NRF2 in neurodegenerative diseases

NRF2 in neurodegenerative diseases

Accepted Manuscript NRF2 in neurodegenerative diseases Dr. Antonio Cuadrado PII: S2468-2020(16)30026-2 DOI: 10.1016/j.cotox.2016.09.004 Reference:...

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Accepted Manuscript NRF2 in neurodegenerative diseases Dr. Antonio Cuadrado PII:

S2468-2020(16)30026-2

DOI:

10.1016/j.cotox.2016.09.004

Reference:

COTOX 7

To appear in:

Current Opinion in Toxicology

Received Date: 27 September 2016 Accepted Date: 30 September 2016

Please cite this article as: A. Cuadrado, NRF2 in neurodegenerative diseases, Current Opinion in Toxicology (2016), doi: 10.1016/j.cotox.2016.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NRF2

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NRF2

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ROS

NRF2

Cytokines

- a-SYN - TAU - APP

Neuronal damage

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- mitochondria - NOX - etc

Protein aggregation

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Oxidative stress

Inflammation

Microglial polarization

a-SYN others

- NFkB - iNOS - IL1 - COX2 - IL6 - etc - TNF

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NRF2 in neurodegenerative diseases Antonio Cuadrado

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Affiliations:

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Running title: NRF2 in neurodegenerative diseases

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Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Investigación Sanitaria La Paz (IdiPaz), Instituto de Investigaciones Biomédicas Alberto Sols UAM-CSIC, and Department of Biochemistry, Faculty of Medicine, Autonomous University of Madrid, Madrid, Spain.

*Correspondence address: Dr. Antonio Cuadrado Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC C/ Arturo Duperier 4 28029 Madrid, Spain E-mails: [email protected] Tel: 34915854383 Fax: 34915854401

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ABSTRACT Neurodegenerative diseases, and degenerative disorders as a whole, share in common the deviation from homeostatic responses related to the control of proteostasis and low-grade

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chronic oxidative, inflammatory, and metabolic stress. These are all crucial events where transcription factor Nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays a very important defensive role. In this paper, biochemical and genetic evidence connecting NRF2

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with neurodegenerative diseases will be discussed, mainly in the context of preclinical mouse models and in patients with Alzheimer’s and Parkinson’s disease. NRF2 can be

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targeted pharmacologically and the most successful drugs to endorse a neuroprotective therapy will be commented, including dimethyl fumarate. Keywords:

oxidative

stress;

proteinopathy;

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neurodegenerative diseases; Alzheimer's disease

neuroinflammation;

neuroprotection;

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Introduction Neurodegenerative diseases represent the most prevalent degenerative disorders of the elderly and have a huge negative impact on patients, care givers and health systems. A

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disease modifying therapy that could stop brain degeneration seems reachable in the next few years if the precise molecular targets are identified. Neurodegenerative diseases, and degenerative disorders as a whole, share in common the deviation from homeostatic

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responses related to the control of proteostasis and low-grade chronic oxidative, inflammatory, and metabolic stress. These are all crucial events where transcription factor

NRF2 function and regulation

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NRF2 plays a very important homeostatic role.

Transcription factor NRF2 regulates the expression of over 250 genes that contain in their promoters the antioxidant response element (ARE) (Fig. 1) [1]. These genes in

phase

I,

II

and

III

detoxification

reactions,

glutathione

and

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participate

peroxiredoxin/thioredoxin metabolism, NADPH production, fatty acid oxidation, iron metabolism, proteasomal and autophagic processes, and gene regulation of other

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transcription factors [1]. Because of its tremendous impact on physiology and pathology it participates in multiple defensive roles and it is also very tightly controlled at the level of

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protein stability by at least two ubiquitin E3 ligase adapters: Kelch-like ECH-associated protein 1 (KEAP1) and beta-transducin repeat containing protein (β-TrCP). These two mechanisms of control provide opportunity for pharmacological regulation of NRF2 (Fig. 1).

