{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T22:13:27Z","timestamp":1780352007286,"version":"3.54.1"},"reference-count":58,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2024,4,4]],"date-time":"2024-04-04T00:00:00Z","timestamp":1712188800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Bloomsbury Colleges University of London PhD Studentship","award":["MR\/T014725\/1"],"award-info":[{"award-number":["MR\/T014725\/1"]}]},{"DOI":"10.13039\/501100000265","name":"New Investigator Research Grant","doi-asserted-by":"publisher","award":["MR\/T014725\/1"],"award-info":[{"award-number":["MR\/T014725\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Neonatal hypoxia-ischemia (HI) affects 2\u20133 per 1000 live births in developed countries and up to 26 per 1000 live births in developing countries. It is estimated that of the 750,000 infants experiencing a hypoxic-ischemic event during birth per year, more than 400,000 will be severely affected. As treatment options are limited, rapidly identifying new therapeutic avenues is critical, and repurposing drugs already in clinical use offers a fast-track route to clinic. One emerging avenue for therapeutic intervention in neonatal HI is to target mitochondrial dysfunction, which occurs early in the development of brain injury. Mitochondrial dynamics are particularly affected, with mitochondrial fragmentation occurring at the expense of the pro-fusion protein Optic Atrophy (OPA)1. OPA1, together with mitofusins (MFN)1\/2, are required for membrane fusion, and therefore, protecting their function may also safeguard mitochondrial dynamics. Leflunomide, an FDA-approved immunosuppressant, was recently identified as an activator of MFN2 with partial effects on OPA1 expression. We, therefore, treated C17.2 cells with Leflunomide before or after oxygen-glucose deprivation, an in vitro mimic of HI, to determine its efficacy as a neuroprotection and inhibitor of mitochondrial dysfunction. Leflunomide increased baseline OPA1 but not MFN2 expression in C17.2 cells. However, Leflunomide was unable to promote cell survival following OGD. Equally, there was no obvious effect on mitochondrial morphology or bioenergetics. These data align with studies suggesting that the tissue and mitochondrial protein profile of the target cell\/tissue are critical for taking advantage of the therapeutic actions of Leflunomide.<\/jats:p>","DOI":"10.3390\/cells13070631","type":"journal-article","created":{"date-parts":[[2024,4,4]],"date-time":"2024-04-04T10:42:03Z","timestamp":1712227323000},"page":"631","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Leflunomide Treatment Does Not Protect Neural Cells following Oxygen-Glucose Deprivation (OGD) In Vitro"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4010-3003","authenticated-orcid":false,"given":"Claire J. 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Dis. Child.-Fetal Neonatal Ed."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.earlhumdev.2010.05.010","article-title":"Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy","volume":"86","author":"Kurinczuk","year":"2010","journal-title":"Early Hum. Dev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"101271","DOI":"10.1016\/j.siny.2021.101271","article-title":"Need for more evidence in the prevention and management of perinatal asphyxia and neonatal encephalopathy in low and middle-income countries: A call for action","volume":"26","author":"Krishnan","year":"2021","journal-title":"Semin. Fetal Neonatal Med."},{"key":"ref_4","first-page":"4901014","article-title":"Plasticity in the Neonatal Brain following Hypoxic-Ischaemic Injury","volume":"2016","author":"Hristova","year":"2016","journal-title":"Neural Plast."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"506320","DOI":"10.1155\/2012\/506320","article-title":"Molecular mechanisms of neonatal brain injury","volume":"2012","author":"Thornton","year":"2012","journal-title":"Neurol. Res. Int."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1056\/NEJMoa1315788","article-title":"Effects of hypothermia for perinatal asphyxia on childhood outcomes","volume":"371","author":"Azzopardi","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1159\/000522317","article-title":"Therapeutic Hypothermia for Neonatal Encephalopathy in Low- and Middle-Income Countries: A Meta-Analysis","volume":"119","author":"Bellos","year":"2022","journal-title":"Neonatology"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/S0165-3806(00)00110-3","article-title":"Subcellular distribution of calcium and ultrastructural changes after cerebral hypoxia-ischemia in immature rats","volume":"125","author":"Gajkowska","year":"2000","journal-title":"Brain Res. Dev. Brain Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/S1474-4422(13)70261-8","article-title":"Mitochondria: Hub of injury responses in the developing brain","volume":"13","author":"Hagberg","year":"2014","journal-title":"Lancet Neurol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1016\/j.freeradbiomed.2005.08.040","article-title":"Free radicals, mitochondria, and hypoxia-ischemia in the developing brain","volume":"40","author":"Blomgren","year":"2006","journal-title":"Free Radic. Biol. