Elsevier

Free Radical Biology and Medicine

Volume 113, December 2017, Pages 244-254
Free Radical Biology and Medicine

Original article
Administration of 5-methoxyindole-2-carboxylic acid that potentially targets mitochondrial dihydrolipoamide dehydrogenase confers cerebral preconditioning against ischemic stroke injury

https://doi.org/10.1016/j.freeradbiomed.2017.10.008Get rights and content

Highlights

  • Inhibition of dihydrolipoamide dehydrogenase (DLDH) triggers preconditioning effect against stroke injury.

  • DLDH preconditioning involves attenuation of DLDH function, but not DLDH content.

  • DLDH preconditioning involves NQO1 upregulation by the Nrf2 signaling pathway.

  • DLDH preconditioning decreases oxidative stress and cell death and hence confers neuroprotection.

Abstract

The objective of this study was to investigate a possible role of mitochondrial dihydrolipoamide dehydrogenase (DLDH) as a chemical preconditioning target for neuroprotection against ischemic injury. We used 5-methoxyindole-2-carboxylic acid (MICA), a reportedly reversible DLDH inhibitor, as the preconditioning agent and administered MICA to rats mainly via dietary intake. Upon completion of 4 week's MICA treatment, rats underwent 1 h transient ischemia and 24 h reperfusion followed by tissue collection. Our results show that MICA protected the brain against ischemic stroke injury as the infarction volume of the brain from the MICA-treated group was significantly smaller than that from the control group. Data were then collected without or with stroke surgery following MICA feeding. It was found that in the absence of stroke following MICA feeding, DLDH activity was lower in the MICA treated group than in the control group, and this decreased activity could be partly due to DLDH protein sulfenation. Moreover, DLDH inhibition by MICA was also found to upregulate the expression of NAD(P)H-ubiquinone oxidoreductase 1(NQO1) via the Nrf2 signaling pathway. In the presence of stroke following MICA feeding, decreased DLDH activity and increased Nrf2 signaling were also observed along with increased NQO1 activity, decreased oxidative stress, decreased cell death, and increased mitochondrial ATP output. We also found that MICA had a delayed preconditioning effect four weeks post MICA treatment. Our study indicates that administration of MICA confers chemical preconditioning and neuroprotection against ischemic stroke injury.

Introduction

Stroke is a leading cause of disability and death in the United States. Fortunately, the brain can be induced to tolerate stroke injury. One promising strategy to achieve this tolerance is called preconditioning [1], [2], [3], wherein exposure to non-injurious stimuli affords protection against subsequent injurious ischemic challenges. Preconditioning in the brain can be triggered not only by brief episodes of ischemia-reperfusion [4], [5] but also by administration of certain chemicals or drugs [6], [7], [8], [9], [10]. As ethical considerations do not allow for the use of brief repeated occlusion of the cerebral arteries to elicit stroke tolerance in humans, animal models of chemical preconditioning have been actively investigated in hopes to develop a clinically-useful approach to stroke preconditioning in humans. Moreover, chemical preconditioning may also be useful as a prophylactic approach to neuroprotection [11], [12]. The reason for this is that while the onset of stroke is often sudden and unpredictable, individuals with a higher than normal risk of stroke could benefit from prior measures that enhance the brain's tolerance to potential ischemic injury. Such is the case for patients who are scheduled to undergo cardiovascular procedures during which the brain needs to be prophylactically protected against possible stroke injury.

Mitochondria are a known target for preconditioning against stroke injury [13], [14], [15]. When oxygen and nutrients supply to the affected area of tissues come to a halt upon ischemia, mitochondrial ATP production is severely decreased. The decrease in ATP content triggers functional impairment of ATP-dependent calcium channels, leading to overload of cellular and mitochondrial calcium [16], [17] that in turn can trigger glutamate excitotoxicity [18], [19]. On the other hand, as cells have to undergo anaerobic respiration in the absence of oxygen, lactate formed from pyruvate via lactate dehydrogenase accumulates, which results in a decrease in cellular pH and closure of mitochondrial permeability transition pore (MPTP) [20]. Upon reperfusion, a sudden resumption of blood flow can over energize mitochondrial respiration, leading to a spike in mitochondrial generation of reactive oxygen species (ROS) and opening of MPTP [21], [22], [23], [24]. This opening can release cytochrome c that then activates cell death pathways and causes tissue infarction [25], [26]. Moreover, ROS production could further accentuate cellular apoptosis as ROS can induce oxidative stress and impair protein functions [27], [28], [29], [30], [31], [32], [33], [34], [35]. Therefore, based on the key roles of mitochondria in cell death and ischemic reperfusion injury, numerous mitochondrial proteins have been assessed or suggested as targets for preconditioning against ischemic stroke injury [14], [36].

