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USP5-C-MAF Axis Regulates Autophagy-Dependent Neuronal Ferroptosis in Spinal Cord Injury Therapeutics
Shiyang Weng1, Kai Wu1, Yinjun Huang1
1Department of Trauma Center, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
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.
Insights
Spinal cord injury (SCI) causes neuronal ferroptosis, mediated by USP5. This protein enhances c-MAF stability, activating autophagy-dependent ferroptosis and worsening SCI outcomes. USP5 is a potential therapeutic target for SCI.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Spinal cord injury (SCI) leads to significant motor function impairment.
- Developing new therapeutic targets for SCI is crucial due to its severe impact.
Purpose of the Study:
- To investigate the role of USP5 in neuronal ferroptosis following spinal cord injury.
- To identify potential therapeutic strategies targeting USP5 for SCI treatment.
Main Methods:
- Constructed a mouse model of SCI and used in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) models.
- Assessed cell viability, neuronal death, autophagy, and ferroptosis using CCK-8, Western blot, immunofluorescence, and electron microscopy.
- Identified protein interactions with USP5 using immunoprecipitation/mass spectrometry (IP/MS) and co-immunoprecipitation (Co-IP).
Main Results:
- Neurons in SCI models exhibited significant ferroptosis, with USP5 upregulation mediating this process.
- USP5 overexpression promoted neuronal ferroptosis, while USP5 knockout reduced it, confirmed in vitro and in vivo.
- USP5 deubiquitinating activity enhanced c-MAF stability, activating autophagy and promoting ferroptosis, thus mediating SCI progression.
Conclusions:
- Ferroptosis is closely linked to spinal cord injury.
- USP5 regulates autophagy-dependent ferroptosis in neurons by stabilizing c-MAF, contributing to SCI.
- USP5 represents a promising therapeutic target for managing spinal cord injury.