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.ceca.2017.05.003","article-title":"Mitochondrial and endoplasmic reticulum calcium homeostasis and cell death","volume":"69","author":"Marchi","year":"2018","journal-title":"Cell Calcium"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"888","DOI":"10.1016\/j.tcb.2019.08.003","article-title":"The Good and the Bad of Mitochondrial Breakups","volume":"29","author":"Sprenger","year":"2019","journal-title":"Trends Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Nair, S., Leverin, A.L., Rocha-Ferreira, E., Sobotka, K.S., Thornton, C., Mallard, C., and Hagberg, H. (2022). Induction of Mitochondrial Fragmentation and Mitophagy after Neonatal Hypoxia-Ischemia. Cells, 11.","DOI":"10.3390\/cells11071193"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"22509","DOI":"10.3390\/ijms160922509","article-title":"Mitochondrial Optic Atrophy (OPA) 1 Processing Is Altered in Response to Neonatal Hypoxic-Ischemic Brain Injury","volume":"16","author":"Baburamani","year":"2015","journal-title":"Int. J. Mol. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.neuroscience.2015.05.078","article-title":"Neuronal hypoxia disrupts mitochondrial fusion","volume":"301","author":"Sanderson","year":"2015","journal-title":"Neuroscience"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2588","DOI":"10.1523\/JNEUROSCI.5832-08.2009","article-title":"Developmental shift of cyclophilin D contribution to hypoxic-ischemic brain injury","volume":"29","author":"Wang","year":"2009","journal-title":"J. Neurosci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2050","DOI":"10.1161\/STROKEAHA.110.589051","article-title":"Neuroprotective effect of Bax-inhibiting peptide on neonatal brain injury","volume":"41","author":"Wang","year":"2010","journal-title":"Stroke"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1159\/000375368","article-title":"Does Caspase-6 Have a Role in Perinatal Brain Injury?","volume":"37","author":"Baburamani","year":"2015","journal-title":"Dev. Neurosci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1002\/ana.22431","article-title":"Genetic inhibition of caspase-2 reduces hypoxic-ischemic and excitotoxic neonatal brain injury","volume":"70","author":"Carlsson","year":"2011","journal-title":"Ann. Neurol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"566","DOI":"10.1038\/pr.2012.15","article-title":"Combined effect of hypothermia and caspase-2 gene deficiency on neonatal hypoxic-ischemic brain injury","volume":"71","author":"Carlsson","year":"2012","journal-title":"Pediatr. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1038\/sj.cdd.4402053","article-title":"Apoptosis-inducing factor is a major contributor to neuronal loss induced by neonatal cerebral hypoxia-ischemia","volume":"14","author":"Zhu","year":"2007","journal-title":"Cell Death Differ."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"108088","DOI":"10.1016\/j.neuropharm.2020.108088","article-title":"Inhibiting the interaction between apoptosis-inducing factor and cyclophilin A prevents brain injury in neonatal mice after hypoxia-ischemia","volume":"171","author":"Rodriguez","year":"2020","journal-title":"Neuropharmacology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1038\/nrd.2018.168","article-title":"Drug repurposing: Progress, challenges and recommendations","volume":"18","author":"Pushpakom","year":"2019","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1016\/j.chembiol.2017.12.001","article-title":"Identification of New Activators of Mitochondrial Fusion Reveals a Link between Mitochondrial Morphology and Pyrimidine Metabolism","volume":"25","author":"Sebastian","year":"2018","journal-title":"Cell Chem. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/s13058-020-01301-x","article-title":"Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer","volume":"22","author":"Humphries","year":"2020","journal-title":"Breast Cancer Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1007\/s10620-009-0737-0","article-title":"Protective effects of leflunomide on intestinal ischemia-reperfusion injury: Leflunomide against intestinal ischemia-reperfusion","volume":"55","author":"Yildiz","year":"2010","journal-title":"Dig. Dis. