Nuclear Factor Erythroid 2 - Related Factor 2 Signaling in Parkinson

Send Orders of Reprints at [email protected] CNS & Neurological Disorders - Drug Targets, 2012, 11, 000-000

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Nuclear Factor Erythroid 2 - Related Factor 2 Signaling in Parkinson Disease: A Promising Multi Therapeutic Target Against Oxidative Stress, Neuroinflammation and Cell Death Hemant Kumar, Sushruta Koppula, In-Su Kim, Sandeep Vasant More, Byung-Wook Kim and Dong-Kug Choi* Department of Biotechnology, Konkuk University, Chungju, 380-701, Korea Abstract: Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder with increased oxidative stress as central component. Till date, treatments related to PD are based on restoring dopamine either by targeting neurotransmitter and/or at receptor levels. These therapeutic approaches try to repair damage but do not address the underlying processes such as oxidative stress and neuroinflammation that contribute to cell death. The central nervous system maintains a robust antioxidant defense mechanism consisting of several cytoprotective genes and enzymes whose expression is controlled by antioxidant response element (ARE) which further depends on activation of nuclear factor erythroid 2-related factor 2 (Nrf2). In response to oxidative or electrophilic stress transcription factor Nrf2 binds to ARE and rescues the cells from oxidative stress and neuroinflammation. Recently, Nrf2 has been utilized as a drug target and some agents are currently under clinical trial. Owing to the potential role of Nrf2 in counteracting oxidative stress and neuroinflammation seen in PD, here we have focused on the molecular mechanism of the Nrf2/ARE antioxidant defense pathway in PD. Further, we also summarize published reports on the potential inducers of Nrf2 that demonstrate neuroprotective effects in experimental models of PD with possible future strategies to increase the transcriptional level of Nrf2 as a therapeutic strategy to provide neuroprotection of damaged dopaminergic neurons in PD.

Keywords: Antioxidant inflammation modulators, antioxidant response elements, apoptosis, nuclear factor erythroid 2-related factor 2, neuroinflammation, oxidative stress, Parkinson’s disease. 1. INTRODUCTION Parkinson’s disease (PD) is one of the most common neurodegenerative disorder and increased oxidative stress is a central component in the pathophysiology of PD. High oxygen consumption and high lipid content make neural tissues more sensitive to oxidative stress than other organs. Aerobic organisms have evolutionary advantages over anaerobic species in their ability to utilize molecular oxygen for effective energy abstraction from organic nutrients. However, with the emergence of mitochondrial respiratory processes, a number of byproducts follow, mainly in the form of reactive oxygen species (ROS) [1, 2], which reacts in a nonspecific manner with nucleic acids, proteins, and membrane lipids, can cause gene mutation, impairment or loss of enzyme activity, and altered cell membrane permeability [3, 4]. Oxidative stress could also be generated by reactive nitrogen species (RNS) and reactive electrophilic species (RES). RNS can directly or indirectly lead to protein S-nitrosylation whereas RES react with the nucleophilic cysteine thiol, leading to Cys-S-adducts. Thus reactive species like ROS, RNS and RES exert their deleterious effects by perturbation of cellular redox homeostasis and target macromolecule of the cell thereby causing oxidative stress. To counteract environmental stress caused by these reactive species, cells have developed adaptive, dynamic *Address correspondence to this author at the Department of Biotechnology, Konkuk University, Chungju, 380-701, Korea; Tel: 82-43-840-3610; Fax: 82-43-840-3872; E-mail: [email protected] 1871-5273/12 $58.00+.00

programs to maintain cellular redox homeostasis through a series of antioxidant molecules and detoxifying enzymes that can provide control by quickly removing or detoxifying. Protective responses and induction of these enzymes require at least three essential components a) cis-elements called antioxidant response elements (AREs) or electrophileresponse elements in their 5- flanking promoter regions, the upstream regulatory sequences of which are present on each gene in either single or multiple copies [5], b) nuclear factor erythroid 2-related factor 2 (Nrf2), the redox-sensitive and principal transcription factor that hetrodimerizes with members of the small musculoaponeurotic fibrosarcoma (Maf) Maf G, Maf K and Maf F, family of transcription factors, is a central regulator in both constitutive and inducible ARE-related gene expression, and recruits the general transcriptional machinery for expression of ARErelated genes [5-7]; and c) Kelch ECH association protein 1 (Keap1), a cytosolic repressor protein that binds to Nrf2, retains it in cytoplasm, and promotes its proteasomal degradation [8]. Thus Keap 1 – Nrf2 – ARE signaling plays a significant role in protecting cells from oxidative stress. Most therapeutic approaches for PD are limited to restoring dopamine levels by targeting at the neurotransmitter or receptor level, either by increasing dopamine levels (e.g. L-dopa or dopamine agonist) or preventing its degradation (MAO-B inhibitors). Even though these approaches try to repair the damage, they do not address the underlying processes such as oxidative stress, neuroinflammation or mitochondrial dysfunction that contribute to cell death. Therefore pharmacological © 2012 Bentham Science Publishers

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CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 8

interventions to mitigate or subvert neuroinflammation or production of reactive species might become therapeutic targets in PD. Earlier reviews on Nrf2 are limited to discuss its role in counteracting mitochondrial dysfunction [9] and therapeutic potential in PD [10]. Here, we extend the approach of Nrf2 as a potential multitherapeutic drug target in PD, with reference to oxidative stress mediated neuroinflammation and cell death. Further, we have summarized the data generated by several groups which links the Nrf2 pathway to PD; and also have discussed potential Nrf2 inducers that demonstrate neuroprotective effects in experimental models of PD. Lastly we summarized possible strategies to increase the transcription of Nrf2 as a therapeutic strategy to provide neuroprotection of nigral dopaminergic neurons in PD. 2. OXIDATIVE STRESS AND PARKINSON DISEASE The movement disorder PD is second most frequently occurring neurodegenerative condition after Alzheimer’s disease (AD), and is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the loss of striatal dopamine content [11, 12]. The possible role of mitochondrial dysfunction, oxidative damage, excitotoxicity, and inflammation in PD has been elaborately studied and well established [13-15]. Although several factors have been proposed for the pathogenesis of PD, oxidative stress via the generation of reactive species are major contributors. In the pathology of neurodegenerative disorders, the generation of ROS harmfully affects proteins, lipids, and nucleic acids [16]. ROS itself regulates redox homeostasis by activating a group of genes and signal transduction pathways [17]. Initial evidence for the existence of oxidative stress in PD came from reports based on post mortem analysis of the brain tissue of PD patients that demonstrated increased levels of oxidized proteins, lipids, and nucleic acids [18-22]. One of the major source of ROS generation comes from alternation of mitochondrial complex I either with direct inhibition by 1-methyl-4phenylpyridinium (MPP+), paraquat, rotenone or indirectly with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in experimental models of PD. Mitochondrial complex inhibition hampers the mitochondrial respiratory chain, which causes incomplete oxygen reduction, there by generating reactive species including deleterious superoxide [23], which further converted to peroxynitrite and then finally to hydroxyl radical through Fenton reaction [24]. Thus, reactive species generated from mitochondrial and/or extra-mitochondrial sources appear to be the main contributors to oxidative stress-mediated neurodegeneration in PD models [11, 23-27]. Dopamine (DA), a relatively unstable molecule in nature and more prone to hydroxyl radical attack, undergoes autooxidation in the nigrostriatal tract system, thereby inducing ROS, and causes damage within the cell as well as from outside [28]. Unstored cytosolic fraction of DA undergoes spontaneous or MAO-B mediated degradation to form 3,4dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as major metabolites and superoxide, hydrogen peroxide (H2O2) and DA quinones (DAQs) as minor metabolites [24, 29]. Auto-oxidation of DA to form DAQs and superoxide radical is catalyzed by the presence of