KEAP1 is a homodimeric E3 ligase adapter that contains several electrophilic and redox-sensitive cysteine residues. Under basal redox conditions, KEAP1 binds one molecule of NRF2 at two amino acid sequences with low (aspartate, leucine, glycine; DLG)

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and high (glutamate, threonine, glycine, glutamate; ETGE) affinity, respectively and thus presents NRF2 to a complex formed by Cullin3/Rbx proteins, leading to its ubiquitination and subsequent proteasomal degradation [2,3]. However, ectopic or endogenous

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electrophiles, modify sulfhydryl groups of specific redox-sensitive cysteines of KEAP1, including C151, C273, and C288 [4]. Following these modifications KEAP1 is no longer capable of presenting this protein for ubiquitination [5]. As a result, newly synthetized

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NRF2 escapes KEAP1-dependent degradation, accumulates in the nucleus, and activates ARE-genes.

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β-TrCP is also a homodimeric E3 ligase adapter that participates in the signaling events related to phosphorylation of NRF2 by the glycogen synthase kinse-3 (GSK-3) [68]. Under basal non-stimulated conditions, this kinase phosphorylates specific serine residues of NRF2 (DSGIS) to create a degradation domain that is then recognized by β-

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TrCP and tagged for proteasome degradation by a Cullin1/Rbx complex. On the other hand, signaling pathways that inhibit GSK-3, either by phosphorylation of its N-terminal domain or by reclusion to multivesicular bodies, prevent the formation of this phosphodegron and

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then NRF2 escapes β-TrCP-mediated degradation [6-8]. The transcriptional activity of NRF2 declines with ageing, which is the main risk

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factor for most neurodegenerative diseases. In rats, the age-dependent decay in transcriptional activity of NRF2 causes progressive loss of glutathione synthesis, which is reversible with lipoic acid [9]. On the other hand, unusually high NRF2 activity is a feature of several long-lived animals, suggesting its role in protection against chronic diseases [10]. Also, pharmacological interventions to activate NRF2 with nordihydroguaiaretic acid [11,12] and a mixture of botanical extracts, protandim [12], extended lifespan in male mice.

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In humans, the premature ageing of Hutchinson-Gilford progeria syndrome is characterized by trapping of NRF2 at the nuclear periphery by progerin, resulting in impaired NRF2

NRF2 in animal models of neurodegenerative diseases

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transcription and chronic oxidative stress production [13].

For a comprehensive discussion about neurodegeneration in NRF2-knockout mice (Nrf2-/-) according to specific diseases see [14]. Here, we will overview the most relevant

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milestones that demonstrate a role of NRF2 in crucial parameters of brain pathophysiology such as oxidative and inflammatory stress, gliosis and proteinopathy.

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It would be expected that NRF2 protects against the oxidative stress that characterizes neurodegenerative diseases. This is indeed the case, as it is best exemplified in cellular and animal models of PD, based on inhibition of the mitochondrial respiratory chain.

The

mitochondrial

complex

I

inhibitors

1-methyl-

4-phenyl-1,2,3,6-

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tetrahydropyridine (MPTP), paraquat and rotenone as well as 6-hydoxydopamine promote a massive release of reactive oxygen species (ROS) in combination with loss of ATP production that leads to selective dopaminergic cell death. Nrf2-/- mice are very susceptible

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to these toxins and at the same time wild type mice (Nrf2+/+) are significantly protected by drugs that activate NRF2 and lead to up-regulation of is targets such as heme oxygenase-1

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(HO-1) and NAD(P)H quinone oxidoreductase (NQO1). For example, in Nrf2+/+ mice, but not in Nrf2-/- mice, the isothiocyanate sulforaphane (SFN) protected against MPTP-induced death of nigral dopaminergic neurons and the neuroprotective effects were accompanied by a decrease in astrogliosis, microgliosis, and release of pro-inflammatory cytokines [14]. However, the clinical value of these observations is largely limited by a biased approach: generating oxidant pathology in an otherwise healthy mouse and then providing a cure in the form of antioxidant NRF2 protection, while good as proof of concept, is of little clinical

