Glutathione transferases and neurodegenerative diseases

Neurochemistry International 82 (2015) 10–18

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Neurochemistry International j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n c i

Review

Glutathione transferases and neurodegenerative diseases Anna Paola Mazzetti, Maria Carmela Fiorile, Alessandra Primavera, Mario Lo Bello * Department of Biology, University of Rome Tor Vergata, Rome, Italy

A R T I C L E

I N F O

Article history: Received 22 July 2014 Received in revised form 23 January 2015 Accepted 27 January 2015 Available online 7 February 2015 Keywords: Oxidative stress Parkinson’s disease Alzheimer’s disease Glutathione Glutathione transferases

A B S T R A C T

There is substantial agreement that the unbalance between oxidant and antioxidant species may affect the onset and/or the course of a number of common diseases including Parkinson’s and Alzheimer’s diseases. Many studies suggest a crucial role for oxidative stress in the first phase of aging, or in the pathogenesis of various diseases including neurological ones. Particularly, the role exerted by glutathione and glutathione-related enzymes (Glutathione Transferases) in the nervous system appears more relevant, this latter tissue being much more vulnerable to toxins and oxidative stress than other tissues such as liver, kidney or muscle. The present review addresses the question by focusing on the results obtained by specimens from patients or by in vitro studies using cells or animal models related to Parkinson’s and Alzheimer’s diseases. In general, there is an association between glutathione depletion and Parkinson’s or Alzheimer’s disease. In addition, a significant decrease of glutathione transferase activity in selected areas of brain and in ventricular cerebrospinal fluid was found. For some glutathione transferase genes there is also a correlation between polymorphisms and onset/outcome of neurodegenerative diseases. Thus, there is a general agreement about the protective effect exerted by glutathione and glutathione transferases but no clear answer about the mechanisms underlying this crucial role in the insurgence of neurodegenerative diseases. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The term oxidative stress (OS) refers to an impairment of intracellular equilibrium between oxidant species, produced by the oxidative metabolism and their efficient removal exerted by the antioxidant defense system. When the oxidant species, e.g. the reactive oxygen species (ROS), are increasing and/or the antioxidant species are diminished there is oxidative stress. Human tissues show varying

Abbreviations: OS, oxidative stress; ROS, reactive oxygen species; CNS, central nervous system; PD, Parkinson’s disease; AD, Alzheimer’s disease; GSH, reduced glutathione; GST, glutathione transferase; GSSG, glutathione disulfide; SN, substantia nigra; BSO, L-buthionine-(S,R)-sulfoximine; CSF, cerebrospinal fluid; MPTP, 1-methyl4-phenyl-1,2,3,6, tetrahydropyridine; JNK, Jun Kinase; MCI, mental cognitive impairment; 4-HNE, 4-hydroxy-2-nonenal; MRP1, multidrug resistance protein 1; GPx, glutathione peroxidase; CDK-5, cyclin dependent kinase-5; RBC, red blood cells; SOD, superoxide dismutase; GCS, gamma glutamyl cysteinyl synthetase; GS, glutathione synthetase; BBB, blood–brain barrier; MRS, magnetic resonance spectroscopy; ADDS, antioxidant and detoxifying defense system; MAPEG, membrane-associated proteins in eicosanoid and glutathione metabolism; PG, prostaglandin; CLIC, chloride intracellular channel proteins; SAPK, stress-activated protein kinase; TRAF2, TNF receptor-associated factor 2; NS, nitrosative stress; GR, glutathione reductase; Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor-erythroid 2 p45-related factor 2; NOS, nitric oxide synthase; ARE, antioxidant responsive element; Prx1, peroxiredoxin 1; APP, amyloid precursor protein. * Corresponding author. Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, Rome 00133, Italy. Tel.: +390672594375; fax: +39062025450. E-mail address: [email protected] (M. Lo Bello). http://dx.doi.org/10.1016/j.neuint.2015.01.008 0197-0186/© 2015 Elsevier Ltd. All rights reserved.

degrees of susceptibility to OS. The central nervous system (CNS) is highly sensitive to OS because of low antioxidant enzyme levels, high content of oxidant substrates and the formation of many ROS (Morozova et al., 2007; Polidori et al., 2007). Neurons are among the most active cells in the oxidative metabolism demanding a fine equilibrium between supply and consumption of both glucose and oxygen. The appearance of OS is a consequence of a disequilibrium between production of ROS and the cell antioxidant defense (Ciccone et al., 2013; Smeyne and Smeyne, 2013) with potential damage to membrane (lipid peroxidation), DNA and mitochondrial complex I (Dringen et al., 2000; Spencer et al., 1994; Sugawara and Chan, 2003). Many studies suggest a crucial role for OS in the first phase of aging (Jha and Rizvi, 2009), or in the pathogenesis of various diseases including neurological ones (Arning et al., 2004; Butterfield et al., 2007; Marco et al., 2004; Markesbery, 1997; Raichle and Gusnard, 2002). 2. Glutathione and oxidative stress in neurodegeneration There is substantial agreement that the unbalance between oxidant and antioxidant species may affect the onset and/or the course of a number of common diseases including Parkinson’s (PD) and Alzheimer’s (AD) diseases. Both these diseases are considered multifactorial pathologies and a number of events may occur simultaneously after OS including mitochondrial dysfunction, accumulation of mutations in mitochondrial DNA, DNA repair

