Elsevier

Brain Research

Volume 1499, 7 March 2013, Pages 145-157
Brain Research

Research Report
Protective effect of lipoic acid against oxidative stress is mediated by Keap1/Nrf2-dependent heme oxygenase-1 induction in the RGC-5 cellline

https://doi.org/10.1016/j.brainres.2012.12.041Get rights and content

Abstract

Oxidative stress plays a key role in neurodegeneration of CNS neurons such as in Alzheimer disease, Parkinson's disease and glaucoma. R-α-lipoic acid (R-LA) has been shown to have a neuroprotective effect through its antioxidant activity. However, the mechanism underlying its neuroprotection is totally unknown in retinal neurons. In this study, we show that R-LA has a dramatic neuroprotective effect against oxidative stress-induced death of the retinal neuronal RGC-5 cell line. We observed that R-LA induces the expression of heme oxygenase-1 (HO-1) by promoting the translocation of NF-E2-related factor 2 (Nrf2) to the nucleus. We examined the mechanism underlying HO-1 induction by R-LA by focusing on downstream signaling pathways. We found that R-LA activates Akt, and HO-1 induction by R-LA (involving Nrf2 translocation to the nucleus) was suppressed by phosphoinositide 3-kinase (PI3K) inhibitors. In addition, R-LA produced reactive oxygen species (ROS), including hydrogen peroxide. Pretreatment with a ROS scavenger or a NADPH oxidase inhibitor suppressed R-LA-induced Nrf2 translocation to the nucleus and HO-1 induction. These results suggest that ROS production triggered by R-LA might modify Kelch-like ECH-associated protein (Keap1), which in turn induces HO-1 expression through the PI3K signaling pathway. Furthermore, R-LA significantly attenuated cell death and accumulation of 4-hydroxy-2-nonenal (4HNE) in the retina induced by optic nerve injury in vivo through an HO-1 activity-dependent mechanism. These data demonstrate for the first time that R-LA exerts a neuroprotective effect against oxidative stress in retinal neurons in vitro and in vivo by inducing HO-1 through Keap1/Nrf2 signaling.

Highlights

► R-LA has a protective effect against oxidative stress in RGC-5 cells. ► R-LA induces expression of the antioxidant HO-1 in RGC-5 cells. ► R-LA induces PI3K-dependent translocation of Nrf2 to the nucleus. ► The trigger for Nrf2 activation is ROS production by R-LA. ► R-LA prevents RGC death and 4HNE accumulation via HO-1 induction after injury.

Introduction

Oxidative stress is thought to be an important mechanism of cell death in various neurodegenerative diseases (Tezel, 2006). The eye has a 3- to 4-fold higher oxygen consumption relative to brain tissue and, consequently, has a higher exposure to various reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals and superoxide anions. Consequently, the eye contains very high quantities of antioxidants, such as superoxide dismutase, catalase, ascorbate and vitamin E (Rao et al., 1985, Rao, 1990, Atalla et al., 1987). Retinal ganglion cells (RGCs) play a key role in integrating visual information and relaying it to the visual centers of the brain. Glaucoma is increasingly recognized as a neurodegenerative disorder, characterized by an accelerated loss of RGCs. Several common mechanisms have been proposed to be involved in loss of RGCs, including ischemia, excitotoxicity and oxidative stress (Quigley et al., 1995, Barber et al., 1998, Lafuente et al., 2001). Glaucoma prevalence and incidence increase exponentially with age (Kong et al., 2009, Chrysostomou et al., 2010).

R-α-lipoic acid (R-LA) functions as an essential cofactor for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (Packer et al., 1997). It has been reported that administration of R-LA improved antioxidant functions and reduced lipid peroxidation in aged rats, in which R-LA showed improvement of antioxidant function and reduction of accumulation of lipid peroxidation endogenous levels of R-LA were diminished during aging (Palaniappan and Dai, 2007). In the retina, Chidlow et al. reported that R-LA protects RGCs from ischemia-reperfusion injury (Chidlow et al., 2002). However, the mechanisms underlying the neuroprotection afforded to RGCs by R-LA remain unclear. Recently, we investigated the protective effect of (1R)-isopropyloxygenipin (IPRG001) on RGC-5 cells and mouse RGCs in vitro and in vivo using various models of oxidative stress, and found a crucial role for Kelch-like ECH-associated protein (Keap1)/NF-E2-related factor 2 (Nrf2) signaling (Koriyama et al., 2010). It has been reported that modification of cysteine residues on Keap1, such as oxidation (Fourquet et al., 2010) and S-nitrosylation (Koriyama et al., 2010), leads to translocation of Nrf2 to the nucleus and the expression of antioxidant genes. Therefore, in the present report, we investigated whether R-LA has a neuroprotective effect on RGC-5 cells and mouse RGCs through this Keap1/Nrf2 signaling. We demonstrate that R-LA transiently generates hydrogen peroxide (H2O2), thereby triggering Keap1/Nrf2 signaling in RGC-5 cells. This activation of Keap1/Nrf2 signaling induces the antioxidative protein, heme oxygenase-1 (HO-1). We show for the first time that the neuroprotective action of R-LA against oxidative stress involves activation of the Keap1/Nrf2 signaling pathway in RGCs in vitro and in vivo.

Section snippets

Protective effect of R-LA against oxidative stress in RGC-5 cells

To evaluate the neuroprotective effect of R-LA, we used the serum deprivation model of oxidative stress injury. We first determined exposure time of R-LA needed to protect RGC-5 cells against serum deprivation-induced injury (Fig. 1A). Serum deprivation induced cell death in 60% of RGC-5 cells within 12 h. The maximum protective effect of R-LA was observed with 4–6 h of pretreatment (in the presence of serum) to serum deprivation; the 0–2 h of pretreatment was ineffective. Next, we determined the

Discussion

This study has several salient findings. First, we showed that R-LA prevents RGC-5 cell death through HO-1 induction via the Keap1/Nrf2 signaling pathway. Second, we found that R-LA-induced nuclear translocation of Nrf2 to nucleus is PI3K/Akt-dependent. Third, R-LA has the ability to increase ROS production and to stimulate nuclear translocation of Nrf2. Finally, we found that R-LA prevents mouse RGCs in vivo from optic nerve injury.

Chemicals

α-Lipoic acid (RS-LA, R-LA and S-LA) was provided by Dr. Tetsuya Konishi (Niigata University of Pharmacy and Applied Life Sciences). α-Lipoic acid was dissolved in phosphate-buffered saline (PBS) containing 0.33% of 5 M NaOH. The phosphatidylinositol 3-kinase (PI3K) inhibitors, WT and LY294002 (LY), and the NADPH oxidase inhibitor, DPI, were purchased from Sigma-Aldrich (St. Louis, MO, USA). An HO-1 inhibitor, SnMP, was obtained from Frontier Scientific Inc. (Logan, UT, USA). The ROS scavenger,

Acknowledgments

We thank Ms. Sachiko Higashi and Ms. Tomoko Kano for their administrative and technical assistance. This work was supported by Grants-in-Aid for Scientific Research to Y.K. (No. 22791651), S.K. (Nos. 22300109 and 23650163).

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    These authors contributed equally to thiswork.

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