Kumar et al.

metals, oxygen or enzyme tyrosinase. DAQs, if not sequestered properly, cyclize to form aminochrome, a highly unstable molecule that leads to depletion of nicotinamide adenine dinucleotide phosphate (NADPH) and release of superoxide. Auto-oxidation of DA may be increased in early stages of PD when DA turnover is increased to compensate for dying dopaminergic neurons [30]. Moreover, novel transgenic mouse model in which striatal neurons were engineered to take up extracellular DA without acquiring regulatory mechanisms found in DA neurons, developed motor dysfunctions and progressive neurodegeneration in the striatum within weeks [31]. The strong electrophilic nature of DAQs makes use of sulfhydryl groups present in many proteins as electron donors with subsequent formation of quinoproteins and disulfide bonds that finally compromise protein function. DAQs reacts with tyrosine hydroxylase (TH) [32], dopamine transporter (DAT) [33], parkin [34] and –synuclein (-syn) [35], and finally converts them into redox-cycling quinoproteins. One probable mechanism to ameliorate the cytotoxic effect of DAQs is sequestration with glutathione (GSH) to form 5-S-glutathionyl-DA, at the expense of GSH, a central molecule to keep redox homeostasis [36]. Although GSH is not the only antioxidant molecule affected in PD, the magnitude of GSH depletion is in correlation with the severity of the disease, and it is also the earliest known indicator of nigral depletion [37]. In addition, iron accumulation also occurs in PD, and the iron content of substantia nigra (SN) is seen to be elevated as compared to aged matched controls with an increase of the Fe (III)/Fe (II) ratio from 2:1 to 1:2 [38]. Increased iron and Fe (II) enhance the conversion of H2O2 to hydroxyl radical via Fenton reaction, thereby increasing oxidative stress. High DAQs, low GSH and high iron levels are the main causes of oxidative toxicity to dopaminergic cells in PD brains. 3. NEUROINFLAMMATION AND CELL DEATH IN PARKINSON’S DISEASE Chronic neuroinflammation is an established source for ROS production in PD. Over-activated microglia contributes to neurodegenerative processes through the production of various neurotoxic factors including free radicals and proinflammatory cytokines [39]. Initial study suggested that the degeneration of dopaminergic neurons in PD was associated with massive microglial activity in SNpc [40], and later the presence of activated microglia in the putamen, hippocampus, transentorhinal cortex, cingulate cortex and temporal cortex were also reported [41]. Recently, in vivo study indicated that microglial activation in PD patients is anatomically widespread in the pons, basal ganglia and frontal and temporal cortical regions, and the level of microglial activation is independent of clinical severity [42]. Activated microglia expresses several enzymes responsible for inflammatory processes. These enzymes contribute to the pathogenesis of various neurodegenerative diseases including PD [43-45]. Increased oxidative stress, neuroinflammation, reduced antioxidant levels, GSH depletion and mitochondrial defects are all known to induce apoptosis in the pathogenesis of PD [46]. Several studies on PD patients have pointed out the potential involvement of apoptosis in pathogenesis of PD [47, 48]. Moreover, p62, a common component of cytoplasmic inclusions in protein aggregation diseases like PD [49], interacts directly with

Nuclear Factor Erythroid 2 - Related Factor 2 Signaling in Parkinson Disease

Keap1. A model in which p62 competes with Nrf2 for interaction with Keap1 is envisaged. Hence, p62 is able to set up a positive feedback loop to activate Nrf2, which in turn stimulates increased transcription of the p62 gene. In this manner, p62 protein contributes to a sustained activation of Nrf2 in response to oxidative and electrophilic stress [50]. 4. NRF2-KEAP1-ARE SIGNALLING Nrf2 pathway is the major pathway that responds to reactive species and redox potentials by activating phase II detoxification enzymes at the transcriptional level [51, 52]. It was first identified by Moi et al. in 1994, as controlling the expression of the -globin gene [52]. It belongs to the cap ‘n’ collar (CNC) family of transcription factors having a distinct basic leucine-zipper motif [7]. Other members from this family are Nrf1, Nrf3, Bach1, and Bach2. Under basal conditions, repressor Keap1 holds Nrf2 in the cytosol along with actin filaments. Unless it is activated, Nrf2 is ubiquitinated by the E3-ubiquitin ligase-like domain of Keap1 in a constitutive manner [51, 53-55], followed by 26S proteasomal degradation [56], and displays a short halflife [8, 57, 58]. Thus repressor protein Keap1 regulates Nrf2 negatively, by promoting its proteasomal degradation through the cullin-3-dependent pathway [59-62]. When redox balance is more toward the oxidative side, as occurs during attacks by electrophilic and/or oxidative stimulus, ROS [6, 63, 64], or RNS [65, 66]. Nrf2 is released from Keap1, gets translocated into the nucleus and binds with ARE in the promoter region of its target genes thereby inducing a battery of cytoprotective genes and antioxidative enzymes [5, 67]. A distinguished feature of Keap1 is its high cysteine content, which makes it an excellent candidate as a sensor for inducer. Stress generated from chemicals or radiation modifies reactive cysteine of Keap1 (C151, C272, and C288), followed by Protein Kinase C (PKC)-mediated phosphorylation at Ser 40, which leads to dissociation of Nrf2 from Keap1 and increased translocation and transcription of Nrf2 dependent genes [55, 68]. Interestingly, some reports suggest that Keap1 shuttles between the nucleus and the cytoplasm via chromosomal region maintenance-1 (Crm1) dependent nuclear export mechanism [69], or Keap1 transiently enters the nucleus and targets Nrf2 for ubiquitylation, thus indicating that both ubiquitylation and degradation occurs in the nucleus [51]. ARE possesses the DNA consensus sequence required for functional activity which characterizes its unique responsiveness to oxidative stress [70], and get activated specifically by chemical compounds with capacity to either undergo redox cycling or by compounds that are metabolically transformed to a reactive or electrophilic intermediate [71]. Apart from maintaining cellular redox homeostasis in stressed conditions, ARE is also responsible for maintaining the basal expression of several genes under normal condition, as reactive species are constantly being generated from aerobic metabolism. Thus, Nrf2, Keap1, Maf proteins and ARE or EpRE, are essential for the antioxidant response [72]. Phosphorylation of Nrf2 at serine and threonine residues by PKC, phosphatidylinositol 3-kinase (PI3K), PKR-like


