Nrf2–ARE pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases

Nrf2–ARE pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases

    Nrf2-ARE Pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases Izaskun Buendia, Pa...

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    Nrf2-ARE Pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases Izaskun Buendia, Patrycja Michalska, Elisa Navarro, Isabel Gameiro, Javier Egea, Rafael Le´on PII: DOI: Reference:

S0163-7258(15)00216-8 doi: 10.1016/j.pharmthera.2015.11.003 JPT 6829

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Buendia, I., Michalska, P., Navarro, E., Gameiro, I., Egea, J. & Le´on, R., Nrf2-ARE Pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases, Pharmacology and Therapeutics (2015), doi: 10.1016/j.pharmthera.2015.11.003

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P&T #22907

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Nrf2-ARE Pathway: an emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases

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Izaskun Buendia,a,b Patrycja Michalska,a,b Elisa Navarro,a,b Isabel Gameiro,a,b Javier Egeaa,b and Rafael León.*a,b a

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Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, 28006-Madrid, Spain. Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, UAM, 28029-Madrid, Spain.

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Abstract: Neurodegenerative diseases (NDDs) are predicted to be the biggest health concern in this century and the second leading cause of death by 2050. The main risk factor of these diseases is aging, and as the aging population in Western societies is increasing, the prevalence of these diseases is augmenting exponentially. Despite the great efforts to find a cure, current treatments remain ineffective or have low efficacy. Increasing lines of evidence point to exacerbated oxidative stress, mitochondrial dysfunction and chronic neuroinflammation as common pathological mechanisms underlying neurodegeneration. We will address the role of the nuclear factor E2-related factor 2 (Nrf2) as a potential target for the treatment of NDDs. The Nrf2-ARE pathway is an intrinsic mechanism of defence against oxidative stress. Nrf2 is a transcription factor that induces the expression of a great number of cytoprotective and detoxificant genes. There are many evidences that highlight the protective role of the Nrf2-ARE pathway in neurodegenerative conditions, as it reduces oxidative stress and neuroinflammation. Therefore, the Nrf2 pathway is being increasingly considered a therapeutic target for NDDs. Herein we will review the deregulation of the Nrf2 pathway in different NDDs and the recent studies with Nrf2 inducers as “proof-of-concept” for the treatment of those devastating pathologies.

Key words: Neurodegenerative diseases, oxidative stress, neuroinflammation, nuclear factor E2-related factor 2, Nrf2, electrophile response elements, phase II antioxidant response, Nrf2-inducers.

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To whom correspondence should be addressed: Rafael León Phone: +34 91 497 27 66; Fax: 91 497 34 53; E-mail: [email protected]

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1.- Introduction .............................................................................................................................. 3 1.2.- The Nrf2-EpRE pathway: positive and negative regulation mechanisms ........................ 5 2. Nrf2-EpRE inducers as potential treatments for neurodegenerative diseases ........................... 9 2.1.- Alzheimer’s Disease: Nrf2-EpRE dysregulation and Nrf2 inducers .............................. 10 2.2.- Parkinson’s disease: Nrf2-EpRE dysregulation and Nrf2 inducers ................................ 16 2.3.- Huntington disease: Nrf2-EpRE dysregulation and Nrf2 inducers ................................ 21 2.4.- Amyotrophic lateral sclerosis: Nrf2-EpRE dysregulation and Nrf2 inducers ................ 25 2.5.- Multiple sclerosis: Nrf2-EpRE dysregulation and Nrf2 inducers................................... 30 3.- Conclusion ............................................................................................................................. 40 Abbreviations .............................................................................................................................. 40 Conflict of interest....................................................................................................................... 42 Acknowledgements ..................................................................................................................... 42 References ................................................................................................................................... 43

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1.- Introduction

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Neurodegenerative diseases (NDDs) are among the most serious and prevalent

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health concerns in modern society. NDDs have become the first cause of dependence,

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with frightening predictions of increasing incidence in the near future. The discovery of genetic mutations in affected family members has dominated the drug development programs in the last two decades. Despite a great effort and inversion in R&D, from both pharmaceutical companies and public resources, has been done, however, none of

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these efforts have been translated into new drug approvals. Most NDDs cases are sporadic, considered as the result of complex relationships between genetic and

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environmental factors. Nevertheless, compelling evidence suggests the existence of common clinical and pathological features among different NDDs that can bring into light new targets for their treatment.

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NDDs such as Alzheimer’s disease (AD), Parkinson´s disease (PD), Huntington

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disease (HD), Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS) or ischemic stroke are more prevalent in elderly, being aging their principal risk factor.

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NDDs progress over many years and lead to disability and premature death. Recent discoveries have demonstrated that NDDs share many additional common pathological events. The most representative are abnormal protein aggregation (Ross & Poirier,

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2005), proteasomal (Halliwell, 2006) or autophagic dysfunction (McCray & Taylor, 2008), inflammation (Zipp & Aktas, 2006), neuronal apoptosis (Okouchi et al., 2007), oxidative stress (Andersen, 2004), mitochondrial dysfunction (Lin & Beal, 2006), and abnormal interactions between neurons and glia that accentuate the inflammatory status (Carnevale et al., 2007). Among them, mitochondrial dysfunction, reactive gliosis and oxidative damage to lipids, proteins and DNA, have been widely described in AD (Butterfield et al., 2001; Shaftel et al., 2008), PD (Alam et al., 1997; Clements et al., 2006), HD (Browne & Beal, 2006), ALS (Goodall & Morrison, 2006b), and ischemic stroke (Olmez & Ozyurt, 2012).

1.1.- Neurodegenerative diseases and oxidative stress as common pathological pathway

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ACCEPTED MANUSCRIPT In general, oxidative stress, mitochondrial dysfunction, inflammation and reactive gliosis are increasingly considered as primary causes of degeneration, although it has to be demonstrated indisputably. Compelling evidence indicates that free radicals

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are extremely important to cause neuronal death (Dasuri et al., 2013). Under

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pathological conditions there is an excessive production or reactive oxygen species (ROS) and reactive nitrogen species (RNS) combined with the failure of detoxifying

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and/or repairing systems leading to a situation of oxidative stress. The main source of these species is the oxidative phosphorylation at the mitochondria, and several enzymes may contribute to the generation of these toxic oxidants. Interestingly, specific

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hallmarks of different NDDs have been found to target mitochondria, contributing to oxidative stress. For example, amyloid beta (A), the most prominent hallmark of AD,

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induces oxidative stress and mitochondrial dysfunction through alteration of the mitochondrial membrane potential (Muller et al., 2010). Moreover, Aβ42 binds cooper (I) ions very effectively (Atwood et al., 2000), the Aβ42-Cu+ complex is able to reduce

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oxygen to generate H2O2 and, thus, free radicals (Jiang et al., 2007). Therefore, the Cu+-

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Aβ42 oligomers complexes are markedly cytotoxic. In PD, -synuclein protein forms aggregates, the main pathological hallmark in

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this disease. This protein has a mitochondrial targeted amino-terminal sequence that can associate with the inner mitochondrial membrane and disrupt complex I function, thus generating oxidative stress (Chinta et al., 2010). In addition, the protein parkin is

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associated to healthy mitochondria (Darios et al., 2003) and its loss of function generates oxidative stress and selective impairment of complex I activity (Muftuoglu et al., 2004). Furthermore, DJ-1, a protein related to early onset of parkinsonism when is mutated (Bonifati et al., 2003), is involved in the response to oxidative stress (Cookson, 2010). DJ-1 can act as a scavenger and also stabilizes the transcriptional factor nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), the master regulator of the antioxidant response, to boost the phase II response (Clements et al., 2006). When DJ-1 accumulates mutations, its protective effect is lost and dopaminergic neurons become more vulnerable to oxidative stress. In HD, increased levels of oxidative stress have been widely reported (AyalaPena, 2013). Although the mechanisms leading to oxidative stress generation need to be completely elucidated, it is envisioned that mutant huntingtin impairs brain mitochondria (Orr et al., 2008). This hypothesis was corroborated experimentally by

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ACCEPTED MANUSCRIPT Hands et al., demonstrating that huntingtin aggregation directly increased ROS production and cell toxicity (Hands et al., 2011). One of the main characteristics of ALS are mutations in the copper-zinc

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superoxide dismutase type 1 (SOD1) protein gene (Rosen et al., 1993). Post-mortem

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and biopsy studies of ALS patients have shown impaired mitochondria and defective respiratory chain complex activity. Overexpression of mutant SOD1 in transgenic mice

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caused disruption of the respiratory chain, decreased mitochondrial Ca2+ trafficking and increased ROS production (Lin et al., 2006). Recently, Pollarri et al. have extensively reviewed the implication of oxidative stress in ALS (Pollari et al., 2014).

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MS is defined as a chronic inflammatory and autoimmune disease. It has been proposed that the inflammatory process, characterized by infiltration of leukocytes and

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production of inflammatory intermediaries, is mediated by ROS production and mitochondrial dysfunction (Witte et al., 2009). Uncontrolled ROS release can promote leukocyte migration and contributes to oligodendrocyte damage (Mahad et al., 2008;

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Smith et al., 1999). In MS, ROS and RNS production is mainly produced by

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macrophages and microglia (van Horssen et al., 2011). Compelling evidence demonstrates de implication of the NADPH oxidase (NOX) family in the oxidative

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burst of macrophages and microglia. These evidences point to NOX activation and ROS formation (mainly superoxide anion radical) as important pathological pathways of macrophage and microglial-induced neuronal and oligodendrocyte injury in MS (Cheret et al., 2008).

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Brain ischemia is considered as an acute case of neurodegeneration. When cerebral ischemia occurs, the oxygen and glucose deprivation (OGD) induces a pathological cascade leading to neuronal death. The main events are Ca2+ overload over the OGD period followed by a high increase of free radical production in the reoxygenation phase (Egea et al., 2007). The functional impairment of mitochondria, caused by the decrease of glucose and oxygen levels, is augmented by a massive production of ROS in the re-oxygenation period raised by the OGD-induced overwork of the NOX enzymes (Radermacher et al., 2013; Rodrigo et al., 2013).

1.2.- The Nrf2-EpRE pathway: positive and negative regulation mechanisms In response to oxidative stress, cells have different defence systems to regulate oxidative stress derived damage. The phase II antioxidant response is considered the most important defence pathway present at cells. It is regulated by the transcription 5

ACCEPTED MANUSCRIPT factor Nrf2, a member of the cap‘n’collar family of basic region-leucine zipper transcription factors. Nrf2 is responsible for both, constitutive and inducible expression of EpRE-regulated genes, such as NAD(P)H quinone oxidoreductase-1 (NQO1), heme

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oxygenase-1 (HO-1), thioredoxins (Trxs), glutathione S-transferase (GST), microsomal

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GSTs (mGST1 and mGST2), γ-glutamylcysteine synthetase (γ-GCS) modifier subunit (GCLm) and catalytic subunit (GCLc), glutathione reductase (GR), superoxide

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dismutase-1 (SOD1), glutathione peroxidase (GPx) and other phase I, II, and III enzymes that conjugate drug metabolites or xenobiotics (Thimmulappa et al., 2002). Under normal conditions, the Nrf2-EpRE system keeps the Nrf2 protein at low

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levels. This is achieved by constitutive synthesis and degradation of Nrf2. The repressor protein Kelch-like ECH-associated protein-1 (Keap1), which is mainly present in the

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cytoplasm, tightly regulates this process. Keap1 binds to an E3 ubiquitin ligase complex (Rbx-1) via cullin-3, a protein adaptor (Kobayashi et al., 2004). This complex promotes Nrf2 ubiquitination and 26S proteasome degradation (Figure 1a) (Dhakshinamoorthy &

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Jaiswal, 2001).

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ACCEPTED MANUSCRIPT B) Oxidative conditions

A) Normal conditions

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- Thiol homeostasis: Txn, TrxR1, Srx1 - Inhibition of inflammation: HO-1

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Figure 1: Summary of the activation/regulation processes of the Nrf2-EpRE pathway. A) Under normal conditions, Nrf2 is sequestered in the cytosol by the repressor protein Keap1. Keap1 facilitates Nrf2 ubiquitination by Cul3 leading to degradation through the 26S proteasome pathway. B) Under oxidative stress conditions, electrophiles or ROS/RNS might react with cysteines present at Keap1, inducing a conformational change that liberates Nrf2, therefore abolishing its degradation (hinge and latch mechanism). Nrf2 can also be liberated by direct inhibition of the interaction Nrf2-Keap1 by small molecules. Thereafter, Nrf2 translocates into the nucleus and subsequently binds to the EpRE elements, forming heterodimers with small Maf protein and starting the transcription of phase II genes. C) nAChRmediated activation of Nrf2-EpRE pathway. nAChR activation leads to the activation of PI3K that leads to the activation of Akt, a kinase that is able to phosphorylate GSK3, thus inhibiting its action. GSK3 act as a negative regulator of Nrf2 by several mechanisms. The first one is the direct phosphorylation of Nrf2 to directly induce its degradation, thus by inhibiting GSK3, Nrf2 translocates into the nucleus to induce the EpRE sequences. Nrf2 can also be activated by ERK that is also activated by nAChR signalling. D) Under chronic inflammation conditions many kinases are continuously activated as the case of GSK3 and P38. In these conditions, GSK3 directly activates Fyn that, in turn, phosphorylates Nrf2, that is then excluded from the nucleus, stopping the expression of phase II genes.

