Evaluation of nuclear factor erythroid 2-related factor 2 gene as a genic
modulator of response to oxidative stress in neurodegenerative diseases.
PhD Program Director Prof. Stefano Del Prato
Tutor:
Prof. Gabriele Siciliano
PhD Candidate:
Dr.ssa Annalisa Lo Gerfo
I ABSTRACT
To maintain redox homeostasis is imperative for normal function of the brain. This process is regulated by antioxidants, detoxifying proteins and other molecules. With age, genetic and environmental risk factors, the oxidative-redox system becomes imbalanced and oxidative stress (OS) ensues through increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The rate of ROS/RNS production eventually overwhelms our endogenous antioxidant defenses leading to the accumulation of oxidative damage such as post-translational modifications of lipids, proteins and DNA/RNA, a common feature of many neurodegenerative diseases. The oxidative modifications affect the physiological functions of these cell components and cause abnormal deposits in neurons and/or glia in diseases such us Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Although it remains hard to understand whether or no OS is the cause or effect of the disease, also because of the multifactorial nature of neuronal death and additive effects of pathogenic mechanisms on neuronal vitality along disease course, the association between oxidative damage and the disease makes therapeutic targeting of the antioxidant systems an attractive option. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a primary sensor and a master regulator of OS via its ability to modulate the expression of hundreds of antioxidant and detoxifying genes. Activation of the Nrf2-ARE pathway has shown benefits in animal models of many neurodegenerative disorders supporting the concept of developing pharmaceuticals to activate the Nrf2-ARE pathway in the brain.
Nrf2 belongs to the Cap’n’collar (Cnc) transcription factor family and is considered the “master regulator” of the antioxidant response since it modulates the expression and the coordinated induction of an array of defensive genes encoding phase II detoxifying enzymes and antioxidant proteins, such as NAD(P)H: quinine oxidoreductases (NQOs), heme oxygenase- 1 (HO-1), the glutathione S-transferase (GST) family, multidrug resistance-associated proteins (Mrps). TheNrf2 is a very unstable protein, typically present in association with its negative regulator Kelch-like ECH-associated protein 1 (Keap1), which acts as a molecular sensor of cellular redox homeostasis disturbance. Under basal condition, Keap1 retains Nrf2 in the cytoplasm, linking this transcriptional factor to the actin cytoskeleton and driving its degradation. Specifically, Keap1 acts as a linker protein between Nrf2 and the Cul3-based E3-ubiquitin ligase complex, promoting Nrf2 ubiquitination and consequent degradation by the 26S proteasome.
II This quenching interaction between the two proteins is a dynamic process controlled by specific intracellular cascades that allow for a fine-tuned regulation of inducible expression of Nrf2 target genes under OS or after exposure to toxic electrophiles. In fact, activation of Nrf2 requires its cytosolic stabilization via oxidative modification of distinct Keap1 cysteine residues and/or Keap1 ubiquitination and proteasomal degradation. It has been largely demonstrated that also Nrf2 phosphorylation facilitates its dissociation from Keap1. Therefore, several signaling pathways, such as the activation of mitogen-activated protein kinase (MAPK) cascade, phosphatidylinositol 3-kinase (PI3K), and protein kinaseC (PKC), favour Nrf2 detachment from its repressors and the consequent translocation to the nucleus. In the nuclear compartment Nrf2 forms a heterodimer with its partner small Maf and binds specific cis-acting antioxidant response element (ARE) sequences, ultimately transactivating a battery of highly inducible cytoprotective genes thus allowing cell to efficiently cope with endogenous stress and exogenous toxicants. Nrf2 has also been shown to modulate the transcription of genes promoting mitochondrial biogenesis, such as mitochondrial transcription factors (TFAM), and consequently to be directly involved in mitochondrial maintenance.
Considering the pivotal defensive role exerted by the Nrf2/ARE pathway, it is evident that the dysregulation of Nrf2-regulated genes offers a logical explanation for the direct and indirect association between OS and several neurodegenerative conditions, such as PD, AD and ALS. AD is probably the most common neurodegenerative disease, accounting for 60% to 70% of cases of dementia with nearly 44 million affected people worldwide, and although its etiology is still unclear, it is characterized by the presence of brain amyloid plaques and neurofibrillary tangles whose accumulation ultimately leads to extensive neuronal loss and progressive decline of cognitive function. They are deposits of proteins distributed throughout the brain of AD patients, particularly in the entorhinal cortex, hippocampus, and temporal, frontal, and inferior parietal lobes. Some of the major risk factors for AD are unhealthy aging in sporadic AD cases, the presence of ApoE-4 alleles in both sporadic and familial AD and genetic factors, such as mutation in amyloid precursor protein (APP) and presenilin-1 (PS1) in familial AD among others. AD brain is characterized by mitochondrial dysfunction, reactive gliosis and oxidative damage to lipids and proteins.
Growing evidence demonstrates that the AD brain is under tremendous OS. A significantly increased HO-1 expression was reported in post-mortem AD temporal cortex and hippocampus compared to aged-matched control. Additionally, an increased Nqo1 activity and expression was found in astrocytes and neurons of AD brain and Nrf2 was predominantly localized in cytoplasm
III in AD hippocampal neurons. Furthermore, there is increased protein oxidation and lipid peroxidation in AD brain when compared to aged matched controls. Recent studies in aged APP/PS1 AD mouse models showed reduced Nrf2, Nqo1, GCL catalytic subunit (GCLC) and GCL modifier subunit (GCLM) mRNA and Nrf2 protein levels.
PD affects more than 1% of the population over 60 years of age and is the second most common neurodegenerative disorder after AD. The majority of cases (90%) are sporadic, while about 10% show monogenic inheritance.
PD is caused by the degeneration of dopaminergic neurons within the substantia nigra pars compacta (SNc) and although there is still no clear explanation for the intrinsic vulnerability of these neurons, it is known that they are more prone and susceptible to OS. Data indicate that OS plays an important role in αSyn proteostasis. As the master regulator of the cellular antioxidant defense system, the Nrf2-ARE pathway is a logical target to examine for neuroprotection against misfolded proteins induced pathology.
Activation of theNrf2-ARE pathway has been shown to be protective against the toxic forms of αSyn in several studies. In SK-N-SH neuroblastoma cells, ferrous iron promotes αSyn aggregation through inhibiting Nrf2 pathway. αSyn aggregation exacerbates ferrous iron-induced oxidative damage, mitochondrial impairment and apoptosis.
Recently studies have identified the importance of astrocytic Nrf2 regulating αSyn proteostasis. Astrocytic over expression of Nrf2 (GFAP-Nrf2) can reduce αSyn aggregates in the central nervous system of a PD mouse model with neuronal over expression of human αSyn mutant A53T. These observations are not due to Nrf2-mediated down regulation of the hαSynA53T transgene levels in the mice.
ALS is a rare adult-onset neurodegenerative disease characterized by the selective degeneration of motor neurons in the motor cortex, brainstem, and spinal cord. Most of the cases (90%) are sporadic (SALS), while the remainder presents a family history (FALS). Although the exact cause of ALS is still unknown, a major step forward in the understanding of the pathogenetic events involved in ALS was provided in 1993 by the observation that mutations in the gene coding for the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1) are carried by the 15– 20% of FALS patients.
