Zinc accumulation in mitochondria promotes ischemia-induced BBB disruption through Drp1-dependent mitochondria fission

https://doi.org/10.1016/j.taap.2019.114601Get rights and content

Highlights

  • Ischemia/reperfusion induces zinc accumulation in endothelial cells (ECs).

  • Zinc accumulation triggers ROS generation in cultured ECs and in cerebromicrovessels of rats.

  • Zinc specifically accumulates in mitochondria, leading to mitochondrial ROS generation.

  • Excessive zinc accumulation results in mitochondria network disruption through Drp-1 pathway.

  • Zinc overload activates MMP-2 and causes ischemia-induced BBB damage.

Abstract

High concentration of zinc has been reported to act as a critical mediator of neuronal death in the ischemic brain. Our previous studies showed that labile zinc accumulates in cerebromicrovessels and contributes to blood–brain barrier (BBB) permeability increase after cerebral ischemia. However, the role of mitochondrial zinc in ischemia-induced BBB permeability alteration is still unclear. In this study, we showed that ischemia/reperfusion induced free zinc accumulation in endothelial cells (ECs), resulting in increased generation of reactive oxygen species (ROS) in both cultured ECs and in microvessels isolated from the brain of ischemic rats. Furthermore, we found that zinc was highly accumulated in mitochondria, leading to mitochondrial ROS generation under the ischemic condition. Moreover, zinc overload in mitochondria resulted in the collapse of the network of mitochondria, which was mediated through Dynamin-related protein-1 (Drp-1) dependent mitochondrial fission pathway. Finally, the zinc overload in mitochondria activated matrix metalloproteinase-2 and led to ischemia-induced BBB permeability increase. This study demonstrated that zinc-ROS pathway in mitochondria contributes to the ischemia-induced BBB disruption via Drp-1 dependent mitochondrial fission pathway.

Introduction

As one of the most abundant metal ion in the brain, zinc has been found to widely distribute in the brain and serve in multiple physiological functions, such as the development of the brain, learning and memory (Kawahara et al., 2014; Ordak et al., 2018; Pochwat et al., 2015). In recent years, it has been demonstrated by us and others that zinc acts as a critical mediator of neuronal death at high concentrations in the ischemic condition (Choi and Suh, 2018; Szewczyk, 2013; Weiss et al., 2000). Most studies of zinc in the brain focused on the role of zinc in the neuronal functions, as zinc vesicles would be released from a subset of glutamatergic terminals of neurons under pathological conditions. Further studies have highlighted the damaging effects of zinc accumulation in neurons after ischemia or neurodegenerative diseases (Pivovarova et al., 2014; Slepchenko et al., 2017; Zhao et al., 2014).

The rise in cytosolic free zinc is accompanied by zinc translocation to mitochondria, which act as zinc-pools to regulate the homeostasis of free zinc in the cytosol. However, the overload of zinc in mitochondria disrupts the function of mitochondria, resulting in reactive oxygen species (ROS) generation and triggering the mitochondria-induced cell death (Dineley et al., 2005; Dong et al., 2015; Slepchenko et al., 2016).

Our recent studies reported that ischemia-induced zinc accumulation in cerebral microvessels led to the disruption of blood–brain barrier (BBB), which is a crucial event for the safety of re-canalization therapy of acute ischemic stroke in clinic (Qi et al., 2016). We reported that zinc overload in microvessels activated matrix metalloproteinases (MMPs), which degraded the tight junction proteins between endothelial cells, and contributed to ischemia-induced BBB permeability increase.

However, the role of mitochondrial zinc in zinc-induced endothelial cell injury is still unclear. Our recent study reported that a positive feedback loop between zinc accumulation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced ROS production amplified the damaging effects of both, and thereby deteriorated brain injury after cerebral ischemia/reperfusion (Zhao et al., 2018). Moreover, it was shown that the Dynamin-related protein-1 (Drp-1) dependent mitochondria fission disrupts the network of mitochondria, leading to mitochondrial fragmentation and dysfunction (Ghasemi et al., 2018; Singh and Sharma, 2017). Therefore, in this study we investigated the role of mitochondria in zinc-induced endothelial cell permeability increase under the ischemic condition.

Section snippets

Cell culture

The mouse brain microvascular endothelial cell line, bEnd3, was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured as in our previous study (Qi et al., 2016). Endothelial cells (ECs) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS (Life technologies, CA, USA) and Antibiotic-Antimycotic (Life technologies, CA, USA) at a humidified atmosphere of 5% CO2/95% air at 37 °C.

Oxygen/glucose deprivation and drug exposure in vitro

Oxygen/glucose deprivation (OGD) exposure was performed to mimic

Ischemia induces zinc overload in cultured brain endothelial cells

To demonstrate the level of free zinc in ECs after ischemia/reperfusion, we stained the cultured ECs, which underwent 45-min or 90-min OGD treatment plus 24-h reperfusion, using NG staining, a specific zinc indicator in cytosol (Fig. 1A). It was shown that zinc signals in normal cells were barely visible. As expected, the intensity of intracellular zinc significantly elevated with 45-min OGD/reperfusion. The fluorescence intensity of cytosol zinc was further increased with 90 min

Discussion

The integrity of BBB in the early stage of ischemic stroke is crucial for the safety of recanalization (Li et al., 2019; Venkat et al., 2017). Endothelial dysfunction contributes to the disruption of BBB in cerebral ischemia, but the mechanisms of endothelial injury are still unclear.

Zinc is critical for neuronal activities in physiological and pathological conditions in the central nerve system (Frederickson et al., 2000). Our previous studies have demonstrated that zinc serve as a critical

Disclosure

None.

Conflict of interest statement

All authors have no conflict to declare.

Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (81571175, 81620108011).

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