Research reportMitophagy is activated in brain damage induced by cerebral ischemia and reperfusion via the PINK1/Parkin/p62 signalling pathway
Introduction
Cerebral ischemia as a leading cause of mortality and disability worldwide, which brings a heavy burden to both society and family. Lack of blood supply for a short time period can result in severe brain damage, and reperfusion following ischemia is believed to contribute to delayed secondary brain damage (Schaller and Graf, 2004; White et al., 2000).
The mitochondria play important role in adenosine triphosphate (ATP) production, calcium homeostasis, reactive oxygen species (ROS) production, and in dire cases, membrane permeability and are involved in multiple signals (Gottlieb, 2011). Recent studies have shown that reperfusion following focal cerebral ischemia induced significant mitochondrial dysfunction such as mitochondrial permeability transition pore opening, mitochondrial morphological damage, Ca2+-induced mitochondrial swelling, and the release of mitochondrial cytochrome c into the cytosol (Dave et al., 2011; Li et al., 2012; Sun et al., 2012). Autophagy, as a highly regulated process, degrades the bulk of cytoplasmic macromolecules and organelles in mammalian cells via the lysosomal system (Rajawat and Bossis, 2008). Specifically, mitophagy is the selective degradation of excess or damaged mitochondria by autophagy, which is an important mechanism of mitochondrial quality control for maintaining normal function of this organelle (Fimia et al., 2013). Mitophagy often occurs following damage or stress to mitochondria, with a number of studies indicating mitophagy is activated after cerebral ischemia/reperfusion (Li et al., 2014; Liu et al., 2013; Yuan et al., 2015).
PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy is the most extensively-studied mechanism underlying mitophagy (Yuan et al., 2015). Although recent studies have suggested that PINK1-Parkin are activated after ischemic brain injury (Zhou et al., 2015), the integrated mechanisms remain largely unknown. Neurons, astrocytes, and endothelial cells are important components in the neurovascular unit (NVU), which interact with each other to maintain the homeostatic microenvironment for neuronal survival and function (Vangilder et al., 2011). Therefore, in the present study, we investigated the characteristics of mitophagy and the role of the PINK1/Parkin/p62 pathway in the context of cerebral ischemia and different time-points of reperfusion, and clarified activation of mitophagy in NVU after ischemia-reperfusion injury.
Section snippets
Reagents
For western blotting and immunofluorescence, primary rabbit monoclonal antibodies for LC3, LC3B(D11), and anti-rabbit antibodies against cytochrome c oxidase subunit IV (COXIV) were purchased from Cell Signaling Technology (Beverly, MA, USA); mouse monoclonal antibodies for VDAC1, LAMP1(H4A3) were from Abcam (HongKong, China), as were rabbit polyclonal antibodies for PINK1, Parkin, and p62. For western blotting, we used mouse monoclonal antibodies for Beclin1 and β-actin (ImmunoWay
Cerebral infarct area and brain damage following MCAO and reperfusion
At 80 min after MCAO, there were hyperintense signals in the left hemispheres on T2 weighted imaging (T2WI) (Fig. 1A.a). However, no obvious abnormal signals were found on T1 weighted imaging (T1WI) (Fig. 1A.b). Large infarct areas in the territory of the left MCA in the diffusion weighted imaging (DWI) and the corresponding apparent diffusion coefficient (ADC) images (Fig. 1A.c and d) were observed. In addition, images showed no significant enhanced blood brain barrier (BBB) permeability in
Discussion
The present study demonstrated that autophagy/mitophagy was activated following cerebral ischemia and at different time-points after reperfusion, reaching a peak at 24 h after reperfusion. The PINK1/Parkin pathway was found to be involved during the course of the resulting pathophysiological processes. In particular, the NVU, a structure involved in the maintenance and homeostasis of neurons, was affected. The results of current study suggest that autophagy/mitophagy was also partially
Conclusion
Our results indicate that mitophagy is activated following ischemic-reperfusion injury and that the PINK1/Parkin/p62 signalling pathway modulates mitophagy induced by ischemia-reperfusion. Moreover, autophagy/mitophagy occurred primarily evident in neurons although it was evident in other important components of the NVU, including astrocytes and to a lesser extent vascular endothelial cells.
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgments
This work is supported by the National Natural Science Foundation of China (Grant No. 81503422).
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2023, Experimental NeurologyCitation Excerpt :Altogether, forced expression of RGD1564534 attenuated brain white matter and small vessel damage in MCAO rats. Evidence exists demonstrating that mitophagy can be induced following cerebral I/R (Lan et al., 2018), and activating mitophagy alleviates nerve injury in cerebral I/R (Yang et al., 2018a, 2018b; Zhang et al., 2020a, 2020b; Cai et al., 2021). We then focused on the effect of RGD1564534 on mitochondrial production.