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interest. Even if antioxidant NRF2 protection is expected, the usefulness of NRF2, merely considered as an antioxidant target, is not clear in humans, because simple antioxidant supplements have provided little benefit or even deleterious effects [15]. One example is

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the use of Coenzyme Q in the QE3 phase 3 trial for PD, which showed no evidence of clinical benefit [16], or the 2CARE for Huntington disease, the largest ever trial for this disease, that was cancelled due to futility (http://huntingtonstudygroup.org/tag/2care/).

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Obviously, antioxidant therapy based on up-regulation of NRF2 has in theory several important advantages over antioxidant supplements. Contrarily to antioxidant treatments,

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NRF2 activation will provide antioxidant metabolism (NADPH, GSH, and thioredoxin) in local places where needed while leaving physiological ROS signaling intact. Besides, the effect on the enzymatic antioxidant defense may be more prolonged than that of short lived small molecules (for a discussion see [15]). In any case, to provide compelling evidence

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that NRF2 is a valid target to slow neurodegenerative progression, it is necessary to determine its protective effect in superior models that reproduce the main hallmark of the human neurodegenerative pathologies which is the proteinopathy associated with α-

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synuclein (α-SYN), β-amyloid, TAU, etc and then the additional hallmarks such as oxidative, inflammatory and metabolic stress.

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Regarding AD, lentiviral overexpression NRF2 in the hippocampus of old mice

expressing mutant amyloid precursor protein (APP) and presenilin 1 (PS1) transgenes was able to prevent spatial learning deficits [17], already suggesting that NRF2 activity is limiting in the diseased brain and that enhanced expression will be protective. Studies in Nrf2-/- mice expressing hallmarks of amyloidopathy [18] or tauopapthy [19,20] further demonstrate that NRF2-deficiency exacerbates oxidative stress, astrogilosis, microgliosis

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and neuroinflammation. Even in the absence of human transgenes, Nrf2-/- mice appear to develop with aging cognitive deficits that correlate with spontaneous TAU aggregation and decreased expression of the autophagy adaptor protein NDP52 [20]. Similarly, NRF2-

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defficiency led to impaired autophagy and aggravated the proteinopathy induced by APP/PS1 [18] and by an adenoassociated vector expressing human TAUP301L [19].

So far, the most reliable mouse model of PD to reproduce human synucleinopathy is

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the stereotaxic delivery to the ventral midbrain of an adeno-associated vector expressing human α-SYN [21]. In this model, Nrf2-/- mice exhibited exacerbated degeneration of

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nigral dopaminergic neurons and increased dystrophic dendrites and α-SYN aggregates, reminiscent of human Lewy pathology, which correlated with impaired proteasome gene expression and activity. Dopaminergic neuron loss was associated with neuroinflammation and gliosis that were intensified in Nrf2-/- mice. In response to exogenously added α-SYN,

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Nrf2-/- microglia exhibited an exacerbated production IL-6, IL-1 and iNOS and reduced phagocytic capacity [22]. As it will be commented on later, Nrf2+/+ mice expressing human α-SYN but not Nrf2-/- mice were rescued by chronic treatment of the NRF2 activator

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dimethyl fumarate (DMF).

Therefore, the homeostatic actions of NRF2 go beyond protection against oxidative

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stress and involve proteostasis in neurons. The role of NRF2 in autophagy was first suggested by the observation that SQTSM1/p62 exhibits an STGE motif that upon serine phosphorylation competes with NRF2 for binding to KEAP1, sending this protein to the autophagosome and unleashing NRF2 from this control [23-25]. Then, it was found that the SQTSM1 gene is positively regulated by NRF2, leading to a feed-forward cycle of autophagy activation [26]. Recently, we have identified NRF2 as a crucial regulator of