Keap1 is an excellent redox sensor of xenobiotics and oxidative stress inside the cells. It has three cysteine residues (Cys151, Cys273 and Cys288), which are crucial for

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ACCEPTED MANUSCRIPT Keap1-dependent ubiquination of Nrf2 (Zhang & Hannink, 2003). Under cellular stress, like ROS or environmental chemicals, oxidative/electrophilic molecules modify cysteine residues of Keap1. These modifications result in conformational changes of

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Keap1 to release Nrf2. As result, the Cullin3/Rbx1-dependent poly-ubiquitination of

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Nrf2 assisted by Keap1 is blocked, Nrf2 is liberated and rapidly translocates into the nucleus. Once in the nucleus, Nrf2 heterodimerizes with small MAF or JUN proteins

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and the complex binds to a cis acting element present in the promoter of its target genes, called anti-oxidant response elements (ARE), also denominated electrophile response elements (EpRE) (Rushmore et al., 1990). This activation mechanism of Nrf2 under

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stress conditions is known as the “hinge and latch” model (Figure 1b) (Tong et al., 2006). Many described Nrf2 inducers are electrophilic compounds that react with

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cysteines present at Keap1. Nevertheless, recently it has been described different molecules able to inhibit the protein-protein interaction between Keap1 and Nrf2. Moreover, the Nrf2 system is regulated by an auto-regulatory loop. Nrf2

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regulates the transcription of Keap1, cullin-3 and Rbx-1 and, in turn, Keap1/cullin-

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3/Rbx-1 degrades Nrf2 (Kaspar & Jaiswal, 2010). The Keap1 complex could be imported into the nucleus where promotes the degradation of nuclear Nrf2 (Niture et al.,

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2009). Further, feedback systems appear to exist since Nrf2 activation induces proteasome expression and activity. It has been observed that Nrf2 activators increase the expression of genes encoding for the proteasome subunits 20S and 19S (Kapeta et al., 2010; Kwak et al., 2003).

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Recently, we have demonstrated the Nrf2-EpRE pathway regulation via nicotinic receptors (Egea et al., 2009; Parada et al., 2014) (Figure 2c) supporting the link between the so-called “cholinergic anti-inflammatory pathway” (Martelli et al., 2014) and the phase II antioxidant response regulated by Nrf2. After receptor activation, Nrf2 might undergo post-translational modifications that promote its nuclear translocation and binding to the EpRE sequences (Figure 2c) (Niture et al., 2010). Several kinases have been shown to differently affect Nrf2 translocation via specific phosphorylation sites. Phosphorylation of Ser40 by atypical PKC iota (aPKC) is one of the best characterized (Bloom & Jaiswal, 2003; Huang et al., 2000; Numazawa et al., 2003). This phosphorylation releases Nrf2 from Keap1 (Bloom et al., 2003), allowing Nrf2 transport to the nucleus. Similar activating effects on Nrf2 translocation to the nucleus have been observed with many other kinases: casein kinase-2 (CK2) (Pi et al., 2007), phosphatidylinositide-3-kinases (PI3K) (Nakaso et al., 2003), c-Jun N-terminal 8

ACCEPTED MANUSCRIPT kinase (JNK) and extracellular regulated kinase (ERK) (Keum et al., 2006), among others. It has also been suggested that mitogen-activated protein kinases (MAPKs)induced phosphorylation of Nrf2 acts through indirect mechanisms that does not affect

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the interaction Keap1-Nrf2 (Sun et al., 2009).

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It is also possible to exert negative regulation of Nrf2 via phosphorylation by kinases (Figure 2d). Several kinases are constitutively activated or over-expressed in

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pathological conditions such as chronic inflammation. For example, glycogen synthase kinase 3-beta (GSK3β) can cause degradation of Nrf2 by the proteasome in a Keap1independent manner. This is achieved by phosphorylation of Nrf2, leading to its

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recognition by an E3 ligase receptor and the F-box protein β-TrCP (Chowdhry et al., 2013). Moreover, GSK3β activation in pathological conditions could phosphorylate

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Fyn, activating this kinase. Then, Fyn regulates Nrf2 via phosphorylation, triggering its nuclear export and degradation of Nrf2 by the proteasome (Jain & Jaiswal, 2007). The MAPK p38 has been shown to stabilize the interaction between Keap1 and Nrf2 thereby

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elevating the breakdown of Nrf2 (Keum et al., 2006). Nrf2 activation is also impaired

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by the NF-κB subunit p65/RelA, which co-imports Keap1 into the nucleus to trap Nrf2 (Yu et al., 2011), and by E-cadherin, which blocks nuclear translocation of Nrf2 in a β-

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catenin-dependent fashion (Kim et al., 2012b). In the other hand, NF-κB is known to suppress the transcription of EpRE-dependent genes (Nair et al., 2008). There exist a cross-talk between Nrf2 and NF-κB revelling the mechanism by which Nrf2 induction exerts an anti-inflammatory effect. Compelling evidence demonstrates the importance

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of Nrf2 in the anti-inflammatory response, as it was demonstrated in Nrf2-K.O. mice. In this model, the inflammatory response to lipopolysaccharide (LPS) was highly exacerbated compared to wild type animals (Innamorato et al., 2008). Recently, it was also demonstrated that Nrf2 also regulates different types of protective proteins such as brain derived neurotrophic factor (Kwak et al., 2003), the anti-apoptotic Bcl-2 (Kwak et al., 2003) and the anti-inflammatory interleukin IL-10 (Otterbein et al., 2000).

2. Nrf2-EpRE inducers as potential treatments for neurodegenerative diseases As described above, oxidative damage, mitochondrial dysfunction and chronic inflammatory status are common pathological pathways widely described in NDDs. The Nrf2-EpRE pathway acts as sensor and regulator of oxidative stress, being tightly regulated in normal conditions. Compelling evidence suggests that this pathway is

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ACCEPTED MANUSCRIPT deregulated in NDDs, and thus it contributes to intensify the advance and severity of the diseases. Moreover, aging is one of the most common risk factors in NDDs. It has been widely demonstrated that oxidative stress is augments during aging due to an increased

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production of oxidative species and the failure of antioxidant defences. In this line,

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compelling data indicates that the ability of Nrf2 to activate the phase II antioxidant response declines with aging, thus, contributing to an exacerbated status of oxidative

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stress (Zhang et al., 2015). For example, the levels of GSH are reduced in specific regions of the central nervous system (CNS) in many NDDs, hence reducing the antioxidant capabilities of neurons, leading to cell dysfunction and/or cell death (Benzi

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& Moretti, 1995; Cooper, 1997). Furthermore, HO-1 is over-expressed in several NDDs such as AD (Schipper et al., 2006), PD (Benzi et al., 1995), ALS (Ferrante et al., 1997)

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and MS (van Horssen et al., 2008), probably, in an effort to restore redox status and to decrease inflammation in those conditions (Cuadrado & Rojo, 2008; Schipper, 2004). Additionally, reactive gliosis is a common pathological hallmark in AD (Wang et al.,

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2000) and PD (van Muiswinkel et al., 2004). We will focus on the deregulation of the

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Nrf2-EpRe pathway in NDDs and the study of different Nrf2 inducers for the treatment

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of those diseases, focusing on AD, PD, HD, ALS, MS and brain ischemia. 2.1.- Alzheimer’s Disease: Nrf2-EpRE dysregulation and Nrf2 inducers Despite the high amount of oxidative damage found in postmortem brains of AD patients, it was found that Nrf2 was predominantly cytoplasmic in hippocampal neurons

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(Ramsey et al., 2007). Furthermore, higher expressions of glial HO-1 and NQO1 were reported in temporal cortex and hippocampus of demented patients compared to agedmatched controls (Schipper et al., 2006; Wang et al., 2000). Additional evidences have been reported in studies performed in a transgenic AD mice model expressing mutated human APP and PS1 genes (APP/PS1 mice), showing reduced NQO1, glutathione synthetic enzymes and Nrf2 levels in hippocampal neurons (Kanninen et al., 2008). Moreover, the Nrf2-EpRE pathway is attenuated at the time of Aβ deposition in the transgenic mice APP/PS1. Importantly, in vitro Nrf2 over-expression protects against neurotoxicity of Aβ. Protection achieved is associated to an increase in the expression of Nrf2 target genes, and the consequent reduction of oxidative stress. In addition, it has been demonstrated that several Nrf2 inducers alleviate cognitive defects in transgenic AD animal models (Frautschy et al., 2001).

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ACCEPTED MANUSCRIPT Contrary to most signalling kinases, GSK3β is active in un-stimulated cells and sensitises cells to different toxic stimuli. Growth factors and neurotrophins activate signalling kinases, including Akt, ERK and PKC, which may phosphorylate GSK3β,

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thereby inactivating it and increasing cell survival (Woodgett, 1994). However, it is not

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known which and how, GSK3β substrates participate in the activation of cell death programs.

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GSK3β participates in many vital metabolic processes in the cell, for example, it negatively regulates the activity of several transcription factors (Jacobs et al., 2012). In 2006, Salazar et al. demonstrated that GSK3β directly phosphorylates Nrf2 inducing a

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decreased activity of this factor and moreover, inducing its nuclear exclusion (Figure 1d). They demonstrated the capacity of active GSK3β to phosphorylate Nrf2 in in vitro and in vivo models (Salazar et al., 2006). In fact, this Nrf2 negative regulation

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mechanism mediated by GSK3β might be related to its pro-apoptotic character and related to the increase of oxidative stress observed during aging. It was further

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demonstrated that exposure of neurons to oxidative stress (H2O2) rapidly induces Nrf2

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nuclear translocation. However, in the same conditions, when GSK3β was constitutively active, it induced Nrf2 nuclear exclusion causing neurons sensitization to oxidative

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stress and increased neuronal death (Rojo et al., 2008). Taking into account the overactivity of GSK3β described in AD patients and the negative regulation of the Nrf2EpRE pathway by this kinase, it has been postulated that drugs directed to inhibit GSK3β and/or to increase phase II response might reduce neuronal loss in AD.

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Summarizing, the Nrf2-EpRE pathway is altered during this disease course. Indeed, patients show reduced nuclear levels of Nrf2 within hippocampal neurons (Ramsey et al., 2007). Thus, Nrf2 inducers are emerging as a valuable tool for the treatment of AD. The most studied family of natural products are polyphenolic compounds, known to be Nrf2 inducers (Magesh et al., 2012). They have been widely tested for the treatment of AD due to their pharmacological properties. Curcumin, (1,7-bis[4hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione)

(1,

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phytochemical derived from Curcuma longa, is one of the most used polyphenolic Nrf2 inducers for the treatment of AD. At molecular level, curcumin contains electrophilic α,β-unsaturated carbonyl groups which could selectively react with nucleophiles such as cysteine-thiols present at Keap1, thus releasing Nrf2 (Balogun et al., 2003). The interest of curcumin for the treatment of AD is based not only on its anti-A properties (Ono et 11

ACCEPTED MANUSCRIPT al., 2004) (Ono et al. showed that curcumin inhibited the formation of Aβ fibrils and destabilized the pre-formed Aβ fibrils95), but also on its antioxidant and antiinflammatory properties and its safety profile (Ringman et al., 2005). Curcumin

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increases lifespan and reduces neurotoxicity in five different genotypes of AD in

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drosophila models (Caesar et al., 2012), and several groups have reported that feeding with curcumin decreases Aβ levels and plaque burden in Tg2576 transgenic AD mice

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model (Begum et al., 2008; Lim et al., 2001; Yang et al., 2005). Curcumin has an active metabolite, tetrahydrocurcumin (THC) (2, Figure 2) which has been reported to improve lifespan of AD mice when they were fed with 0.2 % THC from the age of 13 months

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(average life span of THC-treated mice was 11.7 % higher when compared to controls). Nevertheless, the same study reported no differences in life span when animals were

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treated with THC from the 19th month, revealing the importance of an early treatment (Kitani et al., 2007). Moreover, epidemiological studies have demonstrated that populations with high consumption of this natural product, as Indians and some

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populations in Singapore, showed lower AD incidence and better performance in Mini-

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Mental State Examination (Chandra et al., 2001; Goodall et al., 2006a). Based on these promising findings, curcumin has been tested in humans as drug candidate for AD

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(dietary supplement) (Ono et al., 2004). In this sense, several clinical trials have been performed (NCT00595582, NCT00164749, NCT00099710) or are recruiting patients currently (NCT01811381, NCT01716637, NCT01383161). However, clinical trials results were limited due to curcumin insolubility in water and poor bioavailability. For

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therapeutic applications, curcumin must be combined with other drugs, or new delivery strategies need to be developed (Belkacemi et al., 2011). New derivatives from curcumin are being synthesized in order to improve its therapeutical properties (Chen et al., 2011; Orlando et al., 2012; Yanagisawa et al., 2014). O

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Resveratrol, (5-[(E)-2-(4-hydroxyphenyl)ethenyl] benzene-1,3-diol) (3, Figure

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2), is a natural polyphenolic compound identified in more than 70 species of plants,

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especially abundant in the skin of red grapes, which has been found to activate the Nrf2EpRE pathway (Chen et al., 2005; Hsieh et al., 2006). Focusing on AD pathology,

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resveratrol is able to protect cells against A treatment in vitro and in vivo (Savaskan et al., 2003; Zhang et al., 2014a). Preclinical models of AD showed that resveratrol prevented neurodegeneration via SIRT1, PGC-1α and p53 (Kim et al., 2007).