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endoplasmic reticulum kinase, c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways were shown to regulate Nrf2 transcriptional activity by facilitating the release of Nrf2 from Keap1 [73]. The Nrf2 pathway appears to be regulated positively by ERK and JNK whereas p38 MAPK has both positive and negative regulation [74, 75]. The illustration of regulating Nrf2 pathway by several factors is shown in Fig. (1). 5. GENES REGULATED BY NRF2/ARE PATHWAY AND PARKINSON’S DISEASE Nrf2 acts a regulator of antioxidant defense and detoxification systems, and its activation can repress cellular damage or injury in many different tissues and organs [76]. The gene families regulated by ARE include phase I and II detoxification enzymes, transport proteins, proteasome subunits, chaperones, growth factors, and their receptors, as well as some transcription factors [8, 56, 59, 77]. Phase II metabolizing/detoxifying enzymes and antioxidants such as NAD(P)H:quinone oxidoreductase 1 (NQO-1), heme oxygenase-1 (HO-1), -glutamyl cysteine synthetase catalytic subunit (GCLC), -glutamyl cysteine synthetase modifier subunit (GCLM), glutathione S-transferase A2 (GSTA2), glutathione reductase (GR), thioredoxin reductase (TR), and peroxiredoxin (Prx) [5, 78, 79] are of prime importance in protecting against neuronal cell death caused by oxidative stress and underlying neuroinflammation in PD. These enzymes are expressed in various isoforms and distributed in various organelles and subcellular compartments and cooperatively participate in metabolic reactions that eliminate reactive species at the sites of origin. Nrf2 inducers exhibit their antioxidant/neuroprotective effects by up-regulation of various cytoprotective enzymes and proteins as mentioned. These expressed cytoprotective proteins are referred as the “ultimate antioxidants,” as they have relatively long half-lives. The other advantage is that they are not consumed during their antioxidant actions can catalyze a wide variety of detoxification reactions, and are involved in regeneration of some direct antioxidants. 5.1. Heme Oxygenase-1, Nrf2 Pathway and Parkinson Disease Three isoforms of heme oxygenases (HO) have been characterized HO- 1 (inducible), HO-2 constitutive) and HO-3 (catalytically inactive). HO is a microsomal enzyme that catalyzes stepwise degradation of haem group to biliverdin (rapidly degraded to bilirubin by biliverdin reductase), free iron and carbon monoxide (CO) [80]. HO-1 expression is observed in all cells and tissues subjected to oxidative stress, or reactive species from outside or from inside, as occurs with DAQs, superoxides, activation of nitric oxide synthase, GSH depletion, H2O2, cytokines and xenobiotics [81]. HO-1 expression is controlled at the transcription level by activation of the Nrf2 pathway. Besides many other transcription factors, the promoter region of the HO-1 gene contains a regulatory sequence for two AREs regulated by Nrf2 [82]. HO-1 has been implicated in aging and neurodegenerative disorders including PD. The dopaminergic neurons of SNpc shows moderate increase in HO-1 protein levels and potent immunoreactivity in lewy

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Fig. (1). Schematic illustration of regulation of Nrf2 pathway under basal and stress condition. In basal condition Nrf2 continuously undergoes proteosomal degradation. Disruption of Nrf2-Keap1 association as mediated by electrophiles, free radicals or inducers of Nrf2, leads to diminished rate of proteolysis and thereby enhanced nuclear accumulation of Nrf2 into the nucleus. Nrf2 binds with ARE in the promoter region of its target genes thereby inducing a battery of cytoprotective genes and antioxidative enzymes like heme oxygenase-1 (HO-1), NAD (P) H: quinone oxidoreductase 1 (NQO1), - glutamylcysteine ligase (-GCL), and many others, which results in adaptive response. In addition, phosphorylation of Nrf2 at serine and threonine residues by Kinases such as, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), PKR-like endoplasmic reticulum kinase, c-Jun N-terminal protein kinase (JNK), extracellular signalregulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) is assumed to facilitate the dissociation of Nrf2 from Keap1 and subsequent translocation to the nucleus. DJ-1 is found to inhibit oxidative damage and stabilize Nrf2 by preventing its interaction with Keap 1. P62 also plays role in Nrf2 activation by transporting Keap1 for autophagic degradation and making positive feedback loop with Nrf2. For further details see the main text.

bodies [83]. Increased expression of HO-1 confers an adaptive survival response to oxidative and inflammatory insults in in vitro and in vivo models of PD [84, 85]. SN from PD patient’s exhibit increased expression of HO-1 that correlates with astrogliosis and microgliosis. In response to exogenously added -syn, Nrf2-/- microglia failed to activate the expression of the HO-1 gene [86]. In a recent study, Nrf2-knockout mice showed exacerbated gliosis and dopaminergic nigrostriatal degeneration. On the other hand the severity of gliosis and dopaminergic degeneration in HO1 null mice was neither increased nor reduced after MPTP injection for five consecutive days. Both Nrf2-/- and HO-1-/showed similar free iron deposit, pointing out that these proteins do not contribute significantly to iron accumulation or clearance in MPTP-induced parkinsonism [87].

5.2. NAD(P)H: Quinone Oxidoreductase 1, Nrf2 Pathway and Parkinson Disease One of the factors observed in dopaminergic neurodegeneration in PD is the formation of DAQs [24, 29]. NQO1 is an inducible enzyme, which catalyzes two electron reduction of environmental or internally formed quinones including DAQs to the redox-stable hydroquinone [88, 89]. NQO1 expression and activity are elevated in neurons and astroglia of SN in PD patients [90], and overexpression of NQO1 protects dopaminergic cells against DA-induced cell death [91]. In response to exogenously added -syn, Nrf2-/microglia failed to activate the expression of the NQO1 gene [86]. NQO1 also found to be upregulated in astroglia cells in the SN in response to 6-hydroxy dopamine (6-OHDA) rat model of PD [92]. ARE is required for basal expression as


