ns simulations to measure the distances of G285 in three models: EMP1, EPO, and ML1-R. The ML1-R complex consistently maintained a shorter G285 distance (~ 35 Å), compared to 40–45 Å in EPO and EMP1 complexes (Fig. 5 h), suggesting that ML1-R does not achieve the fully active conformation associated with JAK2 activation. This trend continued over 1,000 ns, with the G285 distance for the ML1-R complex decreasing after 500 ns (Fig. 5 i). These results suggest that ML1-R induces conformational EPOR changes that differ from EPO-induced changes, particularly at the BOX1 motif, potentially differentially influencing JAK2 activation and signaling. ML1-R activates EPOR-mediated signaling pathways differently from reEPO. As the abovementioned data raise the possibility of differential activation patterns for ML1-R and reEPO in EPOR downstream signaling, we examined how reEPO and ML1-R activate EPOR signaling at the same molecular concentration (1 nM) using western blotting (Fig. 6 a). Treatment with reEPO rapidly promoted the upregulation of phosphorylated JAK2 (pJAK2) at 15–30 min and returned it to basal level at 60–90 min. ML1-R also increased the pJAK2 levels but at a lower intensity and more prolonged than reEPO (Fig. 6 b). These data suggest that reEPO and ML1-R exhibit differential kinetics in the activation of JAK2 signaling. Next, we confirmed the activation patterns of Akt and Erk, which are downstream of the JAK2 pathway and may contribute to the neuroprotective effects of reEPO and ML1-R. Whereas reEPO gradually upregulated phosphorylated Akt (pAkt) levels up to 60 min with a decrease at 90 min, ML1-R induced a different pattern, with a faster increase in Akt phosphorylation by 30 min, which began to decrease at 45 min (Fig. 6 c). Both reEPO and ML1-R induced an upregulation of phosphorylated Erk (pERK) within 15 min; however, the pErk levels induced by ML1-R were higher than those induced by reEPO. The pErk levels time-dependently decreased toward baseline (Fig. 6 d). Additionally, we examined the concentration-dependent activation by reEPO and ML1-R (Supplementary Fig. 3a). With increased concentrations, ML1-R promoted Akt and Erk phosphorylation more strongly than reEPO, showing a saturation effect at a concentration of 1 nM (Supplementary Fig. 3b, c). Previous reports demonstrated that the Akt and ERK pathways are important for EPO-mediated protection and proliferation 22 , 23 , 24 . To examine the involvement of ML1-R in the activation of Akt or ERK signaling pathways, we pretreated cells with LY294002 and U0126, which are specific inhibitors of PI3K and MEK, respectively (Fig. 6 e, h). LY294002 and U0126 efficiently blocked the upregulation of pAkt and pErk by reEPO (Fig. 6 f, g) and ML1-R (Fig. 6 i, j). To determine whether ML1-R activates Akt and Erk pathways, we treated HAP1 EPOR-WT and HAP1 EPOR-KO cells with reEPO or ML1-R and checked the patterns of Akt and Erk phosphorylation (Supplementary Fig. 4a). Similar to the results above, ML1-R activated Akt and Erk to a greater extent than reEPO in HAP1 EPOR-WT cells at 15 min, whereas Akt and Erk activation by reEPO and ML1-R were not observed in HAP1 EPOR-KO cells (Supplementary Fig. 4b, c). Furthermore, the neuroprotective effects of reEPO and ML1-R against hypoxia–reoxygenation-induced cell death were significantly inhibited by LY294002 and U0126 (Supplementary Fig. 5a, b). These data suggest that ML1-R induces differential activities in EPOR-mediated signaling pathways compared with reEPO due to changes in binding interactions and geometric structures of the ligand-binding site in the EPOR. ML1-R may promote EPOR downstream signaling by long-lasting EPOR expression in the plasma membrane. EPO-induced EPOR activation is transient because EPO-bound EPORs are rapidly internalized and undergo degradation or recycling for EPOR de-novo synthesis 41 . This suggests that the binding interaction and geometric structural changes of the EPO–EPOR and ML1-R–EPOR complexes may differentially influence the physiological turnover or recycling of EPORs. To demonstrate changes in EPOR expression induced by reEPO and ML1-R, we performed RT-qPCR and immunoblot analyses to determine the total EPOR mRNA (Fig. 7 a) and protein (Fig. 7 b) levels. Following reEPO treatment, total EPOR mRNA levels were reduced by 15 min and recovered after 30 min (Fig. 7 a), and total EPOR protein levels underwent turnover by degradation and synthesis (Fig. 7 b and Supplementary Fig. 6a). Surprisingly, following ML1-R treatment, total EPOR mRNA levels were increased by 30 min (Fig. 7 a), and total EPOR protein levels were maintained for 30–60 min (Fig. 7 b and Supplementary Fig. 6a). Given that EPO-stimulated EPOR internalization and degradation depend on JAK2 activation, these results indicate that ML1-R promotes the upregulation of EPOR expression through long-lasting JAK2 activation, resulting in the maintenance of EPOR levels in the PM. To investigate the turnover of EPORs in the PM following treatment with reEPO and ML1-R, we isolated PM fractions and extracted PM proteins (Fig. 7 c). Following reEPO treatment, the level of EPORs at the cell surface was gradually reduced for 30 min, recovered to basal levels at 60 min, and decreased at 90 min (Fig. 7 d). Interestingly, ML1-R delayed the internalization of EPORs for 60 min and restored the basal level of EPOR expression (Fig. 7 d), whereas Na+/K + ATPase expression remained unchanged (Fig. 7 e). To further examine EPOR expression on the cell surface, we assessed EPOR levels in the PM using a cell-surface biotinylation assay (Supplementary Fig. 7a, b). Similar to the results of PM protein extraction, ML1-R induced delayed EPOR endocytosis for 30 min, which subsequently returned to basal EPOR levels without changes in Na+/K + ATPase levels (Supplementary Fig. 7c, d). According to the immunoblot results, the differences in EPOR levels in the PM between ML1-R and reEPO treatment remained mostly consistent, suggesting that ML1-R may influence EPOR expression or EPOR trafficking. Taken together, our data suggest that ML1-R increases EPOR expression through the EPOR signaling pathway, thereby increasing the amount of EPOR available in the PM to enhance EPO-mediated activation of signaling pathways, which in turn exhibits neuroprotective effects via positive feedback (Fig. 8 ). Thus, ML1-R has the potential as a neuroprotective drug for treating reperfusion injury in ischemic stroke or neurodegenerative diseases. Discussion Using in-vivo and in-vitro systems, we investigated the neuroprotective effects of the partial EPOR agonist ML1-R in comparison with those of reEPO against ischemic stroke and reperfusion brain injury. We previously reported novel EPO-derived small peptides, MK-X and ML1-h3, which exert neuroprotective effects against oxidative stress resulting from glutamate-induced excitotoxicity and hypoxia–reoxygenation without inducing unwanted effects, including cell proliferation 34 , 35 . ML1-R, an ML1 analog based on the C-helix of EPO with low affinity to EPORs 34 , 35 , 36 , is a modified peptide in which leucine residues at both ends of an ML1 subsequence have been substituted with arginine. Because the affinity of small peptides for EPOR can influence various EPOR-mediated effects, including erythropoiesis, cell proliferation, and cell protection, it is important to adjust the binding affinity of modified peptides to EPOR 32 , 33 , 34 , 35 , 36 . Our in-silico results predictions using AlphaFold3 showed that ML1-R has a lower EPOR affinity than EPO, causing changes in the geometry and structure of ML1-R–EPOR complexes. ML1-R may induce geometric changes in the intracellular domains of EPOR known as the JAK2-binding BOX1 motif, potentially leading to differences in EPOR downstream activation. In our previous report, it was unclear how ML1-h3 binding induces geometric changes in full-length EPORs because the structural biological analysis focused only on the extracellular domains of EPORs 35 , 36 . In this study, we analyzed structural changes in full-length EPORs using computational prediction models. Moreover, we observed alterations in JAK2, Akt, and ERK activation. These results allowed us to elucidate the detailed mechanisms underlying the neuroprotective ML1-R effects. ML1-R induced delayed and prolonged JAK2 activation and activated Akt and ERK phosphorylation more strongly and rapidly than reEPO, promoting EPOR production and increased EPOR expression in the PM. Thus, the differential effects of ML1-R on EPOR downstream signaling may be due to geometric alterations in the ML1-R–EPOR complex. Both reEPO and ML1-R inhibited intracellular calcium overload following glutamate-induced excitotoxicity (Fig. 2 b). EPO modulates intracellular calcium levels through the regulation of several calcium-permeable ion channels 42 , 43 , 44 , and we speculated that ML1-R causes similar effects. In our study, the reduction in glutamate-induced intracellular calcium overload by ML1-R was comparable to that observed with reEPO. However, ML1-R more effectively attenuated ROS overproduction caused by glutamate-induced excitotoxicity and hypoxia–reoxygenation (Fig. 2 c and Fig. 3 b). Therefore, the greater increase in cell viability with ML1-R compared to that with reEPO was likely due to the mitigation of ROS generation and mitochondrial dysfunction, which may have contributed more than the alleviation of calcium overload. EPO has well-known anti-apoptotic effects by promoting anti-oxidant synthesis, thereby maintaining the mitochondrial membrane potential 45 . We presume that these combined ML1-R effects contribute to increased cell viability. Various studies have shown that the EPO–EPOR signaling pathway leads to changes in apoptosis markers 46 , 47 , 48 . Our study confirmed these findings for reEPO, with even greater changes observed with ML1-R. Under glutamate-induced excitotoxicity and hypoxia–reoxygenation conditions, the changes in Bax (Fig. 2 g and Fig. 3 f) and cleaved Parp1 (Fig. 2 j and Fig. 3 i) levels were inconsistent; however, the alterations in Bcl-2 (Fig. 2 h and Fig. 3 g) and cleaved Casp3 (Fig. 2 i and Fig. 3 h) expression were consistently stronger with ML1-R than with reEPO. Thus, the changes in Bcl-2 and cleaved Casp3 may contribute more strongly to apoptosis mitigation than the changes in Bax and cleaved Parp1, and the partial binding interaction between ML1-R and EPOR may play a crucial role in producing effects distinct from those of reEPO. Recently, various studies on non-hematopoietic EPO effects have been conducted in various organs, including the central nervous system. In the brain, most cells, including neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells forming the blood–brain barrier, express EPORs 4 . This raises the question of the role of EPO and EPOR in physiological brain processes, as hematopoiesis and proliferation should not occur in the brain. Our study findings confirmed that the total EPOR level exhibited a cyclic pattern of degradation and subsequent production, depending on the duration of reEPO treatment. In contrast, ML1-R treatment favored EPOR production over degradation (Fig. 7 b, c). The increased EPOR expression can be attributed to enhanced production, as indicated by the upregulation of EPOR mRNA expression (Fig. 7 a). However, this is likely the result of various factors, including recycling and reuse of EPORs or reduced EPOR degradation, making it difficult to identify the exact cause. Moreover, partial binding of ML1-R to EPOR may result in delayed and prolonged JAK2 activation, and the cumulative effects of increased EPOR production may lead to stronger activation of various EPOR downstream molecules. We speculate that EPO and EPOR in the brain play crucial roles in enhancing cellular protective effects through EPOR signaling during acutely dangerous conditions like ischemic stroke. This suggests that a positive feedback mechanism may be activated to increase EPOR expression in the PM to promote neuroprotective effects. The EPO activity in diverse tissues suggests that other receptors may interact with EPORs. The erythropoietic