"Systemic administration of NAC can deliver cysteine to the brain and raise GSH levels in the CNS"......


European Journal of Neuroscience

Volume 27 Issue 1 Page 20-30, January 2008

Oxidative stress on EAAC1 is involved in MPTP-induced glutathione depletion and motor dysfunction

* Koji Aoyama,
* Nobuko Matsumura,
* Masahiko Watabe and
* Toshio Nakaki

ABSTRACT:


Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter expressed on mature neurons in the CNS, and is the primary route for uptake of the neuronal cysteine needed to produce glutathione (GSH).

Parkinson's disease (PD) is a neurodegenerative disorder pathogenically related to oxidative stress and shows GSH depletion in the substantia nigra (SN). Herein, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, an experimental model of PD, showed reduced motor activity, reduced GSH contents, EAAC1 translocation to the membrane and increased levels of nitrated EAAC1.

These changes were reversed by pre-administration of n-acetylcysteine (NAC), a membrane-permeable cysteine precursor.

Pretreatment with 7-nitroindazole, a specific neuronal nitric oxide synthase inhibitor, also prevented both GSH depletion and nitrotyrosine formation induced by MPTP. Pretreatment with hydrogen peroxide, l-aspartic acid β-hydroxamate or 1-methyl-4-phenylpyridinium reduced the subsequent cysteine increase in midbrain slice cultures. Studies with chloromethylfluorescein diacetate, a GSH marker, demonstrated dopaminergic neurons in the SN to have increased GSH levels after NAC treatment.

These findings suggest that oxidative stress induced by MPTP may reduce neuronal cysteine uptake, via EAAC1 dysfunction, leading to impaired GSH synthesis, and that NAC would exert a protective effect against MPTP neurotoxicity by maintaining GSH levels in dopaminergic neurons.

INTRO:

Parkinson's disease (PD) is a progressive, late-onset disorder resulting from dopaminergic neurodegeneration in the substantia nigra (SN). Although the precise pathogenesis of PD is still unclear, oxidative stress plays an important role in the underlying mechanism (Halliwell, 1992).

In patients with PD, the SN shows high levels of oxidative by-products (Dexter et al., 1989; Yoritaka et al., 1996; Alam et al., 1997) and iron (Dexter et al., 1987), which can react with hydrogen peroxide (H2O2) via the Fenton reaction to form hydroxyl radicals (Youdim et al., 1989) and low glutathione (GSH) levels (Perry & Yong, 1986; Sian et al., 1994).

GSH plays a critical role in protecting cells from oxidative stress and xenobiotics, as well as maintaining the thiol redox state. However, brain GSH declines with ageing (Maher, 2005), and GSH depletion enhances oxidative stress leading to neuronal degeneration (Schulz et al., 2000; Bharath et al., 2002). Although patients with PD exclusively show GSH loss in the SN, the precise mechanism has not yet been clarified.

GSH is a tripeptide composed of glutamate, cysteine and glycine. Cysteine is the rate-limiting substrate for GSH synthesis in neurons (Dringen et al., 1999). In primary neuron culture, approximately 90% of total cysteine uptake is mediated by sodium-dependent systems, mainly excitatory amino acid transporters (EAATs), also known as system XAG- (Shanker et al., 2001; Chen & Swanson, 2003; Himi et al., 2003b). There are five EAATs, termed GLAST, GLT-1, EAAC1, EAAT4 and EAAT5 (Danbolt, 2001). GLAST and GLT-1 are localized primarily to astrocytes; EAAC1, EAAT4 and EAAT5 to neurons. EAAT4 and EAAT5 are restricted to cerebellar Purkinje cells and the retina, respectively, whereas EAAC1 is widely expressed in the CNS (Maragakis & Rothstein, 2004). Knockdown expression of GLAST or GLT-1 in rats using antisense oligonucleotides increased the extracellular glutamate concentration, whereas EAAC1 knockdown had no effect on extracellular glutamate (Rothstein et al., 1996). Astrocyte glutamate transporters are limited to glutaminergic synapses, whereas EAAC1 is detected diffusely over cell bodies and processes (Rothstein et al., 1994).