ALS is a complex and multifactorial disease characterized by the involvement of several interconnected pathogenic events, such as OS, mitochondrial dysfunction, inflammation, glutamate excitotoxicity, protein misfolding and aggregation, aberrant RNA metabolism, and altered gene expression. In particular, OS is one of the most detrimental contributors of disease
IV onset and progression. In fact, several distinctive oxidation markers have been observed in both nervous and peripheral tissues in SALS and FALS patients. Elevated protein carbonyl and 3-nitrotyrosine levels have been detected in spinal cord and motor cortex from SALS and FALS patients, particularly in large ventral motor neurons. Lipid oxidation has also been identified in motor neurons, astrocytes, and microglia of SALS patients compared to control individuals. Elevated levels of HNE have been detected also in CSF and in sera from ALS patients. Additionally, mitochondrial defects have been reported as a major hallmark in motor neuron degeneration in ALS. These dysfunctions are tightly interrelated with OS cascades, activating overlapping molecular pathways in a vicious cycle of harmful events. Notably, impairment in defensive mechanisms has also been revealed in ALS, including downregulation of members of glutathione S-transferase family, peroxiredoxins, and, in particular, the transcriptional factor Nrf2.
The aim of this research project has been to evaluate a possible association between 653 A> G, -651 G> A and -617 C> functional polymorphisms in the NRF2 promoter gene, and the respective risk of ALS, PD and AD disease and their possible implication in molecular mechanisms of cellular response to oxidative damage. In particular, the following evaluations were assessed:
• the distribution of allelic and genotypic frequencies of the three functional single nucleotide polymorphisms (SNPs) -653 A> G, -651 G> A and -617 C> A in 154 ALS patients, 172 PD patients, 240 AD patients and 186 healthy controls; genotyping was carried out by DNA direct sequencing.
• the plasma levels of some oxidative stress biomarkers in 73 ALS patients, 47 PD patients, 139 AD patients and 68 healthy controls ; in particular, as oxidative damage markers, we evaluated the protein oxidation products (AOPP) and, as non-enzymatic antioxidant markers we evaluated the Antioxidant Iron Reduction Capacity (FRAP) and total plasma thiol groups. The biomarkers levels were carried out by spectrophotometric methods
• the mRNA expression level by Real Time PCR in 13 ALS patients, 15 PD patients and 14 AD patients
• the possible association of the functional polymorphisms in the NRF2 gene promoter with the clinical features of the patients.
V
• the possible association of the functional polymorphisms in the NRF2 gene promoter with
the NRF2 transcript levels and oxidative stress biomarkers
The analysis of AD population shows that the allelic variant -653G is associated with increased risk of disease (OR 1.27 IC95% 1.01-1.59); relative to the polymorphisms -651 G> A and -617 C> A, any significant differences in the genotypic distribution and allelic frequencies of patients with AD compared to the controls has been found. The evaluation of peripheral oxidative stress biomarkers shows a significant decrease in FRAP (p<0.01) and thiol groups levels (p<0.001). We not found any
imbalance in the AOPP level of AD patients compared tocontrols. mRNA expression in individuals
carrying one (AG) or two (GG) mutated alleles of the -653 A>G SNP promoter was significantly decreased (p<0.01) compared to wild-type (AA) carriers at this position, this both for AG and GG carriers.
Analysis of PD population data shows that the allelic variant -653G is associated with increased risk of disease (OR 1.34 IC95% 1.08-1.67); with respect to the polymorphisms -651 G>A and -617 C> A, any significant differences has been found in the genotypic distribution and in allelic frequencies of PD patients compared to the controls. The evaluation of peripheral oxidative stress biomarkers shows a significant decrease in FRAP (p< 0.0001) and thiol groups levels (p<0.001). Any imbalance in the AOPP level of PD patients compared to controls has been found. mRNA expression in individuals carrying one (AG) or two (GG) mutated alleles of the -653 A>G promoter SNP was significantly decreased (p<0.01) compared to wild-type (AA) carriers at this position. Also in this case the difference was significant difference or both AG and GG carriers. Finally, also in PD patients, a correlation between -653G variant, mRNA expression level and oxidative stress biomarkers has been found.
Analysis of ALS population data shows that the allelic -653G variant is associated with increased risk of disease (OR 1.71 IC95% 1.18-2.48); in relation to the polymorphisms -651 G> A and -617 C> A, no significant differences has been found in the genotypic distribution and in the allelic frequencies of patients with ALS compared to the controls. The evaluation of peripheral oxidative stress biomarkers shows a significant increase in AOPP levels (p< 0.001) and a significant decrease in thiol groups levels (p<0.01) in ALS patients; we did not found any imbalance in the FRAP level of ALS patients compared to controls. mRNA expression in individuals carrying one (AG) or two (GG) mutated alleles of the -653 A>G SNP promoter was significantly decreased (p>0.05) compared to wild-type (AA) carriers at this position; this observed between the G non carriers and
VI G carriers, although at of genotypic levels, AG and GG. Finally, the data obtained showed a correlation between -653G variant, mRNA expression level and oxidative stress biomarkers.
The data obteined suggest that the -653G variant in NRF2 promoter gene is a common risk factor for AD or PD and ALS. This variant is always associated to decreased level of NRF2 mRNA as evaluated in peripheral lymphocytes.
All together these underlines that Nrf2-ARE pathway can be one of the molecular mechanisms commonly involved in neurodegeneration, although the different profile of alteration of redox balance indicates that the effects are different in each one of the neurodegenerative disorders.
In any case, conclusive remarks can be assumed in terms of relevance of oxidative stress events as integral part of the pathogenic complex of these diseases.
VII
1. INTRODUCTION ... 1
1.1 Oxidative stress ... 1
1.1.1 Oxidative stress biomarkers ... 4
1.1.1.1 Advanced protein oxidation products (AOPP) ... 4
1.1.1.2 Iron-reducing capacity of plasma (FRAP) ... 5
1.1.1.3 Plasma thiols (-SH). ... 6
1.2 Mitochondria: powerhouse of the cell. ... 7
1.3 Nrf2/ARE pathway ... 11
1.3.1 The Nrf2-ARE pathway and neuroprotection ... 22
1.3.2 Modifications of Nrf2-ARE pathway in neurodegenerative diseases ... 22
1.3.3 Nrf2-ARE pathway and Alzheimer’s disease ... 24
1.3.4 Nrf2-ARE pathway and Parkinson’s disease ... 25
1.3.5 Nrf2-ARE pathway and amyotrophic lateral sclerosis ... 27
2. AIM OF THE WORK ... 29
3. PATIENTS AND METHODS ... 30
3.1 Study design ... 30
3.2 Patient-control materials ... 31
3.3 Methods ... 33
3.3.1 Genotyping of -653 A>G, -651 G>A e -617 C> polymorphisms in the NRF2 gene promoter ... 33
3.3.2 RNA extraction and Real Time PCR analysis ... 35
3.3.3 Plasma oxidative stress markers. ... 36
3.3.3.1 Evaluation of advanced oxidation protein products (AOPP).... 36
VIII
3.3.3.3 Evaluation of plasmatic total thiols (-SH). ... 37
3.3.4 Statistical analyses.... 37
4. RESULTS ... 38
4.1 Genotyping of -653 A>G, -651 G>A e -617 C> polymorphisms in the NRF2 gene promoter ... 38
4.2 mRNA Expression Analysis ... 43
4.3 Plasma oxidative stress markers. ... 45
4.4 Association between -653 A>G SNP, mRNA Expression and oxidative stress biomarkers. ... 47
5. DISCUSSION ... 50
6. CONCLUSIONS ... 60
1
1.INTRODUCTION
1.1 Oxidative stress
The respiratory chain is a very efficient mechanism, but during the step of transporting electrons, it may happen that a small percentage of electrons may prematurely reduce oxygen, forming reactive oxygen species (ROS), which are potentially harmful and dangerous for the cell. ROS are ions or very small molecules that include oxygen ions, free radicals and peroxides, organic and inorganic; they are highly reactive due to the presence of unpaired electrons in the orbital outside and are formed as a natural byproduct of oxygen metabolism and play an important role in cell signaling. ROS is a collective term that includes a wide variety of oxygen free radicals, such as superoxide anion (O2-) and hydroxyl radical (OH-) but also oxygen derivatives that do not contain unpaired electrons, such as peroxide of hydrogen (H2O2) (Uttara et al., 2009).