activity of EPO is mediated by homodimeric receptors 37 , whereas EPO activity in non-erythroid tissue is mediated by a heterodimer consisting of one EPOR monomer and one cytokine β−common subunit (CD131) involved in protection against inflammation and tissue injury 49 . We hypothesized that ML1-R interacts with the EPOR dimer and performed a modeling analysis using AlphaFold3. Previous research confirmed that ML1 sequence regions show weak binding affinity for the EPOR dimer 35 , and in-silico predictions for ML1-R indicated the possibility of partial binding affinity to EPOR. As the EPOR and cytokine βcommon subunit form a heterodimer leading to tissue-protective effects 3 7 , we anticipate that ML1-R interacts with one EPOR and another receptor. Related experimental results will be presented in a future study. In conclusion, we suggest that the partial EPO agonist ML1-R is a promising candidate for neuroprotection, resulting from the alleviation of brain injury caused by ischemic stroke and reperfusion. Research on known EPO derivatives, including ML1-R, has the potential for the development of safer and more effective drugs for various diseases. Therefore, we expect that the neuroprotective effects of ML1-R and its role in counteracting oxidative stress have potential benefits in the treatment of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Methods MCAO and reperfusion The study protocol was approved by the Animal Care and Use Committee of Keimyung University School of Medicine. Briefly, 24 C57/BL6 mice (8 weeks old) were randomly divided into experimental (reEPO: n = 8, ML1-R: n = 8) or vehicle (n = 8) groups and anesthetized by inhalation of 1.5–2.0% isoflurane in a mixture of 70% N 2 O and 30% O 2 . After dissecting the neck skin, the bifurcation of the common carotid artery was exposed. The arteries were separated from the surrounding nerves and connective tissue, and the right external carotid artery was ligated by surgical nylon monofilament insertion. The rectal temperature was maintained at 37.0 ± 0.5°C intraoperatively using a controlled heating pad. At the end of the ischemic period, the filament was removed, and reperfusion was performed with intravenous injection of reEPO (2,000 IU/kg in 0.3 ml saline), ML1-R (0.3 mg/kg in 0.3 ml saline), or vehicle (0.3 ml saline). Animals from each group were sacrificed at the end of the reperfusion period (24 h after reperfusion following MCAO), and their brains were collected for histochemical staining to assess the neuroprotective effects of reEPO and ML1-R. Evaluation of brain injury The brains were rapidly dissected. Coronal sections were cut into 2-mm thick slices. Subsequently, they were stained with 2% TTC in saline for 20 min at 37°C. The infarcted and non-infarcted areas of the TTC-stained brain sections were imaged using a digital camera. The TTC intensity of each hemisphere was analyzed using ImageJ (NIH, Bethesda, MD, USA), and the ratio (MCAO versus non-MCAO) is presented as a percentage. Rat primary cortical neuron culture Embryonic cerebral cortices were obtained from pregnant Sprague–Dawley rats on embryonic day 17. Cerebral cortices were treated with 0.25% trypsin-EDTA (#25200056, Gibco, USA) and chemically and mechanically dissociated into single cells by pipetting. The dissociated cells were plated on plates or coverslips coated with 50 µg/ml poly-D-lysine (P8920, Sigma-Aldrich, St. Louis, MO, USA). They were incubated in neurobasal medium (#21103049, Gibco) containing 2% B27 supplement (#17504044, Gibco), 0.5 mM L-glutamine (#15430614, Gibco), 25 µM glutamic acid (#G1251, Sigma-Aldrich), and 1% penicillin/streptomycin (#15140122, Gibco) at 37°C with 5% CO 2 . The medium was replaced with fresh medium without glutamate every 3–4 days. On day 10, cortical neurons were treated with reEPO, or ML1-R under control, glutamate excitotoxicity, and hypoxia–reoxygenation conditions. Cell culture HAP1 cells are a near-haploid human cell line derived from the KBM-7 chronic myelogenous leukemia cell line. The HAP1 parental cell line (#C631, Horizon Discovery, UK) and HAP1 EPOR-KO cell line (#HZGHC003882c008, Horizon Discovery) were cultured at 37°C and 5% CO 2 in Iscove’s modified Dulbecco’s medium (#12440, Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. Hypoxia and reoxygenation Primary rat cortical neurons were seeded into 96-well plates. The next day, the cultured cortical neurons were placed inside a sealed, airtight container consisting of anaerobic atmosphere generation bags (#68061, Sigma-Aldrich), which can induce a hypoxic atmosphere by absorbing oxygen and generating carbon dioxide within 2.5 h. The primary cortical neurons were maintained under hypoxic conditions at 37°C for 18 h and reoxygenated for 4 h at 37°C and 5% CO 2 . Subsequently, the cultured cortical neurons were treated with reEPO or ML1-R for 18 h before viability analysis. Cell viability To examine the viability of rat primary cortical neurons following the treatment with 30 µM glutamate or hypoxia–reoxygenation, we used calcein-AM (#C3100MP, Invitrogen). Cultured rat cortical neurons were plated in 96-well plates (2.5×10 4 cells per well) and incubated with calcein-AM (3 µM) for 30 min after various treatments. The intensity of the calcein-AM signal was measured using a microplate reader (Ex/Em = 485/535 nm; VersaMax, Molecular Devices, USA). Measurement of calcium concentration, ROS production, and mitochondrial membrane potential To characterize glutamate-induced intracellular calcium increases, calcium levels were observed for 15 min in the presence of ML1-R or reEPO. To quantify intracellular calcium levels, cultured cortical neurons were stained using the Fluo-4 NW Calcium Assay Kit (#F36206, Molecular Probes, USA). Cortical neurons were grown in 96-well plates (3×10 4 per well) for 10 days, then washed with phosphate-buffered saline (PBS) and loaded with 100 µl Fluo-4 dye in assay buffer (Hank’s balanced salt solution and 20 mM HEPES) for 45 min at 37°C with 5% CO 2 . Intracellular calcium levels in the presence of 30 µM glutamate were recorded (Em = 516 nm; VersaMax). Cellular ROS levels were measured using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA; #D399, Invitrogen). Cultured rat cortical neurons plated into 96-well plates (2.5×10 4 cells per well) were incubated with 5 µM H2DCFDA in PBS for 30 min, washed with PBS, and then resuspended in medium for 2 h. After treatment with glutamate (30 µM) or hypoxia–reoxygenation in the presence or absence of reEPO or ML1-R, we monitored the oxidative stress by measuring H2DCFDA fluorescence intensities using a microplate reader (Ex/Em = 485/535 nm; VersaMax). The ROS levels of reEPO and ML1-R were observed after 6 h. Mitochondrial membrane potential was measured using the TMRE-Mitochondrial Membrane Potential Assay Kit (#ab113852, Abcam, Cambridge, UK). Primary cultured cortical neurons plated into 96-well plates (4×10 4 cells per well) were first incubated with culture medium containing 200 nM TMRE for 30 min at 37°C, then rinsed three times with PBS and incubated with PBS/0.2% bovine serum albumin (BSA). The fluorescence signal was quantified using a microplate reader (Ex/Em = 549/575 nm; VersaMax). Immunoblotting and antibodies The cells were lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholic acid, and 0.1% SDS) on ice for 30 min. The lysates (20 µg of protein) were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8–10%), and the proteins were transferred onto polyvinylidene difluoride membranes. The membrane was blocked with 0.1% TBST and 5% non-fat dry milk at 25°C for 30 min. The primary antibodies diluted in 0.1% TBST and 3% BSA included anti-pJAK2 (#3776), anti-JAK2 (#3230), anti-pAKT (#9271), anti-AKT (#9272), anti-pERK (#9101) anti-ERK (#9102), anti-Bax (#2772), anti-cleaved Casp3 (#9661), anti-cleaved Parp1 (#5625, all Cell Signaling Technology), anti-EPOR (#sc-365662), anti-Bcl2 (#sc-783, both Santa Cruz Biotechnology, USA), anti-GAPDH (#MAB374, Merck Millipore Biotechnology, USA), and anti-Na+/K + ATPase (#ab7671, Abcam). After several washes, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies. The proteins were visualized using an enhanced chemiluminescence substrate kit (Pierce, Rockford, IL, USA). Blots were scanned and quantified using ImageJ software. Hoechst staining assay Hoechst 33342 staining was performed to detect apoptosis. Cultured cortical neurons were plated onto a microscope cover glass (5×10 4 cells per well) and allowed to attach for 24 h. The next day, the neurons were maintained in hypoxic conditions at 37°C for 18 h and reoxygenated for 4 h at 37°C and 5% CO 2 . Subsequently, the cultured cortical neurons were treated with reEPO or ML1-R for 18 h before viability analysis. The cells were fixed in freshly prepared 4% paraformaldehyde for 30 min and permeabilized in PBS containing 0.1% Triton X-100 and 2% BSA for 5 min. After washing, the cells were blocked with PBS containing 1% BSA for 1 h and then incubated with a NeuN antibody (#ab7671, Abcam) for 2 h in blocking buffer to detect cortical neurons. Following washing with PBS, cells were incubated with goat anti-rabbit conjugated with Alexa Fluor 488 (#A11008, Invitrogen) secondary antibodies in blocking buffer at 4°C for 16 h. Cells were then washed with cold PBS again before incubation with 5 µg/ml Hoechst 33342 (#H3570, Invitrogen) for 15 min at 37°C in the dark. Finally, the cells were washed with PBS and mounted using mounting medium (#F6182; Sigma-Aldrich). Apoptotic cells were identified using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Plasma membrane protein extraction PM fractions were isolated using a PM protein extraction kit (#ab65400, Abcam), according to the manufacturer’s instructions. Primary cortical neurons (5×10 8 cells) cultured on 100 mm dishes were rinsed twice with ice-cold PBS, immediately incubated with 2 ml of homogenized buffer on ice, and harvested using a cell scraper. Resuspended cells were homogenized in a Dounce homogenizer for 30–50 times on ice and centrifuged at 700× g for 5 min at 4°C. The supernatants were subsequently centrifuged at 10,000× g for 30 min at 4°C. The pellet and supernatant contained the total cellular membrane and cytosolic proteins, respectively. The pellet was resuspended in 200 µl of upper-phase solution and then mixed with 200 µl of lower-phase solution. The samples were incubated on ice for 5 min and centrifuged at 1,000× g for 5 min at 4°C; upper- and lower-phase solutions containing PM and intracellular membrane proteins, respectively, were collected. The extractions were repeated by adding a fresh upper-phase solution to the lower-phase solution and vice versa. The resulting upper-phase solutions from repeated extractions were combined and diluted with 5× volume of water, followed by centrifugation at 15,000× g for 30 min at 4°C. Pellets containing PM proteins were harvested and analyzed using immunoblotting. RNA extraction and RT-qPCR Total RNA was extracted from primary cultured cortical neurons using the RNeasy Plus Mini Kit (#74136, Qiagen, Germany) following the manufacturer’s protocol. mRNA was reverse-transcribed to cDNA using SuperScript IV Reverse Transcriptase (#18090010, Thermo Fisher Scientific). We performed PCRs using Herculase II Fusion DNA Polymerase (#600677, Agilent Technologies). Primers of the following sequences were obtained from Bionics (Daejeon, Republic of Korea): EPOR (forward: CTA TGG CTG TTG CAA CGC GA and reverse: CCG AGG GCA CAG GAG CTT AG) and β-actin (forward: ATC GTG GGC CGC CCT AGC ACC and reverse: CTC TTT AAT GTC ACG CAC GAT TTC). The housekeeping gene β-actin was used as an internal standard. AlphaFold-based structural models The protein sequences for EPOR (P19235) and EPO (P01588) were obtained from the UniProt database 50 . The peptide sequences used in the study were EMP1 (GGTYSCHFGPLTWVCKPQGG) and ML1-R (RHVDKAVSGLRSLTTR). The structural models of EPO, peptides, and EPO–EPOR-peptide complexes were predicted using AlphaFold3 51 . The models generated by AlphaFold3 were further refined and used in subsequent MD simulations. MD simulations The membrane-protein