well as induction of NQO1 gene in response to xenobiotics, antioxidants and oxidants. 5.3. Glutathione, Nrf2 Pathway and Parkinson Disease The tripeptide GSH, which is the most abundant nonprotein thiol in cells [93, 94], is synthesized from glutamate, cysteine and glycine in the cytosol in two steps, each requiring adenosine triphosphate (ATP) hydrolysis; the formation of c-glutamylcysteine, followed by its conjugation to glycine [95, 96]. Glutamate cysteine ligase (GCL) catalyzes the first and rate-limiting step of de novo synthesis, making it a major determinant of overall GSH synthetic capacity [95-97]. GCL is a heterodimer comprising a catalytic subunit and a modifier subunit, which changes the catalytic characteristics of the holoenzyme. GSH is a major component of cellular antioxidant defenses, and reduction of GSH levels is one of the earliest biochemical parameter observed in PD patients [93, 98, 99]. GSH protects cells against ROS and RNS via nonenzymatic reduction, whereas the removal of hydroperoxides requires enzymatic catalysis by glutathione peroxidase [93, 94, 100]. Nrf2 plays a key role in the regulation of cellular GSH homeostasis. Nrf2-/- cells and tissues have low induction of GSH or have low GHS [101, 102], and accumulate greater levels of gamma-glutamylcysteine synthase than wild-type Nrf2 cells [103]. Nrf2 regulates GSH biosynthesizing enzymes (GCLM, GCLC) [104], cysteine/glutamate exchange transporter [105], GPX2 and GST, which use GSH as a cofactor [106], modulates the GSH redox state by regulating GSR and protects the cells against oxidative stress [107]. Interestingly, GCL, coded by Nrf2 responsive gene [108, 109] is unaffected in PD patients at postmortem [98]. However reduce levels of GCL were observed throughout the brain as a consequence of the aging process [110]. 6. ROLE OF NRF2/ARE PATHWAY IN COUNTERACTING OXIDATIVE STRESS, NEUROINFLAMMATION AND CELL DEATH IN PARKINSON DISEASE Identification of gene DJ-1 (PARK7), in familial forms of PD, links oxidative stress to its etiology [14, 66]. The interaction of Nrf2 and DJ-1 is another bridge to support the role of Nrf2 in PD. DJ-1 stabilizes Nrf2 by preventing its interaction with Keap1 and thereby preventing its proteasomal degradation [111]. Moreover DJ-1 deficient mice showed decreased Nrf2 protein expression in response to paraquat [112]. Overexpression of DJ-1 results in increased Nrf2 protein levels, promotes its translocation into the nucleus and enhances its recruitment onto the ARE site in the Trx1 promoter region [113]. Direct evidence of the involvement of the Nrf2/ARE pathway in the pathogenesis of PD came from a postmortem study that examined the expression and localization of Nrf2 in susceptible neuron populations in AD and PD. Nrf2 mediated transcription was not induced in neurons in AD despite the presence of oxidative stress whereas in PD, nuclear localization of Nrf2 was strongly induced compared with age-matched normal controls. Neuronal response to oxidative stress differs with AD and PD cases and this response might be insufficient to protect neurons from degeneration [114]. In another clinical


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study, complete haplotype analysis of the NFE2L2 and KEAP1, the Nrf2 and Keap1 encoding genes in two independent case-control materials showed strong protective effects of an NFE2L2 haplotype in two independent casecontrol materials, indicating that varying efficiency in the oxidative protection by Nrf2 may influence PD pathogenesis [115]. Clinical signs of PD are observed when neuronal degeneration is at an advanced stage, and current treatment options are focused on increasing dopamine levels. As evident by a recent study, Nrf2 activation by tertbutylhydroquinone (tBHQ) and sulforaphane can be protective when activated after the start of a lesion with 6OHDA in organotypic nigrostriatal coculture [116]. Recently, Cook et. al., established a novel cellular model of PD, termed human olfactory neurosphere derived (hONS) cell lines, from olfactory mucosa biopsies from multiple PD patients and healthy controls which express functional and genetic differences in a disease-specific manner. PD patientderived hONS cells showed deficiencies in GSH and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium,inner salt] metabolism, moreover small interfering RNA (siRNA) -mediated ablation of Nrf2 in control donor cells decreased both total GSH content and MTS metabolism to levels detected in cells from PD patients. Furthermore, activation of the Nrf2 pathway in PD hONS cultures restored GSH levels and MTS metabolism to that of control levels [117]. Nrf2-ARE pathway activation by using siRNA directed against Keap1 in primary astrocytes activates Nrf2, increased the levels of Nrf2-ARE driven genes and protected against oxidative stress. Moreover, Keap1 siRNA resulted in a persistent upregulation of the Nrf2-ARE pathway and protection against oxidative stress in primary astrocytes. Additionally, Keap1 siRNA injection into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage [118]. In a report by Cao et. al., several approaches were attempted to increase Nrf2 activity, including chemical induction using tBHQ, Nrf2 over expression or Keap1 siRNA knockdown. It was shown that Nrf2 over expression protects cells against specific types of oxidative stress caused by 6-OHDA, and 3 morpholinosydnonimine (SIN-1), but not against MPP+ [119]. Interestingly, Keap1 knockout (KO) mice were generated with the expectation that the absence of the keap1 gene might produce strong resistance to oxidative and electrophilic stress. However, these deficient mice died postnatally approximately 2 and 3 weeks, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach [120]. Another study utilized embryonic cortical cultures prepared from keap1 KO mice, showed that constitutive activation of Nrf2 by deletion of the keap1 gene leads to the increased resistance of neurons against oxidative stress caused by high concentrations of glutamate and rotenone, and Nrf2 activation caused by keap1 gene deletion leads to neuronal protection [121]. Alternatively, upregulation of the Nrf2 pathway either by over expressing Nrf2 or its DNA-binding dimerization partner, Maf-S, restores the locomotor activity of -syn-expressing flies. Similar benefits were observed upon RNA-interferencemediated down regulation of Keap1, as well as in conditions of keap1 heterozygosity. Consistently, the -syn induced dopaminergic neuron loss is suppressed by Maf-S over