The main source of ROS in vivo is aerobic respiration precisely, although they are also produced by the fatty acids beta-oxidation, by the xenobiotic components metabolism, after the activation of phagocytosis by pathogens. During periods of environmental stress, the ROS levels can increase dramatically, causing significant damage to cell structures. This increase is identified with the term of oxidative stress (OS) (Mancuso et al., 2008).
OS is usually defined as the altered balance between the production of ROS and their removal by cellular antioxidant mechanisms, such us enzymatic scavengers and low-molecular-weight reductants.
The term oxidative stress was introduced for the first time in 1989 by Sies, who defined it as an imbalance between the production of ROS oxygen and antioxidant defense systems. OS identifies a condition in which the normal intracellular balance between oxidizing substances, produced physiologically during metabolic processes, and antioxidant defense systems that have the function of neutralizing them is missing. This disequilibrium causes an increase in the levels of the oxidizing molecules, which, exceeding the threshold value, cause toxicity to the cell (Sompol et al., 2009). This condition could be the result either of an excessive production of ROS, or of a loss of the natural antioxidant defenses, or of both factors (Siciliano et al., 2007). Small doses of ROS are constantly produced in aerobic organisms and low concentrations can be useful in numerous biochemical processes, including cell differentiation, apoptosis, immunity
2 and defense against microorganisms (Iorio, 2007). A too high concentration of ROS are dangerous for the cell for their ability to damage biological macromolecules.
The exposure to free radicals has led the cell to develop a defense mechanisms; in physiological conditions, ROS production is counterbalanced by the antioxidant defense system, thus ensuring redox homeostasis (Rahman, 2007). Antioxidants are molecule able to slow down or prevent the oxidation of other molecules, where oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent capable of receiving them, but these reactions can produce free radicals that trigger a series of chain reactions potentially harmful to the cell (Uttara et al., 2009). Antioxidants stop these chain reactions by removing free radicals and inhibiting other oxidation reactions, oxidizing themselves.
The ROS, can be classified into two categories: the first includes free oxygen radicals (superoxide anion, the hydroxyl radical and the singlet oxygen), which contain an unpaired electron, while the second group includes non-radical species, such as hydrogen peroxide (Rahman, 2007).
Free radicals, as previously described, can be particularly harmful for the cell, attacking and damaging any biological molecule of the organism (Uttara et al., 2009 ).
However, our body has extremely effective defense systems that neutralize ROS and inhibit its oxidizing activity, called antioxidant agents.
An ideal antioxidant should be rapidly absorbed, high efficient in eliminating free radicals and chelating reduced metals, and able to perform its action in water and / or membrane domains [7]. Antioxidant agents can be classified according to their activation times in primary or enzymatic antioxidants, and secondary or non-enzymatic antioxidants (Rahman, 2007).
To the class of enzymatic antioxidants belongs a limited number of proteins such as catalase, glutathione peroxidase and superoxide dismutase, capable of detoxifying the cell from ROS through a series of reactions, in which free radicals lose their reactivity and are transformed into substances that are harmless to the cell. Non-enzymatic antioxidants, on the other hand, can be further subdivided into direct and indirect antioxidants. The former are fundamental in defense against oxidative stress and include ascorbic and lipoic acid, polyphenols and caratenoids; the same cell is able to synthesize a minimal quantity of these molecules. The latter include chelating agents able to bind metals and prevent radical formation (Uttara et al., 2009 ).
Mitochondria use most of available oxygen (85–90%) to produce ATP, but, at the same time, are the major producers of ROS, such as superoxide (O−2) and hydrogen peroxide (H2O2) principally originate by loss of electrons from OXPHOS during oxidative phosphorylation with
3 the consequent incomplete reduction of molecular oxygen (Hernandes and Britto, 2009; Orsini et al., 2006).
Superoxide itself is not greatly dangerous; nevertheless, it can rapidly react with the mild oxidant nitric oxide (NO to generate peroxynitrite (ONOO−) (Pryor and Squadrito, 1995; Mart´ınez and Andriantsitohaina, 2009).
Similarly, H2O2 is a slight oxidant but bit by bit it decomposes to generate the hydroxyl radical . Both ONOO− and damage the function of biomolecules inside the cell. Particularly, ROS attack the backbone and the side chains of proteins determining protein misfolding and aggregation. In addition, they attack nucleic acids, leading to alteration of purine and pyrimidine bases. Moreover, ROS cause lipid peroxidation, producing highly dangerous molecules, such as malondialdehyde, 4-hydroxy-2- trans-nonenal (HNE), acrolein, and thiobarbituric acid reactive substances (TBARSs) (Trachootham et al., 2008). Summarizing, OS causes several interdependent mechanisms leading to cell death. All the human body’s cells are subjected to oxidative stress, but the neurons are particularly affected by oxidative damage of aerobic metabolism. This susceptibility can be attributed on the one hand to their high oxygen requirement and on the other hand to low expression of antioxidant proteins (Halliwell, 2001). Strong production of ROS is associated with deleterious effects on neuronal cell, also exerting crucial roles in regulating specific signaling mechanisms. In particular, ROS are able to activate kinase cascade (Son et al., 2011), to regulate the calcium mobilization and signaling (Yan, et al., 2006; Feissner et al., 2009), to control the expression of antioxidant genes (Ma, 2010; Allen and Tresini, 2000), and, finally, the ROS seem to control the differentiation (Vieira et al., 2011) and neurogenesis (Kennedy et al., 2012) in neural stem cell. OS is a critical gambler in several diseases, including age-dependent neurodegenerative disorders such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). The involvement of OS in several neurodegenerative conditions has been demonstrated by the identification of pathological mutations in genes performing in antioxidant pathways as well as oxidative stress markers in patients’ samples (Halliwell, 2006; Melo et al., 2011; Gandhi and Abramov,2012). Nevertheless, in many cases it is not clear whether this kind of stress is a primary cause or downstream event associated with the progression of the neurodegeneration.
Consequently, a better understanding of ROS involvement in the pathogenesis of neurodegenerative diseases can offer the possibility to identify new targets for neuroprotective therapies.
4
1.1.1 Oxidative stress biomarkers
Oxidative stress are described as involved in many pathology.
Identifying a valid biomarker of this condition could help to understand in which diseases reactive chemical species play a causal role and therefore develop preventive strategies to delay the development of the disease.
A biomarker is defined as a quantifiable substance, particularly resistant to degradation, used as an indicator of a particular biological state, normal or pathological, or as an index of response to a pharmacological therapy (Franzini et al., 2010). According to this definition, oxidative stress markers can be considered both the oxidation products of biomolecules (nucleic acids, lipids, proteins) but also the consumption of antioxidants.
Among the markers able to provide a general Figure of the cellular redox state there are the products of advanced oxidation to proteins (AOPP), the iron-reducing capacity of the plasma (FRAP) and the plasmatic thiols (-SH).
1.1.1.1 Advanced protein oxidation products (AOPP)
Products of advanced protein oxidation (AOPP) are the products of the plasma protein reaction with the chlorinated oxidants produced by myeloperoxidase (MPO) (Noyan et al., 2006). The MPO is responsible for the production of AOPP in various ways both by the activity of 2HOCl and by the action deriving from the enzyme's own capacity.
The direct action of HOCl on plasma proteins is responsible for the production of chlorinated and chloramine proteins (Witko-Sarsat et al., 2014). Some of these intermediates have a very short half-life and are easily hydrolyzed to form aldehydes, ammonia and carbon dioxide resulting in a rapid increase in total carbonyls (Hawkins et al., 1998). The MPO, however, acting directly on tyrosine residues, contributes to the formation of the dimers of Tyr (di-Tyr) whose main addition products to the phenolic ring are 3,5-Chloro-Tyr which give rise to protein aggregation (Fu et al., 2000). Furthermore, MPO is able to convert L-serine to glycolaldehyde, which mediates the formation of carboxy-methyl-lysine, defined as a protein glycation product (AGE). In patients with chronic but non-diabetic kidney disease, an increase in AGEs was observed, which correlated with an increase in AOPPs (Witko-Sarsat et al., 2014).