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Resveratrol showed interesting results in an AD model induced by streptozotocin (STZ) intracerebroventricular (i.c.v.) injection. In this model, resveratrol (30 mg/kg i.p. once

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per day during 8 weeks) reduced phosphorylated tau levels and ERK1/2 signalling elicited in STZ animals via SIRT1 (Du et al., 2014). In this study, authors reported that resveratrol also improved spatial learning capabilities using Water Morris test (Du et al.,

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2014). Similar results were obtained in vitro (it protected mouse cortical neurons

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exposed to Aβ via SIRT1) (Feng et al., 2013) and in vivo (it increased mean life expectancy in SAMP8 mice, reducing amyloid burden, tau hyperphosphorylation and

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cognitive impairment) (Porquet et al., 2013). A phase III clinical trial has been performed with resveratrol to study its effect in mild-moderate AD (NCT00678431) and other clinical trials are actively recruiting participants at the moment (NCT01504854,

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NCT01716637, NCT01219244). It was able to cross the blood brain barrier (BBB) although it was rapidly metabolized, thus lowering its bioavailability (Davinelli et al., 2012). In order to improve resveratrol bioavailability, several derivatives have been reported, as pterostilbene (4, Figure 2), a phenolic derivative that has demonstrated higher efficiency to modulate cellular stress and cognition (Chang et al., 2012; Joseph et al., 2008). Epigallocatechin

gallate,

(EGCG,

[(2R,3R)-5,7-dihydroxy-2-(3,4,5-

trihydroxyphenyl)chroman-3-yl]-3,4,5-trihydroxybenzoate, 5, Figure 2), is the most abundant polyphenol in green tea. EGCG is able to increase Nrf2 nuclear levels in Wistar rats (20 mg/kg) (Magesh et al., 2012; Sriram et al., 2009). It has been reported to promote the non-amyloidogenic processing of Aβ and to ameliorate the AD-related cognitive deficits displayed in different AD mouse models (Jia et al., 2013; RezaiZadeh et al., 2008). For example, 13-month female APP/PS1 mouse treated with 2 or 6

13

ACCEPTED MANUSCRIPT mg/kg/day during 4 weeks showed reduced Aβ42 levels in the hippocampus, improved learning and memory, and reduced TNF-α/JNK signalling (Jia et al., 2013; Rezai-Zadeh et al., 2008). Intraperitoneal (i.p.) injection of 20 mg/kg or orally administered 50 mg/kg

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to APPswe Tg mice, improved working memory, and reduced Aβ oligomerization and

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plaques via promotion of the non-amyloidogenic α-secretase proteolytic pathway (Rezai-Zadeh et al., 2005). Two clinical trials are recruiting participants to elucidate if

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EGCG could delay cognitive decline in both, patients in early stage of AD (NCT00951834) and people with Down´s syndrome (NCT01699711). Ozarowski et al. reported that subchronic administration of Rosmarinus

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officinalis extracts improved long-term memory in rats (Ozarowski et al., 2013). Its main components, carnosol (6, Figure 3) and its acid derivative, carnosic acid (7, Figure

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3), are phenolic diterpenes (Magesh et al., 2012) able to increase Nrf2 nuclear translocation in PC12 cells at different concentrations, being the most potent 10 µM (Martin et al., 2004). It has been demonstrated that CA (10 µM) promotes Nrf2

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translocation to the nucleus (>10 fold increase) and phase II enzymes over-expression,

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(GCL and HO-1) (Satoh et al., 2008). CA shows higher solubility, decreased toxicity and better neuroprotective effect than carnosol (Satoh et al., 2008). Related to AD,

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carnosic acid reduced Aβ1-42 secretion in different cell lines, 71 % in human U373MG astrocytoma cells and 61 % in SH-SY5Y neuroblastoma cells (Meng et al., 2013; Yoshida et al., 2014). It has also shown interesting results in vivo: male Wistar rats were treated with carnosic acid (10 mg/kg) 1 h before being injected with 1.5 nmol/µl of A,

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followed by treatment with 3 mg/kg/day during 14 days. Treated animals showed reduced cell death in CA1 hippocampal region (Rasoolijazi et al., 2013). It also improved performance in the passive shock avoidance-learning test by 90.3 % and short-term spatial memory test by 39 % respect to A treated animals (Rasoolijazi et al., 2013). Gingko biloba extract EGb 761, a mixture of active compounds, is able to induce phase II detoxifying enzymes via Nrf2-EpRE pathway as demonstrated in Hepa1c1c7 and Hepg2 cells (Liu et al., 2007). Its main components are ginkgolide (8, Figure 3) and bilobalide (9, Figure 3). EGb 761 shows anti-apoptotic and Aβ antiaggregant properties in a N2a cell line stably expressing Swedish mutant APP695. At the concentration of 100 g/mL it reduced 86 % Aβ aggregation compared to non-treated cells (Luo et al., 2002). Clinical trials have been conducted with controversial results. A large

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ACCEPTED MANUSCRIPT randomized trial did not show therapeutic benefits (DeKosky et al., 2008) although other clinical trials obtained beneficial effects with this extract (Ihl, 2013). Triterpenoids are among the most potent up-regulators of the Nrf2-EpRE

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pathway proposed to be beneficial for the treatment of AD. Ursolic acid (10, Figure 3),

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extracted from salvia officinalis, and oleanoic acid (11, Figure 3) have been extensively studied as protective agents due to their Nrf2 inducing properties (Li et al., 2013b; Liu

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et al., 2008; Ma et al., 2015). To improve their efficacy, synthetic derivatives were designed obtaining the triterpenoid derivative 2-cyano-3,12-dioxooleana-1,9-dien-28oic acid methyl esther (CDDO-Me or Bardoloxene Methyl) (Liby et al., 2005) (12,

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Figure 3). Administration of CDDO-Me to TG19959 transgenic mice from 1 to 4 months old, improved spatial memory retention, decreased microgliosis, oxidative stress

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and Aβ burden (Dumont et al., 2009). It must be noted that Bardoloxene Methyl was tested in patients with chronic kidney disease, although a high rate of cardiovascular side effects were reported (de Zeeuw et al., 2013). OH HO HOOC

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O

D

OH

HO O

H

O

7: Carnosic acid

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10: Ursolic acid

OH H

9: Bilobalide

O H

OH

H

O

HO

H

R

8: Ginkgolide

OH

H

O

O O

O

R O H

O

O

O O

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HO

O

H

H

6: Carnosol

O

HO

R

O

N

O HO

O O

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11: Oleanoic acid

O

H

H

12: CDDO (Bardoxolone Methyl)

Figure 3: Nrf2 inducers with beneficial effects on Alzheimer’s disease in vivo models and/or tested in human clinical trials

Sulforaphane (13, Figure 4), a natural compound isolated from cruciferous vegetables as broccoli, has the ability to activate the Nrf2-EpRE pathway very efficiently. Sulforaphane (5 M) exerts protection against A-toxicity (15 M) via activation of the phase II antioxidant response in vitro (Lee et al., 2013) (Egea et al., 2015). It was able to ameliorate cognitive deficit in two AD animal models (Kim et al., 2013; Zhang et al., 2014b). AD-lesion was induced by the combination of D-galactose (120 mg/Kg) and aluminium (20 mg/Kg), then, sulforaphane (25 mg/Kg) reduced the number of lessions, decreased the levels of aluminium in brain and reduced the spatial

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ACCEPTED MANUSCRIPT memory deficits and amyloid plaques.129 Clinical trials with sulforaphane are ongoing for other pathologies such as autism spectrum disorder with good results (NCT01474993).

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Sleep disorders are characteristic of AD patients, and melatonin secretion is

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dramatically decreased in these patients, thus, melatonin (14, Figure 4) has been proposed as potential treatment for AD. Melatonin is a neurohormone secreted mainly

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by the pineal gland that also has the ability to induce Nrf2 (Luchetti et al., 2010). Several clinical trials have been performed to study its effect on sleeping and cognition, and most of them have reported beneficial effects (Asayama et al., 2003; Brusco et al.,

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2000; Cardinali et al., 2012). Melatonin metabolism itself could contribute to generate a line of defense against xenobiotics and oxidative stress through phase I detoxification

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components and, more interestingly, through the induction of phase II related genes via Nrf2 transcription factor (Buendia et al., 2015). Treatment of Tg 2576 mice with melatonin (10 mg/kg) from 4-12 or 8-12 months, improves behavioural performance

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(Peng et al., 2013). Long-term melatonin therapy provides cognitive benefits in

of

A

aggregation,

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APP/PS1 Tg mice (100 mg/l in the drinking water) in a way that implies the suppression expression

of

antioxidant

enzymes

and

reduction

of

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pro-inflammatory cytokines (Olcese et al., 2009). Several studies have been performed with melatonin 10 mg/kg i.p.: treatment during 3 weeks after i.c.v. Aβ injection decreased memory impairment and neurodegeneration (Ali et al., 2015); treatment during 1 month prevented memory and synaptic dysfunction produced by D-galactose

AC

and alleviated learning and memory impairment reducing the apoptotic neurons, the Aβ deposits and protecting the cholinergic system in APP695 Tg mice (Ali et al., 2015). O HN O S

N

C

MeO

S

N H

13: Sulforaphane

14: Melatonin

Figure 4: Chemical structure of sulforaphane (14) and melatonin (15), Nrf2 inducers tested as potential treatments for AD and other NDDs.

2.2.- Parkinson’s disease: Nrf2-EpRE dysregulation and Nrf2 inducers PD is defined as a progressive NDD that selectively affects dopaminergic neurons from the substantia nigra pars compacta and also from the locus coeruleus, dorsal motor nucleus of vagus and nucleus basalis of Meynert (Lang & Lozano, 1998;

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ACCEPTED MANUSCRIPT Morroni et al., 2013). It affects 1 % of people over 60 years in industrialized countries and is considered as the second most prevalent neurodegenerative disorder after AD (de Lau & Breteler, 2006; Fereshtehnejad & Lokk, 2014; Nussbaum & Ellis, 2003). The

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failure of the dopaminergic signalling pathway leads to different cardinal motor

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symptoms as tremor, rigidity, bradykinesia and postural instability (Fernandez, 2012; Morroni et al., 2013; Schapira et al., 2014). The most prominent hallmark is the

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deposition of Lewy bodies, which are found in different areas of the brain (Tufekci et al., 2011; Zhang et al., 2013a). During the advanced stages, approximately 80 % of PD patients develop dementia, which is an important predictor of mortality in PD

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population (Aarsland et al., 2003; Fereshtehnejad et al., 2014).

