Elsevier

Brain Research Bulletin

Volume 142, September 2018, Pages 63-77
Brain Research Bulletin

Research report
Mitophagy is activated in brain damage induced by cerebral ischemia and reperfusion via the PINK1/Parkin/p62 signalling pathway

https://doi.org/10.1016/j.brainresbull.2018.06.018Get rights and content

Highlights

  • Mitophagy was activated after cerebral ischemia and reperfusion and reached the highest level at 24 h after reperfusion.

  • Autophagy occurred in diverse components of the neurovascular unit.

  • The PINK1/Parkin/p62 signalling pathway was involved in cerebral ischemia-reperfusion injury.

Abstract

This study examined the course of mitophagy following cerebral ischemia with reperfusion and the role of the PTEN-induced kinase 1 (PINK1)/Parkin/p62 signalling pathway. The middle cerebral artery of male Sprague-Dawley rats was occluded for 90 min and was followed by different time-points of reperfusion. Cerebral infarct areas were detected by 2,3,5-triphenyl tetrazolium chloride staining, while brain damage was observed by haematoxylin and eosin staining. Levels of LC3, Beclin1 and LAMP-1 were estimated by western blots. LC3B location was observed in various cells in the neurovascular unit. In addition, PINK1 accumulation in damaged mitochondria and Parkin/p62 mitochondrial translocation were investigated by double immunofluorescence staining. Finally, the levels of PINK1, Parkin and p62 expression in mitochondrial fractions were estimated by western blots. Cerebral ischemia with different time-points of reperfusion resulted in infarct in the territory of the middle cerebral artery accompanied by overall brain damage. In addition, we found up-regulation of LC3B, Beclin1, and LAMP-1, as well as mitophagy activation after reperfusion, with peak expression of these proteins at 24 h after reperfusion. Electron microscopy and immunofluorescence indicated that LC3B was primarily located in neurons, although lower levels of expression were found in astrocytes and even less in vascular endothelial cells. Moreover, significant increases in PINK1 accumulation in the outer membrane of mitochondria and increased Parkin/p62 mitochondrial translocation were shown at 24 h after reperfusion. These findings suggest that the PINK1/Parkin/p62 signalling pathway was involved in the pathophysiological processes following ischemia and reperfusion.

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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