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impairment (Ciccone et al., 2013), alteration of glutathione (GSH) metabolism and GSH related enzymes (Smeyne and Smeyne, 2013). However, a clear overview of a multiple connection between OS and neurodegeneration is beyond the scope of this article as it deals mainly with GSH and glutathione transferases (GSTs). 2.1. Parkinson’s disease(PD) PD is an age-related neurodegenerative disease clinically associated with impairment of motor and cognitive functions. Despite the etiology of this disease being unknown, all PD patients show in the central and peripheral nervous systems the so-called “Lewy bodies” which are intracellular aggregates of misfolded α-synucleine (Jenner et al., 1992; Owen et al., 1996). The most characteristic anatomic lesion is the loss of dopaminergic neurons located in the pars compacta of substantia nigra (SN). This region is particularly sensitive to OS probably because of the presence of endogenous dopamine, iron and neuromelanine. Moreover, it is apparent that in this area the antioxidant and detoxifying defense system (ADDS) (Fig. 1) is weak because of low levels of GSH (Pearce et al., 1997; Sian et al., 1994) and low activity of γ-glutamylcysteine synthetase (GCS), the enzyme responsible for the de novo synthesis of GSH (Kang et al., 1999). The analysis of SN, in the post-mortem brain of PD patients, has revealed an increase of lipid peroxidation, in particular of malonaldehyde and lipid hydroperoxides along with a substantial reduction of GSH levels. This seems to be specific to this area and this pathology since there is no change in GSH in other areas of the brain or in other neurodegenerative diseases (Di Monte et al., 1992). The association between depletion of GSH and PD onset has been confirmed by in vitro studies carried out in murine cells of neuroblastoma (NS20Y). Treatment of these cells for different times with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GCS, depletes GSH and is responsible for cell death (Andersen et al., 1996). Depletion of GSH, in cells treated with BSO, is accompanied by loss of intracellular connections and of neurites, changes of morphology of the cellular body, reduction of the total cell number,

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block of cell proliferation, DNA fragmentation and eventually cell apoptosis (Nicole et al., 1998). In a genetic study of a Drosophila model of PD it was shown that parkin mutants produced a neurodegenerative phenotype enhanced by loss-of-function mutations of GSTs. Overexpression of these latter genes in these neurons suppressed neurodegeneration and suggested a protective effect of these antioxidant enzymes (Whitworth et al., 2005). In a murine cellular model (HT22 murine hippocampal cells) GSH was depleted by homocysteic acid treatment, at the same time there was an increase in the levels of mRNAs encoding three different proteins: heavy and light ferritin and GST (Morozova et al., 2007). In a search for proteins involved in PD progression by using proteomic approach on postmortem samples of PD patients it was found that GST P1-1 is intimately associated with several critical cellular processes directly related to PD progression (Shi et al., 2009). When comparing postmortem ventricular cerebrospinal fluid CSF from PD and normal control subjects by using two dimensional gel electrophoresis (2D-DIGE) six proteins were found to be different in PD individuals in respect to control ones. Among them there was GST P1-1 and apolipoproteins E and A1 (Maarouf et al., 2012). The molecular mechanisms underlying the regulation of Jun Kinase (JNK) activity under stress conditions were studied in C57BL/6 wild type and GSTP knockout mice, both treated with 1-methyl-4-phenyl1,2,3,6, tetrahydropyridine (MPTP), a potent neurotoxin. The results showed that GSTP knockout mice were more susceptible to the neurotoxic effects of MPTP and indicated that in vivo GST P may act as an endogenous regulator of stress by controlling JNK activity (Castro-Caldas et al., 2012). In addition to GSTs, many other GSHrelated enzymes have been connected with antioxidative defense in the brain and with OS. Especially GSH peroxidases (GPx) are here important because of their role in removing hydroperoxides or by balancing H2O2 homeostasis in signaling cascades (Brigelius-Flohé and Maiorino, 2013). Also the peroxidase activity of some GST isoforms (e.g. class Alpha GST) should be mentioned (Hurst et al., 1998) and in general the antioxidant function of GSTs in the removal of short-lived lipid hydroperoxides that break down to yield

Antioxidant and Detoxifying Defense System

B

A

NO donor binding protein

Cys 1-Prx C

GST P1-1 monomer

GSH

D

GST Z1-1

Fig. 1. GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD (Garcia-Garcia et al., 2012) and AD (Saharan and Mandal, 2014). This picture emphasizes the concept that GSH homeostasis and its cellular functions are mediated by a number of different enzymes involved in the ADDS (Carvalho et al., 2014; Satoh et al., 2014). A. Cys 1-Peroxiredoxin (Cys 1-Prx) removes peroxides by the aid of GSH/GST system (Manevich et al., 2003). B. GST (NO donor binding protein) binds tightly NO carriers at the active site (Cesareo et al., 2005). C. Surface representation of GST P1-1 monomer with bound GSH (courtesy of Prof. Michael Parker). D. GST Z1-1 utilizes GSH for the isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine degradation pathway (Polekhina et al., 2001).