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1007\/s00383-006-1668-x","article-title":"Protective effects of leflunomide against ischemia-reperfusion injury of the rat liver","volume":"22","author":"Karaman","year":"2006","journal-title":"Pediatr. Surg. Int."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1434","DOI":"10.1111\/j.1442-2042.2006.01592.x","article-title":"Prevention of renal ischemia\/reperfusion-induced injury in rats by leflunomide","volume":"13","author":"Karaman","year":"2006","journal-title":"Int. J. Urol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1565","DOI":"10.1016\/j.tiv.2012.04.020","article-title":"Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests","volume":"27","author":"Lundqvist","year":"2013","journal-title":"Toxicol. In Vitro"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Mittelman, D., and Wilson, J.H. (2013). The fractured genome of HeLa cells. Genome Biol., 14.","DOI":"10.1186\/gb-2013-14-4-111"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"26057","DOI":"10.1038\/srep26057","article-title":"Characteristics of Mitochondrial Transformation into Human Cells","volume":"6","author":"Kesner","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.acthis.2017.03.001","article-title":"A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture","volume":"119","author":"Valente","year":"2017","journal-title":"Acta Histochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1016\/j.cmet.2015.05.007","article-title":"The OPA1-dependent mitochondrial cristae remodeling pathway controls atrophic, apoptotic, and ischemic tissue damage","volume":"21","author":"Varanita","year":"2015","journal-title":"Cell Metab."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0092-8674(92)90204-P","article-title":"Multipotent neural cell lines can engraft and participate in development of mouse cerebellum","volume":"68","author":"Snyder","year":"1992","journal-title":"Cell"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Zhu, S., Yan, X., Xiang, Z., Ding, H.F., and Cui, H. (2013). Leflunomide reduces proliferation and induces apoptosis in neuroblastoma cells in vitro and in vivo. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0071555"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e126915","DOI":"10.1172\/jci.insight.126915","article-title":"Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer","volume":"4","author":"Yu","year":"2019","journal-title":"JCI Insight"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Kowluru, R.A., and Alka, K. (2023). Mitochondrial Quality Control and Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24098076"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"875","DOI":"10.1007\/s00431-021-04320-8","article-title":"New possibilities for neuroprotection in neonatal hypoxic-ischemic encephalopathy","volume":"181","author":"Victor","year":"2022","journal-title":"Eur. J. Pediatr."},{"key":"ref_39","first-page":"CD003311","article-title":"Cooling for newborns with hypoxic ischaemic encephalopathy","volume":"2013","author":"Jacobs","year":"2013","journal-title":"Cochrane Database Syst. Rev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1007\/s11910-019-0916-0","article-title":"Therapeutic Hypothermia in Neonatal Hypoxic-Ischemic Encephalopathy","volume":"19","author":"Wassink","year":"2019","journal-title":"Curr. Neurol. Neurosci. Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"199","DOI":"10.3389\/fncel.2017.00199","article-title":"Mitochondria, Bioenergetics and Excitotoxicity: New Therapeutic Targets in Perinatal Brain Injury","volume":"11","author":"Leaw","year":"2017","journal-title":"Front. Cell Neurosci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"468","DOI":"10.1038\/s41589-022-01224-y","article-title":"Small molecule agonist of mitochondrial fusion repairs mitochondrial dysfunction","volume":"19","author":"Guo","year":"2023","journal-title":"Nat. Chem. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1038\/s41392-020-00447-6","article-title":"Small molecule induces mitochondrial fusion for neuroprotection via targeting CK2 without affecting its conventional kinase activity","volume":"6","author":"Zeng","year":"2021","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1126\/science.aao1785","article-title":"MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A","volume":"360","author":"Rocha","year":"2018","journal-title":"Science"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Jones, A., and Thornton, C. (2022). Mitochondrial dynamics in the neonatal brain\u2014A potential target following injury?. Biosci. Rep., 42.","DOI":"10.1042\/BSR20211696"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"120369","DOI":"10.1016\/j.lfs.2022.120369","article-title":"Dual regulating of mitochondrial fusion and Timp-3 by leflunomide and diallyl disulfide combination suppresses diethylnitrosamine-induced hepatocellular tumorigenesis in rats","volume":"294","author":"Abass","year":"2022","journal-title":"Life Sci."