In vivo modulation of the Parkinsonian phenotype by Nrf2

Abstract

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. In this report we show that Nrf2, a transcription factor that regulates the expression of phase 2 and antioxidative enzymes, modulates MPTP neurotoxicity in rodents. Nrf2 knockout and wild-type mice were administered MPTP doses ranging from 20 to 60 mg/kg. Seven days after MPTP administration dopamine transporter (DAT) levels were measured using [¹²⁵I]-RTI-121 quantitative autoradiography as an index of dopamine terminal integrity in the striatum. The results indicate that MPTP administration resulted in a greater loss of DAT levels in the striatum of Nrf2 knockout mice than in wild-type at all MPTP doses tested. Activation of the Nrf2 pathway by oral administration of the Nrf2 inducer 3H-1,2-dithiole-3-thione (D3T) to wild-type mice produced partial protection against MPTP-induced neurotoxicity. The protective effect of D3T was not due to a change in MPTP metabolism since the level of the MPTP metabolite MPP+ was not significantly different in the D3T treated striatum relative to vehicle control. Administration of D3T to Nrf2 knockout mice did not protect against MPTP neurotoxicity suggesting that the Nrf2 pathway is necessary for the D3T-mediated attenuation of MPTP neurotoxicity. This study demonstrates the significance of activating intrinsic antioxidative mechanisms in an in vivo model of neurodegeneration. The in vivo activation of the Nrf2 pathway in the brain may be an important strategy to mitigate the effects of oxidative stress in neurodegenerative disorders and neurological disease.

Introduction

Idiopathic Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects approximately 1% of the United States population above 65 years of age (Bertram and Tanzi, 2005). The etiology of PD is not known; however, genetic (Kurz et al., 2003, Mayeux, 2003) and environmental factors (Tanner et al., 1999) play a significant role. The neuropathological hallmark of PD is the progressive loss of dopaminergic neurons in the substantia nigra resulting in the loss of greater than 70% of dopamine and other dopaminergic terminal markers in the striatum leading to the resting tremor and movement abnormalities characteristic of the disease (Bernheimer et al., 1973, Dauer and Przedborski, 2003, Greenamyre and Hatings, 2004, Hornykiewicz and Kish, 1986). At the molecular level, oxidative stress has been implicated in the neuropathology associated with PD (Gu et al., 1998) and in animal models of PD (Chun et al., 2001, Dobson et al., 2004, Schober, 2004). Importantly, the identification of genes such as DJ-1, Parkin, and UCH-L1 in familial forms of PD further links mitochondrial dysfunction and oxidative stress to its etiology (Dawson and Dawson, 2003, Park et al., 2005). Thus, it has been postulated that bolstering the capacity of a cell to cope with oxidants may be a strategy to prevent the onset of and/or delay the progression of PD. One means of increasing the antioxidative potential of a cell is through activation of NF-E2-related factor 2, Nrf2, which controls the transcription of phase 2 enzymes and other enzymes important to the antioxidative response (Kwak et al., 2001). Under basal conditions, Nrf2 is sequestered in the cytoplasm by the actin binding protein Keap1. Additionally, Keap1 regulates Nrf2 by directing it to the proteosome, thereby facilitating its degradation (Kwak et al., 2003a). Under oxidative conditions, cysteines within the Kelch domain of Keap1 become oxidized, and Nrf2 is released (Kang et al., 2004, Wakabayashi et al., 2004). Nrf2 translocates to the nucleus and heterodimerizes with members of the small Maf protein family. The dimer binds to the antioxidant response element, which is present in the promoter region of enzymes that are critical to the response of the cell to oxidative stressors, and induces transcription of target genes.

The Keap1–Nrf2 system is an intrinsic mechanism of cellular protection against oxidative stress. Sulfhydryl-reactive chemicals such as Michael reaction acceptors, isothiocyanates, and dithiolethiones are inducers of this pathway (Dinkova-Kostova et al., 2002, Itoh et al., 2004). The Nrf2-dependent response of the potent chemoprotective agent 3H-1,2-dithiole-3-thione (D3T) in up-regulating phase 2 and antioxidative enzymes has been demonstrated in vivo (Kwak et al., 2003b). By protecting against oxidative stress, the activation of Nrf2-regulated genes decreases DNA damage and other aspects of the carcinogenic process. Isothiocynates and dithiolethiones have been used in animal models and human clinical trials as chemopreventive agents against liver and gastric carcinogenesis (Fahey et al., 2002, Kensler et al., 2005, Ramos-Gomez et al., 2001).

Recently, the strategy of enhancing the innate cellular capability of managing oxidative stress via the Nrf2 pathway has been extended to neuronal systems. First, it was shown that Nrf2 induction is protective in cultured cells of the central nervous system against oxidative stress (Kraft et al., 2004). Sulforaphane and tert-butylhydroquinone, inducers of Nrf2, was used to boost the capacity of cultured astrocytes to tolerate hydrogen peroxide. Subsequently, it was shown that up-regulation of Nrf2 target genes in glial cells protects neurons from oxidative insult (Shih et al., 2003). Primary cortical neurons exposed to methylphenylpyridinium ion, the ultimate MPTP toxicant, or rotenone were protected by Nrf2 induction (Lee et al., 2003). Over-expression of Nrf2 by intrastriatal infusion of Nrf2-adenovirus-infected astrocytes was shown to be protective against intrastriatal administration of the complex II inhibitor malonate (Calkins et al., 2005). Additionally, these authors report that mice lacking the Nrf2 gene are more susceptible to oxidative insult from complex II inhibition. Activation of Nrf2 has also been shown to be protective in rodent models of cerebral ischemia (Satoh et al., 2006, Shih et al., 2005, Zhao et al., 2006). Deprenyl, a selective monoamine oxidase B inhibitor, has been used in clinical trials to slow the progression of symptoms in patients with Parkinson's disease (Pålhagen et al., 2006). Recently, Nrf2 activation has been identified as an alternative mechanism by which deprenyl slows the progression of Parkinson's disease (Nakaso et al., 2006). Our present findings indicate that genetic ablation of Nrf2 makes mice more sensitive to MPTP neurotoxicity and that treatment of wild-type but not Nrf2 knockout animals with the Nrf2 inducer D3T partially protects against MPTP neurotoxicity.