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secondary electrophiles, including epoxyaldehydes, 2-alkenals, 4-hydroxy-2-alkenals (Hubatsch et al., 1998; Prabhu et al., 2004; Yang et al., 2002). In the context of OS and neurodegeneration oxidation of catecholamines yields aminochrome, dopachrome, noradrenochrome, and adrenochrome that are harmful because they can produce O-2 by redox cycling. These quinone-containing compounds can be conjugated with GSH through the actions of GSTs, a reaction that prevents redox-cycling. O-quinones formed from dopamine can also be conjugated with GSH by GST, and this reaction is similarly thought to combat degenerative processes in the dopaminergic system in human brain (Dagnino-Subiabre et al., 2000). 2.2. Alzheimer’s disease (AD) AD is an age-related disease, among the most common causes of dementia and is clinically associated with impairment of cognitive functions, language deficit, loss of memory and change in behavior. The mental cognitive impairment (MCI) is considered an intermediate step between the normal process of aging and AD. People with MCI show a slow decrease of cognitive functions without signs of dementia. Some of them may maintain this condition while others may develop AD (within 10–20% of all cases). The possible role of GSTs in the etiology of AD disease has been studied by Lovell et al. (1998) in post-mortem samples of AD patients, in comparison with healthy subjects of the same age. They found that GST activity is significantly decreased in all areas of brain of AD patients, particularly in the amygdala, hippocampus and parahippocampal gyrus, inferior parietal lobule and nucleus basalis of Meynert. A significant decrease was also found in the ventricular CSF. Studies in vivo conducted in the synaptosomal fractions of rats treated with strong oxidants and N-acetyl cysteine, a well known precursor of GSH, suggested that elevation of endogenous GSH in neurodegenerative diseases associated with OS may be promising (Pocernich et al., 2000). 4-Hydroxy-2-nonenal (4-HNE), a product of lipid peroxidation, is increased in AD. In the rat cerebral cortex and especially in synaptosomes, under lipid peroxidation, there was a slight increase of GSTs Mu and Pi as well as in AD patients and controls (Sidell et al., 2003). To better define age at onset of AD and PD diseases gene expression studies were performed in the hippocampus obtained from AD patients and controls. Further allelic association studies of four genes differently expressed in AD and control (CTR) revealed that only the GST O1 gene is significantly associated with age and onset of disease. This is consistent with the function of this GST linked with inflammatory processes playing a role in these neurodegenerative diseases (Li et al., 2003). In other postmortem samples of AD brain 4-HNE was found covalently linked to GST A4-4 and multidrug resistance protein 1 (MRP1) (both involved in efficient removal of lipid peroxidation products) (Sultana and Butterfield, 2004). In human postmortem frontal cortex from individuals with MCI, mild AD and late onset of AD (LOAD) respectively, in comparison with healthy subjects, there was a significant decline in antioxidants (GSH, GST, GPx etc.) at the synapses level (Ansari and Scheff, 2010). GST P1-1, as a critical regulator of a cyclin dependent kinase-5 (CDK5) activity, was proposed to effectively modulate CDK5 signaling, eliminate OS and prevent neurodegeneration in human AD brains (Sun et al., 2011). In search for a possible association between OS and MCI a study of possible biomarkers of lipid peroxidation and enzymes of antioxidant defense was conducted in three different populations (MCI, mild AD and healthy aged subjects). Despite an increase in peroxidation and changes of some enzymes there was no clear answer, probably, because of the small size of these groups (29–30 individuals each) (Torres et al., 2011). In another study carried out on erythrocytes (RBC) of elderly people, in comparison with AD patients, it was found that OS was increased in both aging and dementia and exhibited elevated concentrations of hydroperoxides along with a decrease of both GSH/

