Department of Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, Liaoning Province, 116044, PR China.
Department of Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, Liaoning Province, 116044, PR China; Inner Mongolia Autonomous Region People's Hospital, Inner Mongolia Autonomous Region, 010017, PR China.
Free Radic Biol Med. 2024 Nov 1;224:757-769. doi: 10.1016/j.freeradbiomed.2024.09.029. Epub 2024 Sep 20.
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.
We hypothesized that TRIM25 reduces ATAD3A expression by ubiquitinating ATAD3A, promoting mitophagy via the PINK1/Parkin pathway, and aggravating CI/RI.
Rat middle cerebral artery occlusion (MCAO) followed by reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) in PC12 cells were used as animal and cell models, respectively.
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).
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.
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.
脑缺血再灌注损伤（CI/RI）是导致神经元损伤和死亡的复杂过程，其中自噬体参与其发病机制。然而，自噬体在 CI/RI 中的意义仍存在争议。
我们假设 TRIM25 通过泛素化 ATAD3A 来降低 ATAD3A 的表达，通过 PINK1/Parkin 途径促进自噬，从而加重 CI/RI。
大鼠大脑中动脉闭塞（MCAO）后再灌注和氧葡萄糖剥夺再氧合（OGD/R）分别用于动物和细胞模型。
为了评估 CI/R 模型的成功，采用 TTC 和 HE 染色。使用相对检测试剂盒测定血清生化指标。采用 Western Blot 分析评估 ATAD3A、TRIM25 以及自噬相关蛋白（PINK1、Parkin、P62 和 LC3II/LC3I）的表达。采用 QRT-PCR 检测 mRNA 水平。通过 JC-1 染色评估线粒体膜电位。使用 Mitosox Red 试剂盒测定 PC12 细胞中线粒体活性氧水平。此外，通过透射电子显微镜（TEM）观察对自噬体结构进行特征描述。
我们的研究结果显示，在体内和体外 CI/RI 模型中，ATAD3A 的表达下调，TRIM25 的表达上调。Western Blot、JC-1 染色、Mitosox 测定、免疫荧光测定和 TEM 观察等各种实验技术均支持在这两种模型中均发生了 PINK1/Parkin 信号通路介导的自噬。ATAD3A 抑制自噬，而 TRIM25 在 CI/RI 损伤过程中促进自噬。此外，结果表明 TRIM25 通过蛋白酶体途径与 ATAD3A 相互作用并泛素化 ATAD3A，影响 ATAD3A 蛋白的稳定性和表达。
TRIM25 通过破坏 ATAD3A 促进 Pink1/Parkin 依赖性过度自噬，从而加重 CI/RI。靶向 TRIM25 和 ATAD3A 可能为减轻 CI/RI 和相关神经损伤提供治疗策略。