PD etiology is not completely elucidated, however, most reliable theories

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propose environment and/or genetic conditions or combination of both as origin (Calne et al., 1987; Kitada et al., 1998; Tarozzi et al., 2013). Recently, increased oxidative stress, mitochondrial dysfunction, excitotoxicity and neuroinflammation have been

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proposed as the mechanisms leading to neuronal death in PD (Olanow, 2007;

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Yacoubian & Standaert, 2009). It has been demonstrated that defects of the respiratory chain (complex I) induce accumulation of mitochondrial DNA mutations, abnormal

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mitochondrial calcium homoeostasis, defective autophagic removal of mitochondria (mitophagy), and increased oxidative stress leading to PD (Schapira, 2008; Schapira et al., 2014). Furthermore, one of the earliest detectable parameters in PD is the depletion of glutathione observed in the substantia nigra (Andersen, 2004), which leads to a

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selective decrease in mitochondrial complex I activity (Valko et al., 2007). Additionally, current evidence suggests that mitochondrial complex I inhibition may be the central cause of sporadic PD. Also, derangements in complex I cause α-synuclein aggregation, which contributes to the demise of dopaminergic neurons. The augmented oxidative stress in PD has been related to ROS production induced by the metabolism of dopamine and excitatory amino acids (H2O2, superoxide anion radical, NO· and OH·) (Gilgun-Sherki et al., 2001; Uttara et al., 2009; Woo et al., 2014). Consequently, dopaminergic neurons are specifically under high oxidative stress due to their dopamine content. Under stress conditions, they also evoke the production of superoxide and nitric oxide by microglia, thus increasing the oxidative damage (Gu et al., 2005; Tsang & Chung, 2009). Besides, pathogenic mutations in several genes including α-synuclein, LRRK2, parkin, DJ-1 and PINK-1 also play an important role in mitochondrial dysfunction in PD patients (Andersen, 2004). Nrf2 accumulation and 17

ACCEPTED MANUSCRIPT activation of NQO1 and HO-1 are observed in the substantia nigra of PD patients (Lastres-Becker et al., 2012; van Muiswinkel et al., 2004), and it has been reproduced in PD mouse models (Chen et al., 2009; Innamorato et al., 2010), reflecting oxidative

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stress (Yamazaki et al., 2015). Nevertheless, postmortem studies of PD patients have

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demonstrated that Nrf2 translocates into the nucleus. However, the expression of phase II antioxidant genes is not increased as expected, demonstrating the dysregulation of

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this pathway (Ramsey et al., 2007). Thus, the relationship between Nrf2 and PD is well established because (i) Nrf2 activity and effectivity decreases with age and age is one of the main risk factors for PD, (ii) in postmortem PD brains Nrf2 has been localized in

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nuclei although the induction of phase II enzymes is insufficient to protect neurons (Ramsey et al., 2007) (iii) it has been shown that there is a protective Nrf2 haplotype

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which delays PD onset and (iv), Nrf2 KO mice are more susceptible to 6-OHDA and MPTP PD models, and Nrf2 activation protects against different PD models (Lou et al., 2014). Based on all these findings, it has been proposed that intervening oxidative stress

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through the Nrf2-EpRE pathway could be useful in the treatment of PD.

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PD therapies are mainly focused on dopamine replacement. These treatments improve symptoms temporally, without modifying disease progression (Schapira et al.,

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2014). Focusing on oxidative stress, several antioxidant compounds derived have shown to be beneficial either in vitro and in vivo models of PD. In that sense, triterpenoids, analogues of oleanolic acid (11, Figure 4), are a good example of Nrf2 inducers proposed for the treatment of PD. They have demonstrated a

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great ability to inhibit oxidative stress and inflammatory processes by inducing the Nrf2-EpRE signalling pathway (Yang et al., 2009b). To improve their druggability, several synthetic derivatives have been described. Among them, the derivative 2-cyano3,12-dioxooleana-1,9-dien-28-oic acid methylamide (CDDO-MA) (15, Figure 5) showed a marked protection in the acute and chronic 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) mouse models of PD. Structure-activity analysis demonstrated that,-unsaturated carbonyl groups in CDDO-MA are essential for its Nrf2 inducing properties by Michael addition of cys-thiol groups present at Keap-1. It induces a conformational change that activates the phase II response, thus, increasing the expression of genes related to mitochondrial biogenesis, glutathione synthesis and antioxidant enzymes (Beal, 2009; Liby et al., 2007; Yang et al., 2009b). Moreover, improved brain levels of phase II antioxidant enzymes can be achieved with new structural analogues such as the ethylamide (16, TP-319, Figure 5) and 18

ACCEPTED MANUSCRIPT trifluoroethylamide (17, TP-500, Figure 5) of CDDO. Both TP-319 and TP-500 activated the Nrf2 pathway at nanomolar range, whereas sulforaphane and TBHQ induced in the micromolar range. Both, TP-319 and TP-500 (4 µM, twice, 12 h apart by

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oral gavage) have shown to augment mRNA levels of Nrf2 and downstream genes by 5-

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25 fold in liver (GCLc, NQO, HO-1, Gsr6 and 9) and in striatum (NQO and HO-1) in vivo. MPTP administration (10 mg/kg, three times every 2 h) causes 50 % loss of

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striatal dopamine and its metabolites (3,4-dihyroxyphenylacetic acid and homovanillic acid), and also tyrosine hydroxylase in neurons at the substantia nigra 7 days after. TP319 and TP-500 protected against MPTP induced toxicity in a dose dependent manner.

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Furthermore, when the experiment was reproduced in Nrf2 K.O. mice, TP-319 and TP500 were not able to protect against MPTP-induced neuronal loss, confirming the

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implication of Nrf2 as their mechanism of action (Kaidery et al., 2013). The synthetic CDDO based analogues have been protected (Sporn et al., 2008) as potential drugs against inflammatory and oxidative stress-mediated neurological disorders (Joshi &

O H

N

H N

H

HO

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H

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16: R = Me (TP-224) 17: R = Et (TP-319) 18: R = CH 2CF 3 (TP-500)

HO

OH

HO

19: Licochalcone A

OMe

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20: Licochalcone E

N

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OH O

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Johnson, 2012).

H

22: Bromocriptine

Br

23: Tetramethylpyrazine

Figure 5: Nrf2 inducers with beneficial effects on Parkinson’s disease in vivo models

Other Nrf2 inducers that have shown benefits in different PD models are the natural products Licochalcone A (18, Figure 5) and E (19, Figure 5). These phenolic constituents of the roots of licorice species have demonstrated to be antioxidants able to protect neurons from different oxidative stress models (Haraguchi et al., 1998). In addition, both chalcone derivatives also possess anti-inflammatory activity (Cho et al., 2010; Haraguchi et al., 1998; Kim et al., 2012a; Yoon et al., 2005). Focusing on PD, Licochalcone A and E have shown to inhibit superoxide anion generation in xanthine

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ACCEPTED MANUSCRIPT xanthine-oxidase system, showing an IC50 of 2.3 and 4 µg/ml, respectively. In addition, Licochalcone E increased EpRE-luciferase activity by 5.2 ± 1.3 fold in HEK 293T after 6 h of treatment and 1.8 ± 0.2 in SH-SY5Y cells after 6 h of incubation (at 5 µM and 2

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µM, respectively). Therefore, they increased HO-1 and NQO1 expression being able to

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attenuate LPS (0.2 µg/ml)-induced inflammatory response and 6-OHDA (100 µM during 24 h) induced cytotoxicity. The inhibition of Nrf2-induction (using siRNA)

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reversed the neuroprotective and anti-inflammatory responses. It was also demonstrated their activity in vivo, being able to protect against MPTP-induced dopaminergic neurons loss in mice treated during 5 days with 10 mg/kg of compound with a mechanism of

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action dependent of Nrf2 (Haraguchi et al., 1998; Kim et al., 2012a). As previoiusly described, carnosol and carnosic acid, isolated from herbs

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Rosmarinus officinalis and Sage, (6 and 7, Figure3) are Nrf2-EpRE activators also tested in PD in vivo models. Carnosic acid offered interesting neuroprotection in SH-SY5Y cells exposed to 6-OHDA (100 M) (Wu et al., 2015). In addition, carnosic

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acid (5-10 M) protected against toxicity induced by dieldrin (10 M) in cultured

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dopaminergic cells (SN4741) in a concentration dependent maner (Park et al., 2008). This activity was confirmed by the improvement of behavioural performance observed

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in 6-OHDA intrastriatal-injected rats (5 µg/µl) treated with 20 mg/kg, 3 times per week during 3 weeks (Wu et al., 2015). Carnosic acid and carnosol themselves are not electrophilic, but in presence of oxidative stress, both are converted to the

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corresponding o-quinone electrophilic derivatives being able to induce Nrf2 (Satoh et al., 2013). Thus, this property makes these compounds highly safe, because they are not reactive unless oxidative stress is present. Flavonoids is another group of natural products tested as potential treatments for NDDs based on their ability to induce Nrf2. Naringenin, the predominant flavonoid found in grapefruit, has shown neuroprotective profile in PD and AD models (Heo et al., 2004; Zbarsky et al., 2005). Epidemiological studies have pointed to a link between flavonoid dietary intakes with reduced PD risk (Gao et al., 2012). Lou et. al, elucidated its mechanism showing that naringenin increases ARE-luciferase activity 1.5, 2 and 2.5 times at 20, 40 and 80 µM, respectively. Furthermore, after 24 h of treatment it increased HO-1, GCLc and GCLm protein levels, as well as GSH levels. Naringenin was able to reduce neuronal cell death and ROS production (measured by DCFDA fluorescence) by inducing the Nrf2-EpRE pathway, JNK and p39 MAPK. In vivo experiments demonstrated its capacity to protect against 6-OHDA (100 µM) treated 20

ACCEPTED MANUSCRIPT after 6-OHDA injection (given at 70 mg/kg by oral gavage once daily for four consecutive days) (Lou et al., 2014). It is also interesting to highlight a compound currently used for the treatment of

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PD, bromocriptine (21, Figure 5) that has shown relationship with the Nrf2 pathway

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(Lim et al., 2008). This compound was obtained as a dopamine receptors agonist and was the first drug approved as anti-parkinsonian therapy since 1974 (Foley et al., 2004).

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It ameliorates motor deficits by activation of dopamine D2 receptors. Firstly, it was used to treat patients with motor fluctuations as adjuvant therapy to levodopa. Later on, it was used in monotherapy, for the treatment of early stages of the disease (Radad et

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al., 2005). Bromocriptine induces Nrf2 from 0.1 to 5 µM in PC12 cells (1.3 to 3 folds induction) (Lim et al., 2008). It was able to rescue PC12 cells exposed to H2O2 (100

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µM, 24 h) at 5 µM (Lim et al., 2008). Looking at in vivo experiments, this compound (at 5 mg/kg i.p 7 days) completely protected against the decrease of mouse striatal dopamine and its metabolites after i.c.v. injection of 6-OHDA (Riis et al., 1987). It has

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also shown to be protective in mice against MPTP-induced cell death (Kondo et al.,

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1994; Ogawa et al., 1994; Takashima et al., 1999). Bromocriptine has demonstrated to be an antioxidant by inhibiting hydroxyl radical formation and lipid peroxidation in vivo

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(Ogawa et al., 1994; Radad et al., 2005). Furthermore, Nrf2 expression and translocation to the nucleus were augmented in those models by the action of bromocriptine. The neuroprotective mechanism of action is dependent of the PI3K/Akt

2008).

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survival pathway, whereas the dopamine receptors are not involucrated (Lim et al.,

Tetramethylpyrazine (TMP, 22, Figure5) is a natural product purified from Ligusticum wallichii Franchat widely used to treat neurovascular and cardiovascular diseases. Its mechanism of action has been related to the elevation of Nrf2/HO-1 (Kao et al., 2013). Recently, Lu C. et.al have shown that this compound is also able to reduce neuronal damage in the MPTP PD rat model, improve motor deficits and decrease oxidative stress by restoring the reduction of Nrf2 and GCLc expression (Lu et al., 2014). 2.3.- Huntington disease: Nrf2-EpRE dysregulation and Nrf2 inducers It was described as a NDD where neostriatal (putamen and caudate) and cerebral cortex degenerates (Ribeiro et al., 2014). It is a genetic disorder with an autosomal dominant inheritance pattern and it is characterized by cytosine, adenine, and guanine

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ACCEPTED MANUSCRIPT (CAG) repeats expansion in de huntingtin gene (Ayala-Pena, 2013). The consequence is the extension of poly-glutamines at the N-terminus of the huntingtin protein that accumulates, causing abnormal huntingtin aggregates (Zielonka et al., 2014). Clinically,

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the main manifestation of HD is chorea, an irregular and involuntary dance-like gait.

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Finally, motor impairments appear, cognitive decline occurs and therefore, psychiatric symptoms augment as the disease progress. The number of pathogenic CAG repeats

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correlates with the severity and age of onset of the disease (Ayala-Pena, 2013; Kumar et al., 2010; Martin & Gusella, 1986).

Among the alterations described in HD, excitotoxicity is one of the main causes

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of cell death observed in HD (Kumar et al., 2015; Tabrizi et al., 1999). Furthermore, a hyperactive dopaminergic system could contribute to choreic symptoms and is

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implicated in neurotoxicity in HD. Consistent with these alterations, high levels of oxidative stress and mitochondrial dysfunction are the main changes observed in these patients (Browne et al., 2006; Browne et al., 1999).