GSSG ratio and GST activity. However, alterations of these parameters of OS were considered not specific and interpreted as age-related abnormalities (Kosenko et al., 2012). During the aging process, elevated OS levels and decreased antioxidant functions were found in the human hippocampal and frontal cortex and this may render these areas more susceptible to neuronal degeneration (Salminen and Paul, 2014; Venkateshappa et al., 2012). 3. Glutathione The species involved in the defense mechanism include GSH, a molecule responsible of detoxification of many noxious and dangerous compounds (Meister and Anderson, 1983). This biological compound is one of the most efficient and abundant antioxidant species present inside the cell where its concentration ranges between 1 and 10 mM (Dringen, 2000). Inside the cerebrum GSH levels are lower than in other tissues because high concentrations of its precursors can be toxic (Martin and Teismann, 2009). Many post-mortem studies found that GSH content is lower (about 40%) in the hippocampus and cortex than in other regions of the brain of PD patients (Pearce et al., 1997; Sian et al., 1994). Previously, GSH was considered to be mainly localized in the cytosolic compartment, but it has recently been found to be present also in the nuclei (García-Giménez et al., 2013) and mitochondria (Marí et al., 2013) where it plays important antioxidant functions. As the most abundant non-protein thiol it undergoes a series of reactions involving the sulfur atom of the cysteinyl moiety including the redox change between the oxidized and reduced status (GSSG/2 GSH). This ratio is considered a good index of OS. The defense mechanisms exerted by GSH are well known (see for a concise review: Dickinson and Forman, 2002) and are summarized as follows: 1) As an antioxidant agent, GSH may undergo a series of nonenzymatic reactions based on thiol–disulfide exchange. It is substrate in the reactions catalyzed by GPx and GST enzymes; it is involved also in the mixed disulfide exchange with proteins (the reaction is called glutathionylation of proteins), possibly not enzymatically (Popov, 2014) (Fig. 1). Finally, it can act as ROS scavenger by a direct reaction with radicals and the subsequent involvement of superoxide dismutase (SOD). The easiest way to maintain the optimal concentration of GSH is by restoration of reduced form from disulfide. However, when GSH is depleted through detoxification processes or by GSSG excretion (Trauner et al., 1997), the replenishment of GSH may be accomplished mainly by de novo synthesis of GSH. 2) The enzymatic synthesis of GSH occurs in two subsequent demanding energy reactions. The first is between glutamic acid and cysteine and is governed by GCS. The second reaction is between the new formed di-peptide (gamma-glutamyl cysteine) and glycine; this reaction gives rise to GSH and is catalyzed by glutathione synthetase (GS). The committed step occurs during the first reaction, the regulation of GCS being critical for GSH homeostasis. 3) The most relevant notion about the intracellular GSH content is that its concentration is highly responsive to environmental factors including physical stimuli (temperature, low wave radiations), heavy metals, glucose, exogenous compounds (xenobiotics), etc. All these factors may induce the enzymatic synthesis of GSH by acting on the gene coding for GCS. There is a signaling pathway which is obviously activated when the above stimuli are also produced inside the cell (e.g. lipid peroxides products, increase of ROS) (Farr et al., 2014; Hayes et al., 2005). 4) The concentration of GSH in the brain is typically around 1–2 mM, several hundred times greater than seen in the CSF, which is ~4 μM, and in the blood, which is ~2 μM (Aoyama

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and Nakaki, 2013). To maintain this rather disparate concentration ratio, active intracellular GSH synthesis is required. It has been suggested that GSH import from the blood into the brain occurs through a transporter in brain capillaries and endothelial cells; however, the concept of transport of the intact GSH molecule through the blood–brain barrier (BBB) is controversial and the characterization of such a transporter is limited (Carvalho et al., 2014). Therefore, it is debatable whether direct uptake can significantly contribute to GSH levels in brain cells; and probably, the major source of GSH within the brain is re-synthesis, through recycling of its constituents. 5) GSH metabolism in the brain has been reviewed on the basis of studies carried out mainly in culture cells and animals models (Dringen et al., 2000). In the brain, GSH is synthesized by both astrocytes and neurons, and the former appear to contain higher GSH levels than neurons both in vivo and in culture (Dringen, 2000). These two types of cells prefer different extracellular precursors for glutathione and a hypothesis of metabolic interaction between them has been made suggesting that astrocytes may confer protection to neurons against ROS-mediated toxicity also in vivo (Dringen and Hirrlinger, 2003; Muyderman et al., 2004). It is relevant to observe also that de novo synthesis of GSH in the brain depends on the precursor’s availability. While intracellular glutamate and glycine are present at higher level of their Km values toward GCS and GS, respectively, the level of intracellular cysteine is around its Km value, and therefore its concentration is considered the rate-limiting factor for GSH synthesis in the brain (Aoyama and Nakaki, 2013; Dringen et al., 2014). In another study, using postmortem samples, synaptosomal and mitochondrial fractions exhibited lower antioxidant activity compared to cytosol in the frontal cortex from control brains. This indicates the susceptibility of synaptosomes against oxidative damage and that elevated GSH content and related enzyme activities in the frontal cortex of PD brains, compared to controls, might contribute to neuroprotection in PD (Harish et al., 2013). 6) Age-dependent changes in the brain which impact on the overall quality of life are strongly related to GSH changes (Currais and Maher, 2013). Further research focused on better understanding how age affects GSH homeostasis, with a particular emphasis on the key transcription factors involved in GSH metabolism, is needed (Farr et al., 2014). In a very recent review, Saharan and Mandal (2014) discussed the putative role of GSH in AD pathology and explored its potential as an early biomarker for AD. Using a noninvasive magnetic resonance spectroscopy (MRS) technique which allows for quantitation of GSH in a specific region of brain it was possible to establish a more causative association between AD onset/ progression and GSH changes. The potential of GSH as an in vivo biomarker, in other neurological diseases, along with its therapeutic use is also discussed by Carvalho et al. (2014). Therefore, depletion of GSH and the regulation of some GSH related enzymes can be critical events in the physiology and pathology of neuronal cells. Martin and Teismann (2009) consider GSH depletion as one of the most important and early biochemical changes in PD insurgence, then suggest that it is not only a consequence of OS but could also play an active role in PD pathogenesis. PD is considered to be a result of multifactorial processes including oxidative stress, inhibition of mitochondrial complex I, ubiquitin– proteasome dysfunction, and inflammation. Glutathione depletion can inhibit complex I, E1 ubiquitin ligase (E1), and proteasome activity. It can also exacerbate oxidative stress and activate the JNK pathway, leading to an inflammatory response.