system was constructed using the CHARMM-GUI Membrane Builder 52 , 53 , 54 , 55 . A pre-equilibrated lipid bilayer composed of a lipid mixture including cholesterol was generated using default parameters. The proteins obtained from AlphaFold3 were inserted into the membrane based on the default orientation suggested in the OPM 2.0 database 56 . The system was solvated with TIP3P water molecules, and ions were automatically added to neutralize the system and achieve a physiological ionic strength of 0.15 M NaCl using the default ion placement method of CHARMM-GUI. Input files for MD were generated using GROMACS (version 2023.3) 54 with the default settings provided by CHARMM-GUI. MD simulations were carried out using GROMACS with 2.0 fs time steps. The production run was conducted for 100 ns, corresponding to 50 million steps. Trajectory snapshots were recorded every 100 ps, yielding 1,000 frames for the post-simulation analysis. The system was first subjected to energy minimization, followed by equilibration in two phases: constant number of particles, volume, and temperature and constant number of particles, pressure, and temperature. The constant number of particles, pressure, and temperature ensemble was used for the production run at a temperature of 310 K and pressure of 1 bar, employing a V-rescale thermostat and a Parrinello–Rahman barostat for temperature and pressure coupling, respectively. Subsequent analysis of the simulation trajectory was performed using standard GROMACS tools to evaluate the structural and dynamic properties of the protein-lipid system. RMSD, RMSF, and radial distribution functions were calculated to assess stability and interactions within the membrane. Visualization and additional analyses were conducted using VMD 55 and PyMOL 57 to gain structural insights. Quantification and statistical analysis Statistical information is provided in the figure legends. All experiments were performed at least three times using independent cells or animal preparations. Repeats for experiments or cell numbers are given in the figure legends as “n.” Error bars represent ± standard error. To measure statistical significance, we performed one-way ANOVA on ranks using Dunnett’s method or two-way ANOVA followed by Tukey’s multiple comparison test. p < 0.05 was considered significant. All statistical analyses and graph plots were generated using GraphPad Prism software. Declarations Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1I1A3055783; RS-2023-00239274; RS-2024-00354916; 2020R1A6A1A03040516). Author contributions O.H.K, S.J.Y, S.R.L, and C.M. conceived the project and designed the project. J.C.B, and H.J.J. performed middle cerebral artery occlusion and TTC staining. J.S.B. conducted the structural studies and MD simulation using AlphaFold3 and assisted with the RNA-seq studies and bioinformatics analysis. O.H.K. performed all of in-vitro experiments and wrote the manuscript. All authors read and approved the final version of the manuscript. Competing interests Authors declare that they have no competing interests. Additional information Supplementary informationis available for this paper. Data and materials availability The data that support this study are available from the corresponding authors upon reasonable request. A Source Data File is provided. Raw and processed data are available via the Dryad with DOI: 10.5061/dryad.66t1g1k9z. References Bunn HF, Erythropoietin (2013) Cold Spring Harb Perspect Med 3:a011619 Suresh S, Rajvanshi PK, Noguchi CT (2019) The Many Facets of Erythropoietin Physiologic and Metabolic Response. Front Physiol 10:1534 Noguchi CT, Asavaritikrai P, Teng R, Jia Y (2007) Role of erythropoietin in the brain. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5779616","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398749958,"identity":"2e85c9e1-f4cd-4b1a-9d92-d52304a8fb3b","order_by":0,"name":"Oh-Hoon Kwon","email":"","orcid":"","institution":"Convergence Research Advanced Centre for Olfaction, Daegu Gyeongbuk Institute of Science and Technology (DGIST)","correspondingAuthor":false,"prefix":"","firstName":"Oh-Hoon","middleName":"","lastName":"Kwon","suffix":""},{"id":398757200,"identity":"0642b797-d19b-4520-9830-d0f2af366585","order_by":1,"name":"Jinsik 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Moon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYNCCCmYGNijTgEgtZ0BamEnRwtgGUk6sFv5phw8+LpxnbdfHf/4Aw48aBmPzBgJaJG6nJRvP3Jae3CaRzMDYc4zBTOYAAS0G0jlm0rzbDiezSQAdxtvAYCNByGEQLXOAWvgPMzD+JV5Lw2E7NoZkBmagLWYEtYD9wnMsPYFNItngsMwxCWOCWvhnJx98zFNjbS/ff/Dhwzc1NoYzCGmBgcQGIHEAaCuxGhgY7IlXOgpGwSgYBSMOAAAlEjHe6G3XaQAAAABJRU5ErkJggg==","orcid":"","institution":"Convergence Research Advanced Centre for Olfaction, Daegu Gyeongbuk Institute of Science and Technology (DGIST), DGIST","correspondingAuthor":true,"prefix":"","firstName":"Cheil","middleName":"","lastName":"Moon","suffix":""}],"badges":[],"createdAt":"2025-01-07 08:54:48","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5779616/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5779616/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73247617,"identity":"c0c36ba6-d9a5-4f17-9003-9efd091b7728","added_by":"auto","created_at":"2025-01-08 07:22:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1037706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective effects of reEPO and ML1-R against brain injury in murine stroke models and hypoxia––reoxygenation injury of cultured cortical neurons. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic diagram of the murine MCAO-reperfusion model with the injection of vehicle, reEPO, or ML1-R. (\u003cstrong\u003eb\u003c/strong\u003e) Representative images of TTC-stained brain sections in mice following unilateral MCAO and reperfusion with the injection of vehicle, reEPO (2,000 IU/kg), or ML1-R (0.3 mg/kg). MCAO unilaterally affects the left brain hemisphere (scale bar: 1 mm). (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of TTC stainings in MCAO-affected versus contralateral (non-MCAO) hemispheres in vehicle-, reEPO-, and ML1-R-injected mice (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, mean ± SEM, two-way analysis of variance (ANOVA) with Tukey’s test, n=8). (\u003cstrong\u003ed\u003c/strong\u003e) Schematic diagram of hypoxia induction and reoxygenation in cultured neurons with or without reEPO (0.5 IU/ml) or ML1-R (1 ng/ml) administration. (\u003cstrong\u003ee\u003c/strong\u003e) Representative images indicating apoptosis in Hoechst-stained primary cultured cortical neurons after hypoxia–reoxygenation with or without reEPO or ML1-R treatment (scale bars: 20 μm). (\u003cstrong\u003ef\u003c/strong\u003e) Ratio of condensed cells to total cells in cultured neurons (*p\u0026lt;0.05, **p\u0026lt;0.01, ****p\u0026lt;0.0001, mean ± SEM, two-way ANOVA with Tukey’s test, n=9).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/838cd2df025553657dec7bc0.png"},{"id":73247601,"identity":"87037b2d-21b1-43f4-96bd-573f46a43a2e","added_by":"auto","created_at":"2025-01-08 07:22:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":321864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential effects of reEPO and ML1-R on calcium overload, ROS production, mitochondrial dysfunction, viability, and apoptosis caused by glutamate-induced excitotoxicity. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic diagram of glutamate-induced excitotoxicity in primary cultured cortical neurons in the presence or absence of reEPO (0.5 IU/ml unless mentioned otherwise) and ML1-R (1 ng/ml). (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of intracellular calcium levels. Cells were incubated with 30 μM glutamate for 3 min in the presence or absence of reEPO or ML1-R. (\u003cstrong\u003ev\u003c/strong\u003e) Quantification of intracellular ROS production under glutamate-induced stress in primary cultured cortical neurons for 6 h with or without reEPO or ML1-R. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of tetramethylrhodamine ethyl ester perchlorate (TMRE) intensity. Cells were incubated with 30 μM glutamate for 5 h with or without reEPO or ML1-R. (\u003cstrong\u003ee\u003c/strong\u003e) Viability of cultured primary cortical neurons after glutamate treatment for 24 h with or without reEPO or ML1-R. (\u003cstrong\u003ef\u003c/strong\u003e) Immunoblots of Bax, Bcl-2, cleaved Casp3, and cleaved Parp1 in cultured primary cortical neurons after glutamate treatment for 24 h with or without reEPO or ML1-R. (\u003cstrong\u003eg–j\u003c/strong\u003e) Relative Bax, Bcl-2, cleaved Casp3, and cleaved Parp1 levels normalized to GAPDH levels (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, mean ± SEM, ns: non-significant, two-way ANOVA with Tukey’s test, n=5).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/f0fce092745049df525dca05.png"},{"id":73248988,"identity":"d6b0a2c5-481d-4c4f-aae4-b9357a85384d","added_by":"auto","created_at":"2025-01-08 07:30:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":347220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential effects of reEPO and ML1-R on hypoxia–reoxygenation-induced ROS production, mitochondrial dysfunction, viability, and apoptosis. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic diagram of hypoxia–reoxygenation induction in primary cultured cortical neurons with or without reEPO (0.5 IU/ml unless mentioned otherwise) or ML1-R (1 ng/ml) treatment. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of intracellular ROS production in primary cultured cortical neurons following hypoxia–reoxygenation for 6 h with or without reEPO or ML1-R. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of TMRE intensity with or without reEPO or ML1-R under the hypoxia–reoxygenation. (\u003cstrong\u003ed\u003c/strong\u003e) Viability of cultured primary cortical neurons after hypoxia–reoxygenation for 18 h with or without reEPO or ML1-R. (\u003cstrong\u003ee\u003c/strong\u003e) Immunoblots of Bax, Bcl-2, cleaved Casp3, and cleaved Parp1 in cultured primary cortical neurons after hypoxia–reoxygenation with or without reEPO (1 nM) or ML1-R. (\u003cstrong\u003ef–i\u003c/strong\u003e) Relative Bax, Bcl-2, cleaved Casp3, and cleaved Parp1 levels normalized to GAPDH levels (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, mean ± SEM, ns: non-significant, two-way ANOVA with Tukey’s test, n=5).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/39c64d7d6b28d60f4ad024e0.png"},{"id":73247619,"identity":"335602c8-c0cf-4bc6-8aa5-dc30722e05aa","added_by":"auto","created_at":"2025-01-08 07:22:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2577414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJAK2 activation by reEPO and ML1-R and computational prediction of ligand–EPOR complexes. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Immunoblots of JAK2, Akt, and Erk activation by reEPO and ML1-R in HAP1 EPOR-WT and HAP1 EPOR-KO cells. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of pJAK2 in HAP1 EPOR-WT and HAP1 EPOR-KO cells (**p\u0026lt;0.01, mean ± SEM, two-way ANOVA with Tukey’s test, n=4). (\u003cstrong\u003ec, d\u003c/strong\u003e) EPOR structure obtained using AlphaFold3 showing the extracellular (cyan), transmembrane (orange), and intracellular (magenta) domains. The protein sequence uses the same color scheme. Additionally, the BOX1 domain, which is the JAK2 binding site, is highlighted in red. (\u003cstrong\u003ee–g\u003c/strong\u003e) Structure of the EPO–EPOR complex confirmed by X-ray crystallography (PDB ID: 1EER). The complex of EPO, ML1-R, and EPOR was obtained through multimer prediction using AlphaFold3. (\u003cstrong\u003eh\u003c/strong\u003e) The binding site from 1EER with the predicted binding site in the AlphaFold3 model. (\u003cstrong\u003ei, j\u003c/strong\u003e) Ligand-EPOR complex structures obtained using AlphaFold3 showing the extracellular (cyan), transmembrane (orange), and intracellular (magenta) domains. To compare the binding distances between EPO and the peptide, we measured the distances between the L251, L263, and L272 residues of the transmembrane domain. (\u003cstrong\u003ek\u003c/strong\u003e) To obtain more detailed distance data, we examined the distances between transmembrane residues using a 1,000 ns simulation.