5 AOPPs have been studied for the first time in patients with uremia and chronic renal failure undergoing dialysis (Witko-Sarsat et al., 2014; Witko-Sarsat et al., 1996): the dialysis membrane in fact activates neutrophils and causes the production ofa large quantities of ROS (superoxide anion, hydrogen peroxide, hydroxyl radical and hypochlorous acid) which, in view of the inability of the plasma antioxidant system to remove them effectively, induce oxidative stress. Witko-Sarsat and collaborators (1996) found in the blood of these patients high levels of oxidized proteins, distinguishable from the non-oxidized forms thanks to their different spectroscopic characteristics (deriving, for example, from the oxidation of the aromatic groups). The AOPP thus detected were distinguished in those with a molecular mass of 600 kDa and those of 60 kDa. The former are called "high molecular weight" (HMW) and correspond to albumin, which appears to form aggregates deriving from dislofure and / or cross-linking bridges due to the formation of dithyrosin. The second "low molecular weight" group is instead made up of monomeric albumin (Witko-Sarsat et al., 1996). Following these findings, a possible role of AOPPs in the pathogenesis of other diseases has been hypothesised. Their accumulation was then identified in diabetes mellitus.
1.1.1.2 Iron-reducing capacity of plasma (FRAP)
Blood is a tissue composed of cellular components suspended in a liquid called plasma (Cao et al., 1998). The plasma represents about 55% of the blood and its fundamental function is to maintain the volume of the circulating blood constant and give nutritive substances to the tissues and cells. Plasma also collect all waste substances derived from the metabolism of cells and eliminate them through the liver, intestines, kidneys and sweat. In the plasma or in the serum (plasma from which the clotting proteins have been removed) there are a series of molecules with a strong antioxidant power such as enzymes (superoxidodismutase, catalase and glutathione peroxidase); but also macromolecules such as albumin, ceruloplasmin, ferritin and a whole series of small molecules such as L-ascorbic acid (vitamin C), -tocopherol (vitamin E), carotenoids, uric acid and bilirubin (Cao et al., 1998). The antioxidant power of the serum is therefore not a simple sum of the activities of the various antioxidant substances, but a dynamic balance that is influenced by the interactions between the different antioxidant portions. This cooperation between the various antioxidant substances appears to be the greatest protection against the attack by free radicals.
6 In particular, among the molecules mentioned above, uric acid makes the greatest contribution to what is called the antioxidant power of plasma (Koracevic et al., 2001). Uric acid plays therefore a very important role within our organism, as anendogenous antioxidant. In fact, it is able to react with free radicals to form the relatively stable urate radical, which interrupts the radical reactions (Rodonev et al., 2003).
In fact, at physiological concentrations, the urate reduces the methemoglobin formed by the peroxide reaction with the hemoglobin, protects the erythrocytes against lipid peroxidation and lysis.
The methods for the global assessment of the plasma antioxidant power are based on the rationale that the reduction of the concentration / activity of one or more biochemical components responsible for the neutralization of oxidizing species in a given biological system is indicative of an alteration of the oxidative balance. Generally this type of evaluation is carried out on extracellular fluids, in particular on blood (plasma or serum), and constitutes an approach that offers numerous advantages, such as the susceptibility to changes following treatment with antioxidants. Furthermore, the simultaneous dosage of a series of antioxidants is considered a technically valid instrument because many of the components of this defense system work together in concert (Huang et al., 2005). In this work FRAP has been decoded to be used for the evaluation of the plasma antioxidant power.
1.1.1.3 Plasma thiols (-SH).
The thiols represent a qualitatively significant component of the barrier plasma antioxidant. In fact, the sulfhydryl groups of the plasma molecules, such as P-SH proteins, can oppose the propagation of the peroxidative processes by inactivating both alkoxic (RO •) and hydroxyl radicals (HO •).
Considering the event from the stoichiometric point of view, a pair of thiol groups can oxidize a pair of alkoxyl (RO •) or hydroxyl radicals (HO •), yielding to it two electrons (in the form of two hydrogen atoms). In this way both types of radicals are inactivated: the alkoxyl radicals are released as alcohol molecules while the hydroxyl radicals become harmless water molecules. The now oxidized thiol groups, on the other hand, react to each other, generating disulfide bridges. Furthermore, the sulfhydryl groups oxidize counteract the attack of some histo-lesensive free radicals, but when formed in the context of protein molecules, they can have undesired
7 consequences. For example, the formation of a disulfide bridge between the cysteine residues of two different proteins can lead to a kind of "polymerization". If the disulfide bridge, on the other hand, is formed within the same chain, the protein can permanently modify its conformation. In both cases it is possible that the proteins involved in the formation of S-S bonds undergo an alteration of their own functional capabilities. The -SH test is based on the ability of the -SH groups to develop a colored complex which can be determined photometrically when reacting with 5,5-ditiobis-2-nitrobenzoic acid (DTNB) according to the method initially proposed by Ellman in 1959 and subsequently adapted by Hu in 1994. The evaluation of the thiols allows an indirect estimation of the reduced glutathione value in plasma. This molecule, in fact, thanks to its free thiol group, is the main protective mechanism against oxidative stress, being the most powerful antioxidant produced by the body.
1.2 Mitochondria: powerhouse of the cell.
Originally, the primordial eukaryotic cells were unable to use oxygen for metabolic purposes. More than a billion years ago, according to endosymbiosis theory, these eukaryotic cells were colonized by aerobic bacteria. A symbiotic relationship developed and became permanent. The bacteria evolved into mitochondria, thus endowing the host cells with aerobic metabolism, a much more efficient way to produce energy than anaerobic glycolysis. This alliance has facilitated bacteria to the availability of metabolic substrates, now assigned to the host cell, and at the same time has made a new kind of metabolism, much more efficient, eukaryotes: aerobic or oxidative metabolism (Ernster et al.,1988). Mitochondria are cytoplasmic organelles ranging from 1 to 10 μ and are often described as “electrical control units of the cell” because they generate most cell supply of adenosine triphosphate (ATP), used precisely as a chemical energy source from cell. Mitochondria not only perform this function but are also involved in other processes, such as signaling, cell differentiation, death, and also in the cell cycle control and cell growth (Babcock and Wikstrom,1992). The number of mitochondria in a cell varies according to the type of tissue and the body; there are cells with a single mitochondrion and cells with many thousands of mitochondria; these organelles are able to move freely in the cytoplasm and tend to thicken in the points where there is a greater demand for energy.
Mitochondria (Figure 1) contain an outer membrane and an inner membrane composed by phospholipids and proteins, but with different functions; for this particular double-membrane
8 (Di Mauro et al., 2004). The outer mitochondrial membrane fully surrounds the inner membrane, with a small intermembrane space in between. The outer membrane has many protein-based pores that are big enough to allow the passage of ions and molecules as large as a small protein. In contrast, the inner membrane has much more restricted permeability, much like the plasma membrane of a cell. The inner membrane is also loaded with proteins involved in electron transport and ATP synthesis. This membrane surrounds the mitochondrial matrix, where the citric acid cycle produces the electrons that travel from one protein complex to the next in the inner membrane (Di Mauro et al., 2004).
