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Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron γ-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of γ-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrosoyte up to 517% (p < 0.05). Ethanol increased both γ-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the γ-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol-mediated oxidative stress and apoptotic death.
Glutathione (l- γ-glutamyl-l-cysteinylglycine) (GSH) is an intracellular thiol that plays a key role in scavenging reactive oxygen species (ROS) and mitigating oxidative stress. GSH serves multiple functions critical to cell survival, including detoxifying electrophiles, maintaining the essential thiol status of proteins and providing a reservoir for cysteine (Meister and Anderson 1983; DeLeve and Kaplowitz 1990; Suthanthiran et al. 1990).
Cortical astrocytes possess an active, highly regulated GSH homeostasis machinery that typically generates cell GSH content far in excess of that in neurons (Cooper 1997; Schulz et al. 2000). Additionally, astrocytes are in high abundance (Bignami 1991), they establish numerous small contacts with neurons (Rohlmann and Wolff 1996) and they play an important role in maintaining neuron GSH. The initial step in maintenance of neuron GSH homeostasis is efflux of GSH by the astrocyte (Wang and Cynader 2000), with the GSH subsequently hydrolyzed to the CysGly dipeptide by the astrocyte membrane-bound enzyme, γ-glutamyl transpeptidase (γGT) (Dringen et al. 1997). CysGly readily diffuses to adjacent neurons where it is cleaved to its two constituent amino acids at the neuronal surface by the dipeptidase, aminopeptidase N (APN) (Dringen et al. 2000; Dringen et al. 2001). This generates cysteine, which is transported into the neuron by the sodium-dependent alanine-serine-cysteine (ASC) system where its availability is an important determinant of GSH synthesis (Kranich et al. 1996; Wang and Cynader 2000).
A practical consequence of astrocyte-mediated control of neuron GSH homeostasis is protection of the neuron from oxidative damage by reactive oxygen and nitrogen species (Tanaka et al. 1999; Gegg et al. 2003). Reports from numerous laboratories have illustrated that ethanol elicits apoptotic death of neurons in the developing brain (Ramachandran et al. 2001; Olney et al. 2002) and in neuron cultures (de la Monte and Wands 2001; Ramachandran et al. 2003), and there is compelling evidence that ethanol-induced oxidative stress may be an important player in this process (Heaton et al. 2002; Ramachandran et al. 2003). Importantly, augmentation of neuron GSH content can prevent ethanol-mediated oxidative stress in cultured fetal cortical neurons and the ensuing increase in apoptotic death (Ramachandran et al. 2003). Recent studies in our laboratory have illustrated that co-culturing astrocytes with fetal cortical neurons likewise blocks ethanol-mediated oxidative stress, normalizes neuron GSH homeostasis and decreases subsequent apoptotic death (Watts et al. 2005). This strongly supports the concept that the astrocyte-related neuroprotection occurs by maintenance of neuron GSH homeostasis. The following studies extend these observations to delineate the regulatory elements that are activated by this pro-oxidant setting, thereby enhancing the neuroprotective potential of astrocytes. These experiments detail three key points in the γ-glutamyl cycle that are up-regulated by exposure to this pro-oxidant: increased astrocyte GSH efflux, increased activity and expression of astrocyte γGT, and increased activity and expression of neuron APN.
