Ying Luo , Meiling Yao , Rui Wang , Songjie Liao , Jian Yu
{"title":"Netrin-1 binding to UNC5b improves post-stroke neuronal ferroptosis via AMPK-BACH1 pathway","authors":"Ying Luo , Meiling Yao , Rui Wang , Songjie Liao , Jian Yu","doi":"10.1016/j.ejphar.2025.177507","DOIUrl":null,"url":null,"abstract":"<div><div>Ferroptosis contributes to neuronal destruction after ischemic stroke which may be improved by inhibiting BTB domain and CNC homolog 1 (BACH1), a recently recognized ferroptosis facilitator. Axon guidance molecule netrin-1 (Ntn1) functions in neuroprotection against ischemic insult by engaging into its receptor of uncoordinated-5 homolog B (UNC5b) <em>via</em> adenosine 5‘-monophosphate-activated protein kinase (AMPK), which potentially binds to BACH1. Whether Ntn1/UNC5b regulates post-stroke ferroptosis through AMPK-BACH1 pathway remains unclear. Ntn1 supplementation and UNC5b knockdown by siRNA were performed in photo-thrombosis stroke mice and oxygen-glucose deprivation-treated HT22 neurons. AMPK inhibitor BAY3827 and BACH1 activator Leptomycin B (LMB) were administrated. Ferroptosis was determined by ferroptosis-associated proteins (FSP1, GPX4 and ACSL4), Fe<sup>2+</sup>, malondialdehyde and mitochondrial morphology. BACH1 and p-AMPK/AMPK as well as the interaction between them were examined by Western blot and co-immunoprecipitation. Neuronal ferroptosis and the protein levels of BACH1 and p-AMPK were increased after photo-thrombosis and oxygen-glucose deprivation. Ntn1 supplementation or UNC5b knockdown relieved neuronal ferroptosis and neurological impairment with downregulated BACH1 and upregulated p-AMPK, nonetheless, UNC5b knockdown prevented the beneficial role of Ntn1. Both BAY3827 and LMB could reverse the change of ferroptosis caused by Ntn1 where BAY3827 inhibited the effects of Ntn1 to p-AMPK and BACH1 while LMB only inhibited the effect of Ntn1 to BACH1 without p-AMPK, suggesting BACH1 was regulated by AMPK. Co-immunoprecipitation verified that AMPK could physically bind to BACH1. Our results demonstrate UNC5b-evoked neuronal ferroptosis post stroke, and favor that Ntn1 improves post-stroke ferroptosis by its interaction with UNC5b <em>via</em> the AMPK-BACH1 pathway.</div></div>","PeriodicalId":12004,"journal":{"name":"European journal of pharmacology","volume":"998 ","pages":"Article 177507"},"PeriodicalIF":4.2000,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"European journal of pharmacology","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0014299925002614","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PHARMACOLOGY & PHARMACY","Score":null,"Total":0}
引用次数: 0
Abstract
Ferroptosis contributes to neuronal destruction after ischemic stroke which may be improved by inhibiting BTB domain and CNC homolog 1 (BACH1), a recently recognized ferroptosis facilitator. Axon guidance molecule netrin-1 (Ntn1) functions in neuroprotection against ischemic insult by engaging into its receptor of uncoordinated-5 homolog B (UNC5b) via adenosine 5‘-monophosphate-activated protein kinase (AMPK), which potentially binds to BACH1. Whether Ntn1/UNC5b regulates post-stroke ferroptosis through AMPK-BACH1 pathway remains unclear. Ntn1 supplementation and UNC5b knockdown by siRNA were performed in photo-thrombosis stroke mice and oxygen-glucose deprivation-treated HT22 neurons. AMPK inhibitor BAY3827 and BACH1 activator Leptomycin B (LMB) were administrated. Ferroptosis was determined by ferroptosis-associated proteins (FSP1, GPX4 and ACSL4), Fe2+, malondialdehyde and mitochondrial morphology. BACH1 and p-AMPK/AMPK as well as the interaction between them were examined by Western blot and co-immunoprecipitation. Neuronal ferroptosis and the protein levels of BACH1 and p-AMPK were increased after photo-thrombosis and oxygen-glucose deprivation. Ntn1 supplementation or UNC5b knockdown relieved neuronal ferroptosis and neurological impairment with downregulated BACH1 and upregulated p-AMPK, nonetheless, UNC5b knockdown prevented the beneficial role of Ntn1. Both BAY3827 and LMB could reverse the change of ferroptosis caused by Ntn1 where BAY3827 inhibited the effects of Ntn1 to p-AMPK and BACH1 while LMB only inhibited the effect of Ntn1 to BACH1 without p-AMPK, suggesting BACH1 was regulated by AMPK. Co-immunoprecipitation verified that AMPK could physically bind to BACH1. Our results demonstrate UNC5b-evoked neuronal ferroptosis post stroke, and favor that Ntn1 improves post-stroke ferroptosis by its interaction with UNC5b via the AMPK-BACH1 pathway.
期刊介绍:
The European Journal of Pharmacology publishes research papers covering all aspects of experimental pharmacology with focus on the mechanism of action of structurally identified compounds affecting biological systems.
The scope includes:
Behavioural pharmacology
Neuropharmacology and analgesia
Cardiovascular pharmacology
Pulmonary, gastrointestinal and urogenital pharmacology
Endocrine pharmacology
Immunopharmacology and inflammation
Molecular and cellular pharmacology
Regenerative pharmacology
Biologicals and biotherapeutics
Translational pharmacology
Nutriceutical pharmacology.