The matrix, under an electron microscope, is similar to a homogeneous gel of granules with paracrystalline structure, formed mainly by calcium salts, ribosomes (of dimensions considerably smaller than those found in the cytosol) with a structure similar to that of bacteria and of double-stranded circular DNA molecules. It also contains numerous proteins involved in the Krebs cycle, in the beta oxidation of fatty acids and in the metabolism of amino acids.They perform numerous tasks, such as pyruvate oxidation, the Krebs cycle, and metabolism of amino acids, fatty acids, and steroids, but the most crucial is probably the generation of energy as adenosine triphosphate (ATP), by means of the electron-transport chain and the oxidative-phosphorylation system (the “respiratory chain”) (Fig. 1).
9 Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA (Mancuso et al., 2008). This genetic material is known as mitochondrial DNA or mtDNA. The human mtDNA is a 16,569-bp, double-stranded, circular molecule containing 37 genes (Fig. 2). Of these, 24 are needed for mtDNA translation (2 ribosomal RNAs [rRNAs] and 22 transfer RNAs [tRNAs]), and 13 encode subunits of the respiratory chain: seven subunits of complex I (ND1, 2, 3, 4,4L, 5, and 6 [ND stands for NADH dehydrogenase]), one subunit of complex III (cytochrome b), three subunits of cytochrome c oxidase (COX I, II, and III), and two subunits of ATP synthase (A6 and A8). Mitochondrial genetics differs from mendelian genetics in three major aspects: maternal inheritance, heteroplasmy, and mitotic segregation (Mancuso et al., 2008) .
Figure 2. The map of the human mitochondrial genome.. (Di Mauro et al., 2003)
Mitochondria can be divided by binary splitting similar to bacterial cell division, but unlike bacteria, these organelles can also merge with each other. The rules of the mitochondrial division differ among different eukaryotes; in unicellular eukaryotes, for example, growth and division are phases closely linked to the cell cycle, for example a mitochondrion can divide simultaneously with the nuclear division (Mancuso et al., 2008). In multicellular eukaryotes, in humans, for example, mitochondria can replicate their DNA regardless of the phase of the cell cycle in which the cell is located, and replicate according to the cellular energy requirement.
10 When the energy requirement of a cell is high, the mitochondria grow and divide, when the energy requirement is low, the mitochondria are destroyed or inactivated (Mancuso et al., 2008). The mitochondria, by means of the mitochondrial respiratory chain (OXPHOS), and through the process of oxidative phosphorylation, fulfill the requirements of ATP and therefore of energy of the cell (Hatefi, 1985). The respiratory chain consists of a series of electron carriers (complexes), most of which are integral proteins of the inner membrane, containing prosthetic groups associated to proteins able to accept and donate one or two electrons (Hatefi, 1985). The electron carrier complexes are four types: complexes I, II, III, and IV, in which two mobile electron carriers are to be added: cytochrome c and coenzyme Q. Respiratory chain transporters are arranged spatially in order of decreasing redox potential, so as to ensure the passage of electrons through the entire chain, when they flow from NADH or from FADH2 to O2. ATP synthesis entails two coordinated processes (Fig.3). First, electrons (actually hydrogen ions derived from NADH and reduced flavin adenine dinucleotide in intermediary metabolism) are transported along the complexes to molecular oxygen, thereby producing water. At the same time, protons are pumped across the mitochondrial inner membrane (i.e., from the matrix to the intermembrane space) by complexes I, III, and IV. ATP is generated by the influx of these protons back into the mitochondrial matrix through complex V (ATP synthase), the world’s tiniest rotary motor.
Figure 3. Schematic view of the mitochondrial respiratory chain, showing nuclear DNA-encoded ( blue) and mtDNAencoded red) subunits. Protons (H+) are first pumped from the matrix to the intermembrane space through
complexes I, III, and IV, then flow back into the matrix through complex V to produce ATP. Coenzyme Q (CoQ) and cytochrome c (Cyt c) are electron carriers. (Di Mauro et al., 2003)
11
1.3 Nrf2/ARE pathway
The transcription factor Nrf2, also known as NF-E2-related factor 2, plays a vital role in maintaining cellular homeostasis, especially upon the exposure of cells to chemical or oxidative stress, through its ability to regulate the basal and inducible expression of a multitude of antioxidant proteins, detoxification enzymes and xenobiotic transporters. In addition, Nrf2 contributes to diverse cellular functions including differentiation, proliferation, inflammation and lipid synthesis and there is an increasing association of aberrant expression and/or function of Nrf2 with pathologies including cancer,neurodegeneration and cardiovascular disease. The activity of Nrf2 is primarily regulated via its interaction with Keap1 (Kelch-like ECH-associated protein 1), which directs the transcription factor for proteasomal degradation.
Although it is generally accepted that modification (e.g. chemical adduction, oxidation, nitrosylation or glutathionylation) of one or more critical cysteine residues in Keap1 represents a likely chemico-biological trigger for the activation of Nrf2, unequivocal evidence for such a phenomenon remains elusive. An increasing body of literature has revealed alternative mechanisms of Nrf2 regulation, including phosphorylation of Nrf2 by various protein kinases (PKC, PI3K/Akt, GSK-3b, JNK), interaction with other protein partners (p21, caveolin-1) and epigenetic factors (micro-RNAs -144, -28 and -200a, and promoter methylation). These and other processes are potentially important determinants of Nrf2 activity, and therefore may contribute to the maintenance of cellular homeostasis.
Several lines of evidence in the literature suggest that the reactive chemical species and electrophilic substances can have an important role in inducing different causative mechanisms of various pathologies such as tumorigenesis, diseases affecting the cardiovascular system, central nervous system, and peripheral nervous system (Kohen and Nyska, 2002; Nguyen et al., 2003). The human body, in order to neutralize these toxic substances, has developed a plethora of defense mechanisms (Sykiotis and Bohmann, 2010). Between the several mechanisms, the Nrf2-ARE pathway is now considered the most regulator of cellular defense mechanisms against oxidative stress (Johnson and Johnson, 2015). Nrf2 be up to the Cap‘n’collar (Cnc) transcription factor family and is considered the leader of the antioxidant response since it regulates the expression of several defensive genes (Jung and Kwak, 2010; Petri et al., 2012). Nrf2, initially identified as a regulator of gene expression for beta-globins (Moi et al., 1994), in the nucleus
12 modulates the transcriptional activation of its target genes by binding to a nucleotide sequence called Antioxidant Response Element (ARE), identified for the first time in the regulatory regions of the genes encoding glutathione S-transferase (GST) and for NAD [P] H: quinone oxidoreductase-1 (NQO1) in rats and mice (Rushmore et al., 1991). ARE are enhancer containing a 5'-TGACnnnGC-3 'consent sequence present, sometimes also in multiple copies, at the level of the promoter of genes called ARE-dependent. Among the Nrf2-regulated genes we find those that code for antioxidant enzymes and for phase II detoxification enzymes, such as heme oxygenase-1 (HO-1), NQO1, catalase, SOD and proteins involved in synthesis and in the metabolism of glutathione (some proteins of the Glutathione-S-transferase family (GSTs) and the γ-glutamyl cysteine ligase (γ-GCL)) (Kundu and Surh, 2008). The involvement of Nrf2 in both basal and inducible expression of ARE-dependent genes makes it the main regulator of the antioxidant response and a modulator of several apparently independent processes, such as the immune and inflammatory response, tissue remodeling and cell proliferation (Hyberston et al., 2011).