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1007\/s00018-022-04595-6","article-title":"Mfn2-mediated mitochondrial fusion promotes autophagy and suppresses ovarian cancer progression by reducing ROS through AMPK\/mTOR\/ERK signaling","volume":"79","author":"Ashraf","year":"2022","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1186\/s12931-023-02634-9","article-title":"Role of mitochondrial fusion proteins MFN2 and OPA1 on lung cellular senescence in chronic obstructive pulmonary disease","volume":"24","author":"Li","year":"2023","journal-title":"Respir. Res."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.chembiol.2018.02.014","article-title":"Flaming Mitochondria: The Anti-inflammatory Drug Leflunomide Boosts Mitofusins","volume":"25","author":"Pellattiero","year":"2018","journal-title":"Cell Chem. Biol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.tox.2018.02.003","article-title":"Mitochondrial dysfunction induced by leflunomide and its active metabolite","volume":"396\u2013397","author":"Xuan","year":"2018","journal-title":"Toxicology"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1897","DOI":"10.2147\/DDDT.S252626","article-title":"Leflunomide Inhibits Proliferation and Induces Apoptosis via Suppressing Autophagy and PI3K\/Akt Signaling Pathway in Human Bladder Cancer Cells","volume":"14","author":"Cheng","year":"2020","journal-title":"Drug Des. Devel Ther."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Maremanda, K.P., Sundar, I.K., and Rahman, I. (2021). Role of inner mitochondrial protein OPA1 in mitochondrial dysfunction by tobacco smoking and in the pathogenesis of COPD. Redox Biol., 45.","DOI":"10.1016\/j.redox.2021.102055"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Maiwald, C.A., Annink, K.V., R\u00fcdiger, M., Benders, M., van Bel, F., Allegaert, K., Naulaers, G., Bassler, D., Kleberma\u00df-Schrehof, K., and Vento, M. (2019). Effect of allopurinol in addition to hypothermia treatment in neonates for hypoxic-ischemic brain injury on neurocognitive outcome (ALBINO): Study protocol of a blinded randomized placebo-controlled parallel group multicenter trial for superiority (phase III). BMC Pediatr., 19.","DOI":"10.1186\/s12887-019-1566-8"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1552","DOI":"10.21037\/tp-23-253","article-title":"A narrative review on treatment strategies for neonatal hypoxic ischemic encephalopathy","volume":"12","author":"Korf","year":"2023","journal-title":"Transl. Pediatr."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"709301","DOI":"10.3389\/fnsyn.2021.709301","article-title":"Current Therapies for Neonatal Hypoxic-Ischaemic and Infection-Sensitised Hypoxic-Ischaemic Brain Damage","volume":"13","author":"Tetorou","year":"2021","journal-title":"Front. Synaptic Neurosci."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"e20160191","DOI":"10.1542\/peds.2016-0191","article-title":"High-Dose Erythropoietin and Hypothermia for Hypoxic-Ischemic Encephalopathy: A Phase II Trial","volume":"137","author":"Wu","year":"2016","journal-title":"Pediatrics"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1056\/NEJMoa2119660","article-title":"Trial of Erythropoietin for Hypoxic-Ischemic Encephalopathy in Newborns","volume":"387","author":"Wu","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"113400","DOI":"10.1016\/j.jpeds.2023.113400","article-title":"Safety of High Dose Erythropoietin Used with Therapeutic Hypothermia as Treatment for Newborn Hypoxic-Ischemic Encephalopathy: Secondary Analysis of the HEAL Randomized Controlled Trial","volume":"258","author":"Juul","year":"2023","journal-title":"J. Pediatr."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/7\/631\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:23:34Z","timestamp":1760106214000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/7\/631"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,4,4]]},"references-count":58,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["cells13070631"],"URL":"https:\/\/doi.org\/10.3390\/cells13070631","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,4,4]]}}}