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expression or keap1 heterozygosity. This model provides a genetically accessible in vivo system to evaluate the potential of additional Nrf2 pathway components and regulator as therapeutic targets, and validates the sustained up-regulation of the Nrf2 pathway as a neuroprotective strategy against PD [122]. Thus, substantial evidence utilizing several approach showed the involvement of oxidative stress in Nrf2 activation and their role in PD. As we know, neuroinflammation also contributes to the cascade of events leading to neuronal degeneration in PD [39]. These mechanisms include microglial activation [4042], astrogliosis, and cytokine release [43-45] which eventually lead to cell death [46-48]. A study by Rojo et. al., showed critical role of Nrf2 in regulating microglial dynamics and neuroinflammation in experimental models of PD. Nrf2-/- mice, with hypersensitivity to oxidative stress showed more severe dopaminergic dysfunction, profound lesion in SNpc-striatum axis, and more astrogliosis and microgliosis than that to wild type Nrf2+/+ littermates in response to chronic MPTP treatment (20 mg/kg for 4 weeks). Nrf2 plays a crucial role in tuning the balance between classical and alternative microglial activation, thereby modulating microglial dynamics and identifying Nrf2 as a molecular target to control microglial function in PD [123]. Genetically engineered ARE-hPAP reporter mice, Nrf2-/mice and GFAP-Nrf2 transgenic mice were injected with subchronic 30 mg/kg MPTP to evaluate the role of Nrf2 mediated neuroprotection. It was observed that Nrf2-ARE signaling, Nrf2 and NQO1 expression, and NQO1 activity were decreased in the striatum, but increased in the SNpc in ARE-hPAP mice, as revealed by histochemical staining and hPAP activity after MPTP treatment. A greater fraction of TH was lost in the striatum and SNpc of Nrf2-/- mice compared with Nrf2+/+ mice after MPTP treatment and over expression of Nrf2 conferred resistance to MPTP. GFAPNrf2 (+) mice were completely protected from striatum TH loss, and the extent of astrogliosis and microglial activation were also dramatically attenuated in the GFAP-Nrf2 (+) mice compared to GFAP-Nrf2 (-) after MPTP treatment [84]. A new animal model based on stereotaxic delivery of an adeno-associated viral vector (rAAV) for expression of human -syn in the ventral midbrain of Nrf2-/- mice was presented. Nrf2-/- mice exhibited exacerbated degeneration of nigral dopaminergic neurons and increased dystrophic dendrites, reminiscent of lewy neurites, which correlated with impaired proteasome gene expression and activity. Furthermore, intensified neuroinflammation and gliosis were observed in Nrf2-/- mice. Nrf2-/- microglia failed to activate the expression of two anti-inflammatory genes, HO-1 and NQO1 in response to exogenously added -syn. Impaired Nrf2 response correlated with a shift in the microglial activation profile, towards increased production of proinflammatory markers, IL-6, IL-1 and iNOS [86]. Sulforaphane, a potent Nrf2 activator at 50 mg/kg, protects against the sub-acute model of MPTP by increasing Nrf2 protein levels in basal ganglia, and up-regulates phase II antioxidant enzymes HO-1 and NQO1 in wild-type mice, but not in Nrf2-knockout mice. SFN provided protection against MPTP-induced death of nigral dopaminergic neurons (Nissl and TH positive), and neuroprotective effects were accompanied by decrease in astrogliosis, and microgliosis, and the release of pro-inflammatory cytokines [124].

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Interestingly, Nrf2 gene ablation increases sensitivity to MPTP. Measurement of striatal DAT levels using quantitative autoradiography in Nrf2 knockout and wild-type mice for 7 days after acute MPTP dosing ranging from 20 to 60 mg/kg suggests that Nrf2 knockout mice express a significantly greater loss of DAT levels in the striatum than wild-type mice [125]. Furthermore, oral administration of 3H-1,2-dithiole-3-thione (D3T), an inducer of the Nrf2 pathway, to wild-type mice either at 0.5 mmol/kg on days 5, 3 and 1 prior to MPTP or a single 0.5 mmol/kg dose of D3T 2 days prior to 40 mg/kg dose of MPTP administration, provided partial protection against MPTP-induced neurotoxicity [125]. In another study, Nrf2 protected neurons in mixed primary neuronal cultures containing both astrocytes (~10%) and neurons (~90%) through coordinate up-regulation of ARE-driven genes. Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin MPP+ and rotenone induced apoptosis compared to Nrf2+/+ neurons. Nrf2+/+ neuronal cultures had higher expression levels of genes encoding detoxification enzymes, antioxidant proteins, calcium homeostasis proteins, growth factors, neuronspecific proteins, and signaling molecules compared to Nrf2-/- neuronal cultures. Furthermore, Nrf2-/neurons were more venerable against cytotoxic effects of ionomycin (a calcium ionophore), and 2, 5-(di- t-butyl)-1, 4 hydroquinone (dtBHQ) (an endoplasmic reticulum Ca2+ATPase inhibitor) compared to Nrf2+/+ neurons. Adenoviral vector-mediated over expression of Nrf2 recovered AREdriven gene expression in Nrf2-/- neuronal cultures and rescued Nrf2-/- neurons from rotenone or ionomycin induced cell death [126]. Human dopaminergic neuroblastoma SH-SY5Y cells exposed to the paraquat indicate that apoptosis signalregulating kinase 1 (ASK1) is a crucial effector of ROSinduced cell death, and points to Nrf2, the master regulator of redox homeostasis, and its target thioredoxin (Trx), Nrf2/Trx axis as a specific target for therapeutic intervention [127]. Another study, showed endogenous Nrf2 expression is critical to control the extent of cellular damage due to the neurotoxin 6-OHDA in both N27 cells culture and in the living animal. Cultured neurons from Nrf2 null embryos exposed to 6-OHDA show increased apoptotic cells as well as attenuated cellular viability as compared to cells that can engage an Nrf2-ARE response. Nrf2/ mice demonstrate lesions almost twice the size of wild-type animals after 6OHDA injections in striata [128]. Thus, Nrf2 plays a pivotal role in acute [101, 129] as well chronic inflammation [123]. In motor neurons, Nrf2 activation critically modulates apoptotic pathways [128, 130-132]. Recent study showed that Nrf2 protein up-regulates antiapoptotic protein Bcl-2 and prevents cellular apoptosis [133]. These reports points the role of Nrf2 as multi therapeutic target to counteract oxidative stress, inflammation and neuronal cell death as summarized in Table 1. 7. NRF2/ARE PATHWAY AS A THERAPEUTIC TARGET FOR TREATMENT OF PARKINSON’S DISEASE Several compounds utilize the Nrf2 pathway to show neuroprotective effects by increasing or up -regulating


Table 1.


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Role of Nrf2/ARE Pathway in PD

Model

Intervention

NRF2/ARE Pathway and PD

Ref.

DJ-1 deficient mice

Paraquat

Decreased Nrf2 protein expression in response to paraquat.

[112]

susceptible populations

In PD, nuclear localization of Nrf2 was strongly induced whereas Nrf2-mediated transcription was not induced in neurons in AD.

[114]

Clinical Study

in AD and PD

Clinical Study

PD and age matched control Swedish and Polish cases

NFE2L2 and KEAP1, the Nrf2 or Keap1 encoding genes, KEAP1 showed no association with PD whereas NFE2L2 haplotype influences risk of PD in two discrete Caucasian case.

[115]

Organotypic Nigrostriatal Cocultures

6-OHDA

Nrf2 activation rescue dopaminergic neurons from 6-OHDA-induced toxicity in nigrostriatal organotypic cocultures.