Nrf2 belongs to a family of transcription factors of the Cap'n'Collar-basic leucin zipper type (CNC-bZIP), comprising other 3 members: p45 NFE2, Nrf1 and Nrf3 (Moi et al., 1994; Sykiotis and Bohmann, 2010). The protein contains 605 amino acids and has a molecular weight of 67.7 kDa; the analysis of the amino acid sequence allowed to identify seven domains, highly conserved along the evolutionary scale, called domains Nrf2-ECH homology (Neh) (Itoh et al, 1995). The Neh1 domain contains the CNC-bZIP motif, thus defined for the high homology with the bZIP domain of the CNC transcription factor of Drosophila melanogaster (Mohler et al, 1991); the bZIP motif consists of a basic DNA-binding region, necessary for the binding of Nrf2 with the ARE sequences, and of a structural leucine hinge region that allows the formation of the dimer between Nrf2 and the Small musculoaponeurotic fibrosarcoma proteins (Maf). Furthermore, the Neh1 domain, interacting with the ubiquitin UbcM2-E2 complex, regulates the stability of the protein (Plafker et al, 2010). The Neh2 domain negatively regulates the protein by interacting with Kelchlike erythroidcellderived protein with the CNC homology (ECH) -associated protein 1 (Keap1) through the DLG and ETGE motifs (Moi et al., 1994; Niture et al, 2014; Dinkova-Kostova et al., 2015). The Neh3 domain allows the transactivation of Nrf2 by interaction with the chromatin remodeling protein CHD6 (Nioi et al, 2005). Neh4 and Neh5 domains are also responsible for the transactivation of Nrf2 by binding with transcription coactivators such as CREB binding protein (CBP) (Katoh et al, 2001). The Neh6 domain has two regions, called DSGIS and DSAPGS, constituting a platform for the binding,
phosphorylation-13 dependent, of the β-transducing-repeat-containing protein (β-TrCP), one of the proteins involved in the degradation of Nrf2 (McMahon et al, 2004). Recently, the Neh7 domain has been identified interacting with the retinoic acid α receptor (RARa) and repressing the expression of the Nrf2 target genes (Wang et al, 2013). The main regulatory mechanism of Nrf2 has been described by Itoh and collaborators (1999) and involves Keap1 acting as transcription factor suppressor; Keap1, in fact, is a cytoplasmic protein that, by binding Nrf2 at the Neh2 domain, prevents its translocation to the nucleus and therefore access to the promoters of the genes containing the ARE elements (Itoh et al, 1999); this repression is essential when, in the absence of adequate stress stimulation, it is not necessary to induce gene activation. Keap1 thus allows to regulate the intracellular localization of Nrf2 between cytoplasm and nucleus (McMahon et al., 2004; Itoh et al, 2003; Zhang and Hannink, 2003). Keap1 presents, between the amino-terminal region (NTR) and the terminal carboxylic region (CTR), an intermediate region (IVR), a double glycine repeats domain (DGR) responsible for binding Keap1 with Nrf2 and cytoskeletal actin or with myosin VII, a Bric-a-brac domain Tramtrack Broad-complex (BTB) necessary for protein dimerization, such that two neighboring BTB domains are able to bind Cullin-3 (Kang et al., 2004; Keum and Choi, 2014) (Fig. 2).
Figure 4. Keap-1 domains (Keum e Choi, 2014)
Two models have been proposed to explain the mechanism by which Keap1 regulates the activity of Nrf2. According to the "hinge and latch" model (Fig. 3) there is, at the same time, a high affinity interaction between the ETGE motif of Nrf2 and the DGR domain of a Keap1 monomer (hinge) and a low affinity interaction between the domain DLG of Nrf2 and the DGR domain of the other monomer (latch). This last link, unlike the first one, is regulated by the presence of inductors of Nrf2 (Uruno and Motohashi, 2011). Keap1, thanks to the BTB domain, acts as an adapter for the Cullin3 / Ring Box1 E3-ubiquitin ligase complex, and its close interaction with Nrf2 allows the binding of the ubiquitin ligase E3 to the N-terminal portion of Nrf2; in this way Keap1 contributes to a constant polyubiquitination of Nrf2, at the level of lysine residues present in the ETGE and DLG domains, inducing its degradation in the
14 proteasome 26 S (Zhang et al., 2004). It has been hypothesized that in the presence of Nrf2 inducers the low affinity binding DLG-DGR is lost; this rupture creates a conformational change that interferes with the degradation process of Nrf2 proteasome-dependent. In this situation Nrf2 accumulates in the cytoplasm, translocates into the nucleus and activates the transcription of ARE-dependent genes (Keum et al., 2014).
Figure 5. Adjustment of Nrf2 dependent on Keap1 according to the "hinge and latch" model (Keum e Choi, 2014).
Recently Baird and colleagues (2014) studied the molecular interaction between Nrf2 and Keap1 in vitro. The results of this work have shown that the Keap1 / Nrf2 complex exists in two different conformations: an open conformation, in which Nrf2 is linked only to the DGR of a Keap1 monomer, through the high affinity motif ETGE, and a closed conformation in which both the DLG and the ETGE motives are linked respectively to the DGR domain of each Keap1 protein of the dimer (Baird et al., 2014). The researchers then proposed the model called "conformational cycling model" or "cyclic sequential attachment and regeneration model" (Fig. 4) according to which Nrf2, under basal conditions, binds sequentially to the Keap1-Keap1 dimer, first through the motif High affinity ETGE, then through the low affinity DLG motif. In this conformation Nrf2 undergoes ubiquitination and consequent degradation in the proteasome. The Keap1-Keap1 dimer thus, free from the bond with Nrf2, is regenerated and is therefore able to bind to the newly translated Nrf2, thus starting a new cycle.
15 Figure 6. Adjustment of Nrf2 dependent on Keap1 according to the "conformational
cycling model"; in red the ETGE domain, in blue the DLG domain (Dinková-Kostova e Ambramov, 2015).
The Nrf2 inducers promote the stabilization of an "altered" closed conformation (Baird et al., 2014); in fact, their link to the Keap1 cysteines leads to a conformational change that negatively modifies its ability to function as an adapter for the Cullin3 / Ring Box1 E3-ubiquitin ligase complex. In this condition, the newly synthesized Nrf2 is not able to bind Keap1, due to the lack of available Keap1-Keap1 dimers; Nrf2 is therefore free to move into the nucleus and activate the expression of ARE-dependent genes (Keum et al., 2014: Baird et al., 2014). The activation of Nrf2 seems to depend on mechanisms that increase the stability of the transcription factor, leading to its accumulation in the cell. The protein, in fact, under basal conditions has a short half-life which, in the presence of inducers, increases from 7-15 min to 30-100 min (Nguyen et al., 2003: McMahon et al., 2004). These inducers may be endogenous molecules, such as reactive species (ROS and RNS), prostaglandins and NO, or exogenous agents, such as heavy metals, electrophilic compounds, xenobiotics and phytochemicals, for example genistein, quercetin, curcumin and sulforafhane (SFN). Such inductors differ greatly in structure, even if they share some chemical properties; for example, they are able to modify thiol residues at the level of Keap1 cysteines by oxidation, reduction or alkylation. The chemical change in these sensor residues causes a conformational change such as to induce the dissociation of Nrf2 from Keap1 (Motohashi and Yamamoto, 2004; Wakabayashi et al., 2004). The functional significance
16 of the different cysteine residues of Keap1 was examined by site-specific mutagenesis experiments (Zhang and Hannink, 2003; Yamamoto et al., 2008); the results suggest that different chemicals can bind to different cysteine residues and thus the Keap1-Nrf2 system is able to respond to a wide range of stimuli (Suzuki et al., 2013). In particular, some cysteine residues (Cys 151, Cys 273, Cys 288) have been identified as direct sensors for electrophiles and oxidants (Fig. 5); Keap1 can therefore be considered the sensor of oxidative stress (Dinkova-Kostova and Kostov, 2012).
Figure 7. Adjustment of Nrf2 dependent on Keap1. In basal conditions Nrf2 is sequestered in the cytoplasm by a homodimer of Keap1 which facilitates ubiquitination and degradation of Nrf2 in the proteasome. In the presence of
inducers reacting with specific cysteine residues of Keap1, we obtain the release of Nrf2 and its nuclear translocation. In the nucleus, Nrf2 heterodimizes with small Maf proteins and binds to the antioxidant response
element (ARE), activating the expression of a battery of cytoprotective genes (Espinosa-Diez et al., 2015).
