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

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.