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Alterations of II, III and IV mitochondrial complexes have been widely

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described in HD post-mortem brains (Colle et al., 2015; Gan & Johnson, 2014). Hence, 3-nitropropionic acid (3-NP) and/or malonate have been used to inhibit mitochondrial

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complex II, leading to striatal medium spiny neurons degeneration as mouse model of HD (Beal, 1994; Brouillet et al., 2005). As result of this inhibition, the electron transport chain is disrupted, leading to extensive oxidative stress. In this line, several groups of research have concluded that alterations in Nrf2 pathway are implicated in the

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development of HD (van Roon-Mom et al., 2008). It has been demonstrated that the induction of the Nrf2-EpRE pathway resulted in neuroprotection against malonateinduced toxicity (Jin et al., 2013). It is also known that activation of Nrf2 by cystamine reduces the toxicity elicited by 3-NP on in vitro and in vivo models (Colle et al., 2015; Gan et al., 2014; Jin et al., 2013). Nowadays, there is no cure for HD, and current treatments are designed to alleviate HD symptoms (Jakel & Maragos, 2000). In summary, memantine, minocycline (Nrf2 inducer (Kuang et al., 2009) that selectively inhibits M1 polarization of microglia (Kobayashi et al., 2013)) (23, Figure 6), tetrabenazine (24, Figure 6), cysteamine (25, Figure 6), ethyl-eicosapentaenoic acid (Ethyl-EPA, 26, Figure 6), coenzyme Q10 (Nrf2 inducer) (Li et al., 2015) (27, Figure 6) and HDAC inhibitors are used in clinic. Among them, coenzyme Q10 is currently in phase II and minocycline, memantine, cysteamine and ethyl-EPA are in phase I clinical studies (Kumar et al., 2015). 22

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O

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OH

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MeO HO N

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24: Tetrabenazine

23: Minocycline

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25: Cysteamine

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26: Ethyl-EPA

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OH

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OH

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NH 2 O

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27: Coenzyme Q10

Figure 6: Products from natural or synthetic sources in clinical use or clinical trials for the treatment for

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HD.

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Focusing on the Nrf2 pathway, stimulation of the endogenous antioxidant machinery could be a potential therapeutic intervention for the treatment of HD. Triterpenoids, particularly analogues of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid

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(CDDO) (12, Figure 3) have shown to be neuroprotective in different diseases (Yang et

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al., 2009b). It has been demonstrated that these triterpenoids conferred protection against 3-NP neurotoxicity, DNA and proteins oxidation, and disrupted glutathione

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regulation and lipid peroxidation. Also in a mice model of HD, those compounds decreased oxidative stress and neuron atrophy and improved motor function and longevity (Yang et al., 2009b). For example, CDDO methyl amide (CDDO-MA, 14,

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Figure 5) induced Nrf2 translocation to the nucleus (1 µM, 8 h) and increased mRNA Nrf2 levels between 4.5 folds, along with other EpRE genes, such as NQO1, GR and HO-1 (Yang et al., 2009b). It showed interesting neuroprotective activity (800 mg/kg diet during 1 week) against striatal neurons oxidative damage induced by 3-NP (50 mg/kg/day for 7 days) (Yang et al., 2009b). Another derivative of the family, 3-cyano3,12-dioxooleana-1,9-dien-28-oic acid-trifluoroethylamide (CDDO-TFEA, 17, Figure 5) gave, also, interesting results in a N171-82Q transgenic mice model of HD. It was provided in the diet (100 mg/kg) starting at 30 days of age up to 4 months of age, being able to increase mice survival (19.4 % respect to control animals). It also attenuated striatal atrophy and reduced neuronal loss. This neuroprotective effect was related to a decrease in oxidative stress and increased NQO1 expression (40 % increase) and HO-1 (20 % increase) in skeletal muscle, striatum and brown adipose tissue (Stack et al., 2010).

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S

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28: Probucol

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29: Succinobucol (AGI-1067)

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Figure 7: Nrf2 inducers with beneficial effects on Huntington’s disease in vivo models.

Probucol (28, Figure 7), a phenolic lipid-lowering agent with antioxidant properties, has been extensively studied. It exerts protective properties in different

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diseases such as hypercholesterolemia or artherosclerosis (Buckley et al., 1989; Yamashita & Matsuzawa, 2009). Additionally, it has demonstrated to be protective in

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AD, PD and HD models (Champagne et al., 2003; Colle et al., 2012; Ribeiro et al., 2013). Focusing on HD, probucol has shown to be neuroprotective form 10 and 30 µM

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against neuronal damage, ROS production and lipid peroxidation in striatal slices exposed to 1 mM of quinolinic acid and 1 mM 3-NP (Colle et al., 2012). Probucol at 1,

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3 and 10 µM protected against 3-NP induced mitochondrial dysfunction in mitochondria-enriched synaptosomes. It blocked ROS production (measured by DCFDA fluorescence) and lipid peroxidation observed in this model and it showed

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neuroprotection against TBHQ induced mitochondrial dysfunction (Colle et al., 2013). More recently, succinobucol (also named AGI-1067, 29, Figure 7), an analogue of probucol, has also exhibited the hypocholesterolemic, anti-inflammatory, and antioxidant properties, with reduced adverse effects compared to probucol (Kunsch et al., 2004). Recently, its neuroprotective profile has been tested in HD in vitro models showing promising results (Colle et al., 2013; Colle et al., 2015). More in detail, succionobucol has shown neuroprotective activity in SH-SY5Y cells treated with 1 mM of 3-NP, through a mechanism of action that involves rapid Nrf2 translocation to the nuclei, GCL expression and GSH increase (Colle et al., 2015). There are also evidences showing that dimethyl fumarate (DMF) (37, Figure 9) (an orally bioavailable fumaric acid ester) causes significant improvements in motor functions in HD mice model by activating the Nrf2 signalling pathway (Ellrichmann et al., 2011; Mrowietz et al., 1999). DMF treatment (30 mg/kg twice daily via oral gavage

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ACCEPTED MANUSCRIPT four weeks after birth up to 12 weeks after treatment initiation) attenuated motor impairment in R612 HD mice model. DMF also preserved morphologically intact neurons in striatum and motor cortex by increasing Nrf2 in neurons (Ellrichmann et al.,

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2011).

2.4.- Amyotrophic lateral sclerosis: Nrf2-EpRE dysregulation and Nrf2 inducers

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ALS is an adult-onset NDD that affects the spinal cord motor neurons, brainstem and motor cortex, leading to progressive muscle atrophy and long term disability (Rowland & Shneider, 2001). Approximately 10 % of ALS cases are familiar and 20 %

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are caused by mutations in Cu/Zn-superoxide dismutase 1 (SOD1) (Rosen et al., 1993). Nevertheless, the mechanisms underlying neuronal death observed in sporadic ALS are

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still elusive, although several studies implicate neuroinflammation, protein aggregation, glutamate excitotoxicity, mitochondrial dysfunction and oxidative stress in its development (Contestabile, 2011). Focusing on oxidative stress, there is substantial

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evidence of oxidative damage in brain and spinal cord of both familial and sporadic

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ALS patients, as well as in animal models of ALS (Barber & Shaw, 2010). Taking into account the evidences of extensive oxidative damage in ALS, the activation of natural

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antioxidant systems of cells seems to be a promising target for the treatment of this disease. Postmortem studies have demonstrated that the expression of Nrf2 related genes is diminished in motor neurons expressing mutant SOD1 (Pehar et al., 2007) in spinal cord of ALS patients (Sarlette et al., 2008). It has also been observed that Nrf2

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mRNA and protein levels are reduced in neurons of ALS patients, while Keap1 mRNA expression was increased in the motor cortex (Sarlette et al., 2008), suggesting a dysfunction of the Nrf2-EpRE pathway in this disease. Compelling evidence suggest that the high toxicity of mutSOD1 may induce the downregulation of the Nrf2 pathway revelling a close mutSOD1-Nrf2 relationship (Milani et al., 2013). Vargas et al. (Vargas et al., 2008) demonstrated that Nrf2 induction on astrocytes expressing mutant hSOD1 (a widely used in vivo model of ALS (Gurney et al., 1994)) preserved motor neurons, and extended the survival of transgenic hSOD1 mice. It has been demonstrated that, under stress conditions, motor neurons release fibroblast growth factor-1 (FGF-1) to induce astrocyte activation triggering the expression of nerve growth factor (NGF) and inducing Nrf2. However, prolonged astrocyte stimulation by SOD-mediated oxidative stress and FGF-1 leads to progressive neurodegeneration via disruption of normal neuron-glia interactions deregulating the 25

ACCEPTED MANUSCRIPT Nrf2-EpRE pathway (Pehar et al., 2005). These evidences suggest the potential use of Nrf2 induction as a suitable strategy for the treatment of ALS. Currently, riluzole, a sodium channel blocker (Schwartz et al., 2002) and

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glutamate release inhibitor (Wang et al., 2004) is the only drug approved for ALS (30,

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Figure 8), although it barely extends ALS patients survival up to three months (Rowland et al., 2001). Regarding oxidative stress as one of the mechanisms involved in

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ALS pathophysiology, several antioxidant molecules (N-acetylcysteine, seleginine (Jung & Kwak, 2010) and vitamin E) have been tested in clinical trials with limited or no beneficial effect (Barber et al., 2010; Orrell et al., 2008).

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Curcumin (1, Figure 1), a potent Nrf2 inducer, has shown promising results for the treatment of ALS. Jiang et al. reported that curcumin (10 μM) was able to increase

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Nrf2 nuclear translocation inducing the expression of it target genes HO-1 (3.4 fold increase), NQO1 (3.3 fold increase) and GCLc (2.5 fold increase) in primary spinal cord astrocytes (Jiang et al., 2011). Curcumin reduced H2O2-induced ROS production

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(measured by DCFDA fluorescence), oxidative damage and mitochondrial dysfunction

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in primary Nrf2+/+ astrocytes, but not in Nrf2-/- astrocytes. These results indicate that the cytoprotective effect of curcumin in astrocytes is due to an up-regulation of the Nrf2

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pathway.

An in vitro screening study allowed Barber and co-workers to identify caffeic acid phenethyl ester (CAPE) (31, Figure 8), as Nrf2-inducer with neuroprotective properties against ALS (Barber et al., 2009). At a concentration of 10 μM, CAPE was

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able to protect NSC34 cells (a motor neuron-like cell line stably transfected with human mutant G93A-SOD1), against menadione-induced oxidative stress (10 μM menadione). The same concentration of CAPE significantly increased primary motor neuron survival against oxidative stress induced by trophic factors and antioxidants deprivation, in comparison to vehicle treated cells. Interestingly, the observed neuroprotection was related to the Nrf2-EpRE induction since it was able to double Nrf2-EpRE reporter gene at 9.5 μM. In this work, CAPE also showed good CNS penetrating properties in vivo affording good BBB crossing capability in rats (Barros Silva et al., 2013). An important example is DL-3-n-butylphthalide (DL-NBP, 32, Figure 8), the synthetic racemic mixture of the celery-isolated L-3-n-butylphthalide (L-NBP), showed interesting results in G93A-hSOD1 transgenic mice (Feng et al., 2012). Several studies have demonstrated the neuroprotective properties of DL-NBP against oxidative stress (Huang et al., 2010; Li et al., 2009), neuronal apoptosis (Chang & Wang, 2003; Li et al., 26

ACCEPTED MANUSCRIPT 2010), and β-amyloid-induced cytotoxicity (Peng et al., 2008) as well as the ability to ameliorate cognitive decline in an in vivo model of AD (Peng et al., 2010; Peng et al., 2009). Respect to ALS, post-onset oral administration of DL-NBP to G93A-SOD1

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transgenic mice prevented weight loss and substantially extended survival (42 %) in

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comparison to vehicle-treated animals (Feng et al., 2012). Treatment with DL-NBP also lowered the muscular function decline of G93A-SOD1 mice, as indicated by the

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electromyographic parameters and the rotarod tests performed. DL-NBP notably preserved motor neurons respect to the vehicle-treated animals. Neuroinflammation is known to contribute to ALS progression, especially in the late state of the disease

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(Hooten et al., 2015). Thus, these researchers also studied the effect of DL-NBP on astrocytosis and microglial activation in the G93A-SOD1 transgenic mice by

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immunohistochemistry. DL-NBP-treated G93A-SOD1 mice markedly decreased microglial activation compared to vehicle-treated littermates. In order to elucidate the underlying mechanism of DL-NBP-derived neuroprotection in this ALS model, they

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studied the expression of Nrf2, HO-1, NF-κB p65 and TNF-α in wild type G93A-SOD1,

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and G93A-SOD1 DL-NBP-treated mice. DL-NBP increased the expression of Nrf2 and HO-1 while it reduced the translocation of NF-κB and the expression of TNF-α.

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Altogether, these results suggest that the DL-NBP neuroprotective capability against ALS is via Nrf2-induction, making this compound an attractive candidate for the treatment of this disease.