[116]

hONS cell lines, from PD patients

siRNA against Nrf2

hONS cells showed deficiencies in glutathione and MTS metabolism and Nrf2 pathway activation restored these effects. siRNA-mediated ablation of Nrf2 in control donor cells decreased both total glutathione content and MTS metabolism.

[117]

Primary astrocytes and mice

siRNA against Keap1 and MPTP

Keap1 knockdown activates Nrf2 in astrocytes, increased the levels of Nrf2- ARE driven genes and protected against oxidative stress. Keap1 siRNA injected into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage.

[118]

SH-SY5Y cells

6-OHDA, SIN-1, and MPP+

Nrf2 over expression using tBHQ or Keap1 siRNA knockdown provides resistance against oxidative damage caused by 6-OHDA and SIN-1 but not against MPP+.

[119]

-syn-expressing flies

Nrf2 or Maf-S upregulation

Up-regulation of the Nrf2 pathway either by over expressing Nrf2 or its DNAbinding dimerization partner, Maf-S, restores the locomotor activity of -synexpressing flies.

[122]

Nrf2-/- mice

MPTP

Nrf2 plays a crucial role in tuning the balance between classical and alternative microglial activation, thereby modulating microglial dynamics.

[123]

ARE-hPAP reporter mice, Nrf2-/- mice and GFAP-Nrf2

MPTP

MPTP treatment decreased Nrf2/ARE signaling, Greater fraction of TH was lost in striatum and SNpc of Nrf2-/- mice after MPTP treatment. GFAP-Nrf2 (+) mice was completely protected from striatum TH loss and extent of astrogliosis and microglial activation (Iba-1) were also dramatically attenuated in the GFAPNrf2(+) mice as compare with GFAP-Nrf2 (-) after MPTP treatment.

[84]

Nrf2-/- mice

stereotaxic delivery of rAAV for expression of human -syn in the ventral midbrain of mice

Impaired Nrf2 response correlated with a shift in the microglial activation profile, towards increased production of proinflammatory markers, IL-6, IL-1 and iNOS.

[86]

Nrf2 Knock out vs wild type

MPTP

Sulforaphane, protects against MPTP toxicity by increasing Nrf2 protein levels in basal ganglia, upregulation of HO-1 and NQO1 enzymes and decrease in astrogliosis, microgliosis, and release of pro-inflammatory cytokines.

[124]

Knockout mice

MPTP

Nrf2 knockout mice express a significantly greater loss of DAT levels in the striatum than wild-type mice following MPTP treatment. D3T, an Nrf2 inducer when injected in wild mice provided partial protection against MPTP-induced neurotoxicity.

[125]

Primary neuronal culture

MPP, Rotenone, Ionomycin, and dtBHQ

Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin (MPP+ or rotenone) induced apoptosis and more venerable against cytotoxic effects of ionomycin and dtBHQ as that to Nrf2 +/+ neurons.

[126]

SH-SY5Y cells

paraquat

Apoptosis signal-regulating kinase 1 is a crucial effector of ROS-induced cell death and Nrf2/Trx axis as a specific target for therapeutic intervention.

[127]

N27 cell lines, primary culture and Mice

6-OHDA

Loss of Nrf2-mediated transcription exacerbates vulnerability to the neurotoxin 6OHDA both in vitro and in vivo.

[128]

Transgenic Drosophila flies

flies that overexpress the human synuclein protein, flies that bears a homozygous loss-of-function mutation in the parkin gene.

Decaffeinated Coffee and nicotine free tobacco but not caffeine and nicotine showed neuroprotective role in fly PD models through activation of the cytoprotective transcription factor Nrf2.

[139]

cytoprotective genes or enzymes in experimental models of PD. Apomorphine, a dopamine D1/D2 receptor agonist, used in clinics for PD, has been shown to increase the intracellular antioxidative potential in SH-SY5Y cells via the Nrf2-ARE pathway [134]. Deprenyl, a selective MAO-B inhibitor, used

in clinics to slow the progression of symptoms in patients with PD. Recently, Nrf2 activation has been identified as an alternative mechanism by which deprenyl slows the progression of PD [135]. Deprenyl up-regulates the expression and activity of NQO1, attenuates the increase in

8


the quinoprotein levels in MPP+- treated PC12 cells, and protects from oxidative damage by triggering the Nrf2/ARE pathway. Moreover, its effect on NQO1 up regulation was greatly attenuated in Nrf2 siRNA transfected cells. The activation of Nrf2/ARE signaling by deprenyl in PC12 cells is independent of MAO-B inhibition [136]. Bromocriptine, a dopamine agonist used clinically for PD therapy, upregulates the expression and activity of NQO1, attenuates the increase in the protein-bound quinone in H2O2-treated PC12 cells, protects PC12 cells against oxidative damage and increases the expression and nuclear translocation of Nrf2. The Nrf2related cytoprotective and antioxidative effects of bromocriptine are PI3K/Akt pathway-dependent, and are independent of dopamine receptor activation [137]. Rosiglitazone, a PPAR agonist, attenuated ROS formation induced by MPP+ in SH-SY5Y cells, concurrent with an upregulation of glutathione-S-transferase independent of PPAR activation [138]. In an another study, decaffeinated coffee and nicotine free tobacco but not caffeine and nicotine showed neuroprotective role in strain of flies that bears a homozygous loss-of-function mutation in the parkin gene through activation of the cytoprotective transcription factor Nrf2 [139]. Apart from these compounds several other compounds such as Erythropoitein [140], Artesunate [141], Kahweol [142], Luteolin [132] Ginsenoside Rb1 [143], CDDMO-MA [144], MGF24 [145], Eriodictyol [146], Licochalcone E [147], Fibroblast growth factor 9 [148] and Metallothionein-III [149] showed neuroprotective effects by increasing the Nrf2 level through different pathways, as summarized in Table 2. 8. FUTURE STRATEGIES AND POSSIBLE APPROACHES TO TARGET PARKINSON DISEASE BY NRF2 ACTIVATION ARE has a core sequence of TGACNNNGC, which is necessary for its transcriptional response [70]. Drug discovery approaches utilizing core sequence in the promoter region could be a possible way to induce cytoprotective enzymes and genes. Almost all currently known ARE inducers are indirect inhibitors of Keap1–Nrf2 interaction, and they bind with the sulfhydryl groups of cysteines in Keap1 by oxidation or alkylation [150]. Compounds that have propensity towards sulfhydryl groups are potent inducers of ARE activity. Inducers that can increase the expression of cytoprotective genes are classified into 10 chemically distinct classes: (i) Oxidizable phenols and quinones; (ii) Michael acceptors (olefins or acetylenes conjugated to electron-withdrawing groups); (iii) Isothiocyanates; (iv) Thiocarbamates; (v) Trivalent arsenicals; (vi) Dithiolethiones; (vii) Hydroperoxides; (viii) Vicinal dimercaptans; (ix ) Heavy metals; and ( x ) Polyenes [72, 151] (Fig. 2). Several phytochemicals, have been suggested as Nrf2 inducers [152]. Nrf2 activity declines with age [108], which is the main risk factor for PD. Transcriptional activity of Nrf2 can be restored pharmacologically in old animals [153], by activating the Nrf2 pathway with various inducers. In following text we have discussed the most relevant approaches which can be utilized to induce Nrf2 expression. Up regulation of Nrf2 pathway by quinones have been discussed elsewhere [10].