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4. Glutathione transferases GSTs are ubiquitous enzymes belonging to the GSH-mediated Antioxidant and Detoxifying Defense System (ADDS) (Fig. 1) (Hayes et al., 2005). A well known function of GSTs is to promote the conjugation of the sulfur atom of glutathione to an electrophilic center of endogenous and exogenous toxic compounds, thereby increasing their solubility and excretion (Habig and Jakoby, 1981). A possible role as ligandins has been proposed also (Litwack et al., 1971). 1. GSTs are grouped into four structurally distinct enzyme families (cytosolic, mitochondrial, membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) and fosfomycin resistance proteins (Board and Menon, 2013; Hayes et al., 2005). Each family is subdivided in classes and inside each class there may be different isoforms. The most studied superfamily is that of the cytosolic enzymes sub-divided, on the basis of their N-terminal amino acid sequence, into at least seven classes (Alpha, Mu, Pi, Teta, Sigma, Zeta, Omega) each class being further subdivided into different isoforms. While among the classes the degree of homogeneity is low (about 27%), inside each class the degree of homogeneity between the isoforms is about 80–90%. Most of these enzymes are composed of two identical subunits and show a common fold despite a low degree of similarity. The letter accompanying the achronymus (GST) indicates the class and the number identifies the subunit, e.g. GST P1-1, GST of Class Pi with two identical subunits. Each subunit contains a complete active site composed of one site of binding for GSH (Gsite) and one site which binds a number of hydrophobic substrates (H site) adjacent to the G site (Wilce and Parker, 1994). Despite a low grade of similarity among the classes they show a common folding with two distinct domains. For updated reviews about the structure and the properties the reader is referred to Oakley (2011) and Wu and Dong (2012). Their functions will be briefly described below. The mitochondrial GST superfamily is composed, until now, of one class: GST K1-1, in humans and mice. Despite its solubility, like that of cytosolic enzymes, this protein contains a three-dimensional fold similar to that of a prokariotic disulfide bond forming oxidoreductase (DsbA). Sequence analysis and structural studies revealed their distinct and ancient evolutionary origin, but its physiological role has not been clearly identified (Aniya and Imaizumi, 2011; Morel and Aninat, 2011). The MAPEG enzymes are a superfamily of microsomal enzymes i.e. membrane proteins. Microsomal glutathione transferase 1 (MGST1) is the most extensively characterized GST within the MAPEG family and constitutes 3% of the endoplasmic reticulum protein in rat liver and 5% of the outer mitochondrial membrane. Although MGST1 is a membrane bound trimer that is structurally distinct it shares the same broad and overlapping substrate specificity as the cytosolic GSTs. The structure and function of MGST1 and the other MAPEG enzymes have been the subject of several reviews and are not discussed further here (Jakobsson et al., 1999; Morgenstern et al., 2011). Finally, a further distinct family of transferases exists, represented by the bacterial fosfomycin resistance proteins FosA and FosB (Rigsby et al., 2005). 2. The historical functions of conjugation and ligandines of cytosolic enzymes have been now updated to include other enzymatic functions. The Alpha class GST exhibits keto-steroid isomerase activity, in particular GST A3-3 catalyzes the isomerization of endogenous Δ5-3-ketosteroids such as Δ5-androsten-3,17-dione and Δ5-pregnane-3,20-dione to Δ4-androsten-3,17-dione and Δ4pregnane-3,20-dione, respectively (Benson et al., 1977; Johansson and Mannervik, 2002). The Zeta Class is involved in the degradation of tyrosine (Blackburn et al., 1998; Board and Anders, 2011). In humans GST M2-2 and GST M3-3 but not GST M4-4