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macroautophagy gene expression and its relevance in AD, using a mouse model of AD that combines expression of human APPV717I and TAUP301L transgenes with presence or absence of NRF2 [27]. We screened the chromatin immunoprecipitation database ENCODE for two

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proteins, MAFK and BACH1 that bind the NRF2-regulated enhancer ARE. Using a script generated from the JASPAR's consensus ARE sequence, we identified 27 putative AREs in 16 autophagy related genes. Twelve of these sequences were validated as NRF2 regulated

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AREs in 9 autophagy genes by additional chromatin immunoprecipitation assays and quantitative real-time PCR on human and mouse cells after NRF2 activation with SFN.

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Mouse embryo fibroblasts (MEFs) of NRF2-/- mice exhibited reduced expression of these autophagy genes, which was rescued by an NRF2-expressing lentivirus. Nrf2-/- mice coexpressing APPV717I and TAUP301L, exhibited more intracellular aggregates of these proteins together with reduced neuronal levels of SQSTM1, NDP52, ULK1, ATG5 and

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GABARAPL1. Also, co-localization of APPV717I and TAUP301L with the NRF2-regulated autophagy marker SQSTM1 was reduced in the absence of NRF2. Astroglia, as a metabolically active and nurturing cell, play crucial roles in brain

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homeostasis and they express high levels of NRF2 [28]. The initial studies conducted with a reporter mouse carrying placental alkaline phosphatase gene under the control of

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promoter containing a core ARE led to the discovery that astrocytes are very sensitive to NRF2 activation and participate in protection against MPTP [29] and human α-SYNA53T [30]. After the latter realization that NRF2 participates in regulation of intermediate metabolism [31], it is reasonable to suggest that up-regulation of NRF2 in astrocytes is a crucial homeostatic defense in the injured brain. Besides, the gaps left after neuronal death are filled with astrocytes and therefore, astrocytosis also reflects long-term accumulative

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damage. Several studies have shown reduced astrocyte scares in neurodegenerative brains of animals protected with NRF2 activators in models of AD or PD [19,21,32,33]. These findings are consistent with a reduction in neuronal death.

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Microglia also have a crucial role in remodeling of nerve tissue. Resting microglia may be activated towards a classical pro-inflammatory or an alternative wound-healing phenotype and this dynamics is strongly influenced by the redox state [34]. The

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transcription factor Nuclear factor kappa B cells (NFκB), master regulator of proinflammatory genes, is activated by high levels of ROS and therefore, it has been suggested

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that NRF2 counteracts the pro-inflammatory response of microglia through control of redox homeostasis [35]. In connection with neurodegeneration, Nrf2-/- mice submitted to daily inoculation of MPTP for 30 days exhibited a more severe dopaminergic dysfunction than Nrf2+/+ littermates accompanied by increased astrogliosis and microgliosis, as determined

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by an increase in messenger RNA and protein levels for GFAP and F4/80, respectively. Inflammation markers characteristic of classical microglial activation, COX-2, iNOS, IL-6, and TNF were also increased and, at the same time, anti-inflammatory markers such as

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FIZZ-1, YM-1, Arginase-1, and IL-4 were decreased [32]. Very recently a new mechanism of direct inhibition of inflammation by NRF2 has been reported, in which NRF2 suppresses

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macrophage inflammatory response by blocking directly the expression of proinflammatory cytokine genes such as IL-6. It seems that NRF2 does so, not through binding to ARE but to the proximal promoter of the cytokine gene preventing recruitment of RNA polymerase II [36].