Kumar et al.

8.1. Terpenoids Natural terpenoid provides an important scaffold for new drug development [154]. Oleanolic acid is a natural terpenoid and inducer of the Nrf2 pathway. Synthetic triterpenoids, derivatives of 2-cyano-3, 12-dioxooleana-1, 9dien-28-oic acid (CDDO), have been found to be potent inducers of Nrf2 [144, 155]. Dopaminergic neuroprotection by methyl ester of CDDO (CDDO-Me) involved inhibition of microglial derived cytokine production and indirect inhibition of TNF-dependent pro-apoptotic pathways and may have therapeutic potential to modify the course of neurodegenerative disease like PD [155]. Subsequently, oral administration CDDO methyl amide (CDDO-MA) resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological - syn accumulation and oxidative damage in mice [144]. 8.1.1. Antioxidant Inflammation Modulators (AIMs) Antioxidant Inflammation Modulators (AIMs) are preventive and therapeutic agents for conditions involving oxidative stress and inflammation. Several groups have filed patents for the use of oleanolic acid and its derivatives as AIMs. Recently, Abbott collaborated with Reata pharmaceuticals to jointly develop and commercialize a series of second-generation oral AIMs which act by potently activating Nrf2. Bardoxolone methyl (also known as “RTA 402” and “CDDO-Me”), a synthetic triterpenoid and potent inducer of the Nrf2 pathway [156], is one of the compounds undergoing phase III clinical trials for chronic kidney disease in patients with type 2 diabetes mellitus [157]. AIMs, found to be most potent activators of Nrf2, binds to proteins responsible for sensing the redox balance, turning on master regulator genes that promote inflammation and turning on a counter-balancing transcription factor that resolves inflammation [158]. 8.2 Electrophilic Compounds Electrophilicity is a common property of most known ARE inducers due to their ability to become electrophilic quinones upon auto-oxidation. However, not all electrophiles can regulate ARE activity. The biological effects of electrophiles vary, and can be therapeutic or toxic depending on chemical structure. The major classes of electrophilic compounds are represented by enone and catechol types [159]. Cellular distribution in the brain of enone and quinone-type electrophilic compounds may be different. Enone-type electrophiles such as electrophilic neurite outgrowth-promoting prostaglandin (NEPPs) are taken up preferentially into neurons and bind in a thiol-dependent manner to Keap1 [160]. In contrast, catechol (hydroquinone) - type electrophiles, such as tBHQ, preferentially act on astrocytes [161] and modulate ARE driven cytoprotective effects in astrocytes. Carnosic acid obtained from Rosmarinus officinalis accumulates in both non-neuronal and neuronal cells. Thus, it exerts actions on both types of cells [159]. One approach to target chronic neurodegenerative disorder is by utilizing pro-electrophilic compounds that remain non reactive until they reach site of action and then convert to electrophilic compounds [159]. Interestingly, strongylophorine-8, a pro-electrophilic from marine sponges, showed neuroprotective effects through the Nrf2 signaling


Table 2.


9

Summary of Compounds Showing Neuroprotective Effects Through Nrf2/ARE Pathway Up-Regulation in PD

Compound

Class

NRF2/ARE Pathway and PD

Ref.

Apomorphine

Dopamine Agonist

Apomorphine stimulated the translocation of Nrf2 into the nucleus and transactivation of ARE. The expression of HO-1 was dose dependently induced by Apomorphine in SH-SY5Y cells.

[134]

Deprenyl

MAO-B inhibitor

Deprenyl upregulates the expression and activity of NQO1, attenuates the increase in the quinoprotein levels in MPP+-treated PC12 cells, and protects from oxidative damage via the Nrf2-mediated upregulation of NQO1 involving both PI3K/Akt and Erk pathways.

[136]

Bromocriptine

Dopamine Agonist

Bromocriptine upregulates the expression and activity of NQO1, attenuates the increase in the protein-bound quinone in H2O2-treated PC12 cells, and protects against oxidative damage through PI3K/Akt dependent pathway independent of dopamine receptor activation.

[137]

Rosiglitazone

PPAR agonist

Rosiglitazone attenuated ROS formation induced by MPP+ in SH-SY5Y cells concurrent with an upregulation of glutathione-S-transferase independent of PPAR activation

[138]

Decaffeinated Coffee and nicotine free tobacco

Unknown

Decaffeinated Coffee and nicotine free tobacco but not caffeine and nicotine showed neuroprotective role in fly PD models through activation of the cytoprotective transcription factor Nrf2.

[139]

Erythropoietin

Cytokine hormone

Erythropoietin induces nuclear translocation of Nrf2 and upregulates HO-1 expression through the activation of PI3K, MAPK, and Nrf2 pathways in SHSY5Y cells.

[140]

Artesunate

Derivative of artemesinin

Artesunate activates Nrf2-ARE system, upregulates HO-1 in LPS stimulated BV-2 microglial cell through ERK dependent pathway.

[141]

Kahweol

Diterpene

Pretreatment of SH-SY5Y cells with kahweol significantly reduced 6-OHDA induced generation of ROS, caspase-3 activation, and subsequent cell death. It also up-regulated HO-1 expression, via the PI3K and p38/Nrf2 signaling pathways.

[142]

Ginsenoside Rb1

Phytoestrogen

Ginsenoside Rb1 inhibits 6-(OHDA)-induced oxidative injury via the G1/PI3K/AktNrf2 signaling pathway in SH-SY5Y cells.

[143]

CDDO-MA

Synthetic triterpenoid

CDDO-MA provides significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, -synuclein accumulation and oxidative damage in mice through Nrf2/ARE pathway.

[144]

MGF24

Derivative of mechano-growth factor (MGF)

MGF24 provides neuroprotection against 6-OHDA induced apoptosis through HO-1 upregulation via PKC/Nrf2 pathway.

[145]

Eriodictyol

Flavonoid

Eriodictyol induced the nuclear translocation of Nrf2, enhanced the expression of HO-1 and -GCS, and increased the levels of intracellular glutathione thereby protects PC12 cells against H2O2 induced oxidative stress.

[146]

Licochalcone E

Retrochalcone

Licochalcone E protects BV-2 microglial cells and mice against LPS and MPTP respectively, with up-regulation of HO-1 and NQO1 through Nrf2-ARE pathway.

