Acquired inhibition of microRNA-124 protects against spinal cord ischemia-reperfusion injury partially through a mitophagy-dependent pathway

Abstract

Objective: Mitophagy results in selective clearance of damaged mitochondria. We investigated whether mitophagy was involved in the neuroprotection by inhibiting microRNA (miRNA)-124 on ischemic spinal cords.

Methods: Inhibition of miRNA-124 was conducted by intrathecal injection of lentivirus vectors containing antagomiR-124. Spinal cord ischemia was induced in rats by crossclamping the descending aorta just distal to the left subclavian artery for 14 minutes. Hind-limb motor function was assessed with the motor deficit index (MDI). Lumbar spinal cords were harvested for ultrastructural, histologic examinations, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining. Mitophagy was evaluated by expressions of beclin-1 and LC3-II in mitochondria. Expressions of inhibitory member of the apoptosis-stimulating proteins of p53 family, p53, beclin-1, LC3-II, and miRNA-124 were measured by Western blot and quantitative real-time polymerase chain reaction. Mitophagy was inhibited by the antagonist of 3-methyladenine.

Results: Compared with control animals, antagomiR-124 significantly inhibited expressions of miRNA-124 (P < .01) and p53 (P < .05) and enhanced expressions of inhibitory member of the apoptosis-stimulating proteins of p53 family, becline-1 and LC3-II (P < .01, respectively) in spinal cords. MDI at 6, 12, 24, and 48 hours after reperfusion were markedly lower in antagomiR-124 group (P < .01, vs control group, respectively). More motor neurons and less apoptotic cells were detected in lumbar spinal cords of antagomiR-124 group (P < .01 vs control group). Administration of 3-methyladenine completely abolished enhancements of mitochondrial becline-1 and LC3-II by antagomiR-124 (P < .01 vs antagomiR-124 group) and partially inhibited effects of antagomiR-124 on MDI, number of motor neurons, and apoptotic cells (P < .01 or < .05 vs control group and antagomiR-124 group, respectively).

Conclusions: Inhibition of miRNA-124 exerts neuroprotection on spinal cords against ischemia-reperfusion injury, possibly by induction of mitophagy and antiapoptotic effects.

Keywords: apoptosis; ischemia-reperfusion; microRNA-124; mitophagy; spinal cord.