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- Materials and methods
Alcohol consumption during pregnancy impairs brain development. These neurotoxic effects may be on astrocytes (Aschner and Allen 2000), as well as on the size and distribution of neuronal populations, and neuronal differentiation and migration (West et al. 1984; Little et al. 1989; Goodlett et al. 1991; Miller 1995). Earlier studies in our laboratory (Ramachandran et al. 2001) illustrated that in utero ethanol exposure enhanced apoptotic cell death in the fetal brain, which might be mediated by oxidative stress and 4-hydroxynonnenal formation in the mitochondria. These studies demonstrated that maternal ethanol intake can accelerate neuronal apoptotic cell death in whole fetal brain, a setting with a low astrocyte content. Subsequent research has shown that in cultured cortical neurons, ethanol rapidly elicits oxidative stress-mediated apoptotic death (Figs 2 and 3), and that this can be mitigated by GSH supplementation (Ramachandran et al. 2003) and by co-culture with astrocytes (Watts et al. 2005). The current experiments detail an astrocyte-mediated system, which maintains neuronal GSH homeostasis in the presence of ethanol, and they flag key components of the system that are up-regulated by this drug. Elimination of this protective capacity by inhibition of γGT or APN illustrates that it is this system which provides protection of neurons from the ethanol-mediated oxidative challenge (Fig. 9). Significant to the developmental neurotoxicity of ethanol is that the vast majority of cortical astrocytes do not appear until the first postnatal month in rats and mice, and they can only be detected around embryonic day 16 (Qian et al. 2000). Thus, the emergence of astrocytes in the developing brain may be an important determinant of effects of duration of ethanol exposure and timing of dosage on neurotoxic responses to this compound (Watts et al. 2005).
Ethanol and GSH synthesis machinery in the astrocyte
Central to this neuroprotective role of astrocytes is stability of the cells' GSH-generating machinery. In our studies, ethanol had little effect on astrocyte GSH content (Fig. 4), even with 6 days exposure to 4 mg/mL ethanol. This is unlike ethanol effects on neuronal GSH content (Fig. 1) and it likely reflects a highly stable and resilient astrocytic GSH homeostasis machinery. These studies have found astrocytes to be much more resistant to E-mediated effects than neurons, a phenomenon which may be due to the higher GSH content and subsequent protection from oxidative stress-mediated damage (Ramachandran et al. 2003). This is supported by the lower baseline content of GSH in neurons in our culture setting, typically ranging from 2.5 to 3.7 mmoles/mg DNA, while in astrocytes it is between 5.5 and 6 mmoles/mg DNA (Fig. 4). A rather remarkable finding in the current studies is that, when exposed to the mild pro-oxidant, ethanol, astrocyte GSH content remains stable, even in the presence of striking increases in GSH efflux (Fig. 9). This reflects either a prodigious baseline capacity to synthesize the compound and/or compensatory systems that up-regulate synthesis. We found no consistent evidence of an ethanol-related up-regulation of the two enzymes which synthesize GSH. Rather, γ-glutamyl cysteine ligase and glutathione synthetase activities either remained unchanged or were decreased (Figs 6 and 7), suggesting little or no change in availability of substrates for the synthesis of GSH. γ-Glutamyl cysteine ligase is the rate-limiting enzyme in the synthesis of GSH and is subject to multiple post-translational regulatory events, including feedback inhibition by GSH and availability of the substrate, cysteine (Huang et al. 1993; Soltaninassab et al. 2000). The above experiments illustrate that, even when γ-glutamyl cysteine ligase activity is reduced by almost 40% (Fig. 6) by 4 mg/mL ethanol at 4 days of exposure, GSH efflux is strongly enhanced (Fig. 8). One final factor in the maintenance of net astrocyte GSH content is the increase in glutathione reductase in response to ethanol (Fig. 5). Compared with control values, these were statistically significant for up to 2 days of ethanol exposure, increasing their maximum activity within 1 day of treatment. These elevated activities remained constant for the entire 6 day regimen, although there was also a time-dependent increase in control samples. The mechanism underlying the latter phenomenon is unclear. Thus, the maintenance of astrocyte GSH content in the presence of such an elevated efflux may reflect both a high baseline GSH synthesis capacity and an up-regulation of glutathione reductase activity.
