Netrin-1 binding to UNC5b improves post-stroke neuronal ferroptosis via AMPK-BACH1 pathway

Ying Luo; Meiling Yao; Rui Wang; Songjie Liao; Jian Yu

doi:10.1016/j.ejphar.2025.177507

Netrin-1 binding to UNC5b improves post-stroke neuronal ferroptosis via AMPK-BACH1 pathway

Eur J Pharmacol. 2025 Jul 5:998:177507. doi: 10.1016/j.ejphar.2025.177507. Epub 2025 Mar 12.

Authors

Ying Luo¹, Meiling Yao², Rui Wang², Songjie Liao³, Jian Yu⁴

Affiliations

¹ Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University; Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases; National Key Clinical Department and Key Discipline of Neurology, No.58 Zhongshan Road 2, Guangzhou, 510080, China; Department of Neurology, The Affiliated Hospital of Southwest Medical University, Laboratory of Neurological Diseases and Brain Function, Luzhou, 646000, China.
² Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University; Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases; National Key Clinical Department and Key Discipline of Neurology, No.58 Zhongshan Road 2, Guangzhou, 510080, China.
³ Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University; Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases; National Key Clinical Department and Key Discipline of Neurology, No.58 Zhongshan Road 2, Guangzhou, 510080, China. Electronic address: liaosjie@mail.sysu.edu.cn.
⁴ Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University; Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases; National Key Clinical Department and Key Discipline of Neurology, No.58 Zhongshan Road 2, Guangzhou, 510080, China. Electronic address: yujian@mail.sysu.edu.cn.

PMID: 40086580
DOI: 10.1016/j.ejphar.2025.177507

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.

Keywords: AMPK; BACH1; Cerebral ischemia; Ferroptosis; Ntn1; UNC5b.

MeSH terms

AMP-Activated Protein Kinases* / metabolism
Animals
Basic-Leucine Zipper Transcription Factors* / metabolism
Cell Line
Ferroptosis* / drug effects
Male
Mice
Mice, Inbred C57BL
Netrin Receptors* / genetics
Netrin Receptors* / metabolism
Netrin-1* / metabolism
Neurons* / drug effects
Neurons* / metabolism
Neurons* / pathology
Signal Transduction
Stroke* / metabolism
Stroke* / pathology

Substances

Netrin-1
Netrin Receptors
AMP-Activated Protein Kinases
Basic-Leucine Zipper Transcription Factors
Ntn1 protein, mouse