As described before, triterpenoids are an advanced example of Nrf2 inducers

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tested in different models of NDDs demonstrating the potential use of the Nrf2-EpRE pathway as target.125 In ALS, two derivatives, CDDO-EA, (16, Figure 5) and CDDOTFEA, (17, Figure 5) have shown neuroprotective properties in in vitro and in vivo models (Neymotin et al., 2011). CDDO-TFEA (300 nM) was able to induce Nrf2 in mutant G93A-hSOD1 NSC34 cells. Western blot experiments also demonstrated overexpression of Nrf2-dependent proteins HO-1 (7 fold increase), NQO1 (3 fold increase) and glutathione-S-transferase (GST) (11 fold increase), in comparison to DMSO treated cells. Both CDDO-EA and CDDO-TFEA reduced ROS (measured by DCFDA fluorescence) production in a dose-dependent manner in wild-type fibroblast, but this reduction was abolished in Nrf2-deficient fibroblasts, demonstrating the dependence on Nrf2-induction. Furthermore, the Nrf2-inducing capability of CDDO-EA and CDDOTFEA was also observed in G93A-SOD1 transgenic mice. The expression and nuclear translocation of Nrf2 was notably increased in the spinal cord of these mice after 27

ACCEPTED MANUSCRIPT treatment with 400 mg/kg in food of each triterpenoid, as well as the levels of Nrf2EpRE dependent proteins. Finally, oral administration of both triterpenoids to G93ASOD1 Tg mice increased survival compared to control groups up to 15 % in the pre-

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symptomatic model and up to 43 % in post-symptomatic model, respectively (Neymotin

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et al., 2011). Taking into account that riluzole only extends survival up to 3 months in ALS patients, these results indicate that the triterpenoids CDDO-EA and CDDO-TFEA

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are interesting candidates for the treatment of ALS. Compound

N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy)

acetamide (CPN-9, 33, Figure 8) (Kanno et al., 2012) has demonstrated interesting

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activities. The treatment of differentiated SH-SY5Y cells with 40 M CPN-9 considerably increased Nrf2 expression and nuclear translocation, as well as mRNA and

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protein levels of HO-1, NQO1 and glutamate-cysteine ligase modifier subunit (GCLm). In wild-type mice, oral administration of 0.5 mg/kg of CPN-9 increased 1.5 times HO-1 mRNA levels in lumbar spinal cord. Post-onset treatment of H46R-hSOD1 transgenic

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mice with 0.1 and 0.5 mg/kg of CPN-9 noticeably slowed down disease progression

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(Kanno et al., 2012). Treated mice preserved motor performance and showed decreased motor neuron loss in the spinal cord, in comparison to vehicle-treated mice.

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Furthermore, post-onset treatment with 0.5 mg/kg of CPN-9 prolonged lifespan of transgenic mice (16 %). Despite its promising neuroprotective activity, CPN-9 exhibits poor aqueous solubility, low BBB permeability and certain toxicity (Tanaka et al., 2014).

To

improve

CPN-9

pharmacokinetic

profile,

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[mesityl(methyl)amino]-N-[4-pyridin-2-yl)-1H-imidazol-2-yl]

derivative

2-

acetamide

trihydrochloride (WN1316) (34, Figure 8) was developed (Tanaka et al., 2014). Its pharmacokinetic profile was improved compared to CPN-9, with a BBB permeability in mice almost 40-fold higher. This compound was two folds better neuroprotectant than CPN-9 against menadione-induced oxidative stress in differentiated SH-SY5Y cells at 6 μM. It also showed remarkable neuroprotection against other oxidative insults such as α-naphtoquinone and 6-OHDA. Compound WN1316 was able to induce the Nrf2-ARE phase II antioxidant response as part of its mechanism of action. Furthermore, 10 and 100 μg/kg of WN1316 preserved motor performance when administrated in the late symptomatic stage in two mouse models of ALS (H46R SOD1 and G93A SOD1 transgenic mice) compared to vehicle treated mice. Regarding the neuroinflammation observed in ALS, treatment of H46R SOD1 mice with 10 μg/kg WN1316 decreased spinal cord microglial and astrocytic activation, as well as production of IL-1β and 28

ACCEPTED MANUSCRIPT iNOS. Finally, the effect of WN1316 (1 and 10 μg/kg) on survival was assessed in the two mentioned mouse models of ALS. In comparison to CPN-9, lower post-onset doses of WN1316 were able to increase the life span of both transgenic mice (25 % in

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SOD1H46R mice and 15 % in SOD1G93A mice), confirming the enhancement of CPN-9

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neuroprotective properties.

Mead and co-workers identified S[+]-apomorphine (35, Figure 8) and

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andrographolide (36, Figure 8), two natural products isolated from the herb Andrographis paniculata (Chakravarti & Chakravarti, 1951), as promising Nrf2 inducers for the treatment of ALS (Mead et al., 2013). Apart from the Nrf2-reporter

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CHO cell line (EC50 = 0.74 and 25.9 μM, respectively), both compounds were able to induce Nrf2, increasing the expression of HO-1 and NQO1 in the astrocyte cell line C6

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and in primary mouse astrocytes, principally S[+]-apomorphine. The enhancement of HO-1 and NQO1 expression was also observed in astrocytes carrying the G93A-SOD1 mutation. Both compounds were also able to increase intracellular and extracellular

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levels of GSH in primary astrocytes, and to reduce menadione-induced motor neuron

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loss in co-cultures of primary mouse motor neurons on an astrocyte feeder layer. These results are in line with the motor neuron-protective role of astrocytes against oxidative

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stress observed in ALS (Barber et al., 2010). Due to its pharmacological profile, S[+]apomorphine was studied in an in vivo model of ALS in this study. In hSOD1G93A transgenic mice, a daily dose of 5 mg/kg of 35 showed decreased levels of oxidative stress (measured by DCFDA fluorescence) and improved motor function. It was able to

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increase HO-1 and NQO1 expression and to decrease oxidized-GSH levels in the spinal cord of these mice, as well as to delay their motor performance decline after 60 days of treatment, in comparison to vehicle-treated mice. Finally, S[+]-apomorphine (4 μM) also protected human fibroblasts from both sporadic and mutant SOD1I113T ALS patients against menadione-induced oxidative stress. O F 3CO

HO

HO

S NH 2 N

O

O

HO

HO

H

O

31: Caffeic acid phenetyl esther 32: DL-3-n-buthylphthalide

30: Riluzole

O

HO OH O O

O

N N H

33: CPN-9

S

N N

N N H

O OH

N H

34: WN1316

29

N

36: Andrographolide N

H

35: s(+)-apomorphine

ACCEPTED MANUSCRIPT Figure 8. Chemical structures of natural and synthetic Nrf2 inducers tested in ALS.

Several natural compounds known to possess Nrf2 inducing capability, such as

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resveratrol (Javkhedkar et al., 2015), EGCG (Wang et al., 2015a; Wang et al., 2015b),

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rosmarinic acid (Domitrovic et al., 2013), carnosic acid (Satoh et al., 2008) and lipoic acid (Pilar Valdecantos et al., 2015), have also demonstrated to be neuroprotective

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against ALS in different models. The administration of rosemary extract, in which rosmarinic and carnosic acid are present, to G93A-hSOD1 transgenic mice improved motor performance and prolonged survival to 23.1 days at 3 mg/kg (rosmarinic acid)

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and to 20.6 days at 0.3 mg/kg (carnosic acid) respect to control conditions (14.3 days survival after disease onset) (Shimojo et al., 2010). Several authors have demonstrated that EGCG provides neuroprotection against H2O2-induced oxidative stress in G93A

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mutant motor neurons (Koh et al., 2004), and its ability to delay motor dysfunction and

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increase lifespan in the G93A-hSOD1 mouse model (Koh et al., 2006; Xu et al., 2006).

2.5.- Multiple sclerosis: Nrf2-EpRE dysregulation and Nrf2 inducers

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MS is a chronic inflammatory autoimmune disease characterized by lymphocyte infiltration, macrophage and microglial activation, and appearance of demyelinating

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plaques (Frohman et al., 2006; Wootla et al., 2012). These are mostly observed in the white matter of spinal cord and brain but also in the grey matter, associated with T cell mediated inflammation and axonal injury (Lassmann, 2010; Lassmann et al., 2007). T

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cells are activated in the peripheral immune system to generate high levels of Th1 and Th17 cytokines (pro-inflammatory cytokines such as IFN-gamma, TNF-α) (JadidiNiaragh & Mirshafiey, 2011; Lovett-Racke et al., 2011), that infiltrate to the CNS leading to inflammation and myelin damage (Wu & Alvarez, 2011). To counteract this damage, Th2 and Threg cells liberate anti-inflammatory cytokines (such as IL-10, IL-4, IL-5 and TGF-β) (Fitzgerald et al., 2007; Khan & Smith, 2014; Payne et al., 2012). In autoimmune diseases like MS, an imbalance between Th1/Th17 and Th2/Treg factors is the principal mechanism that triggers the pathogenesis of the disease (Jager & Kuchroo, 2010). The whole inflammatory process is accompanied by BBB disturbance, which correlates with disease severity (Alvarez et al., 2011; Bennett et al., 2010), and also by the expression of adhesion molecules (VCAMs) and chemokines (CCLs) that facilitate lymphocyte infiltration that can further activate more T cells that will drift toward Th1/Th17 (Holman et al., 2011), thus exacerbating the inflammatory status.

30

ACCEPTED MANUSCRIPT The main characteristic is the infiltration of leukocytes that produce proinflammatory mediators and ROS. Excessive ROS/RNS generation (mainly superoxide anion radical, NO·, ONOO–, H2O2 and OH·) contributes to leukocyte migration,

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oligodendrocyte damage and axonal degeneration (Smith et al., 1999). Mitochondrial

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injury has been identified as an important molecular mechanism involved in MS pathology (Mahad et al., 2008; Witte et al., 2010). It can induce tissue damage by three

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ways: energy failure, induction of apoptosis and/or increasing ROS production. Activated microglia and macrophages produce inflammatory mediators including NO and ROS, which contribute to the development and progression of the disease. ROS can

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target the respiratory chain complexes, and induce the opening of the mitochondrial permeability transition pore, triggering an apoptotic cascade and cellular damage

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(Lassmann & van Horssen, 2011). Furthermore, oxidative stress induces BBB permeability. Thus, oxidative damage may be playing an important role mediating mitochondrial injury and exacerbating the neuroinflammatory status. Indeed, oxidative

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injury in oligodendrocyte and axons of MS patients correlates with demyelination,

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neuroinflammation and neurodegeneration (Haider et al., 2011). Recently, the roles of Nrf2 and DJ-1, an important protein for Nrf2 stabilization,

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are getting increasing attention in the pathogenesis of MS (van Horssen et al., 2010). DJ-1 and Nrf2 protein expression are increased in cerebral spinal fluid of patients with relapsing-remitting MS (Hirotani et al., 2008). In fact, up regulated levels of Nrf2regulated antioxidant proteins (NQO1, HO1) have been found in active demyelinating

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MS lesions, predominantly localized in infiltrating macrophages and reactive astrocytes (van Horssen et al., 2008; van Horssen et al., 2011). DJ1 levels, were increased in astrocytes in both active and chronic inactive MS lesions (van Horssen et al., 2010). Furthermore, induction of experimental autoimmune encephalomyelitis (EAE) in an Nrf2-knockout mouse, an established model for MS, increased the severity of the disease, augmented the number of lesions and increased number of infiltrating immune cells (Johnson et al., 2010). Importantly, oligodendrocytes damaged at the lesions have relatively low levels of Nrf2. Therefore, low levels of Nrf2 or impaired Nrf2 activation in

oligodendrocytes

might

be

a

cause

of

selective

susceptibility

under

neuroinflammatory conditions due to the high vulnerability of those oligodendrocytes to oxidative stress (Juurlink et al., 1998).