In this paper, we describe our findings that mitochondrial dihydrolipoamide dehydrogenase (DLDH) could be a target for chemical preconditioning against ischemic stroke injury. DLDH is a family member of flavin-dependent, pyridine dinucleotide oxidoreductases [37]. It is the third component of α-ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex, and branched chain amino acid dehydrogenase complexes. DLDH is also involved in the glycine cleavage system [38], [39]. Each of these complexes or pathways is fundamental for mitochondrial bioenergetics and cell survival. Yet, mouse with a loss of 50% DLDH protein content is viable and fertile [40], suggesting that DLDH function could be inhibited to an extent to which no harm ensues. Our main findings indicate that dietary inhibition of DLDH for 4 weeks using chow supplemented with 5-methoxyindole-2-carboxylic acid (MICA), a specific inhibitor of DLDH function [41], [42], [43], [44], significantly decreased infarct volume after transient middle cerebral artery occlusion (tMCAO), while no detrimental effects on food intake, body weight gain, blood glucose concentrations, and mitochondrial electron transport chain activities were detected in the absence of stroke. The preconditioning mechanism in the absence of stroke appeared to involve decreased DLDH activity and increased NAD(P)H: ubiquinone oxidoreductase-1 (NQO1) expression via activation of the Nrf2 signaling pathway. This mechanism was also found to operate upon stroke after MICA feeding. Our data indicate that decreased oxidative stress and apoptosis and increased mitochondrial ATP output are involved in stroke neuroprotection induced by MICA/DLDH preconditioning.

Section snippets

Animals

Young male Sprague-Dawley rats (approximately 3 months old) were used in this study. The use of animals was approved by Institutional Care and Use Committee of University of North Texas Health Science Center and the protocol was in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. Rats were randomly grouped for MICA groups and control groups.

Chemicals and reagents

5-methoxyindole-2-carboxylic acid (MICA) was purchased from Fisher Scientific (Hanover Park, IL). Lipoamide, BSA, nitro-blue

MICA administration via injection induces ischemic tolerance

We initially tested whether administration of MICA via intraperitoneal injection would increase the brain's ischemic tolerance or impart preconditioning after tMCAO. In these studies, rats received MICA injection (200 mg/kg, I.P.) [42] once per day for seven days. Following tMCAO performed 24 h after the last MICA injection, brain infarction volume was measured densitometrically after histochemical staining with 2,3,5-triphenyltetrazolium chloride (TTC) [68]. Results in Fig. 1A and B show that

Discussion

The major findings of the present study are that mitochondrial DLDH, involved in four metabolic pathways, may serve as a preconditioning target for neuroprotection against ischemic stroke injury and that MICA administered via dietary feeding does not exhibit detectable toxicity on the animals. The underlying mechanisms of MICA preconditioning are likely multiple, but decreased DLDH activity and NQO1 upregulation via Nrf2 signaling may contribute to the observed preconditioning effects, which

Conclusions

The present study has provided evidence that MICA, potentially targeting mitochondrial DLDH, confers preconditioning effect on neuroprotection against ischemic stroke injury. MICA administration via dietary intake did not show detectable metabolic toxicity on the animals that were studied. The likely mechanisms underlying this neuroprotective effect are decreased DLDH function and augmented Nrf2 signaling process that is well-known for its role in cytoprotection [91], [93], [94], [95], together

Acknowledgements

This work was supported by National Institute of Neurological Disorders and Stroke, the National Institutes of Health (Grant number: R01NS079792).

Conflict of interest

The authors declare no conflict of interest.

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