Regulatory responses to ethanol
Increased GSH efflux
A predominant fraction of GSH in brain is in astroglial cells, and these cells appear to provide extracellular antioxidant protection through its efflux (Dringen et al. 1999b; Shroeter et al. 1999). Figure 8 shows that ethanol can strikingly increase GSH release, especially at the highest ethanol concentration. Such a setting is not without precedent, as Sagara et al. (1996) have reported that efflux of GSH can be increased in astrocytes by oxidative stress. Ethanol is a pro-oxidant and its up-regulation of GSH efflux may be an important mechanism by which astrocytes protect neurons from ethanol-related oxidative damage. How this increased efflux occurs remains to be determined, but it could reflect an effect of ethanol and/or oxidative stress on the transporter protein, MRP1. Hirrlinger et al. (2002) have reported that the ATP-dependent, multidrug-resistant protein 1 (MRP1) expressed in astrocytes mediates export of GSH, GSSG and glutathione conjugates. Also, it is possible that ethanol elicited some degree of non-specific damage to the astrocyte plasma membrane that resulted in leakage of GSH. However, these ethanol regimens do not decrease trypan blue exclusion (Watts et al. 2005), nor do they increase Lactate dehydrogenase (LDH) leakage (data not shown). A final point is that astrocyte protection of neurons occurs within 24 h of exposure, a period during which 2.5 mg/mL ethanol did not enhance astrocyte GSH efflux. This suggests that, at least at the lower level of ethanol, baseline efflux is sufficient to provide neuroprotection.
Up-regulation of γGT
In addition to astrocyte GSH efflux, two other systems are in play that provide the neuron with the rate-controlling GSH precursor, cysteine (Dringen et al. 1999a). The first step in this ‘γ-glutamyl cycle’ is the extracellular degradation of GSH to the CysGly dipeptide by γGT (Meister and Anderson 1983; Hanigan 1998). γGT is a membrane-bound enzyme with its active site on the extracellular surface of the plasma membrane (Hammond et al. 2001). It is highly expressed in the central nervous system, largely on astrocytic end feet (Cambier et al. 2000). Concomitant with GSH efflux from astrocytes, there is a rapid up-regulation of astrocyte γGT activity and expression of its protein (Figs 10a and b). The increase in enzyme activity corresponds with increased expression of the γGT light chain (Fig. 10b) and the rapidity of this up-regulation is conspicuous. The latter occurs within 1 h of exposure to 4 mg/mL ethanol (data not shown) and it continues through at least 4 days of ethanol treatment (Fig. 10b). As with the increased GSH efflux, ethanol-generated oxidative stress may be a key player in this up-regulation. Oxidative stress increases γGT activity and its protein and mRNA (Kugelman et al. 1994). Its synthesis and activity are altered by a variety of agents, including ethanol, growth factors and hormones such as glucocorticoids, retinoic acid and thyroid hormones (Garcion et al. 1999). This step of the γ-glutamyl cycle may be a vital one with respect to maintenance of neuron GSH homeostasis, since its inhibition can prevent astrocyte-induced enhancement of neuron GSH content (Dringen et al. 1999a). Interestingly, γGT appears to play an important role in development, since mice deficient in this enzyme, while appearing normal at birth, grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels are elevated sixfold and 2500-fold, while GSH levels are markedly reduced in eye, liver and pancreas, and plasma cysteine levels are reduced by 80% (Lieberman et al. 1996).
Up-regulation of APN
A third point of ethanol up-regulation is an increase of activity and expression of APN on the neuronal plasma membrane. The CysGly dipeptide generated from extruded GSH by γGT is a precursor for neuron GSH (Dringen et al. 1999a). Recent reports have illustrated that in neurons, this occurs not directly but subsequent to CysGly hydrolysis to its constituent amino acids by APN (Dringen et al. 2001). This reaction is mediated by APN on the outer surface of the neuron plasma membrane and generates cysteine that is imported as a rate-limiting precursor for GSH synthesis (Dringen 2000). Effects of ethanol on this ectopeptidase is an unexplored area. Membrane-bound proteases are widely distributed in cell systems and their expression is highly regulated. These roles are varied and include immune function, embryonic development, regulation of synaptic plasticity, cell growth regulation and regulation of apoptosis (Sedo and Malik 2001). In the brain, APN, which possesses cysteinylglycinase activity, has been localized in microglia, astrocytes and neurons in a variety of brain structures, including the cerebral cortex (Barnes et al. 1994; Lucius et al. 1995; Noble et al. 2001). The current experiments illustrate that exposure of neurons to ethanol increased both the APN enzyme activity and its expression on the neuron plasma membranes (Figs 11a and b). Clearly, such an up-regulation should enhance the production of cysteine and subsequent synthesis of GSH.