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have been shown to catalyze the isomerization of prostaglandin (PGH2) to PGE2 (Beuckmann et al., 2000), while the isomerization of PGH2 to PGD2 is catalyzed by Sigma class GSTs (Jowsey et al., 2001). In addition, some GSTs including GST O11, GST A1-1, GST M2-2 and chloride intracellular channel proteins (CLIC-2) regulate ryanodine receptor Ca2+ channels (Board et al., 2004; Dulhunty et al., 2001; Liu et al., 2009). Other GSTs such as GST P1-1 may regulate stress-activated protein kinase (SAPK) signaling pathways by direct binding to JNK (Adler et al., 1999; Elsby et al., 2003) or TNF receptor-associated factor 2 (TRAF2) (Wu et al., 2006). Similar regulatory functions have been suggested for GST M1-1 (Cho et al., 2001; Dorion et al., 2002) and GST A1-1 (Romero et al., 2006). In response to OS glutathionylation of proteins is considered as a primary line of defense. This reversible modification reaction can be performed nonenzymatically, but it has proposed that GST P1-1 may enhance this modification of thiol proteins during OS and/or nitrosative stress (NS) (Townsend et al., 2009). There is, indeed, a vast range of glutathionylated proteins involved in metabolism (Fratelli et al., 2003), cytoskeletal remodeling (Wang et al., 2001a, 2001b), ion channel modulation (Terentyev et al., 2008), apoptosis (Velu et al., 2007) and epigenetic DNA modification (De Luca et al., 2011). Because of their high affinity and specificity for GSH, GSTs are structurally well equipped to accommodate diverse GSH-bound substrates such as glutathionylated proteins. The possibility that GSTs may bind to glutathionylated proteins in vivo and potentially influence their function was proposed by Irving Listowsky (2005) but it has not explored further (Board and Menon, 2013). It has been shown that GST P1-1 (Fig. 2) can serve as a cellular NO donor and/or carrier (Cesareo et al., 2005; Lo Bello et al., 2001; Vasieva, 2011) potentially regulating the activities of other thiol redox-modifying enzymes (e.g. glutathione reductase (GR)). 3. The GST superfamilies feature a great number of polymorphic genes (see for an updated review Board and Menon, 2013), but to date the most studied are those belonging to the cytosolic superfamily and include single nucleotide substitutions or gene

Cys47

Cys101

Fig. 2. The GSH and GST P1-1 systems collaborate to maintain oxidative homeostasis against insurgence of neurodegeneration (Smeyne and Smeyne, 2013). Ribbon representation of the crystallographic structure of GST P1-1 monomer with its most reactive cysteines, Cys 47 and Cys 101. In red are represented alpha helices, in blue beta sheet; the transparency is a surface representation of the protein (courtesy of Dr. Daniele Di Marino). It is suggested that some pleiotropic functions of this enzyme (e.g. regulation of kinase signaling, glutathionylation of selected proteins and removal of excess iron and NO) rely on their redox properties and in this way GST P1-1 could act as a redox sensor (Zhang et al., 2014).

deletions. Many studies have attempted to establish a correlation between GST polymorphisms and onset/outcome of some neurodegenerative diseases: GST P1-1: The interaction of some GST P1-1 polymorphisms such as GSTP1-114Val with cigarette smoke may favor the insurgence of PD (Deng et al., 2004). Moreover, GST P1-1 polymorphisms are associated with an increased risk of PD following pesticide exposure (Longo et al., 2013; Smeyne et al., 2007). On the other hand the presence of the allelic variant *C may affect the reduction of cognitive functions in certain AD individuals and seems to be responsible for an increased susceptibility for late onset AD (Bernardini et al., 2005; Spalletta et al., 2007). Among the GST variants the V allele of GSTP1 may be a risk factor for AD, mainly in the presence of apoE 4 allele (Pinhel et al., 2008). Recent studies suggest that GST P1-1 is involved in Cdk5 regulation by the modulation of its expression in AD patients and therefore prevents neurodegeneration (Sun et al., 2011). GST T1-1: Deletion of GSTT1 is associated with an increased susceptibility to contract Parkinson’s disease, motor neuron disease and Alzheimer’s disease (Ghosh et al., 2012; Singh et al., 2008; Stroombergen and Waring, 1999). Therefore, the presence of GSTT1 may indicate protection from the disease. GST M1-1: This is the major isoform of Mu class expressed in the brain, after the GST Pi and GST Alpha classes (Smeyne et al., 2007). It is localized in neurons and astrocytes (Abramovitz et al., 1988) and has a protective role in dopaminergic neurons (Vilar et al., 2007). The presence of GST M1-1 delays the onset of PD. In fact, the onset of the disease was an average age of 68 years among patients carrying the GSTM1 gene, compared to the average age of 57 years among those with null genotype (Ahmadi et al., 1999; Perez-Pastene et al., 2007). Also with regard to AD, the null genotype is a risk factor (Piacentini et al., 2012a). The association between PD and pesticide exposure is enhanced in the presence of null GSTM1 gene (Pinhel et al., 2013). A different subunit of GST Mu class (GSTM3) found in the brain of postmortem AD patients was the second most abundant GST subunit in the brain. The authors suggested that deposition of this protein in the tissue could originate from cross-links produced by OS (Tchaikovskaya et al., 2005). GST O1-1/GST O2-2: Genetic linkage studies suggest that GSTO1 influences the age of onset of Alzheimer’s disease and Parkinson’s disease. Polymorphisms of GSTO1 in fact alter the structure of this enzyme, and consequently its ability to detoxify; this could increase the risk of AD. It was seen that the GSTO1 Asp polymorphism influences the age of onset for AD (Kölsch et al., 2004). GSTO1 and GSTO2 are located on chromosome 10q, and recent studies have shown that there is a genetic linkage between chromosome 10q and AD (Reitz et al., 2012). This leads to the hypothesis that class Omega GSTs are involved in AD. In particular, GST O1-1 E155del might play a role in AD because this deletion causes a deficiency of active enzyme in vivo (Zhou et al., 2011). This would lead to a deficiency of antioxidant activity of GST O1-1, to a regulation of interleukin-1β and in turn to a lack of biotransformation of arsenic, responsible for the hyperphosphorylation of protein tau and overtranscription of the amyloid precursor protein, both being involved in the formation of neurofibrillary tangles and brain amyloid plaques (Gong and O’Bryant, 2010). Consequently, there is a weakened protection of brain tissue against OS, an increase in inflammation and in the neurotoxic effect of arsenic (Piacentini et al., 2012b). GST O2-2 is involved in the cycle of ascorbate; therefore, any mutation on this gene may influence the age of onset of PD and AD (Schmuck et al., 2005). For further studies on these relatively ancient member of the cytosolic GST superfamily the reader is referred to Board (2011).