An essential aspect of normal brain function is the bidirectional communication between neurons and neighboring nerve cells. Consistent with its supporting role, overexpression of NRF2 in astrocytes leads to release of reduced glutathione that protects

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neurons in vitro and in the hSOD1 model of amyotrophic lateral sclerosis (ALS) [37]. Primary microglial cultures stimulated with conditioned medium from MPP+-treated dopaminergic cells, activate their inflammatory response and, very importantly, NRF2-

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defficient microglia exhibit an imbalance towards the classical pro-inflammatory activation [32]. With regard to specific signaling molecules, the chemokine fractalkine (CX3CL1) is released by injured neurons and interacts with its cognate receptor, which in the brain is

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exclusively expressed in microglia. Interestingly, CX3CL1 released by TAU-injured neurons activates in microglia the phosphatidylinositol 3 kinase/Akt pathway leading to

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inhibition of glycogen synthase kinase-3. As a result, NRF2 escapes degradation by GSK3/β-TrCP and up-regulates its target genes including heme oxygenase 1 [19]. By this mechanism, neurons prevent pro-inflammatory over-activation of microglia. NRF2 in human pathology

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In hippocampal neurons of AD patients, NRF2 was predominantly cytoplasmic, suggesting impaired capacity of these neurons to reduce proteotoxic and oxidative stress through NRF2-dependent transcription of cytoprotective genes [38]. Other studies,

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including ours, found that APP- and TAU-injured neurons expressed increased levels of NRF2 and its target SQSTM1, probably as a compensatory mechanism to clear these toxic

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proteins through autophagy [19,27]. Others also found up-regulation of HO-1, GCLM and SQSTM1 in AD brains [39] as well as NQO1 [40,41] and HO-1 [42]. It is now speculated that the NRF2 signature, including autophagy and oxidant defense, may change during disease progression.

In nigral dopaminergic neurons, NRF2 is located in the cytoplasm, whereas in agematched PD patients, it is found in the nucleus [38] and the NRF2 signature, represented by

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expression of NQO1 [22,43], and HO-1 [44] is up-regulated, suggesting an attempt of brain protection through this pathway [22,45]. In postmortem samples of PD patients, the cytoprotective proteins associated with NRF2 expression, NQO1 and SQSTM1, were partly

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sequestered in Lewy bodies, suggesting impaired neuroprotective capacity of the NRF2 signature [22].

Many single nucleotide polymorphisms have been identified in coding and non-

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coding regions of the NRF2 coding gene, NFE2L2 [46-49] and some have been associated with risk of chronic pulmonary, gastrointestinal, autoimmune diseases [46]. Although

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formal proof of change in NRF2 activity is lacking for most polymorphisms, several haplotypes in the promoter region have been associated with increased or decreased risk of AD, PD and ALS, as well as with other comorbidities and risk factors. In AD, the only genetic study conducted so far did not find evidence of genetic

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predisposition to this disease but one haplotype allele was associated with 2 years earlier age at AD onset, therefore suggesting that common variants of the NFE2L2 gene may affect AD progression [50]. In amyotrophic lateral sclerosis, one haplotype in NFE2L2,

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including three functional promoter SNPs previously linked with high NRF2 protein expression, was associated with 4 years later disease onset [51] but another study did not

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find evidence association [52].

The genetic association with PD has been analyzed in more detail. An initial case-

control study discovered a protective haplotype made of three SNPs in the NFE2L2 promoter that either delayed onset of disease (Swedish cohort with 165 cases and 190 controls) or reduced risk (Polish cohort with 192 cases and 192 controls) [53]. In a followup study, the same team extended these observations to four new independent European patient-control materials [54]. However, in a Taiwanese population (480 cases and 526

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controls) this association was not replicated [55], suggesting disparity in ethnicities and environmental factors. An alternative approach to assess gene-environment interaction was conducted in an Australian case-control study (1378 cases and 1338 controls) where PD-

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cells derived from olfactory mucosa were submitted to smoke or pesticide exposure. In this study, several susceptibility and age-at-onset modifying SNPs were identified [56]. Functional studies are now needed to explore these results further.