Nrf2 can also be modulated by other mechanisms; for example, the protein kinase C (PKC), activated by intracellular signal transducers or by the ROS itself, is able to phosphorylate the serine 40 residue of Nrf2, compromising its binding to Keap1 (Osburn and Kensler, 2008; Huang et al., 2002). The different activation pathways can therefore regulate the translocation of the transcription factor into the nucleus, within which Nrf2 binds to the ARE elements, upstream of its target genes, activating their transcription. To interact with the ARE elements, Nrf2 must form a heterodimer with one of the Small Maf Proteins. The ARE sequence is present in the promoter of a large number of genes that have in common the coding of proteins involved in
17 protection against oxidative stress (Dinkova-Kostova et al., 2015). It has been shown that under physiological conditions, Nrf2 affects mitochondrial membrane potential, on the oxidation of fatty acids, on the availability of respiration substrates (NADH and FADH 2 / succinate) and on the synthesis of ATP; under oxidative stress the activation of Nrf2 counteracts the increase of ROS production in the mitochondria (Dinkova-Kostova et al., 2015; Nioi et al., 2003). Furthermore, although the primary response to low ROS levels is modulated by the three-way co-operation, mediated by Nf-kB, AP1, and MAPK, a sudden increase in it causes activation of the Nrf2-Keap1 pathway which induces an increase in defenses antioxidants, necessary to minimize oxidative damage (Espinosa-Diez et al., 2015). Considering the key role in regulating these important cellular functions, the Nrf2 transcription factor has been studied in diseases in which oxidative stress involvement is recognized, such as neurodegenerative diseases. Studies in MS mouse models have shown that the absence of the protein, in NRF2 knockout mice, worsens the clinical phenotype (Johnson, 2010), while the Nrf2-dependent induction of antioxidant genes represses the production of IL-17 and other pro-inflammatory mediators thus exerting a neuroprotective effect (Pareek et al., 2011). It has been observed that the use of Nrf2 inducers in the diet of mice Huntington Disease (HD) models leads to upregulation of ARE-dependent genes leading to reduction of oxidative stress, improvement of motor disability and increase in longevity (Stack et al., 2010). Several studies performed on mouse models of Alzheimer Disease (AD) have shown that activation of the Nrf2-ARE pathway, mediated by the use of inducers, improve cognitive dysfunction (Kim et al., 2013), the conservation of spatial memory (Dumont et al., 2009) and reduce oxidative stress levels (Eftekharzadeh et al., 2010). It has been proven that the use of Nrf2 inducers in Parkinson Disease (PD) mouse models provides protection against oxidative insults (Siebert et al., 2009). Vargas and co-workers developed a transgenic mice strain (GFAP-Nrf2) that overexpresses the NRF2 gene by inserting it under the control of the hGFAP gene promoter, selectively expressed in astrocytes The researchers observed an increase in Nrr2 mRNA and protein in astrocytes compared to non-transgenic mice. This increase induces the high expression of two ARE-dependent genes, controlled by the transcription factor Nrf2, GCLC and GCLM, coding for the catalytic and regulatory subunits of the γ-GCL, respectively. The consequent increase in the production of this enzyme, critical for the synthesis of glutathione, leads to a 2-fold increase in total glutathione (GSH + GSSG), both of that contained within astrocytes and that released to neurons. It was also observed that the increase in antioxidant levels is associated with a better cellular response of astrocytes to oxidative stress induced by treatment with tert-butyl hydroperoxide. The researchers then created
18 a murine bitransgenic model (hSOD1G93A / GFAP-Nrf2) with mutation in SOD1, the first gene recognized as causative of familial forms of amyotrophic lateral sclerosis (ALS)and overexpression of Nrf2. The authors then compared the survival rate of motoneurons in different mouse strains: control mice, hSOD1G93A mice, hSOD1G93A / GFAP-Nrf2 mice. This analysis showed a 40% reduction in the survival of the motoneurons of hSOD1G93A mice compared to controls, attributed to still unknown mechanisms of toxicity induced by the mutated SOD1. The reduction in survival was not found in the motor neurons of bitransgenic mice. Vargas and co-workers therefore concluded that the overexpression of Nrf2 in glial cells directly increases resistance to oxidative stress in astrocytes and indirectly, through increased secretion of glutathione, the ability of motoneurons to neutralize the toxic effects caused by the mutated SOD1(Vargas et al., 2008). In another study, the analysis of Nrf2 and Keap1 expression was performed in autopsy samples of primary motor cortex and spinal cord of ALS patients. The results showed a reduction of Nrf2 mRNA and of the protein within the neurons of the patients compared to the controls, while no changes were found in the levels of the Keap1 mRNA and of the protein itself (Sarlette et al., 2008). Furthermore, colocalization of Keap1 was observed within intracellular inclusions present in motoneurons of patients with ALS (Tanji et al., 2013). Nrf2 is a very unstable protein, typically present in association with its negative regulator Kelch-like ECH-associated protein 1 (Keap1), which acts as a molecular sensor of cellular oxidative stress. Under basal condition, Keap1 restrains Nrf2 in the cytoplasm leading to its degradation. Particularly, Keap1 acts as a connection protein between Nrf2 and the Cul3-based E3-ubiquitin ligase complex, promoting Nrf2 ubiquitination and consequent degradation by the 26S proteasome (Kobayashi and Yamamoto, 2005; Villeneuve et al., 2010). Activation of Nrf2 involves its cytosolic stabilization; specific cysteine residues (Cys 151, Cys 273, and Cys 288) have been identified as direct sensors for electrophiles and oxidants; chemical modifications in these sensor residues cause a conformational change that produce the dissociation of Nrf2 from Keap1. Nrf2, detached from his repressors, translocates to the nucleus. Here, Nrf2 heterodimerises with small masculoaponeurotic fibrosarcoma (Maf) proteins which in turn facilitate the binding of Nrf2 to the Antioxidant Response Element (ARE), a cis-acting enhancer sequence (TCAG/CXXXGC) in the promoter region of Nrf2-regulated genes (Son et al., 2011; Yan et al., 2006). These Nrf2-regulated genes can be classified into phase II xenobiotic metabolizing enzymes antioxidants, molecular chaperones, DNA repair enzymes, and anti-inflammatory response proteins (Feissner et al., 2009) and they reduce reactive compounds such as electrophiles and free radicals to less toxic intermediates whilst increasing the ability of the
19 cell to repair any damage ensued. Importantly, Nrf2 has been shown to possess an ARE sequence within its own promoter region providing a platform for Nrf2 to initiate its own transcription further enhancing the adaptive cell defence response (Son et al., 2011). Following its nuclear import, Nrf2 recruits transcriptional machinery to effectively transactivate the ARE-driven genes. This machinery includes co-activators such as receptor associated coactivator (RAC3) which initiates the transactivation domain of Nrf2 whilst the presence of other co-regulators such as CREB binding protein (CBP), coactivator-associated arginine methyltransferase (CARM1) and protein arginine methyl-transferase (PRMT1), further enhance the ability of RAC3 to initiate the transactivation domain (Ma, 2010). What is evident, however, is that Nrf2 plays a major role in health and disease and it is not surprising that Nrf2 is considered to be a potential therapeutic target. The development of a number of Nrf2 inducers as possible pharmacological agents without a complete knowledge of the workings of this pathway and its regulation heightens the need to further our understanding and to determine whether activation of Nrf2 would be beneficial in both the shortand long-term.