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/4ba4a9af8ac31692e2a4a861.png"},{"id":73248989,"identity":"a6fbbc03-71be-4f45-81d1-6c2823ae4112","added_by":"auto","created_at":"2025-01-08 07:30:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":506771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMD simulation of the JAK2 activation site in EPO–EPOR and ML1-R–EPOR complexes. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) The predicted structure showing JAK2 binding to the EPO–EPOR complex, specifically binding at the intracellular membrane region. (\u003cstrong\u003eb–e\u003c/strong\u003e) Comparison of RMSD values when EPO or ML1-R bind to EPOR. EPO shows an average RMSD of approximately 0.5 nm, whereas ML1-R shows RMSD values of 1.0–1.5 nm. The RMSD values of residues L278, I282, and W283, which are critical for JAK2 activation, were also measured. (\u003cstrong\u003ef, g\u003c/strong\u003e) RMSF values of the BOX1 motif during 1,000 ns simulations were measured using Dimer. (\u003cstrong\u003eh\u003c/strong\u003e) To examine the distances between BOX1 motifs, the distance between G285 residues was measured. A 100 ns simulation was performed using EMP1, EPO, and ML1-R. (\u003cstrong\u003ei\u003c/strong\u003e) We subsequently compared the distances between G285 residues of EPO–EPOR and ML1-R–EPOR complexes during 1,000 ns simulations.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/684e1164ada95ebb4b61de93.png"},{"id":73247614,"identity":"2968aaed-9ac4-4cd2-81b6-8d931df7fb99","added_by":"auto","created_at":"2025-01-08 07:22:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":493460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential effects of reEPO and ML1-R on EPOR-mediated signaling. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Time-series immunoblots of pJAK2, JAK2, pAkt, and pErk upon reEPO or ML1-R treatment. (\u003cstrong\u003eb–d\u003c/strong\u003e) Relative pJAK2, pAkt, and pErk levels normalized to JAK2, total Akt (tAkt), and total Erk (tErk) levels, respectively (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, mean ± SEM, one-way ANOVA with Dunnett’s test, n=5). (\u003cstrong\u003ee\u003c/strong\u003e) Immunoblots of reEPO-induced Akt and Erk activation in the presence or absence of LY294002 (50 mM) or U0126 (20 mM). (\u003cstrong\u003ef, g\u003c/strong\u003e) Relative pAkt and pErk levels normalized to tAkt and tErk levels, respectively (*p\u0026lt;0.05, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, mean ± SEM, ns: non-significant, two-way ANOVA with Tukey’s test, n=5). (\u003cstrong\u003eh\u003c/strong\u003e) Immunoblots of ML1-R-induced Akt and Erk activation in the presence or absence of LY294002 (50 mM) and U0126 (20 mM). (\u003cstrong\u003ei, j\u003c/strong\u003e) Relative pAkt and pErk levels normalized to tAkt and tErk levels, respectively (**p\u0026lt;0.01, ****p\u0026lt;0.0001, mean ± SEM, ns: non-significant, two-way ANOVA with Tukey’s test, n=5).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/8076f684d496326ba582bb8f.png"},{"id":73247615,"identity":"d01a7e03-68fb-4056-a24a-0d5c36f09697","added_by":"auto","created_at":"2025-01-08 07:22:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":396626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential effects of reEPO and ML1-R on EPOR expression in the PM. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Time-series RT-qPCR products of EPOR from total mRNA following treatment with reEPO or ML1-R (*p\u0026lt;0.05, one-way ANOVA with Dunnett’s test, n=5). (\u003cstrong\u003eb\u003c/strong\u003e) Relative levels of total EPOR normalized to GAPDH levels (Supplementary Fig. 6a; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, mean ± SEM, one-way ANOVA with Dunnett’s test, n=5). (\u003cstrong\u003ev\u003c/strong\u003e) Time-series immunoblots of EPOR and Na+/K+ ATPase extracted from the PM following reEPO or ML1-R treatment. (\u003cstrong\u003ed, e\u003c/strong\u003e) Relative levels of EPOR and Na+/K+ ATPase in the PM normalized to GAPDH levels (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, mean ± SEM, one-way ANOVA with Dunnett’s test, n=5).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/206ce3c1b30c56bb0b1f293e.png"},{"id":73247618,"identity":"d8325ab3-2262-4b31-94a8-5a668902d9c3","added_by":"auto","created_at":"2025-01-08 07:22:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":532147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic summarizing the differential effects of ML1-Rand reEPO treatment on neuroprotection.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/3a462ce9b6d37c938b2d392d.png"},{"id":73250265,"identity":"d8ba2464-ed36-40d8-88e4-5a3069f90d38","added_by":"auto","created_at":"2025-01-08 07:47:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6891899,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/cf03eaf0-d737-41e6-834a-5d746ebe716c.pdf"},{"id":73247599,"identity":"950e4a09-a6d4-47ce-87fb-fc0ae0a30fc4","added_by":"auto","created_at":"2025-01-08 07:22:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2404445,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-5779616/v1/dd2dcd2b78353f3af4811590.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eThe partial erythropoietin receptor agonist ML1-R is a potent neuroprotective drug with a distinct signaling profile.\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eErythropoietin (EPO), a well-known hypoxia-inducible growth factor, promotes the proliferation, survival, and differentiation of erythroid progenitor cells into erythrocytes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This hematopoietic function of EPO is widely recognized; however, in other organs, including the brain, kidney, heart, and muscle, it plays nonhematopoietic roles under various stress conditions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNeurons and astrocytes also produce EPO, and the EPO receptor (EPOR) is expressed in brain tissue cells including neurons, astrocytes, microglia, and oligodendrocytes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. EPO is related to neuroprotection, neurogenesis, and regeneration. EPO gene expression is regulated by hypoxia-inducible factor-1\u003csup\u003e5, 6, 7\u003c/sup\u003e. EPO and EPOR are upregulated in the brain under pathological conditions in neurological diseases as a neuroprotective response to brain injury\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Because EPORs mediate anti-apoptotic and anti-oxidant effects, EPO has been proposed as a drug target for various neuronal diseases including ischemic stroke\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Stroke is a complex and dynamic brain disease that can lead to disability and mortality. The unique physiological properties of the brain make it extremely sensitive to blood supply changes; it is the organ with the highest energy demand in the body, making continuous oxygen and nutrient supply through blood flow crucial. Ischemic stroke, caused by occlusion of blood flow to the brain, is the most common type of stroke\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Such an occlusion disrupts the balance of oxygen and nutrients required for energy generation in the brain, resulting in the loss of physiological functions and cellular homeostasis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Ischemic conditions rapidly deplete energy, inducing an imbalance in electrochemical gradients and leading to neuronal depolarization. This neuronal depolarization triggers intracellular calcium overload, reactive oxygen species (ROS) generation, mitochondrial dysfunction, and massive glutamate-induced excitotoxicity and neuronal death\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although reperfusion to restore blood flow is very important, reperfusion also substantially upregulates oxidative stress and mitochondrial damage, resulting in potentially lethal reperfusion injury of the brain\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Thus, pharmacological alleviation of reperfusion injury is critical for the treatment of ischemic stroke. Although some drugs that inhibit glutamate release or cell death pathways have been developed, most clinical trials failed because of unexpected adverse effects. Therefore, the development of neuroprotective drugs against ischemic stroke and reperfusion brain injury is still urgently needed.\u003c/p\u003e \u003cp\u003eProtective EPO effects involve the activation of multiple downstream signaling pathways. When EPO binds to EPORs, their interaction induces the autophosphorylation of Janus kinase 2 (JAK2), which subsequently activates the signal transducer and activator of transcription 5 (STAT5), protein kinase B (Akt), and extracellular signal-regulated kinases 1/2 (Erks) pathways, which are involved in cell survival and proliferation\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These pathways induce anti-oxidative, anti-cytotoxic, anti-apoptotic, and anti-inflammatory effects\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThus, EPO has been suggested as a neuroprotective drug in treating neurological diseases including stroke, epilepsy, Parkinson\u0026rsquo;s disease, and Alzheimer\u0026rsquo;s disease\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, studies of using EPO for neuroprotection have shown its limitations including erythropoietic and tumorigenic adverse effects\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, EPO-based strategies that alleviate these adverse effects are required to treat ischemic stroke, reperfusion brain injury, and neurodegenerative diseases. Various EPO analogs have been developed to overcome these unwanted effects\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Structural biology studies have shown that EPO consists of four α-helices (A, B, C, and D) clustered through hydrophobic interactions\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A single EPO molecule binds to a dimeric EPOR consisting of the high-affinity EPOR1 and low-affinity EPOR2. Small peptides derived from EPO subregions involved in EPOR binding may selectively mediate non-hematopoietic protective effects. Distinct from the binding of the entire EPO molecule, their interactions with EPOR may differentially activate downstream pathways, potentially allowing targeted neuroprotection without unwanted proliferative and erythropoietic effects\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our previous research suggests that novel peptides derived from the helix C of EPO, MK-X and ML1-h3, have neuroprotective effects without affecting cell proliferation via differential activation of EPOR-mediated signaling pathways\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Modification of the binding structure or EPOR affinity of EPO derivatives may provide important insights into the development of EPO-based neuroprotective strategies without unwanted erythropoietic and tumorigenic effects.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to develop a new peptide, ML1-R, based on the C-helix of EPO and identify its molecular mechanisms in relation to EPOR neuroprotective effects. Our study suggests that ML1-R, a novel EPOR-binding partial agonist, is a therapeutic strategy for protection against ischemic stroke, reperfusion brain injury, and neurodegenerative disorders.