ACOD1 Facilitates p62-Mediated Keap1 Degradation for Nrf2 Activation to Reduce Neuroinflammation and Oxidative Stress Post Spinal Cord Contusion

Background: Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing terrible neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 and releasing Nrf2; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI.Methods: Adult male C57BL/6 ACOD1-/- mice and their wildtype (WT) littermates were subjected to a moderate thoracic spinal cord contusion. Degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR technique, western blot, oxidization determination, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Locomotor function was evaluated with the Basso Mouse Scale and footprint assay.Findings: Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 pathway was down-regulated; deficiency of ACOD1 exacerbated dysfunction of neurohistology and neuroethology in the SCI mice. Supplement of exogenous Ita or 4-octyl itaconate instead reduced p62 phosphorylation and inhibited autophagy. Besides, ACOD1 was capable of interacting with p-p62 and enhanced Nrf2 activation, further endonuclear Nrf2 promoted transcription of ACOD1 gene.Interpretation: Here we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting the anti-inflammation and anti-OS effects in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI rather than exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita.Funding: The study was supported by the Natural Science Foundation of Jiangsu Province (Grant No. SBK2022022488), the Chinese Traditional Medicine Technology Development Project of Taizhou City (Grant No. TZ202208), the 2022 Lifting Project for Young Scientific and Technological Talents Funded by Jiangsu Association of Science and Technology (Grant No. TJ-2022-033), and the China Postdoctoral Science Foundation (CPSF; Grant No. 2022M721680).Declaration of Interest: The authors declare that they have no competing interests.Ethical Approval: The project was approved by the Ethics Committee of Taizhou People’s Hospital of Nanjing Medical University and the procedure was obeyed by the guidelines of the National Institutes of Health Animal Laboratory Animal Care and Use Guidelines.