Elsevier

Neuroscience

Volume 468, 1 August 2021, Pages 68-74
Neuroscience

Research Article
Ischemia Injury induces mPTP opening by reducing Sirt3

https://doi.org/10.1016/j.neuroscience.2021.06.003Get rights and content

Highlights

  • Sirt3 levels was decreased in the brain of mice subjected to MCAO, associated with elevated levels of VDAC1 and ANT1.

  • VDAC1 and ANT1 was elevated in in vitro stroke model.

  • Sirt3 overexpression attenuated SGD induced apoptosis via downregulated VDAC1 and ANT1.

Abstract

Mitochondrial permeability transition pore (mPTP) opening is critical to mitochondrial apoptosis during ischemic injury. Sirtuin 3 (Sirt3) is a mitochondrial deacetylase known to play a major role in stress resistance and cell death. Our previous studies have shown that Sirt3 activates superoxide dismutase 2 and forkhead box O3a to reduce cellular reactive oxygen species. However, it is unclear the interaction between Sirt3 and mPTP and the roles they play in ischemic stroke. We used the middle cerebral artery occlusion (MCAO) model, a mouse model of stroke, to examine Sirt3 and mPTP-related protein levels. We then applied lentivirus packaged Sirt3 overexpression in HT22 cells, a mouse hippocampal neuronal cell line, to investigate the underlying mechanism. We found Sirt3 protein level was decreased in the penumbra area in MCAO mice, along with an increase in mPTP related proteins, namely voltage-dependent anion channel 1 (VDAC1) and adenine nucleotide translocator 1 (ANT1). Sirt3 overexpression suppressed the increase in VDAC1, ANT1 and cleaved caspase 3 that were induced by the serum and glucose deprivation (SGD) condition. Our studies suggest that ischemic injury induced mPTP opening and apoptosis by reducing Sirt3. It helps to identify new therapeutic targets for ischemic stroke.

Introduction

Stroke is one of the leading causes of death and physical disability worldwide (Panel et al., 2018). About 80% of strokes are ischemic stroke (Lapchak and Zhang, 2017). Mitochondria regulate cell energy homeostasis and cell death. Mitochondrial dysfunction is one of the hallmarks in neuronal death through calcium overload and mitochondrial permeability transition pore (mPTP) opening (Sims and Muyderman, 2010). mPTP opening is critical to mitochondrial apoptosis during ischemic injury (Chanoit et al., 2011, Kinnally et al., 2011). mPTP is composed of Cyclophilin D (CypD) in the mitochondrial matrix, voltage-dependent anion channel 1 (VDAC1) across the outer mitochondrial membrane, and adenine nucleotide translocator 1 (ANT1) in the inner mitochondrial membrane (Halestrap and Davidson, 1990, Szabó et al., 1993, Baines et al., 2005, Kalani et al., 2018).

There are seven mammalian sirtuins (Sirt1-7). Sirt3, 4, and 5 are mitochondrial sirtuins. Sirt3 plays an important role in regulating mitochondrial metabolism, such as amino acid metabolism, the urea cycle, the tricarboxylic acid cycle, fatty acid oxidation, oxidative phosphorylation and mitochondrial dynamics(Samant et al., 2014, Papa and Germain, 2017). Sirt3 is a mitochondrial deacetylase known to play a major role in stress resistance and in cell death (Castillo et al., 2019, Li et al., 2019a, Pillai et al., 2010, Sundaresan et al., 2009, Wu et al., 2020). Sirt3 inhibited mPTP opening in Huntington’s disease (Cheng et al., 2016), amyloid-β related oxidative stress injury (Jiang et al., 2017a), doxorubicin-induced brain cortex and cerebellum mitochondrial toxicity (Marques-Aleixo et al., 2016), seizure (Cheng et al., 2016, Amigo et al., 2017) and sepsis-associated encephalopathy (Jiang et al., 2017b). Sirt3 protected against neuronal ischemia by inhibiting apoptosis (Feng et al., 2018, Zhao et al., 2019). Our previous studies found that Sirt3 activate superoxide dismutase 2 and forkhead box O3a to reduce cellular reactive oxygen species (ROS) levels (Yin et al., 2015). However, it is unclear if there is a relationship between mPTP opening and Sirt3 in ischemic stroke. In this study, we hypothesize that ischemia injury induces mPTP opening by reducing Sirt3. To investigate the expression of Sirt3 and mPTP related proteins in ischemic stroke, we established the MCAO mice model. To explore the relationship between Sirt3 and mPTP opening, we overexpressed Sirt3 to measure mPTP and apoptosis proteins under ischemic condition in vitro in HT22 cells.

Section snippets

Animals

C57BL/6 male mice (7–8 weeks, 22–24 g) were obtained from the SPF Biotechnology Co. Ltd. (Beijing, China). All mice were housed in a temperature and humidity-controlled vivarium, with food and water ad libitum. Approval was granted by the Ethics Committee of Beijing Tiantan Hospital. A total of twenty-three mice were used in this study, two animals died during MCAO operation and after ischemia. Three animals were excluded according to the neurologic function. Efforts were performed to minimize

Changes in protein levels of Sirt3, VDAC1 and ANT1 in the penumbra area.

Cerebral blood flow (CBF) was assessed by laser speckle contrast imaging (LSCI) (RWD Life Science Co., Ltd, Shenzhen, China). Blood perfusion was monitored by LSCI at the end of 60-minute occlusion (Fig. 1A, B). It showed that the CBF at the ischemic hemisphere was decreased about 58.86% compared to the contralateral hemisphere (p < 0.001). In the penumbra area, transmission electron microscopy showed that mitochondria of neurons were swelling and formed vacuolation. Both inner and outer

Discussion

Stroke has multifaceted pathology. Mitochondrial pathways are involved in the pathophysiology of stroke, however the role of Sirt3 in ischemic stroke is unclear. Our previous studies have shown that Sirt3 activates superoxide dismutase 2 and forkhead box O3a to reduce cellular ROS levels (Yin et al., 2015). The protective effect of Sirt3 was also demonstrated in oxygen and glucose deprivation (OGD)-induced neuronal injury via promoting autophagy through the AMPK-mTOR pathway in vitro (Dai et

Funding

This work was supported by Beijing Science and Technology Plan / Brain Science and Research North Science Center Project (Z181100001518005) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB39000000).

Ethical approval

All animal experiments were conducted according to the authorization from the Ethics Committee of Beijing Tiantan Hospital.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to thank Qi Zhang and Cuiping Zhang for their help in transmission electron microscope.

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