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eML1-R efficiently inhibits ischemia-reperfusion brain injury in a stroke model and apoptosis of primary cultured cortical neurons following hypoxia\u0026ndash;reoxygenation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the potential of ML1-R as a novel drug with neuroprotective effects against ischemia-reperfusion brain injury, we induced transient ischemia via middle cerebral artery occlusion (MCAO) in mice for 1.5 h, followed by reperfusion and human recombinant EPO (reEPO) or ML1-R injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). After 24 h, MCAO-reperfusion significantly induced severe unilateral infarction in the brain, as confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The average TTC intensity on the MCAO-treated side was \u0026lt;\u0026thinsp;56% of that on the contralateral side in vehicle-injected mouse brains. Treatment with reEPO efficiently decreased the infarction area, and ML-1R administration strongly inhibited MCAO-reperfusion-induced brain injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the in-vitro protective effects of ML1-R against apoptosis under hypoxia\u0026ndash;reoxygenation conditions, we conducted a Hoechst staining assay with cultured primary cortical neurons. These neurons were maintained under hypoxic conditions for 18 h and then reoxygenated for 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The neurons were then kept with or without reEPO or ML1-R for 18 h, and the ratio of apoptotic cells to total cells was determined using the Hoechst staining assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Similar to the in-vivo results, treatment with ML1-R substantially reduced the number of apoptotic neurons compared to treatment with reEPO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Taken together, in-vivo and in-vitro experiments indicated that ML1-R has better neuroprotective effects than reEPO.\u003c/p\u003e \u003cp\u003e \u003cb\u003eML1-R prevents glutamate-induced neuronal cell death by suppressing calcium overload, oxidative stress, and mitochondrial dysfunction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing ischemia, energy depletion occurs within minutes, leading to the failure of energy-dependent cellular homeostasis and maintenance of brain function. Severe energy depletion under ischemic conditions disrupts the electrochemical gradient of the plasma membrane (PM), leading to depolarization and excessive glutamate release. This causes calcium influx through glutamate receptors, resulting in glutamate-induced excitotoxicity\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To understand the neuroprotective effects of ML1-R, we examined in cultured neurons with or without reEPO or ML1-R treatment various processes that are critical for cellular homeostasis under glutamate-induced excitotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Intracellular calcium levels increased after 3 min of glutamate treatment. Both reEPO and ML1-R similarly inhibited calcium influx (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Moreover, reEPO and ML1-R decreased glutamate-induced ROS levels; ML1-R more strongly inhibited ROS generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). As calcium overload and oxidative stress cause mitochondrial dysfunction in glutamate-induced excitotoxicity, we also measured the mitochondrial membrane potential. Both reEPO and ML1-R suppressed the glutamate-induced reduction in mitochondrial membrane potential, but the effect of ML1-R was more pronounced than that of reEPO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). ML1-R also substantially decreased the loss of cell viability compared to reEPO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). These data indicate that ML1-R has neuroprotective effects by regulating calcium overload and ROS production, thereby mitigating mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMitochondrial damage can activate caspase-dependent and caspase-independent cell death pathways\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Glutamate treatment increased the expression of Bax, cleaved caspase 3 (Casp3), and cleaved Parp1, known apoptosis marker proteins, whereas ML1-R efficiently suppressed their expression and increased Bcl-2 levels which inhibits Bax activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-j). Overall, except for cleaved Parp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej), ML1-R reversed the upregulation of pro-apoptotic markers more strongly than reEPO and effectively restored Bcl-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). These results indicated that ML1-R effectively prevents cellular dyshomeostasis and the activation of cell death pathways following glutamate-induced excitotoxicity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eML1-R protects neurons against hypoxia\u0026ndash;reoxygenation effects by inhibiting oxidative stress, mitochondrial dysfunction, and apoptosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrompt restoration of cerebral blood flow is crucial in ischemic stroke, but the restoration of blood flow can induce reperfusion injury\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To understand the protective effects of ML1-R against reperfusion brain injury in vitro, we assessed cellular processes under hypoxia\u0026ndash;reoxygenation conditions in the presence or absence of reEPO or ML1-R in primary cultured cortical neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). ML1-R inhibited ROS generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), mitochondrial dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), and the loss of neuronal viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) more strongly than reEPO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies reported that hypoxia\u0026ndash;reoxygenation induces caspase-dependent and caspase-independent cell death\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Immunoblotting showed that hypoxia\u0026ndash;reoxygenation upregulated Bax, cleaved Casp3, and Parp1 levels and down-regulated Bcl-2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-i). ML1-R reversed the expression of apoptosis markers more effectively than reEPO under hypoxia\u0026ndash;reoxygenation conditions, significantly increasing Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) and reducing cleaved Casp3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) and cleaved Parp1 levels to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), except for Bax (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). We also assessed the cell proliferative effects of high reEPO and ML1-R concentrations because proliferative EPO functions may induce oncogenicity. We found that ML1-R did not induce the proliferation of PC12 and SH-SY5Y cells, whereas high doses of reEPO significantly increased cell proliferation (Supplementary Fig.\u0026nbsp;1a, b). Taken together, these results indicate that ML1-R suppresses hypoxia\u0026ndash;reoxygenation-induced cell death by alleviating ROS production and mitochondrial dysfunction, resulting in neuroprotection without proliferative effects.\u003c/p\u003e \u003cp\u003eAs ML1-R exhibited a stronger neuroprotective effect than reEPO and significantly influenced cellular homeostasis and the expression of apoptosis-related proteins, gene expression in EPOR signaling pathways might be differentially activated by ML1-R and reEPO. RNA-seq analysis revealed distinct gene expression profiles for reEPO- and ML1-R-treated primary cultured cortical neurons (Supplementary Fig.\u0026nbsp;2a). ML1-R was found to influence EPOR-mediated signaling pathways, with a particular impact on gene expression related to the PI3K-Akt pathway rather than the JAK-STAT pathway. Specifically, genes associated with the PI3K-Akt pathway showed greater up-regulation or down-regulation (Supplementary Fig.\u0026nbsp;2b, c). This differential modulation of EPOR-mediated pathways likely explains the superior neuroprotective effects of ML1-R, promoting cell survival without inducing cell proliferation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eML1-R differentially activates EPOR-mediated JAK2 signaling by changing the geometric structure and binding interaction of EPOR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePreviously, we developed other peptides derived from the C-helix of EPO that retained the neuroprotective effects of EPO while preventing cell proliferation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Among these, ML1-R was identified as a promising candidate because of its ability to activate EPOR-mediated signaling without inducing proliferation. To confirm whether ML1-R directly activates EPOR signaling, we treated HAP1 EPOR wild-type (WT) and knockout (KO) cells with either reEPO or ML1-R and assessed JAK2 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Immunoblotting showed that ML1-R promoted JAK2 activation more than reEPO in HAP1 EPOR-WT cells after 15 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, in HAP1 EPOR-KO cells, JAK2 phosphorylation was not observed following reEPO or ML1-R treatment. Thus, the neuroprotective effects of ML1-R are induced by activating the JAK2 pathway downstream of EPORs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the structural differences contributing to JAK2 activation, we performed in-silico modeling and molecular dynamics (MD) simulations to compare the predicted structures of the EPO\u0026ndash;EPOR and ML1-R\u0026ndash;EPOR complexes. To investigate the structural changes in EPOR, we used AlphaFold3 to model these complexes during signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-g). The predicted binding sites matched those in the known EPO\u0026ndash;EPOR crystal structure (PDB ID: 1EER)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, thus validating the accuracy of the models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Dimerization is mainly mediated by the transmembrane domain of the EPOR and its membrane-spanning leucine zipper\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. To investigate the structural changes in ligand\u0026ndash;EPOR complexes, we targeted L251 located in the upper region, L263 in the middle region, and L272 in the lower region of the EPOR transmembrane domain and measured the distances between these residues. In the EPO-bound model, distances were 9.1, 8.1, and 11.9 \u0026Aring;. They increased in the ML1-R-bound model to 11.0, 11.7, and 12.2 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, j). These findings suggest that ML1-R binding results in a greater separation of transmembrane helices, indicating a weaker binding affinity. During a 1,000 ns simulation, EPO and ML1-R induced opposing distance patterns, suggesting distinct structural effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). These results indicate that the weaker binding affinity of ML1-R may contribute to structural changes in EPOR, potentially influencing differential signaling and neuroprotective effects.\u003c/p\u003e \u003cp\u003eFurther MD simulations over 1,000 ns showed that the ML1-R\u0026ndash;EPOR complex exhibited greater structural variability with higher root-mean-square deviation (RMSD) values than the EPO\u0026ndash;EPOR complex. Structural changes were analyzed in 41 residues from transmembrane and juxtamembrane EPOR domains, and simulations were conducted to observe structural changes in the receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Over the 1,000 ns simulation, the RMSD values of the overall structure of the ML1-R complex was more than twice as high as that of the EPO complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Moreover, the RMSD values of the key residues involved in JAK2 activation (L278, I282, and W283) in the ML1-R complex were higher than those in the EPO complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). These results indicate that the key residues involved in JAK2 activation are structurally more unstable in the ML1-R\u0026ndash;EPOR complex than in the EPO\u0026ndash;EPOR complex. Root-mean-square fluctuation (RMSF) analysis of the BOX1 motif, which is the JAK2 binding site, showed distinct fluctuation patterns for ML1-R and EPO. RMSF analysis revealed that one side exhibited similar behavior for ML1-R and EPO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), whereas the other side exhibited reduced fluctuations in the ML1-R complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). The MD simulation results confirmed that when ML1-R binds to EPOR, it may partially bind and induce conformational changes, resulting in differential JAK2 activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the activated EPOR, the switch tryptophan residue W283 shows a distance of 45 \u0026Aring; when forming a dimer\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To confirm the distance change, we measured simulated distances between the Cα carbons of G285 located in the central BOX1 motif sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) when EPO and ML1-R are bound. EMP1, an EPO-agonistic peptide, has been confirmed experimentally\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, we predicted ligand\u0026ndash;EPOR complexes and conducted 100 ns simulations to measure the distances of G285 in three models: EMP1, EPO, and ML1-R. The ML1-R complex consistently maintained a shorter G285 distance (~\u0026thinsp;35 \u0026Aring;), compared to 40\u0026ndash;45 \u0026Aring; in EPO and EMP1 complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), suggesting that ML1-R does not achieve the fully active conformation associated with JAK2 activation. This trend continued over 1,000 ns, with the G285 distance for the ML1-R complex decreasing after 500 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). These results suggest that ML1-R induces conformational EPOR changes that differ from EPO-induced changes, particularly at the BOX1 motif, potentially differentially influencing JAK2 activation and signaling.