These findings suggest that clearing extracellular glutamate is not a major role of EAAC1. EAAC1 can also transport cysteine at a rate comparable to that of glutamate, with an affinity 10–20-fold higher than that of GLAST or GLT-1 (Zerangue & Kavanaugh, 1996).

A recent study demonstrated age-dependent neurodegeneration with decreased GSH content, increased oxidant levels and increased susceptibility to oxidative stress in EAAC1-deficient mice (Aoyama et al., 2006). Notably, these EAAC1-deficient mice also showed an age-dependent decrease in neuronal number in the SN (Chan et al., 2005; Berman et al., 2007). However, to our knowledge there have been no studies examining EAAC1 in any of the PD models.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known as an exogenous neurotoxin, which induces mitochondrial dysfunction leading to increased oxidative stress, dopamine depletion in the striatum and parkinsonism (Langston et al., 1983).

Herein, we report that oxidative stress may reduce neuronal cysteine uptake via EAAC1 leading to impaired GSH synthesis in the MPTP mouse model of PD.

DISCUSSION:

Neurons rely mainly on extracellular cysteine for GSH synthesis (Dringen et al., 1999), because neurons have no means of direct GSH uptake. Extracellular supplies of the other amino acids, glutamate and glycine, do not increase GSH synthesis (Almeida et al., 1998; Dringen et al., 1999), as intracellular concentrations are already sufficient (Dringen, 2000). Cystine is an oxidized form of two cysteines with a disulfide linkage and is utilized as a substrate for GSH synthesis in some cell types (Bannai & Kitamura, 1980).

However, mature neurons utilize cysteine but not cystine for GSH synthesis (Sagara et al., 1993; Kranich et al., 1996). Because cystine is not an EAAC1 substrate (Kanai & Hediger, 1992) and mature neurons do not have cystine transporters, i.e. system xc–, which is expressed in regions facing the CSF, a role in redox buffering of the cysteine/cystine balance in the CSF has been suggested (Sato et al., 2002).

Recently, mice lacking this cystine transporter were reported to show no changes in brain GSH contents (Sato et al., 2005). Therefore, the availability of cysteine, but not other amino acids, is critically important for neuronal GSH synthesis.

Previous reports demonstrated that glutamate transporters are vulnerable to oxidative stress and that glutamate uptake is inhibited by preincubation with peroxynitrite or H2O2in vitro (Trotti et al., 1998). However, little is known about the influence of oxidative stress on the capacity of EAAC1 to function as a cysteine transporter.

Previous studies have suggested that glutamate uptake is regulated by the redox state of sulfydryl groups on cysteine residues of EAAC1 and that oxidation of the ‘redox site’ by H2O2 decreases glutamate uptake (Trotti et al., 1997a, b). Our data from acute slice culture experiments demonstrate that preincubation with H2O2 reduces subsequent cysteine uptake in the midbrain. Similarly, the marked reduction of cysteine uptake observed in the presence of LAβHA, but not DHK, suggests that EAAC1 is the primary cysteine transporter in the midbrain, as has been demonstrated in the hippocampus (Aoyama et al., 2006). These findings suggest that oxidative stress may impair neuronal GSH synthesis via EAAC1 dysfunction in the midbrain.

Peroxynitrite is a potent oxidant generated by the reaction between superoxide anion and nitric oxide (Kuhn et al., 2004), and plays a major role in MPTP neurotoxicity (Schulz et al., 1995). MPTP neurotoxicity is dependent on its metabolism to MPP+, which can specifically enter dopaminergic neurons via the dopamine transporter (Javitch et al., 1985) to inhibit complex I of the mitochondrial respiratory chain, and thus leads to reactive oxygen species production and ATP depletion (Tipton & Singer, 1993). Dopamine transporter-deficient mice preserved nearly normal striatal dopamine levels when exposed to neurotoxic doses of MPTP (Gainetdinov et al., 1997), while dopamine transporter over-expressing mice showed enhanced MPTP neurotoxicity (Donovan et al., 1999). These indicate that MPTP is a neurotoxin specific to dopaminergic neurons in vivo. In this study, we showed pretreatment with MPP+ to decrease subsequent cysteine uptake in midbrain slices and SH-SY5Y cells, and also to reduce GSH levels in dopaminergic neurons of the SN. These results indicate that MPTP neurotoxicity would be enhanced by inhibiting neuronal cysteine uptake leading to impaired GSH synthesis.