The protein primarily responsible for the regulation of Nrf2 is Kelch-like ECH-associated protein 1 (Keap1), which forms a homodimer responsible for sequestering Nrf2 in the cytosol, thereby rendering it inactive. It is an association between Keap1 and the actin cytoskeleton which prevents this complex entering the nucleus, limiting basal activity of the transcription factor (Jung and Kwak, 2010). Additionally, Keap1 facilitates the Cul3-mediated poly-ubiquitination of Nrf2 leading to its proteasomal degradation. Whilst Keap1 seems to be the major mechanism by which Nrf2 levels are tightly controlled in the cell, recent research would suggest that the pathway is highly complex and supports a multi-faceted defence system.
The NRF2 (or NFE2L2) gene (gene ID: 4780) maps on chromosome 2q31.2 and consists of five exons and four introns (Marzec et al., 2007). It is considered an evolutionarily conserved gene given the high sequence homology found in many species. NRF2 is highly polymorphic presenting a mutagenic frequency of 1 base every 72 bp; numerous gene variants were found in different ethnic groups in the coding region of the gene, in the introns and in the promoter region (Cho et al., 2015). Various studies have evaluated the possible relationship of these polymorphic variants with pathologies linked to oxidative stress, given the function of Nrf2 in inducing cellular antioxidant response. Regarding the coding region of the gene, a single variant in exon 1 has been associated with the risk of developing chronic obstructive pulmonary disease (COPD) (Hua et al., 2010). Particular attention was paid to the SNPs present in the introns and in the promoter region, since they could be involved, respectively, in the alteration of mRNA splicing
20 and in the transcriptional regulation of the gene (Cho et al., 2015). In particular, three single nucleotide polymorphisms (SNPs), present in the gene promoter, have been studied; these SNPs consist of a basic change of cytosine → adenine (C> A), guanine → adenine (G> A) and adenine → guanine (A> G) respectively in position -617 (rs6721961), -651 (rs6706649) and - 653 (rs35652124) from the beginning of the transcription site [87]. The Ensembl browser provides the frequencies of the minor alleles (MAF) of the three SNPs -653A> G, -651C> A and -617C> A, estimated respectively 0.38, 0.06 and 0.15 and reports the linkage disequilibrium (LD) data relative to the SNPs using information from the "1000 Genomes" project. The data show a strong LD between SNPs -653A> G and -651C> A (D '= 1.000 / r2 = 0.056) and between -653A> G and -617C> A (D' = 0.999 / r2 = 0.056 ) in the Tuscan population; our study sample has the same geographical provenance. Marzec and collaborators have demonstrated the localization of these polymorphisms using the Electrophoretic mobility shift assay (EMSA). SNP -617 C> A is located at the level of the ARE-like element, a binding site for Nrf2, responsible for the self-regulation of the NRF2 gene itself; the SNPs -651 G> A and -653 A> G are located in the stress-response element (StRE) region where the myeloid zinc finger-1 transcription factor (Mzf1) is bound (Marzec et al., 2007). Using Transciption Factor Motif Analysis (TRASFAC), it was observed that nucleotide changes are able to modify the stability of binding of transcription factors to the respective target sequences; indeed, changes in sites -653 / -651 and -617 could alter the consensus recognition sequence for Mzf1 and for Nrf2 respectively and influence the transcription of NRF2. To determine if the presence of polymorphic variants were able to influence gene expression, the authors used the luciferase reporter gene, transfected into pulmonary epithelial cells. They observed a 4-fold gene expression when its transcription was under control of the wild-type NRF2 promoter compared to a construct with the deletion promoter of the three SNPs. Furthermore, the basal luciferase activity was superior with the wild type promoter compared to the expression obtained with 3 different promoters, each bearing a single polymorphic variant (-653G, -651A or -617A) (Marzec et al., 2007). A similar study was conducted by Marczak and collaborators (2012) on endothelial cells obtaining similar results. The authors have also observed, by stimulating the cells with different substances such as H2O2, an increase in activity, compared to basal conditions, both when the promoter is of the wild-type type, and when it presents one of the three polymorphic variants. Moreover, gene expression was generally attenuated in the constructors of one of the three variants with respect to the wild-type construct, both in basal conditions and, to a greater extent, in induced stress (Fig. 9) (Marczak et al., 2012).
21 Figure 8. Localization of SNPs -653A> G, -651G> A and -617C> A in the NRF2 gene promoter; luciferase activity
with the different constructs of the NRF2 promoter, both in basal conditions (vehicle) and in stress induced with various substances (Marczak et al., 2012).
According to the authors, the polymorphic variants influence the transcription of NRF2 leading to lower protein production with consequent attenuation of the transcription of its ARE-dependent target genes (Marczak et al., 2012; Marzec et al., 2007). These data contrast with those reported by the Genotype-Tissue Expression portal (GTEx, http: //www.gtexportal.org, Broad Institute), a database created to evaluate how genomic variants can modify gene activity. To this end, the GTEx Consortium has proposed to associate the genome-wide association studies (GWAS) with the RNA sequencing of more than 50 post-mortem tissues of over 400 donors. The data reported by GTEx do not show significant variations in the expression of the NRF2 gene evaluated in the various tissues, as a function of the SNPs -653A> G, -651G> A and -617C> A (GTEx Consortium, 2015). Based on the evidence of the Marzec and Marczak groups respectively, several studies have evaluated the role of the three polymorphisms present in the NRF2 promoter in some diseases for which a pathogenic role of oxidative stress has been hypothesised. These variants were associated with the onset or clinical phenotype of lung diseases (Marzec et al., 2007; Shaheen et al., 2010), cardiovascular disease (Marczak et al., 2012), breast carcinoma (Hartikainen et al., 2012), gastrointestinal diseases (Arisawa et al., 2008) and neurodegenerative diseases (Von Otter et al., 2010; Von Otter et al., 2014; Bergström et al., 2014). In particular, studies conducted on patients with PD have found a protective effect
22 of the GAGCAAAA haplotype, due, according to the authors, to the simultaneous presence of the common alleles at the three functional promoter polymorphisms (AGC): this haplotype was associated with a lower risk of developing PD in both a Polish and a Swedish population and, in the latter, it also seems to delay the age of onset of 4.6 years (Von Otter et al., 2010). Subsequently, the same researchers in a meta-analysis study, extended to Italian, Maltese and German patients, confirmed the protective effects of the GAGCAAAA haplotype and identified four individual SNPs associated with the age of onset of the disease; specifically, the allelic variant -653G anticipates the onset of the 1.1-year PD for allele (Von Otter et al., 2014).Finally, Bergstrom and collaborators (2014), in a cohort of Swedish patients with SLAs, have shown that the GAGCAGA haplotype is associated with a 4-year delay in the age of onset of the disease, confirming the important role of Nrf2 also in this pathology.
1.3.1 The Nrf2-ARE pathway and neuroprotection
The protective effect of Nrf2 against neurodegeneration due to oxidative stress has been well studied. The Nrf2 transcription factor induces the expression of a variety of cytopreventive and detoxification enzymes, which will confer protection in neurodegenerative disorders. The target genes of Nrf2 have been involved in the regulation of glutathione (GSH), antioxidant proteins/enzymes, drug-metabolising enzymes or drug transporters, proteasome subunits, pentose phosphate pathway enzymes and enzymes involved in nucleotide synthesis. The central nervous system is sensitive to oxidative stress, which confers pathological features, including the accumulation of aberrant protein aggregates, microglial activation and mitochondrial dysfunction. These pathological processes will generate ROS, which in turn, cause oxidative stress and damage to lipids, proteins and DNA. These pathophysiological events encompass a wide variety of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS).
1.3.2 Modifications of Nrf2-ARE pathway in neurodegenerative diseases
Abnormalities of Nrf2-ARE pathway were observed in several models of disease-aging dependent and in degenerative disorders; changes of Nrf2-ARE pathway cause ROS accumulation and therefore increase of oxidative damage to biological macromolecules. Several