31

ACCEPTED MANUSCRIPT The use of Nrf2-EpRE pathway as target in MS pathology has been recently validated by the approval of dimethyl-fumarate (DMF, 37, Figure 9) by the FDA and the EMA as first-line therapy for relapsing-remitting MS (Xu et al., 2015). DMF has

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been used for long as treatment of psoriasis. DMF (BG12-commercial formulation,

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Tecfidera®) is able to shift from Th1 to Th2 cells (de Jong et al., 1996). After intake, DMF is hydrolyzed to monomethyl fumarate (MMF, 38, Figure 9), the active metabolite

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that is able to induce Nrf2 in different models as astrocytes (over 15 fold increase of HO-1-mRNA levels at 10 M) (Lin et al., 2011). Its mechanism of action is related to its ability to increase GSH levels, inhibit NF-B translocation thus, decreasing NF-B

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dependent genes expression (inflammatory cytokines, chemokines and adhesion molecules). DMF was found to inhibit the progression of EAE in C57 mice. It inhibited

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macrophage inflammation in spinal cord and decreased expression of the proinflammatory cytokine IL-1 (Schilling et al., 2006). Effects on microglial environment were studied given the inflammatory function of microglia in CNS. DMF (10 µM)

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reduced iNOS mRNA level (27 %), and reduced mRNA levels of the pro-inflammatory

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cytokines IL-1β (67.5 %), IL-6 (65.3 %) and TNF-α (64.5 %). The inhibition of cytokine production by DMF involved the ERK and the Nrf2 pathway (Wilms et al.,

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2010). In vivo studies on EAE mice showed that DMF neuroprotective effect in spinal cords was associated with myelin and axon preservation with a mechanism mediated by the Nrf2 pathway. In Nrf2 deficient murine motoneuron cultures, the protective effect of

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DMF was completely abolished (Linker et al., 2011). Clinical trials with DMF have demonstrated that DMF significantly reduces the number of lesions (Schimrigk et al., 2006) and in a phase II study, treatment with 240 mg three times daily reduced the total number of new lesions by 69 % and the annualized relapse rate by 32 % respect to placebo group (Schimrigk et al., 2006). Phase III clinical trials (DEFINE and CONFIRM) demonstrated an annualized relapse rate reduction of 53 % and 48 % at 240 mg dose twice or three times daily, respectively. Furthermore, the risk of confirmed disability progression was also reduced to 28 % over the 2-year period of the study. Nevertheless, the best outcome was the reduction of new or enlarging hypertense lesions by up to 85 %, and the gadolinium-enhanced lesions by up to 94 % (Fox et al., 2012; Gold et al., 2012).

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ACCEPTED MANUSCRIPT COOMe

37: Dimethyl fumarate

MeOOC

COOH

38: Monomethyl fumarate N

S

N H

H H

O

O

H

N

OH

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O

S

T

MeOOC

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39: s-Allylmercapto-N-acetylcysteine 40: Matrine

Figure 9. Chemical structures of fumarate esters used as treatment of MS and Nrf2 inducers tested in MS.

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In 2014 N. Savion et al. reported a molecule called S-allylmercapto-Nacetylcysteine (ASSNAC) (39, Figure 9), a conjugate of the hydrophobic residue of allicin, S-allylmercaptan with N-Acetylcysteine (NAC) (Izigov et al., 2011), that was

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able to activate the Nrf2 pathway due to thiol reaction with cysteines present at Keap1. Human neuroblastoma SH-SY5Y cells treated with ASNNAC showed a dose dependent

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increase in GSH (max. 2.4 fold at 0.2 mM). It also decreased tert-butyl hydroperoxideinduced cytotoxicity (25 % decrease at 0.2 mM) (Savion et al., 2014). Thus ASSNAC is

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able to induce nuclear translocation of Nrf2, up-regulate GSH levels and protect cells from oxidative stress generated by tert-butyl hydroperoxide. More interestingly,

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ASSNAC was tested in the EAE mouse model, animals that received a 50 mg/kg/day dose showed a reduction of paralysis symptoms (0.4 score vs. 1.4 score of controls), an increase in Nrf2 nuclear content in liver samples (3.6 fold compared to non-treated

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samples) and increased GSH levels in brain and spinal cord samples. Matrine (40, Figure 9) is a natural alkaloid extracted from the herb Radix Sophorae Flaves. In 2011, matrine was found to decrease the clinical scores of EAE, CNS infiltration of inflammatory cells, demyelination, and IL17 and IL23 production (Zhao et al., 2011). Furthermore, matrine also suppressed the expression of molecules involved in immune cell migration, including chemokines (CCL5, CCL3) and adhesion molecules (ICAM-1, VCAM-1). It also inhibited expression of TLR4/MD-2 pathway (important for Th17 cell activation and differentiation) (Kan et al., 2013; Reynolds et al., 2012). As previously mentioned, severity of EAE is associated with the extent of BBB permeability (Alvarez et al., 2011). Another study revealed that matrine reduces inflammatory infiltration to CNS by protecting the BBB basement membrane and tight junction proteins (Zhang et al., 2013b). Matrine treatment in EAE in vivo model also reduced glutamate levels and increases GABA contents in cerebral cortex, thus, it could 33

ACCEPTED MANUSCRIPT be regulating glutamate-mediated excitotoxicity (Kan et al., 2014). In addition, matrine treatment up-regulated production of IL-4, IL-5 and IL-10, indicating that induction of Th2 cells is an important mechanism of action of matrine in EAE. Finally, matrine

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enhanced the levels of Nrf2 and HO-1 in the spinal cords of treated animals. Matrine

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(250 mg/kg) increase nuclear-Nrf2 positive cells in spinal cords of treated animals (6 fold increase) and the expression of HO-1 (5 fold increase) respect to basal conditions.

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These evidences, indicates that it could be blocking the oxidative stress in EAE through this pathway, contributing to stop progression of EAE (Liu et al., 2014). Curcumin (1, Figure 2) has also been tested as potential treatment for MS (Gao

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et al., 2013; Sahin et al., 2012; Zeng et al., 2015). Its immunomodulatory and antiinflammatory properties have also been addressed in several models, and are mostly

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mediated by its ability to inhibit COX-2, iNOS, and the NF-B transcription pathway (Menon & Sudheer, 2007). In 2009, the potential capacity of curcumin for the treatment of MS was studied in the animal model of EAE. It reduced clinical severity of EAE,

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expressed as the mean maximum clinical score (MMCS) from 3.71 on day 12 (control

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group) to 2.75 when rats were treated with 100 mg/kg/day curcumin, and even to 1.65 when treated with 200 mg/kg/day curcumin. It also reduced the number of inflammatory

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cells infiltrating into the spinal cord. Curcumin reduced EAE severity, by decreasing IL6, IL-23 production, suppressing the IL-6, IL-23 activated STAT3 pathway, which results in the inhibition of proliferation and differentiation of Th17 cells (Xie et al.,

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2009). Furthermore, curcumin has demonstrated in the EAE model to be able to reduce IL-17 production, as well as TNF-α generation, accompanied by up-regulation of Il-4, IL-10 and CD4+CD25+-Foxp3+ Treg cells (suggesting that curcumin augments Th2/Treg responses in EAE) production in the CNS (Kanakasabai et al., 2012). Lico A (18, Figure 5) has also been tested for the treatment of MS, showing immunosuppressive properties as it was able to: i) inhibit pro-inflammatory cytokine production (TNFα, IL-1β, IL-6, IL-10 and IFN-γ) in LPS stimulated human peripheral blood mononuclear cells (Liu et al., 2014), ii) reduce LPS-mediated NO production by inhibition of iNOS expression in RAW 264.7 cells (Furusawa et al., 2009) and iii) inhibit activation of STAT3 which is a transcription factor essential for differentiation of Th-17 (Barfod et al., 2002). Lico A (9 M) showed also protection against oxidative stress in primary human fibroblasts by increasing nuclear translocation of Nrf2, accompanied by an increase in HO-1 (10-fold induction of mRNA after 12 h treatment),

34

ACCEPTED MANUSCRIPT GCL unit (2 fold induction of mRNA), GSH/GSSG ratio (due to an increase in GSH levels) and a decrease in ROS production (measured as DCFDA fluorescence) (Kuhnl et al., 2015). Lico A (30 mg/kg/day) attenuated clinical scores of EAE and reduced the

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production of cytokines (TNFα, IFN-γ, and IL-17). In addition, Lico A was able to

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suppress free radicals production (NO and H2O2) in splenocytes of EAE animals after in

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vitro treatment (Fontes et al., 2014).

Triterpenoids derived from the natural compound oleanolic acid (11, Figure 3) has been also tested for MS. Oleanoic acid studies on the EAE model showed decreased clinical scores of the disease. It was also demonstrated that oleanoic acid (50 mg/kg)

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decreased Th1 cytokines and adhesion molecules production (56 % TNFα reduction and 80 % ICAM1 reduction in spinal cord tissue), and increased Th2 cytokines, as IL-10

in

the

CNS

(minimal

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(approximately 2 fold increase in serum samples). It effectively reduced cell infiltration CD11b/Mac-1

immunoreactivity,

markers

for

macrophages/microglia), as well as in BBB disturbance (Martin et al., 2010). Besides,

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synthetic triterpenoids derived from oleanoic acid showed neuroprotective effects

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through up-regulation of the Nrf2-EpRE pathway (Liby et al., 2005; Liby et al., 2007) including an AD transgenic model (Dumont et al., 2009) and ALS (Neymotin et al.,

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2011). The neuroprotective mechanism of three CDDO derivatives, CDDO-Me, (12, Figure 3), CDDOEA (16, Figure 5) and CDDO-TFEA (17, Figure 5), on the EAE model was studied. They effectively ameliorated the clinical signs of EAE (dosage of

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100 L injection of 100 nM concentration, 5 times every 48 h), decreased Th1 and Th17 cytokines, diminished expression of iNOS to basal conditions and up-regulated HO-1 expression (over 3 fold respect to control conditions), consistent with the Nrf2-EpRE pathway up-regulation (Pareek et al., 2011). As detailed before, a balance exists between HO-1 and IL-17 levels, thus Nrf2 expression not only enhances global suppression of inflammatory response and has cytoprotective effects, it also suppresses disease associated cytokines, especially IL-17 levels. Sulforaphane (13, Figure 4) is known for its antioxidant and anti-inflammatory properties related to its potent Nrf2 inducing effect (Boddupalli et al., 2012). It inhibits progression of EAE in mice through activation of the Nrf2 pathway, reducing oxidative stress (measured as decrease in malondialdehyde level reduction) and inflammation and by protecting BBB integrity. Studies on the mechanism of action of sulforaphane revealed that it enhanced the expression of HO-1 and NQO1 through the Nrf2-EpRE

35

ACCEPTED MANUSCRIPT pathway up-regulation, thus reducing the levels of oxidative stress. It also reduced T cell mediated immunity and inflammation. Furthermore, it reduced demyelination and CNS infiltration, and inhibited production of MMP-9 (metalloproteinase that degrades

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extracellular matrix proteins), which contributes to BBB preservation (Li et al., 2013a).

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2.6.- Ischemic stroke: Nrf2-EpRE dysregulation and Nrf2 inducers

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Ischemic stroke occurs when a blood clot is formed; this occludes arteries in the brain reducing the oxygen supply to the tissue. Three main mechanisms lead to cell death (in neurons, glia and vascular elements) in ischemic brain injury: excitotoxicity

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and ionic imbalance, oxidative/nitrosative stress and apoptotic-like cell death (Lo et al., 2003). Brain arteries blockade sets an anoxic state in the brain decreasing energy production. Then membrane is depolarized triggering the release of glutamate, which

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activates NMDA receptors inducing calcium overload and cell death (Kumar et al., 2014). This promotes mitochondrial ROS/RNS production (superoxide anion radical, OH· and NO·) that overwhelms antioxidant defenses causing tissue damage (Pradeep et

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al., 2012). Furthermore, restoration of blood flow (reperfusion phase) increases tissue

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oxygenation leading to an exacerbated production of ROS. Moreover, metalloproteases are activated by free radicals and degrade structural

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proteins of the vascular wall and BBB. This BBB disruption occurs early in the ischemic stroke and can extent progression of brain damage (Sandoval & Witt, 2008). Furthermore, after stroke there is an increase in cellular adhesion molecules that are

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known to facilitate adhesion and migration of leukocytes (Frijns & Kappelle, 2002), that infiltrate to the injured brain and exacerbate the damage by releasing more ROS (mainly superoxide anion radical produced mostly by NADPH oxidase metabolism) and inducing inflammation (Yilmaz & Granger, 2008). The oxidative stress over the ischemia and reperfusion phases leads to cell death, thus prevention and control of oxidative stress in cerebral ischemia/reperfusion injury is a promising therapeutic target (Liu et al., 2015). Recent findings demonstrate that interactions between p62 and the Nrf2-EpRE signaling pathway play a key role in preventing oxidative injury and inflammation (Barone & Feuerstein, 1999) during cerebral ischemia/reperfusion in rat transient middle artery occlusion (tMCAO) (Wang et al., 2013). Some of the Nrf2-EpRE inducers described above have also been tested in ischemia models, being of interest: curcumin (Yang et al., 2009a) (1, Figure 2), sulforaphane (Zhao et al., 2006) (13, Figure 4), ursolic acid (10, Figure 3) (Li et al.,

36

ACCEPTED MANUSCRIPT 2013b), and carnosic acid (Satoh et al., 2008) (7, Figure 3). All these natural compounds afforded interesting results in ischemia in vivo models. In general, all of them were able to reduce infarct volume by a mechanism dependent of Nrf2 activation.