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4. The expression of GSTs in the brain has been reported in a number of papers featuring normal and degenerated tissues. The most common GSTs are the cytosolic enzymes belonging to the Mu, Pi and Alpha classes (Smeyne et al., 2007) but cellular localization indicates that GST Mu and Pi are present in both astrocytes and neurons, while only GST P1-1 is present in A9 DA neurons of SN (Smeyne et al., 2007). Previously, it has been shown that GST expression in rat and human brains is age-dependent and GST P1-1 is the only isoform expressed in the human fetal liver while all the above three class GSTs are present in the adult of rat (Cammer et al., 1989), mouse (Tansey and Cammer, 1991) and human brains (Carder et al., 1990). In a model of transgenic mice with symptoms of neurodegeneration there were changes in the expression of GST P1-1 at the level of both mRNA and protein suggesting that SOD1G93A mutation reduces its detoxification efficiency (Kazmierczak et al., 2011). High levels of GST P1-1 in endothelial cells and glial cells/astrocyte correlate to medical intractable epilepsy, suggesting that this enzyme contributes to resistance to treatment (Shang et al., 2008). In another study about glioma patients, refractory to epilepsy treatment it was found that multidrug resistance phenotype was associated with P-glycoprotein and GST P1-1 (Calatozzolo et al., 2012). Some reports dealing with human brain tumor tissues and cells pointed out to downregulation of GST P1-1 (Chen et al., 2012; Wahid et al., 2013). In murine HT22 hippocampal cells treated with a glutathione-depleting drug, there was an increase in levels of the mRNAs encoding heavy (H) and light (L) ferritin and GST P2-2 (Morozova et al., 2007). In another study in primary cultured rat cerebral cortical cells lithium treatment not only increased GSTM1 mRNA levels, but also increased GSTM3, M5 and A4 mRNA levels (Shao et al., 2008). Human GSTM3 was the second most abundant GST subunit in the brain, as demonstrated by northern blot and HPLC analyses. In the neurodegenerated brain of AD patients it was found by immunocytochemistry that this subunit is associated with neuritic plaques, neurofibrillary tangles and microglia in the region of the hippocampus (Tchaikovskaya et al., 2005). 5. The GST substrates in the brain can be tentatively divided into endogenous and exogenous substrates. Among the first ones there are the o-quinones, as oxidation products of catecholamines, which can contribute to redox cycling, toxicity and apoptosis. All these physiological substrates are efficiently conjugated by GST M1-1 and GST M2-2 (Baez et al., 1997). As endogenous substrates we should also mention the products of lipid peroxidation, such as 4-HNE, as physiological compounds efficiently removed by class Alpha GST (GST A4-4) (Hubatsch et al., 1998; Xie et al., 1998). Finally, oxidation of nucleotides yields base propenals, such as adenine propenal, and hydroperoxides that can be detoxified by GST also in the brain (Hayes et al., 2005). Dieckhaus et al. (2001) reported that GSTs A1-1, M1-1, and P1-1 are active in the detoxification of 2-phenylpropenal, a reactive Felbamate metabolite. In this case, this latter propenal is a metabolite of an exogenous substrate, a drug used in the treatment of refractory epilepsy. In principle, most exogenous substrates (xenobiotics) belong to drugs that are used in tumor and/or neurological disorders (e.g. schizophrenia, bipolar syndrome etc.) treatment. Specifically, we cannot rule out the possibility that the actual used drugs against AD or PD can be recognized as substrates of brain GSTs or at least inducers of GSH-related enzymes via Kelch-like ECH-associated protein 1/nuclear factorerythroid 2 p45-related factor 2 (Keap1/Nrf2) pathways (Higgins and Hayes, 2011). A large number of putative drugs or drugmolecular targets are cited in the recent literature including nitric oxide synthase (NOS) inhibitors (Maher et al., 2014), pyruvate (Isopi et al., 2014), extracellular matrix molecules (Berezin et al.,