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Genome-wide analysis of NRF2 binding sites and its possible disease-associated regulatory SNPs with changes in NRF2 binding appears to be very informative [57]. In

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regard to neurodegenerative diseases, TAU pathology is a hallmark of AD but also of several neurodegenerative diseases including PD [58]. TAU has seven major isoforms formed by alternative splicing of exons 2, 3, and 10 and inclusion of exon 3 in the protein has been reported to be protective, decreasing both propensity to aggregation [59] and

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amyloid-beta toxicity [60]. Very interestingly, integrating genome-wide maps of AREs with disease-susceptibility loci, a highly ranked disease-risk and functional SNP has been found at the first intron of the TAU gene [61]. This SNP displayed complete linkage

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disequilibrium with a highly protective allele identified in multiple GWASs of progressive supranuclear palsy, PD, and corticobasal degeneration.

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Pharmacological regulation of NRF2. A promising therapy Both GSK-3/β-TrCP and KEAP1 are druggable targets and provide a means to

phenocopy the small increases in NRF2 activity that might provide the above mentioned genetic variations of NRF2 and AREs. GSK3 inhibitors were developed a couple of decades ago mainly in the context of AD, since GSK-3 was identified as a crucial TAU kinase. However, most biopharmaceutical pipelines have been closed nowadays and

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therefore the possibility of its intervention remains unexplored. On the other hand, the regulation of the NRF2/KEAP1 system has become a goldmine for pharmacologists and nutritionists. However, from a clinical perspective, it would be useful to distinguish

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between “redox biology” studies, aimed at providing proof-of-concept information and “redox medicine”, whose purpose is clinical translation of basic findings. Thus, regarding neurodegenerative diseases only a few compounds have been demonstrated to cross the

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blood brain barrier and provide suitable pharmaconinetics/pharmacodynamics properties to activate NRF2 in the brain. The most promising results have been obtained with:

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sulforaphane (SFN) [62], synthetic triterpenoids [63] and dimethyl fumarate (DMF). SFN has been used so far in at least 32 clinical studies of chronic diseases such as cancer, asthma, autism, schizophrenia, chronic kidney disease, type 2 diabetes, and cystic fibrosis, but not in neurodegenerative diseases. Synthetic triterpenoids, were developed to

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target NRF2 as antioxidant modulators of inflammation. Initially, their use raised serious concerns due to the deleterious effects of CDDO-methyl ester (bardoxolone methyl) in diabetic nefropathy, which were not related to NRF2 but most likely with an off-target

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alteration of endothelin signaling [64]. Once overcome the initial safety concerns, a new impulse is now given to a second generation synthetic oleanane triterpanoid RTA408 which now

being

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is

tested

in

Friedreich’s

ataxia

(https://clinicaltrials.gov/ct2/show/NCT02255435). The most successful case reported so far in targeting NRF2 is this dimethyl ester

derivative of fumaric acid. DMF crosses the gastrointestinal barrier, after which it is converted into monomethyl fumarate (MMF) [65]. Both fumaric acid esters covalently modify KEAP1, leading to nuclear accumulation of NRF2 and up-regulation of the NRF2 transcriptional signature [66]. The immune modulatory effect of DMF has been exploited in

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autoimmune diseases such as psoriasis [67], lupus erythematosus [68], asthma, and arthritis [69]. Very importantly, DMF is an approved drug for relapsing–remitting multiple sclerosis [70], becoming one of the most successful drugs ever developed by the biopharmaceutical

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industry. The success of DMF with autoimmune diseases with strong inflammatory component suggests that neurodegenerative diseases and in general chronic diseases might benefit from repositioning this drug. In a recent preclinical study of PD [22], daily oral

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gavage of DMF protected nigral dopaminergic neurons against α-SYN toxicity and decreased astrocytosis and microgliosis. This protective effect was not observed in Nrf2-/-

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mice. In vitro studies indicated that this neuroprotective effect was correlated with altered regulation of autophagy markers SQTSM1 and LC3 in dopaminergic cells, microglia and astrocytes, and with a shift in microglial dynamics toward a less pro-inflammatory and a more wound-healing phenotype. It must be noted however, that not all effects of DMF are

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mediated though inhibition of KEAP as we have found KEAP-independent activation of NRF2 in MEFs of KEAP-knockout mice. Moreover, some evidence is being accumulated to indicate that some anti-inflammatory effects of DMF might be NRF2-independent [71].