\u003c/p\u003e \u003cp\u003e \u003cb\u003eML1-R activates EPOR-mediated signaling pathways differently from reEPO.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs the abovementioned data raise the possibility of differential activation patterns for ML1-R and reEPO in EPOR downstream signaling, we examined how reEPO and ML1-R activate EPOR signaling at the same molecular concentration (1 nM) using western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Treatment with reEPO rapidly promoted the upregulation of phosphorylated JAK2 (pJAK2) at 15\u0026ndash;30 min and returned it to basal level at 60\u0026ndash;90 min. ML1-R also increased the pJAK2 levels but at a lower intensity and more prolonged than reEPO (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These data suggest that reEPO and ML1-R exhibit differential kinetics in the activation of JAK2 signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we confirmed the activation patterns of Akt and Erk, which are downstream of the JAK2 pathway and may contribute to the neuroprotective effects of reEPO and ML1-R. Whereas reEPO gradually upregulated phosphorylated Akt (pAkt) levels up to 60 min with a decrease at 90 min, ML1-R induced a different pattern, with a faster increase in Akt phosphorylation by 30 min, which began to decrease at 45 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Both reEPO and ML1-R induced an upregulation of phosphorylated Erk (pERK) within 15 min; however, the pErk levels induced by ML1-R were higher than those induced by reEPO. The pErk levels time-dependently decreased toward baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Additionally, we examined the concentration-dependent activation by reEPO and ML1-R (Supplementary Fig.\u0026nbsp;3a). With increased concentrations, ML1-R promoted Akt and Erk phosphorylation more strongly than reEPO, showing a saturation effect at a concentration of 1 nM (Supplementary Fig.\u0026nbsp;3b, c).\u003c/p\u003e \u003cp\u003ePrevious reports demonstrated that the Akt and ERK pathways are important for EPO-mediated protection and proliferation\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To examine the involvement of ML1-R in the activation of Akt or ERK signaling pathways, we pretreated cells with LY294002 and U0126, which are specific inhibitors of PI3K and MEK, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, h). LY294002 and U0126 efficiently blocked the upregulation of pAkt and pErk by reEPO (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef, g) and ML1-R (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, j). To determine whether ML1-R activates Akt and Erk pathways, we treated HAP1 EPOR-WT and HAP1 EPOR-KO cells with reEPO or ML1-R and checked the patterns of Akt and Erk phosphorylation (Supplementary Fig.\u0026nbsp;4a). Similar to the results above, ML1-R activated Akt and Erk to a greater extent than reEPO in HAP1 EPOR-WT cells at 15 min, whereas Akt and Erk activation by reEPO and ML1-R were not observed in HAP1 EPOR-KO cells (Supplementary Fig.\u0026nbsp;4b, c). Furthermore, the neuroprotective effects of reEPO and ML1-R against hypoxia\u0026ndash;reoxygenation-induced cell death were significantly inhibited by LY294002 and U0126 (Supplementary Fig.\u0026nbsp;5a, b). These data suggest that ML1-R induces differential activities in EPOR-mediated signaling pathways compared with reEPO due to changes in binding interactions and geometric structures of the ligand-binding site in the EPOR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eML1-R may promote EPOR downstream signaling by long-lasting EPOR expression in the plasma membrane.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEPO-induced EPOR activation is transient because EPO-bound EPORs are rapidly internalized and undergo degradation or recycling for EPOR de-novo synthesis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This suggests that the binding interaction and geometric structural changes of the EPO\u0026ndash;EPOR and ML1-R\u0026ndash;EPOR complexes may differentially influence the physiological turnover or recycling of EPORs. To demonstrate changes in EPOR expression induced by reEPO and ML1-R, we performed RT-qPCR and immunoblot analyses to determine the total EPOR mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) levels. Following reEPO treatment, total EPOR mRNA levels were reduced by 15 min and recovered after 30 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), and total EPOR protein levels underwent turnover by degradation and synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;6a). Surprisingly, following ML1-R treatment, total EPOR mRNA levels were increased by 30 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), and total EPOR protein levels were maintained for 30\u0026ndash;60 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;6a). Given that EPO-stimulated EPOR internalization and degradation depend on JAK2 activation, these results indicate that ML1-R promotes the upregulation of EPOR expression through long-lasting JAK2 activation, resulting in the maintenance of EPOR levels in the PM. To investigate the turnover of EPORs in the PM following treatment with reEPO and ML1-R, we isolated PM fractions and extracted PM proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Following reEPO treatment, the level of EPORs at the cell surface was gradually reduced for 30 min, recovered to basal levels at 60 min, and decreased at 90 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Interestingly, ML1-R delayed the internalization of EPORs for 60 min and restored the basal level of EPOR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), whereas Na+/K\u0026thinsp;+\u0026thinsp;ATPase expression remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). To further examine EPOR expression on the cell surface, we assessed EPOR levels in the PM using a cell-surface biotinylation assay (Supplementary Fig.\u0026nbsp;7a, b). Similar to the results of PM protein extraction, ML1-R induced delayed EPOR endocytosis for 30 min, which subsequently returned to basal EPOR levels without changes in Na+/K\u0026thinsp;+\u0026thinsp;ATPase levels (Supplementary Fig.\u0026nbsp;7c, d). According to the immunoblot results, the differences in EPOR levels in the PM between ML1-R and reEPO treatment remained mostly consistent, suggesting that ML1-R may influence EPOR expression or EPOR trafficking.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, our data suggest that ML1-R increases EPOR expression through the EPOR signaling pathway, thereby increasing the amount of EPOR available in the PM to enhance EPO-mediated activation of signaling pathways, which in turn exhibits neuroprotective effects via positive feedback (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Thus, ML1-R has the potential as a neuroprotective drug for treating reperfusion injury in ischemic stroke or neurodegenerative diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUsing in-vivo and in-vitro systems, we investigated the neuroprotective effects of the partial EPOR agonist ML1-R in comparison with those of reEPO against ischemic stroke and reperfusion brain injury. We previously reported novel EPO-derived small peptides, MK-X and ML1-h3, which exert neuroprotective effects against oxidative stress resulting from glutamate-induced excitotoxicity and hypoxia\u0026ndash;reoxygenation without inducing unwanted effects, including cell proliferation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. ML1-R, an ML1 analog based on the C-helix of EPO with low affinity to EPORs\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, is a modified peptide in which leucine residues at both ends of an ML1 subsequence have been substituted with arginine. Because the affinity of small peptides for EPOR can influence various EPOR-mediated effects, including erythropoiesis, cell proliferation, and cell protection, it is important to adjust the binding affinity of modified peptides to EPOR\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our in-silico results predictions using AlphaFold3 showed that ML1-R has a lower EPOR affinity than EPO, causing changes in the geometry and structure of ML1-R\u0026ndash;EPOR complexes. ML1-R may induce geometric changes in the intracellular domains of EPOR known as the JAK2-binding BOX1 motif, potentially leading to differences in EPOR downstream activation. In our previous report, it was unclear how ML1-h3 binding induces geometric changes in full-length EPORs because the structural biological analysis focused only on the extracellular domains of EPORs\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this study, we analyzed structural changes in full-length EPORs using computational prediction models. Moreover, we observed alterations in JAK2, Akt, and ERK activation. These results allowed us to elucidate the detailed mechanisms underlying the neuroprotective ML1-R effects. ML1-R induced delayed and prolonged JAK2 activation and activated Akt and ERK phosphorylation more strongly and rapidly than reEPO, promoting EPOR production and increased EPOR expression in the PM. Thus, the differential effects of ML1-R on EPOR downstream signaling may be due to geometric alterations in the ML1-R\u0026ndash;EPOR complex.\u003c/p\u003e \u003cp\u003eBoth reEPO and ML1-R inhibited intracellular calcium overload following glutamate-induced excitotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). EPO modulates intracellular calcium levels through the regulation of several calcium-permeable ion channels\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and we speculated that ML1-R causes similar effects. In our study, the reduction in glutamate-induced intracellular calcium overload by ML1-R was comparable to that observed with reEPO. However, ML1-R more effectively attenuated ROS overproduction caused by glutamate-induced excitotoxicity and hypoxia\u0026ndash;reoxygenation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Therefore, the greater increase in cell viability with ML1-R compared to that with reEPO was likely due to the mitigation of ROS generation and mitochondrial dysfunction, which may have contributed more than the alleviation of calcium overload. EPO has well-known anti-apoptotic effects by promoting anti-oxidant synthesis, thereby maintaining the mitochondrial membrane potential\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We presume that these combined ML1-R effects contribute to increased cell viability.