USP5-C-MAF Axis Regulates Autophagy-Dependent Neuronal Ferroptosis in Spinal Cord Injury Therapeutics

Objective: Spinal cord injury (SCI) is a severe secondary injury that often results in impaired motor function, imposing a significant burden on both individuals and society. Therefore, there is an urgent need for new therapeutic targets and strategies to address this challenge.

Methods: To construct a mouse model of SCI, an aneurysm clip was used in vivo to clamp the abdominal aorta below the left renal artery in C57BL/6J mice. After 60 min, the aneurysm clip was removed to restore blood flow. In vitro, primary neuronal cells were subjected to OGD/R to mimic the conditions of SCI. Cell viability was assessed using the CCK-8 assay, and the levels of neuronal death, Autophagy, and Ferroptosis were determined using a combination of WB, IF, and transmission electron microscopy. IP/MS and Co-IP techniques were employed for the identification and validation of proteins interacting with USP5.

Results: Neurons exhibit significant Ferroptosis in the SCI mice. USP5 is markedly upregulated in SCI neurons and mediates neural Ferroptosis. In vitro experiments demonstrate that overexpression of USP5 promotes neuronal Ferroptosis, whereas knockout of USP5 significantly reduces it, with consistent results observed in vivo. Notably, the upregulation of USP5 expression markedly increases the accumulation of autophagosomes and autophagic flux in neurons, which may represent a potential mechanism by which USP5 mediates neuronal Ferroptosis. Further investigations utilizing IP/MS and Co-IP confirmed the interaction between USP5 and c-MAF. Additionally, Western blot analysis revealed that USP5, through its deubiquitinating enzyme activity, enhances c-MAF protein stability, thereby activating Autophagy and subsequently promoting neuronal Ferroptosis.

Conclusion: In summary, our results indicate a close relationship between Ferroptosis and SCI. USP5 regulates c-MAF expression through deubiquitination, thereby activating autophagy-dependent Ferroptosis in neurons and mediating the progression of SCI. USP5 may serve as a potential therapeutic target for SCI.