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An interesting Nrf2 inducer tested for ischemic stroke DL-NBP (32, Figure 8). It

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is able to reduce brain damage, neuronal cell death, brain edema and to protect the BBB (Zhang et al., 2006). In vivo models of ischemia have demonstrated the neuroprotective

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effect of this compound. DL-3-NBP post-administration (100 mg/kg, ip) 1h after MCAO reduced almost 50 % of infarct volume. It reduced caspase activation and improved mitochondrial function. Results on post-stroke treatment with NBP might

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indicate that it could be used after acute and sub-acute treatments of stroke (Li et al., 2010). Moreover, clinical trials conducted have demonstrated its efficacy; DL-3-NBP

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(200 mg, 3 times/day) significantly improved neurological performance in the NIH Stroke Survey score (Cui et al., 2013; Jia et al., 2015). This compound was approved in 2010 for the treatment of ischemic stroke patients by the State Food and Drug

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Administration of China.

HO

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O

OH OH

OH MeO

O

O

O HO

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COOH

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41: 11-keto-b-boswellic acid

O

HO

HO

OH

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42: Osthole

43: (-)-Epicatechin

OH OH OH O

OH

HO OH

O

OH

44: Eriodictyol-7-O-glucoside

O

45: Senkyunolide I

Figure 10. Chemical structures of natural and natural-derivatives Nrf2 inducers tested in cerebral ischemia.

Another example is the 11-keto-β-boswellic acid (KBA, 41, Figure 10), a pentacyclic triterpenoid isolated from extracts of Boswellia Serrata. KBA reduced myocardial infarct size through antioxidant mechanisms related to the Nrf2 pathway and prevention of inflammatory cascade (Elshazly et al., 2013). In the MCAO model, KBA (25 mg/kg, i.p.) effectively reduced the infarct size (from 37.1 ± 5 % I/R rats, to 23.2 ± 2.9 % I/R+KBA rats), and attenuated oxidative stress, through the Nrf2 pathway as Nrf2

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ACCEPTED MANUSCRIPT and HO-1 expression was augmented (2 and 4 folds respectively) 48 hours after ischemia in cortical tissue (when treated with KBA, compared to I/R rats) (Ding et al., 2014).

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Osthole (42, Figure 10) is a natural product extracted from diverse plants.

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Studies on the two-vessel occlusion (2VO) animal model (permanent occlusion of bilateral common carotid arteries), showed that osthole attenuated cognitive deficits and

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neuronal damage used at 20 mg/kg (Ji et al., 2010). Moreover, in a global cerebral ischemia model osthole showed anti-oxidant effect by increasing SOD activity and decreased malondialdehyde (MDA) levels. Rat treatment with osthole also decreased

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BBB disruption, probably through the decrease in ROS production. It also increased GSH levels in a MCAO model, from 18 U/mg protein to 32, after 24 h of treatment (40 mg/kg i.p., 30 min before MCAO) (Chao et al., 2010), and reduced total infarct volume

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(60 % reduction). In the HT22 cells, osthole (50 M) increased Nrf2 and HO-1 levels (3 and 1.5 folds respectively), indicating that the protective effect of osthole on brain

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ischemia is probably mediated by the Nrf2-EpRE pathway (Chen et al., 2015).

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As previously described, polyphenolic derivatives are a prominent example of antioxidant Nrf2 inducers tested in a wide variety of neurodegenerative models showing

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good properties. Two of them, (-)-epicatechin (EC, 43, Figure 10) and EGCG (5, Figure 2) were tested in a model of cerebral ischemia being able to reduce cellular damage. Both flavonoids were tested in the MCAO model. They were able to reduce infarct size and to promote behavioural recovery depending on Nrf2 (Han et al., 2014; Lee et al.,

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2004; Lim et al., 2010; Shah et al., 2010). EGCG showed neuroprotective effect against cerebral ischemia in rats subjected to MCAO, it reduced cerebral infarction size to 34.5 ± 7.8 % at a 25 mg/kg dose and up to 9.9 ± 3.2 % at a 50 mg/kg dose when compared to the saline treated control group (45.6 ± 5.3 % infarct size). It also reduced markers of lipid peroxidation, reduced MDA and augmented GSH levels (Choi et al., 2004). In addition EGCG was able to reduce MMP-9 activity, thus contributing to preserve BBB integrity after ischemic injury (Park et al., 2010). EGCG was further evaluated on the MCAO model to determine whether the neuroprotective effect was dependent on the Nrf2 pathway. Compared to control animals, those subjected to I/R injury showed increased Nrf2 levels by 1.5 fold, and treatment with EGCG 40 (mg/kg) increased the Nrf2 levels by 4.5 fold. Consistently, Nrf2-driven genes (HO-1, GCLc and GCLM) were up-regulated by EGCG treatment (Han et al., 2014).

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ACCEPTED MANUSCRIPT Another interesting flavonoid studied is eriodictyol-7-O-glucoside (E7G, 44, Figure 10), purified from the Chinese herb Dracocephalum rupestre, used to treat inflammation. This natural flavonoid is a well-described Nrf2-EpRE inducer that

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confers neuroprotection in different toxic stimuli (Hu et al., 2012). At the concentration

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of 20 M, E7G increased the levels of Nrf2 protein, HO-1, NQO1 and -GCS (2, 2, and 3 folds respectively). It was able to reduce astrocyte death in a concentration dependent

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manner from 10 to 80 M and this protection was directly related to the Nrf2-induction properties. Finally, E7G effectively reduced infarct volume in an in vivo ischemia model. Rats were pre-treated with 30 mg/kg for five days and subjected to focal cerebral

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ischemia. E7G improved neurological deficit by 30 % and significantly reduced infarct volume by 35 %. Its ability to protect against ischemia was related to a reduction in

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apoptotic cells and the induction of Nrf2, HO-1 and γ-GCS (Jing et al., 2013). Senkyunolide I (SEI, 45, Figure 10), a natural product derived from the Chinese herb Rhizoma Chuanxiong, protected HepG2 cells from oxidative damage through a

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mechanism dependent on HO-1 overexpression. It reduced ROS production (measured

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as DCFDA fluorescence), MDA production, and cell death induced by H2O2; importantly, SnPP (inhibitor of HO-1), significantly attenuated these effects (Qi et al.,

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2010). Taking into account the high damage induced by oxidative stress in ischemia/reperfusion and the possible induction of the Nrf2 pathway by SEI, it was evaluated on the MCAO model. SEI reduced the infarct volume (27.31 % ± 2.40 %)

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compared to vehicle group (42.20 % ± 8.62 %) and reduced neurological scores. SEI reduced oxidative stress simultaneously by reducing lipid peroxidation (decreased formation of MDA), and by up-regulating defense enzymes (SOD). This protective effect was mediated by activation of the Nrf2-EpRE pathway, as confirmed by the increase in the ratio of nuclear Nrf2/cytosolic Nrf2 at increasing doses of SEI. Furthermore, SEI administration increased the ratio Bcl-2 (anti-apoptotic)/Bax (proapoptotic) and down-regulated levels of caspase 3 and caspase 9, both involved in the apoptotic pathway. Thus SEI protective effect after IR injury is related to antioxidant properties mediated by the Nrf2 pathway, and its anti-apoptotic properties (Hu et al., 2015).

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ACCEPTED MANUSCRIPT 3.- Conclusion NDDs develop as complex networks of pathological events that are interconnected among them to generate feed-back processes leading to increased

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damage and neuronal death. Although different proteins and cellular systems are

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affected in each NDD, several common pathological pathways are common to all of them. In general, oxidative stress, mitochondrial dysfunction and neuroinflammation are

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common processes that have been widely described in all NDDs. In recent years, the failure or the dysregulation of the principal cellular response to oxidative stress and neuroinflammation, the Nrf2-EpRE phase II response, has been described. Compelling

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evidence suggests that oxidative stress increases the damage in NDDs and the deregulation or failure of the phase II response accelerates the decline of the patients

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suffering from those diseases. Therefore, the activation of the Nrf2-EpRE pathway has been pointed as a key target for the development of new drugs for NDDs. In this sense, natural products are an important source of Nrf2 inducers that have been widely studied

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over the last years. The use of Nrf2 inducers in many animal models of NDDs serves as

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“proof-of-concept” of this important target to find a real disease-modifying drug. Compounds here reviewed have demonstrated highly interesting results in in vitro and

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in vivo models, protecting neurons, decreasing the accumulation of aberrant proteins and increasing life span of different mouse models, among others. All this encouraging results have been related to the antioxidant and Nrf2 inducing properties of the products

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evaluated. In conclusion, Nrf2 inducers have given the “proof-of-concept” to use the Nrf2-EpRE pathway as key target in NDDs and give an excellent starting point to develop clinical trials. These compounds also bring the opportunity to use these compounds as templates for the development of medicinal chemistry projects to improve their pharmacological properties.

Abbreviations A AD AKT ALS APP ARE ASSNAC BBB CA

amyloid- Alzheimer’s disease protein kinase B Amyotrophic Lateral Sclerosis amyloid precursor protein anti-oxidant response elements S-allylmercapto-N-acetylcysteine blood brain barrier carnosic acid 40

ACCEPTED MANUSCRIPT Ca2+ CAPE CCLs CDDO-MA CDDO-Me

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calcium (II) ion caffeic acid phenethyl ester (C-C motif) ligand CDDO methyl amide 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl esther (Bardoloxene Methyl) CDDO-TFEA 3-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-trifluoroethylamide CK2 casein kinase-2 CPN-9 N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy) acetamide CNS central nervous system DJ-1 or PARK7, Parkinson disease protein 7 DL-NBP DL-3-n-butylphthalide DMF dimethyl fumarate EAE experimental autoimmune encephalomyelitis EC (-)-epicatechin EGCG (-)-Epigallocatechin gallate E7G eriodictyol-7-O-glucoside EMA european medicines agency EpRE electrophile response elements ERK extracellular regulated kinase FGF-1 fibroblast growth factor-1 FDA food and drug adminstration GCLm glutamate-cysteine ligase modifier subunit GCLc glutamate-cysteine ligase catalytic subunit γ-GCS γ-glutamylcysteine synthetase GSK-3β glycogen synthase kinase 3-beta GPx glutathione peroxidase GR glutathione reductase GSH glutathione GST glutathione S-transferase HD Huntington disease HO-1 heme-oxygenase-1 H2O2 Hydrogen peroxide i.c.v. intracerebroventricular injection IFN-gamma interferon-gamma IL-1β interleukin-1 beta iNOS inducible nitric oxide synthase i.p. intraperitoneal injection JNK c-Jun N-terminal kinase LPS lipopolysaccharide LRRK2 Leucine-rich repeat kinase 2 MAPKs mitogen-activated protein kinases MDA malondialdehyde mGST1 microsomal GSTs 1 mGST2 microsomal GSTs 2 MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MS Multiple Sclerosis NAC N-acetylcysteine NDDs neurodegenerative diseases

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nuclear factor-κB nerve growth factor NADPH oxidase 3-nitropropionic acid NAD(P)H quinone oxidoreductase-1 nuclear factor E2-related factor 2 N-methyl-D-aspartate receptor Nitric oxide radical 6-hydroxydopamine Hydroxyl radical oxygen and glucose deprivation Peroxinitrite tumor protein p53 parkinson´s disease Peroxisome proliferator-activated receptor gamma coactivator 1-alpha phosphatidylinositide-3-kinases PTEN-induced kinase 1 protein kinase C preseniline 1 11-keto-β-boswellic acid Kelch-like ECH-associated protein-1 reactive oxygen species reactive nitrogen species Cu/Zn-superoxide dismutase 1 sirtuin 1 Senkyunolide I thioredoxins tetrahydrocurcumin Tumor necrosis factor-α thiobarbituric acid reactive substances T helper 1 cell T helper 17 cell rat transient middle artery occlusion T helper 2 cell regulatory T cells vascular cell adhesion molecules two-vessel occlusion animal model 2-[mesityl(methyl)amino]-N-[4-pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride

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NF-κB NGF NOX 3-NP NQO1 Nrf2 NMDA NO· 6-OHDA OH· OGD ONOO– p53 PD PGC-1α PI3K PINK-1 PKC PS1 KBA Keap1 ROS RNS SOD1 SIRT1 SEI Trxs THC TNF-α TBARS Th1 Th17 tMCAO Th2 Treg VCAMs 2VO WN1316

Conflict of interest The authors declare no conflicts of interest. Acknowledgements We are grateful to Financial support from European Commission-ERC, People (Marie Curie Actions) FP7 under REA grant agreement n° PCIG11-GA-2012-322156 to R.L.; Spanish Ministry of Health (Instituto de Salud Carlos III) (grant PI14/00372) and

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ACCEPTED MANUSCRIPT Miguel Servet (CP11/00165) to R. L., and (CP14/00008) to J. E.. I. B. and P.M. thanks MECD for FPU fellowships (AP2010-1219 and FPU13/03737 respectively). E. N. and I. G. thanks UAM for FPI fellowships. We would also like to thank “Fundación Teófilo

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Hernando” for its continued support.

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