[147]

Fibroblast growth factor 9 (FGF9)

Growth factor

FGF 9 upregulates -GCS and HO-1 expression and provide protection in cortical and dopaminergic neurons from MPP+ induced oxidative insult.

[148]

Metallothionein III

Metal binding protein

Metallothionein-III prevented the accumulation of ROS in SH-SY5Y cells challenged with 6-OHDA by a mechanism that involves PI3K and ERK kinase/Nrf2 dependent induction by regulating HO-1 gene expression.

[149]

Luteolin

Flavone

Luteolin protects rat neural PC12 and glial C6 cells from MPP+. This protection critically depends on the activation of Nrf2 since down regulation of Nrf2 by siRNA completely abrogates the protection of luteolin in vitro.

[132]

Sulforaphane

Organosulfur compound

Sulforaphane, protects against MPTP toxicity by increasing Nrf2 protein levels in basal ganglia and upregulation of HO-1 and NQO1 enzymes and decrease in astrogliosis, microgliosis, and release of pro-inflammatory cytokines.

[124]

pathway [162]. Other important electrophilic compounds are sulforaphane from cruciferous vegetables [163], allicin [164], and garlic organosulfur compounds [165]. These approaches could be utilized to target neuroinflammation. 8.3. Inhibition of Negative Regulators of Nrf2 Apart from cytosolic inhibitor Keap1, several mechanisms are involved in down-regulation of the Nrf2

pathway, decreasing transcriptional activity of Nrf2 either directly or indirectly. Glycogen synthase kinase 3 (GSK3), which is a serine/threonine kinase, is active in resting un-stimulated cells and participates in neuronal death [166]. GSK-3 mediates phosphorylation of Nrf2, prevents nuclear localization and co-expression of active GSK-3 prevents binding and activation of AREs located in phase II gene promoters [167]. GSK-3 promotes the cytosolic localization of Nrf2, inhibits the transcriptional activity and

10


Kumar et al.

pathway might provide rationale to target various neurodegenerative disorders including PD. Initially, Nrf2 was thought to be a regulator of the antioxidant enzymes, but later, it became clear from various studies that Nrf2 participates in prevention, control and repair/regeneration of damaged cell through activating several related cytoprotective genes called as “Nrf2 battery”. Nrf2 production appears inversely proportional to age. Interestingly this can be restored pharmacologically in animals, so this system has to be stimulated in order to provide desired protection against the everyday increasing load of oxidants. The dosage or level of expression of this gene should be crucial for beneficial effects. Currently, this pathway is being utilized in clinical trials for chronic kidney disease and multiple sclerosis which provide substantial evidence of Nrf2 importance. Targeting the Nrf2 pathway might provides a possible approach for neuroprotection either by reducing oxidative stress or underlying neuroinflammation seen in PD. Fig. (2). Inducers that can increase the expression of cytoprotective genes are classified into 10 chemically distinct classes: (i) Oxidizable phenols and quinones; (ii) Michael acceptors (olefins or acetylenes conjugated to electron-withdrawing groups); (iii) Isothiocyanates; (iv) Thiocarbamates; (v) Trivalent arsenicals; (vi) Dithiolethiones; (vii) Hydroperoxides; (viii) Vicinal dimercaptans; (ix ) Heavy metals; and ( x ) Polyenes.

blocks the antioxidant and cytoprotective functions of Nrf2 [74]. The role of GSK-3 in in-vitro and in-vivo PD models has been well established [168, 169]. Moreover, neurons located in the SN and the upper pons of PD patients showed increased activity of GSK-3 [170]. Interestingly, GSK-3 is activated in the presence of -syn by phosphorylation of the kinase at Tyr 216, leading to Tau hyperphosphorylation at Ser-262 and Ser-396/404. -syn is absolutely necessary for activation of GSK-3. Interestingly, SH-SY5Y cells lacking -syn, or in -syn-/- mice, did not show increases in phosphorylated GSK-3 levels or changes in Tau hyperphosphorylated at Ser-262 or Ser-396/404 despite treatment with MPP+/MPTP [171]. Another factor which correlates GSK-3 and Nrf2 with PD is aging. Progressive increase in GSK-3 [172], and decline in Nrf2 transcriptional activity have been reported with aging [108]. Fyn, a Src kinase family member, acts as a negative regulator of Nrf2 [173]. Activated GSK-3 phosphorylates Fyn at threonine residues, leading to nuclear localization of Fyn [174]. Interestingly, once Fyn is localized inside the nucleus, it phosphorylates tyrosine residue 568 of Nrf2, which leading to a Crm-1 mediated nuclear export and degradation of Nrf2 [173]. Moreover, Fyn phosphorylates tyrosine residue 125 of -syn and might be involved in pathogenesis of PD [175]. Another transcription factor Bach1 is ubiquitously expressed and competes with Nrf2, leading to negative regulation of the ARE, and the balance of Nrf2 versus Bach1 inside the nucleus influences up or downregulation of ARE-mediated gene expression [176]. 9. SUMMARY AND CONCLUSIONS The etiopathology of PD mainly involves oxidative stress and neuroinflammation that cause dopaminergic cell death. Activation and regulation of multifunctional genes by Nrf2

ABBREVIATIONS 6-OHDA AIMs

= 6-hydroxydopamine = Antioxidant Inflammation Modulators ARE = Antioxidant response element DAQs = Dopamine quinones ERK = Extracellular signal-regulated kinase GCLC = Glutamate cysteine ligase catalytic subunit GCLM = Glutamate cysteine ligase modifier subunit GFAP = Glial fibrillary acidic protein GSH = Glutathione GSK-3 = Glycogen synthase kinase 3 HO-1 = Hemeoxygenase-1 hPAP; ARE-hPAP = ARE-driven human placental alkaline phosphatase Keap1 = Kelch-like ECH-associated protein Maf = Musculoaponeurotic fibrosarcoma MAPK = Mitogen-activated protein kinase MPP+ = 1-methyl-4-phenylpyridinium MPTP = 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine MTS = [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium,inner salt] NADPH = Nicotinamide adenine dinucleotide phosphate NEPP = Neurite outgrowth-promoting prostaglandin NQO1 = NAD (P) H quinone oxidoreductase-1 Nrf2 = Nuclear factor erythroid 2-related factor 2 PD = Parkinson’s disease RES = Reactive oxygen species RNS = Reactive nitrogen species


ROS siRNA TP –syn

= = = =

Reactive oxygen species Short interfering RNA Triterpenoids -synuclein


[20] [21]

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS This work was supported by Konkuk University in 2012. REFERENCES [1]

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[42]

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Received: June 12, 2012


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Revised: August 30, 2012

Accepted: August 31, 2012