Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis

Abstract

Background Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive. Methods The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/LC3 I, TNF-α, IL-1β, etc., were detected under normal or Drp1 interference conditions. Results The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P < 0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P < 0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P < 0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/LC3 I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P < 0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P > 0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4 – 5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P < 0.05). Furthermore, TNF-α and IL-1β increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen–glucose deprivation/reoxygenation (OGD/R) conditions (P < 0.05). Conclusions CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.

Oxidative-Damaged Mitochondria Activate GABARAPL1-Induced NLRP3 Inflammasomes in an Autophagic-Exosome Manner after Acute Myocardial Ischemia

“…However, these studies have primarily focused on inflammation and signaling cascade, and few have focused on the aberrantly expressed genes related to oxidative-damaged mitochondria and oxidative stress in AMI. Our study elucidated the process of vascular inflammation caused by myocardial ischemia from the perspective of mitochondrial quality imbalance and preliminarily explained the mechanism through the autophagic-exosome manner [ 23 ] in the cardiovascular system.…”

Identification of MMP9 as a Novel Biomarker to Mitochondrial Metabolism Disorder and Oxidative Stress in Calcific Aortic Valve Stenosis

“…The ROS fluorescence was excited by a 488 nm laser and emission was collected at 501–563 nm. Quantification for the ROS assay was conducted using ImageJ to measure the FITC fluorescence intensity [ 5 , 6 ].…”

“…The JC-1 aggregate fluorescence was excited by a 633 nm laser and emission was collected at 558-617 nm. Quantification for mitochondrial membrane potential was carried out to measure the fluorescence intensity ratio of JC-1 aggregate/JC-1 monomer [ 5 , 6 ].…”

“…Mitochondria, the main organelles of aerobic respiration, are of great significance for maintaining the normal physiological function of cells [ 5 , 6 ]. Under normal conditions, the mitochondrial quality indices—including mitochondrial dynamics, functions, and metabolism—are in dynamic balance [ 7 ].…”

Dihydroartemisinin Reduces Irradiation-Induced Mitophagy and Radioresistance in Lung Cancer A549 Cells via CIRBP Inhibition

“…Based on previous research, the mitochondrial division inhibitor 1 (Mdivi-1) was extensively studied and found to reduce dynamin-related protein 1 (Drp1) levels and excessive mitochondrial fission, and can inhibit mitophagy through mitochondrial-targeting decreasing PINK1, PARK2 and LC3II levels, both in vivo and in vitro [ 11 , 13 , 14 , 15 ]. In order to clarify the effect of mitophagy on the radioresistance of A549R cells, Mdivi-1 was used in this study.…”