Ubiquitination of Atad3a by Trim25 Exacerbates Cerebral Ischemia-Reperfusion Injury Via Regulating Pink1/Parkin Signaling Pathway-Mediated Mitophagy

Abstract

Background: Cerebral ischemia-reperfusion injury (CI/RI) is a complex process leading to neuronal damage and death, with mitophagy implicated in its pathogenesis. However, the significance of mitophagy in CI/RI remains debated. Hypothesis: We hypothesized that TRIM25 reduces ATAD3A expression by ubiquitinating ATAD3A, promoting mitochondrial autophagy via the PINK1/Parkin pathway, and aggravating CI/RI.

Study Design: Rat middle cerebral artery occlusion (MCAO) followed by reperfusion and oxygen35 glucose deprivation and reoxygenation (OGD/R) in PC12 cells were used as animal and cell models, respectively.

Methods: To evaluate the success of the CI/R modeling, TTC and HE staining were employed. The determination of serum biochemical indexes was carried out using relative assay kits. The Western Blot analysis was employed to assess the expression of ATAD3A, TRIM25, as well as mitophagy-related proteins (PINK1, Parkin, P62, and LC3II/LC3I). The mRNA levels were detected using QRT-PCR. Mitochondrial membrane potential was assessed through JC-1 staining. Mitosox Red Assay Kit was utilized to measure mitochondrial reactive oxygen species levels in PC12 cells. Additionally, characterization of the mitophagy structure was performed using transmission electron microscopy (TEM).

Results: Our findings showed down-regulation of ATAD3A and up-regulation of TRIM25 in both in vivo and in vitro CI/RI models. Various experimental techniques such as Western Blot, JC-1 staining, Mitosox assay, Immunofluorescence assay, and TEM observation supported the occurrence of PINK1/Parkin signaling pathway-mediated mitophagy in both models. ATAD3A suppressed mitophagy, while TRIM25 promoted it during CI/RI injury. Additionally, the results indicated that TRIM25 interacted with and ubiquitinated ATAD3A via the proteasome pathway, affecting ATAD3A protein stability and expression.

Conclusion: TRIM25 promoted Pink1/Parkin-dependent excessive mitophagy by destabilizing ATAD3A, exacerbating CI/RI. Targeting TRIM25 and ATAD3A may offer therapeutic strategies for mitigating CI/RI and associated neurological damage.

Note:
Funding declaration: This work was supported by the Scientific Research Fund of Liaoning Provincial Education Department [grant number LJKFZ20220251] and the National Natural Science Foundation of China [grant number 81502992, 30770297].

Conflict of Interests: None.

Ethics statement: This study was approved by the Ethics Committee of Dalian Medical University, Dalian, China (SCXK 2018-0003).

Keywords: Cerebral ischemia-reperfusion injury, mitophagy, PINK1/Parkin pathway, TRIM25, ATAD3A