A previous report demonstrated MPTP neurotoxicity to be attenuated in nNOS-deficient mice (Przedborski et al., 1996). Therefore, it is important to elucidate the mechanism underlying peroxynitrite-mediated neurotoxicity in the MPTP model. Nitrotyrosine is a permanent marker of peroxynitrite attack on proteins (Beckman, 1994) and is found in post mortem PD brain samples (Good et al., 1998). In the MPTP model, tyrosine nitration inactivates TH, a key dopamine synthesis enzyme, and is found in α-synuclein, a major component of Lewy bodies (Kuhn et al., 2004). Peroxynitrite can oxidize cysteine residues and/or nitrate tyrosine residues on glutamate transporters, and thereby impair their function (Trotti et al., 1997b, 1998). To date, no direct evidence of EAAC1 nitration has been obtained. In this study, we found an increased amount of nitrotyrosine on EAAC1 and colocalization at the plasma membrane in the SN of MPTP-treated mice. Pretreatment with 7-NI, a selective inhibitor of nNOS, prevented the nitrotyrosine formation and GSH depletion induced by MPTP. These results may explain the decrease in GSH, which occurs with EAAC1 dysfunction in the midbrains of MPTP-treated mice. A 30% decline in total GSH appears to be rather large for a partial impairment of neuronal EAAC1 by oxidative stress. Because GSH contents may vary among TH-positive neurons, TH-negative neurons and glial cells, the apparently large decline in total GSH might suggest a predominant GSH distribution in TH-positive neurons in the midbrain.

A previous study demonstrated that MPTP administration decreased the striatal GLT-1 level after 21 days (Holmer et al., 2005). In our study, total EAAC1 amounts were unchanged, while amounts of EAAC1, though nitrated, on the plasma membrane were increased in the midbrains of MPTP-treated mice. Redistribution of EAAC1 to the membrane surface has been demonstrated to be regulated by protein kinase C, particularly protein kinase C subtype α (Davis et al., 1998).

Indeed, protein kinase C activation induced by peroxynitrite has been identified in fibroblasts (Bapat et al., 2001), pulmonary artery endothelial cells (Phelps et al., 1995) and the myocardium (Pagliaro et al., 2001). Interestingly, in this study, treatment with 7-NI, which blocks peroxynitrite formation, did not influence the EAAC1 redistribution to the membrane surface induced by MPTP. This redistribution might be induced by signals other than peroxynitrite. Further study is needed to elucidate the mechanism of the trafficking system in this model.

NAC acts as a precursor for GSH synthesis by supplying cysteine (De Vries & De Flora, 1993) and activates the GSH cycle (De Flora et al., 1991). NAC enters the cell readily (Mazor et al., 1996) and is then deacetylated to form l-cysteine regardless of whether EAAC1 is present (Himi et al., 2003a; Aoyama et al., 2006). NAC exerts a direct chemical effect as an antioxidant, although with less potency than that of GSH (Hussain et al., 1996).

Systemic administration of NAC can deliver cysteine to the brain and raise GSH levels in the CNS (Pocernich et al., 2000). Therefore, NAC would exert its protective effects against oxidative stress mainly by serving as a substrate for GSH synthesis. Our slice culture results showed NAC to act as an effective precursor for GSH synthesis in dopaminergic neurons. There are no reports demonstrating GSH-related protective effects of NAC against MPTP neurotoxicity using behavioral, biochemical or histochemical analysis in vivo, although one study demonstrated NAC treatment to restore the striatal dopamine level in MPTP-treated mice (Perry et al., 1985).

Our present results demonstrate that NAC pre-administration ameliorates motor dysfunction in addition to restoring GSH levels in MPTP-treated mice. We also found the nitrotyrosine level on EAAC1 to be reduced in the midbrains of NAC/MPTP-treated mice as compared with MPTP-treated mice. Although whether NAC would be clinically beneficial in PD is as yet unknown, its low toxicity and ease of administration warrant further investigation of this compound.