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Somatic mutations in KEAP1 and NRF2 that result in constitutive overexpression of the NRF2 signature are associated with a variety of tumors, indicating that NRF2 confers a

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selective advantage to cancer cells probably because they can stand a high level of reactive oxygen species that is generated during proliferation [72,73]. It is therefore possible that NRF2 is a double edge word that on one hand might prevent neurodegeneration but on the other might increase cancer risk [73,74]. However, at least three arguments strongly favor pharmacologic targeting NRF2 in neurodegenerative diseases: 1) the levels of NRF2 activation obtained pharmacologically are very low, in the range of 1.5 to 2 fold increase,

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compared to those of gene mutations, 2) contrary to gene mutations, pharmacologic regulation is reversible, 3) studies in humans with SFN, synthetic triterpenoids and DMF have not led to increased incidence of cancer.

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CONCLUSION

Evidence form animal models and genetic associations indicate that a pharmacologic

therapy to stop or slow progression of neurodegenerative diseases might be achieved with

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one single hit on transcription factor NRF2. This protein provides homeostatic responses

inflammatory and metabolic stress.

ACKNOWLEDGEMENTS

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against the main hallmarks of these disorders including proteotoxic, oxidative,

This paper was funded by SAF2013-43271-R of the Spanish Ministry of Economy and

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LEGEND TO FIGURE Figure 1. Regulation of NRF2 protein stability and effect on homeostatic responses in the brain. A, NRF2 protein degradation is regulated by two ubiquitinating systems: GSK-3/β-

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TrCP/Cul1 and KEAP1/Cul3 that target the Neh2 and Neh6 domains of NRF2, respectively [8]. Both systems are amenable to pharmacologic regulation. B, NRF2 activates expression of about 250 genes involved in in phase I, II and III detoxification reactions, glutathion and

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peroxiredoxin/thyoredoxin metabolism, NADPH production, fatty acid oxidation, iron metabolism, proteasomal and autophagic processes and gene regulation of other

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transcription factors [1]. The figure points those processes that may be most relevant in neurodegenerative diseases. C, neuronal damage induces microglial activation which, if not properly controlled, may lead to further neuronal damage. Pharmacological intervention

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with NRF2 activators should target both neuronal damage and microglial over-activation.

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GSK-3 inhibitor

Cul3

Cul1

GSK-3

KEAP1

b-TrCP NRF2

Neh2

MAF

ARE

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Anti-oxidant metabolism

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- NADPH - GSH - Trx - NQO1 - HO-1

C NRF2

Oxidative stress

Protein aggregation - a-SYN - TAU - APP

Neuronal damage

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- mitochondria - NOX - etc

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NRF2

Neh6

Ubiquitin

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NRF2

NRF2

Neh2

Neh6

Ubiquitin

B

SH SH SH

SH SH SH

Dimethyl fumarate other eletrophiles

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A

Autophagy degradation

- SQTSM1 - NDP52 - ULK1 - ATG5 - GABARAPL1

AntiInflammatory markers - IL4 - Sphk2 - ARE-independent inhibition of cytokine expression

ROS

NRF2

Cytokines

Inflammation

Microglial polarization

a-SYN others

- NFkB - iNOS - IL1 - COX2 - IL6 - etc - TNF

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HIGHLIGHTS: 1. NRF2 protects against oxidative, inflammatory and proteotoxic stress in preclinical models of neurodegeneration.

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2. NRF2 haplotypes associate with risk or protection against neurodegeneration.

3. Pharmacologic regulation of NRF2 is achieved by inhibiting KEAP1 and GSK3/βTrCP.

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4. Dimethyl fumarate, already in in use for treatment of multiple sclerosis, might be

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repurposed for treatment of neurodegenerative diseases.