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2014), ginger components (Azam et al., 2014), cholinesterase inhibitors (Otto et al., 2014), anti-inflammatory drugs (Wang et al., 2015), insulin (Sebastião et al., 2014), etc. We should also mention a possible role of certain bio-metals involved in memory enhancement (zinc) or associated with amyloid precursor protein (APP) (copper) in AD (Parker et al., 2012). Thus, it is expected that, at least for the most important potential drugs, future studies will be performed on their role as verified GST substrates and/ or ADDS inducers and their real effect on neurodegenerative diseases.

5. Concluding comments Many studies suggest a crucial role for oxidative stress in the first phase of aging, or in the pathogenesis of various diseases including neurological ones. Particularly, the role exerted by glutathione and glutathione-related enzymes (e.g. Glutathione Transferases) in the nervous system appears more relevant, this latter tissue being much more vulnerable to toxins and OS than other tissues such as liver, kidney or muscle. Samples available from postmortem PD patients suggest an increased lipid peroxidation with a significant reduction of GSH levels in these individuals. On the other hand in vitro studies, carried out in cellular models to make GSH depletion or to overexpress some GSTs, confirm that the presence of GSH and GSTs is critical to counteract the loss of dopaminergic neurons in PD patients. GST activity is also significantly diminished in CSF and all areas of AD patients’ brain along with an increase of 4-HNE, a product of lipid peroxidation. The way how GSH exerts its protective role has been explained above but it is noteworthy to underline that GSH action is mediated by an array of enzymes involved in the ADDS (Fig. 1). Indeed, as recently suggested by Flohé (2013), the relevance of GSSG/GSH redox potential is poor as it deals with small rate constants of spontaneous reactions as compared with enzyme-catalyzed reactions and their kinetic parameters. Many studies have demonstrated that cytosolic GSTs are an integral part of a dynamic and interactive defense mechanism that protects against cytotoxic electrophilic chemicals and allows adaptation to exposure to oxidative stress (Hayes et al., 2005). Increased evidence suggests that, at least, cytosolic GSTs metabolize many endogenous and foreign compounds that stimulate expression of the antioxidant responsive element (ARE)-gene battery through the Keap1/Nrf2 pathway (Satoh et al., 2014). Therefore, these enzymes may indirectly control the level of other ADDS enzymes and possibly chaperones, inflammation- and apoptosis-associated proteins. Whether modulation of certain signaling pathways by GSTs is relevant to the human brain and could be associated with neurological diseases still remains to be demonstrated, but certainly warrants further studies. Most studies reported in the literature suggest a role of protection for some GST polymorphic genes (e.g. the lack of GSTT1 and/ or GSTM1 genotypes or the presence of polymorphic variants of GSTP1 or GSTO1) for the insurgence of neurological diseases. In general, it has been found that individual GST genes do not make a major contribution to susceptibility to other diseases as studies of knockout genes in mice show a compensatory response by induction of other coordinated ADDS enzymes (Lim et al., 2004). Whether this is true or not in human brain remains to be established. Further studies could give new advances not only in terms of susceptibility to the insurgence of degenerative diseases, but also in terms of efficacy of therapeutic drugs or adverse drug reactions. Recent advances in the treatment of neurodegenerative diseases, based on GSH delivery systems (Cacciatore et al., 2012) or better on enhancement of nuclear localization of Nrf2 transcription factor and level of GST (Farr et al., 2014), may be promising results (Fig. 3).

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Oxidave Stress ↑

Altered GSH Metabolism Anoxidant Therapeucs

GSH Depleon and GST Acvity ↓ GSH ↑ and GSTs expression ↑ via Keap1/Nrf2 pathways Neurodegenerave Disorders Fig. 3. Scheme 1. A simplified scheme is depicted showing that a raised oxidative stress may produce, along with other important events, GSH depletion and decrease of GST activity leading eventually to the insurgency of neurodegeneration. The replenishment of GSH and the recovery of GST enzymes by indirect antioxidants, which regulate the Keap1/Nrf2 pathway, may be beneficial for stopping or delaying the onset of neurodegenerative diseases.

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