\u003c/p\u003e \u003cp\u003eVarious studies have shown that the EPO\u0026ndash;EPOR signaling pathway leads to changes in apoptosis markers\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Our study confirmed these findings for reEPO, with even greater changes observed with ML1-R. Under glutamate-induced excitotoxicity and hypoxia\u0026ndash;reoxygenation conditions, the changes in Bax (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) and cleaved Parp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei) levels were inconsistent; however, the alterations in Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) and cleaved Casp3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) expression were consistently stronger with ML1-R than with reEPO. Thus, the changes in Bcl-2 and cleaved Casp3 may contribute more strongly to apoptosis mitigation than the changes in Bax and cleaved Parp1, and the partial binding interaction between ML1-R and EPOR may play a crucial role in producing effects distinct from those of reEPO.\u003c/p\u003e \u003cp\u003eRecently, various studies on non-hematopoietic EPO effects have been conducted in various organs, including the central nervous system. In the brain, most cells, including neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells forming the blood\u0026ndash;brain barrier, express EPORs\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This raises the question of the role of EPO and EPOR in physiological brain processes, as hematopoiesis and proliferation should not occur in the brain. Our study findings confirmed that the total EPOR level exhibited a cyclic pattern of degradation and subsequent production, depending on the duration of reEPO treatment. In contrast, ML1-R treatment favored EPOR production over degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c). The increased EPOR expression can be attributed to enhanced production, as indicated by the upregulation of EPOR mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). However, this is likely the result of various factors, including recycling and reuse of EPORs or reduced EPOR degradation, making it difficult to identify the exact cause. Moreover, partial binding of ML1-R to EPOR may result in delayed and prolonged JAK2 activation, and the cumulative effects of increased EPOR production may lead to stronger activation of various EPOR downstream molecules. We speculate that EPO and EPOR in the brain play crucial roles in enhancing cellular protective effects through EPOR signaling during acutely dangerous conditions like ischemic stroke. This suggests that a positive feedback mechanism may be activated to increase EPOR expression in the PM to promote neuroprotective effects.\u003c/p\u003e \u003cp\u003eThe EPO activity in diverse tissues suggests that other receptors may interact with EPORs. The erythropoietic activity of EPO is mediated by homodimeric receptors\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, whereas EPO activity in non-erythroid tissue is mediated by a heterodimer consisting of one EPOR monomer and one cytokine β\u0026minus;common subunit (CD131) involved in protection against inflammation and tissue injury\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. We hypothesized that ML1-R interacts with the EPOR dimer and performed a modeling analysis using AlphaFold3. Previous research confirmed that ML1 sequence regions show weak binding affinity for the EPOR dimer\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and in-silico predictions for ML1-R indicated the possibility of partial binding affinity to EPOR. As the EPOR and cytokine βcommon subunit form a heterodimer leading to tissue-protective effects\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e7\u003c/sup\u003e, we anticipate that ML1-R interacts with one EPOR and another receptor. Related experimental results will be presented in a future study.\u003c/p\u003e \u003cp\u003eIn conclusion, we suggest that the partial EPO agonist ML1-R is a promising candidate for neuroprotection, resulting from the alleviation of brain injury caused by ischemic stroke and reperfusion. Research on known EPO derivatives, including ML1-R, has the potential for the development of safer and more effective drugs for various diseases. Therefore, we expect that the neuroprotective effects of ML1-R and its role in counteracting oxidative stress have potential benefits in the treatment of neurodegenerative disorders such as Alzheimer\u0026rsquo;s disease and Parkinson\u0026rsquo;s disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMCAO and reperfusion\u003c/h2\u003e \u003cp\u003e The study protocol was approved by the Animal Care and Use Committee of Keimyung University School of Medicine. Briefly, 24 C57/BL6 mice (8 weeks old) were randomly divided into experimental (reEPO: n\u0026thinsp;=\u0026thinsp;8, ML1-R: n\u0026thinsp;=\u0026thinsp;8) or vehicle (n\u0026thinsp;=\u0026thinsp;8) groups and anesthetized by inhalation of 1.5\u0026ndash;2.0% isoflurane in a mixture of 70% N\u003csub\u003e2\u003c/sub\u003eO and 30% O\u003csub\u003e2\u003c/sub\u003e. After dissecting the neck skin, the bifurcation of the common carotid artery was exposed. The arteries were separated from the surrounding nerves and connective tissue, and the right external carotid artery was ligated by surgical nylon monofilament insertion. The rectal temperature was maintained at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C intraoperatively using a controlled heating pad. At the end of the ischemic period, the filament was removed, and reperfusion was performed with intravenous injection of reEPO (2,000 IU/kg in 0.3 ml saline), ML1-R (0.3 mg/kg in 0.3 ml saline), or vehicle (0.3 ml saline). Animals from each group were sacrificed at the end of the reperfusion period (24 h after reperfusion following MCAO), and their brains were collected for histochemical staining to assess the neuroprotective effects of reEPO and ML1-R.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of brain injury\u003c/h3\u003e\n\u003cp\u003eThe brains were rapidly dissected. Coronal sections were cut into 2-mm thick slices. Subsequently, they were stained with 2% TTC in saline for 20 min at 37\u0026deg;C. The infarcted and non-infarcted areas of the TTC-stained brain sections were imaged using a digital camera. The TTC intensity of each hemisphere was analyzed using ImageJ (NIH, Bethesda, MD, USA), and the ratio (MCAO versus non-MCAO) is presented as a percentage.\u003c/p\u003e\n\u003ch3\u003eRat primary cortical neuron culture\u003c/h3\u003e\n\u003cp\u003eEmbryonic cerebral cortices were obtained from pregnant Sprague\u0026ndash;Dawley rats on embryonic day 17. Cerebral cortices were treated with 0.25% trypsin-EDTA (#25200056, Gibco, USA) and chemically and mechanically dissociated into single cells by pipetting. The dissociated cells were plated on plates or coverslips coated with 50 \u0026micro;g/ml poly-D-lysine (P8920, Sigma-Aldrich, St. Louis, MO, USA). They were incubated in neurobasal medium (#21103049, Gibco) containing 2% B27 supplement (#17504044, Gibco), 0.5 mM L-glutamine (#15430614, Gibco), 25 \u0026micro;M glutamic acid (#G1251, Sigma-Aldrich), and 1% penicillin/streptomycin (#15140122, Gibco) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was replaced with fresh medium without glutamate every 3\u0026ndash;4 days. On day 10, cortical neurons were treated with reEPO, or ML1-R under control, glutamate excitotoxicity, and hypoxia\u0026ndash;reoxygenation conditions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHAP1 cells are a near-haploid human cell line derived from the KBM-7 chronic myelogenous leukemia cell line. The HAP1 parental cell line (#C631, Horizon Discovery, UK) and HAP1 EPOR-KO cell line (#HZGHC003882c008, Horizon Discovery) were cultured at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in Iscove\u0026rsquo;s modified Dulbecco\u0026rsquo;s medium (#12440, Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHypoxia and reoxygenation\u003c/h3\u003e\n\u003cp\u003ePrimary rat cortical neurons were seeded into 96-well plates. The next day, the cultured cortical neurons were placed inside a sealed, airtight container consisting of anaerobic atmosphere generation bags (#68061, Sigma-Aldrich), which can induce a hypoxic atmosphere by absorbing oxygen and generating carbon dioxide within 2.5 h. The primary cortical neurons were maintained under hypoxic conditions at 37\u0026deg;C for 18 h and reoxygenated for 4 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, the cultured cortical neurons were treated with reEPO or ML1-R for 18 h before viability analysis.\u003c/p\u003e\n\u003ch3\u003eCell viability\u003c/h3\u003e\n\u003cp\u003eTo examine the viability of rat primary cortical neurons following the treatment with 30 \u0026micro;M glutamate or hypoxia\u0026ndash;reoxygenation, we used calcein-AM (#C3100MP, Invitrogen). Cultured rat cortical neurons were plated in 96-well plates (2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well) and incubated with calcein-AM (3 \u0026micro;M) for 30 min after various treatments. The intensity of the calcein-AM signal was measured using a microplate reader (Ex/Em\u0026thinsp;=\u0026thinsp;485/535 nm; VersaMax, Molecular Devices, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of calcium concentration, ROS production, and mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eTo characterize glutamate-induced intracellular calcium increases, calcium levels were observed for 15 min in the presence of ML1-R or reEPO. To quantify intracellular calcium levels, cultured cortical neurons were stained using the Fluo-4 NW Calcium Assay Kit (#F36206, Molecular Probes, USA). Cortical neurons were grown in 96-well plates (3\u0026times;10\u003csup\u003e4\u003c/sup\u003e per well) for 10 days, then washed with phosphate-buffered saline (PBS) and loaded with 100 \u0026micro;l Fluo-4 dye in assay buffer (Hank\u0026rsquo;s balanced salt solution and 20 mM HEPES) for 45 min at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Intracellular calcium levels in the presence of 30 \u0026micro;M glutamate were recorded (Em\u0026thinsp;=\u0026thinsp;516 nm; VersaMax).\u003c/p\u003e \u003cp\u003eCellular ROS levels were measured using 2\u0026rsquo;,7\u0026rsquo;-dichlorodihydrofluorescein diacetate (H2DCFDA; #D399, Invitrogen). Cultured rat cortical neurons plated into 96-well plates (2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well) were incubated with 5 \u0026micro;M H2DCFDA in PBS for 30 min, washed with PBS, and then resuspended in medium for 2 h. After treatment with glutamate (30 \u0026micro;M) or hypoxia\u0026ndash;reoxygenation in the presence or absence of reEPO or ML1-R, we monitored the oxidative stress by measuring H2DCFDA fluorescence intensities using a microplate reader (Ex/Em\u0026thinsp;=\u0026thinsp;485/535 nm; VersaMax). The ROS levels of reEPO and ML1-R were observed after 6 h.\u003c/p\u003e \u003cp\u003eMitochondrial membrane potential was measured using the TMRE-Mitochondrial Membrane Potential Assay Kit (#ab113852, Abcam, Cambridge, UK). Primary cultured cortical neurons plated into 96-well plates (4\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well) were first incubated with culture medium containing 200 nM TMRE for 30 min at 37\u0026deg;C, then rinsed three times with PBS and incubated with PBS/0.2% bovine serum albumin (BSA). The fluorescence signal was quantified using a microplate reader (Ex/Em\u0026thinsp;=\u0026thinsp;549/575 nm; VersaMax).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting and antibodies\u003c/h2\u003e \u003cp\u003eThe cells were lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholic acid, and 0.1% SDS) on ice for 30 min. The lysates (20 \u0026micro;g of protein) were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8\u0026ndash;10%), and the proteins were transferred onto polyvinylidene difluoride membranes. The membrane was blocked with 0.1% TBST and 5% non-fat dry milk at 25\u0026deg;C for 30 min. The primary antibodies diluted in 0.1% TBST and 3% BSA included anti-pJAK2 (#3776), anti-JAK2 (#3230), anti-pAKT (#9271), anti-AKT (#9272), anti-pERK (#9101) anti-ERK (#9102), anti-Bax (#2772), anti-cleaved Casp3 (#9661), anti-cleaved Parp1 (#5625, all Cell Signaling Technology), anti-EPOR (#sc-365662), anti-Bcl2 (#sc-783, both Santa Cruz Biotechnology, USA), anti-GAPDH (#MAB374, Merck Millipore Biotechnology, USA), and anti-Na+/K\u0026thinsp;+\u0026thinsp;ATPase (#ab7671, Abcam). After several washes, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies. The proteins were visualized using an enhanced chemiluminescence substrate kit (Pierce, Rockford, IL, USA). Blots were scanned and quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHoechst staining assay\u003c/h2\u003e \u003cp\u003eHoechst 33342 staining was performed to detect apoptosis. Cultured cortical neurons were plated onto a microscope cover glass (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well) and allowed to attach for 24 h. The next day, the neurons were maintained in hypoxic conditions at 37\u0026deg;C for 18 h and reoxygenated for 4 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, the cultured cortical neurons were treated with reEPO or ML1-R for 18 h before viability analysis. The cells were fixed in freshly prepared 4% paraformaldehyde for 30 min and permeabilized in PBS containing 0.1% Triton X-100 and 2% BSA for 5 min. After washing, the cells were blocked with PBS containing 1% BSA for 1 h and then incubated with a NeuN antibody (#ab7671, Abcam) for 2 h in blocking buffer to detect cortical neurons. Following washing with PBS, cells were incubated with goat anti-rabbit conjugated with Alexa Fluor 488 (#A11008, Invitrogen) secondary antibodies in blocking buffer at 4\u0026deg;C for 16 h. Cells were then washed with cold PBS again before incubation with 5 \u0026micro;g/ml Hoechst 33342 (#H3570, Invitrogen) for 15 min at 37\u0026deg;C in the dark. Finally, the cells were washed with PBS and mounted using mounting medium (#F6182; Sigma-Aldrich). Apoptotic cells were identified using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlasma membrane protein extraction\u003c/h2\u003e \u003cp\u003ePM fractions were isolated using a PM protein extraction kit (#ab65400, Abcam), according to the manufacturer\u0026rsquo;s instructions. Primary cortical neurons (5\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells) cultured on 100 mm dishes were rinsed twice with ice-cold PBS, immediately incubated with 2 ml of homogenized buffer on ice, and harvested using a cell scraper. Resuspended cells were homogenized in a Dounce homogenizer for 30\u0026ndash;50 times on ice and centrifuged at 700\u0026times;\u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C. The supernatants were subsequently centrifuged at 10,000\u0026times;\u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C. The pellet and supernatant contained the total cellular membrane and cytosolic proteins, respectively. The pellet was resuspended in 200 \u0026micro;l of upper-phase solution and then mixed with 200 \u0026micro;l of lower-phase solution. The samples were incubated on ice for 5 min and centrifuged at 1,000\u0026times;\u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C; upper- and lower-phase solutions containing PM and intracellular membrane proteins, respectively, were collected. The extractions were repeated by adding a fresh upper-phase solution to the lower-phase solution and vice versa. The resulting upper-phase solutions from repeated extractions were combined and diluted with 5\u0026times; volume of water, followed by centrifugation at 15,000\u0026times;\u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C. Pellets containing PM proteins were harvested and analyzed using immunoblotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from primary cultured cortical neurons using the RNeasy Plus Mini Kit (#74136, Qiagen, Germany) following the manufacturer\u0026rsquo;s protocol. mRNA was reverse-transcribed to cDNA using SuperScript IV Reverse Transcriptase (#18090010, Thermo Fisher Scientific). We performed PCRs using Herculase II Fusion DNA Polymerase (#600677, Agilent Technologies). Primers of the following sequences were obtained from Bionics (Daejeon, Republic of Korea): EPOR (forward: CTA TGG CTG TTG CAA CGC GA and reverse: CCG AGG GCA CAG GAG CTT AG) and β-actin (forward: ATC GTG GGC CGC CCT AGC ACC and reverse: CTC TTT AAT GTC ACG CAC GAT TTC). The housekeeping gene β-actin was used as an internal standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAlphaFold-based structural models\u003c/h2\u003e \u003cp\u003eThe protein sequences for EPOR (P19235) and EPO (P01588) were obtained from the UniProt database\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The peptide sequences used in the study were EMP1 (GGTYSCHFGPLTWVCKPQGG) and ML1-R (RHVDKAVSGLRSLTTR). The structural models of EPO, peptides, and EPO\u0026ndash;EPOR-peptide complexes were predicted using AlphaFold3\u003csup\u003e51\u003c/sup\u003e. The models generated by AlphaFold3 were further refined and used in subsequent MD simulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMD simulations\u003c/h2\u003e \u003cp\u003eThe membrane-protein system was constructed using the CHARMM-GUI Membrane Builder\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. A pre-equilibrated lipid bilayer composed of a lipid mixture including cholesterol was generated using default parameters. The proteins obtained from AlphaFold3 were inserted into the membrane based on the default orientation suggested in the OPM 2.0 database\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The system was solvated with TIP3P water molecules, and ions were automatically added to neutralize the system and achieve a physiological ionic strength of 0.15 M NaCl using the default ion placement method of CHARMM-GUI. Input files for MD were generated using GROMACS (version 2023.3)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e with the default settings provided by CHARMM-GUI. MD simulations were carried out using GROMACS with 2.0 fs time steps. The production run was conducted for 100 ns, corresponding to 50\u0026nbsp;million steps. Trajectory snapshots were recorded every 100 ps, yielding 1,000 frames for the post-simulation analysis. The system was first subjected to energy minimization, followed by equilibration in two phases: constant number of particles, volume, and temperature and constant number of particles, pressure, and temperature. The constant number of particles, pressure, and temperature ensemble was used for the production run at a temperature of 310 K and pressure of 1 bar, employing a V-rescale thermostat and a Parrinello\u0026ndash;Rahman barostat for temperature and pressure coupling, respectively. Subsequent analysis of the simulation trajectory was performed using standard GROMACS tools to evaluate the structural and dynamic properties of the protein-lipid system. RMSD, RMSF, and radial distribution functions were calculated to assess stability and interactions within the membrane. Visualization and additional analyses were conducted using VMD\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and PyMOL\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e to gain structural insights.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical information is provided in the figure legends. All experiments were performed at least three times using independent cells or animal preparations. Repeats for experiments or cell numbers are given in the figure legends as \u0026ldquo;n.\u0026rdquo; Error bars represent\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. To measure statistical significance, we performed one-way ANOVA on ranks using Dunnett\u0026rsquo;s method or two-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. All statistical analyses and graph plots were generated using GraphPad Prism software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1I1A3055783; RS-2023-00239274; RS-2024-00354916; 2020R1A6A1A03040516). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eO.H.K, S.J.Y, S.R.L, and C.M. conceived the project and designed the project. J.C.B, and H.J.J. performed middle cerebral artery occlusion and TTC staining. J.S.B. conducted the structural studies and MD simulation using AlphaFold3 and assisted with the RNA-seq studies and bioinformatics analysis. O.H.K. performed all of in-vitro experiments and wrote the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary informationis available for this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support this study are available from the corresponding authors upon reasonable request. A Source Data File is provided. Raw and processed data are available via the Dryad with DOI: 10.5061/dryad.66t1g1k9z.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBunn HF, Erythropoietin (2013) Cold Spring Harb Perspect Med 3:a011619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuresh S, Rajvanshi PK, Noguchi CT (2019) The Many Facets of Erythropoietin Physiologic and Metabolic Response. Front Physiol 10:1534\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoguchi CT, Asavaritikrai P, Teng R, Jia Y (2007) Role of erythropoietin in the brain. Crit Rev Oncol Hematol 64:159\u0026ndash;171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabie T, Marti HH (2008) Brain protection by erythropoietin: a manifold task. Physiol (Bethesda) 23:263\u0026ndash;274\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaiese K, Li F, Chong ZZ (2004) Erythropoietin in the brain: can the promise to protect be fulfilled? 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CCP4 Newsl Protein Crystallogr 40(1):82\u0026ndash;92\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Daegu Gyeongbuk Institute of Science and Technology (DGIST)","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Erythropoietin, Erythropoietin receptor, Ischemic stroke, Reperfusion brain injury, Partial agonist","lastPublishedDoi":"10.21203/rs.3.rs-5779616/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5779616/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eErythropoietin (EPO) is a glycoprotein that stimulates red blood cell production in the bone marrow and protects neurons from oxidative stress, making it a potential treatment for various neurological diseases. However, EPO analogs often lead to side effects like excessive erythropoiesis and tumor growth. In this study, we aimed to develop ML1-R, a peptide derived from the C-helix of EPO, to enhance neuroprotection while minimizing adverse effects. By modifying amino acids that interact with EPO receptors (EPORs), ML1-R activated EPORs differently from recombinant EPO (reEPO). ML1-R provided stronger neuroprotection than reEPO without promoting cell proliferation. In a murine stroke models and in-vitro neuron cultures, ML1-R reduced brain injury and prevented neuronal death caused by glutamate-induced excitotoxicity and hypoxia-reoxygenation. AlphaFold3 computational analysis showed distinct binding affinity and geometric structures between ML1-R\u0026ndash;EPOR and EPO\u0026ndash;EPOR complexes. ML1-R prolonged JAK2 activation and activated Akt/Erk signaling in distinct patterns, increasing EPORs on cell surface membranes. This reduced apoptosis and alleviated calcium overload, reactive oxygen species generation, and mitochondrial dysfunction induced by glutamate-induced excitotoxicity and hypoxia\u0026ndash;reoxygenation. In conclusion, these findings highlight ML1-R as a promising candidate to treat ischemic stroke, reperfusion brain injury, and neurodegenerative diseases.\u003c/p\u003e","manuscriptTitle":"The partial erythropoietin receptor agonist ML1-R is a potent neuroprotective drug with a distinct signaling profile.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 07:22:49","doi":"10.21203/rs.3.rs-5779616/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4398bbb-2919-46c1-a188-9a8fa69c6f7e","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42481421,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[],"updatedAt":"2025-01-08T07:22:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 07:22:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5779616","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5779616","identity":"rs-5779616","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}