Perspectives in Pharmacology Pharmacology and Toxicology of Astrocyte-Neuron Glutamate Transport and Cycling

The interaction between astrocytes and neurons is examined from the standpoint of glutamate neurotoxicity. The review details 1) the distribution of glutamate transporters on astrocytes and neurons, provoking a reformulation of the interdependence between these two cell types in removing extracellular glutamate and preventing excitotoxic injury; 2) the potential involvement of aberrant glutamate transporter function in the etiology of neuropathological conditions; 3) the role of astrocyte-neuron interaction in widely divergent aspects of brain energetics; 4) the role of astrocytes in the process of glutamate recycling within the context of anesthetic treatment with pentobarbital and thiopental. High extracellular concentrations of the amino acid neurotransmitter, glutamate, can be neurotoxic. Its effects are terminated by re-uptake into neurons or astrocytes, with astrocytes responsible for a major part of glutamate uptake in the brain. Glutamate released into the synaptic cleft can depolarize neurons via specific receptors. Its action is terminated by its uptake from the synaptic cleft, mostly by Na dependent uptake systems that are located on both astrocytes and neurons. Anion conductance is also associated with activation of the glutamate transporters, but it is not coupled to glutamate movement and varies widely for the different transporters. The generated electrogenic gradient translocates glutamate against a several thousand-fold concentration gradient, maintaining optimal glutamate concentrations in the extracellular fluid. Although essential for normal functioning of the central nervous system (CNS), increased extracellular glutamate levels are neurotoxic (Choi, 1992). Over-activation of neuronal N-methyl-D-aspartate receptors (NMDAR) by exogenous glutamate is associated with increased influx of Na and Ca ions and the ensuing neuronal death (Choi, 1992). Increased intracellular Na alters membrane potentials, produces neuronal swelling due to osmotically obliged water movement, and eventually causes cellular lysis. Intracellular Ca overload, in addition to the osmotic stress and cell swelling, leads to stimulation of numerous Ca -activated enzymes, such as the protease calpain I that degrade cellular structural proteins (Siman et al., 1989). Additionally, Ca overload activates phospholipases leading to breakdown of the cellular membrane and subsequent release of endonucleases that degrade DNA. The Ca -mediated activation of phospholipase A2 releases arachidonic acid, increasing production of reactive oxygen species (ROS) (Lafon-Cazal et al., 1993). These ROS, in combination with peroxynitrate and other free radicals that are generated by the activity of nitric oxide synthetase (Dawson et al., 1991), lead to peroxidation of lipid, cellular lysis, and eventual cell demise. Arachidonic acid and ROS (Volterra et al., 1994) inhibit excitatory amino acid (EAA) transporter function limiting removal of extracellular glutamate, thus producing increased NMDAR stimulation, further production of arachidonic acid and ROS, and greater inhibition of EAA transport. This feed-forward This review was supported by the Research Council of Norway (to U.S.), SINTEF Unimed Foundations (to U.S.), the Normox NorFa grant (to U.S.), the Department of Physics, Norwegian University of Science and Technology (NTNU) (to H.Q.), and U.S. Public Health Service grants from the National Institute of Environmental Health Sciences (NIEHS 07331 and 10563) (to M.A.). ABBREVIATIONS: CNS, central nervous system; NMDAR, N-methyl-D-aspartate receptor; ROS, reactive oxygen species; GLT1, glutamate transporter 1; EAA, excitatory amino acid; GSH, glutathione; GLAST, glutamate/aspartate transporter; ALS, amyotrophic lateral sclerosis; TCA, tricarboxylic acid; GABA, -aminobutyric acid; dB-cAMP, dibutyryl cyclic AMP. 0022-3565/02/3011-1–6$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 900068/972761 JPET 301:1–6, 2002 Printed in U.S.A. 1 at A PE T Jornals on M ay 1, 2017 jpet.asjournals.org D ow nladed from NMDARand Ca -mediated cycle eventually leads to neuronal death (Choi, 1992). In addition to Ca -dependent ROS-mediated neuronal lysis, elevated levels of extracellular glutamate inhibit the uptake of cystine, a precursor of glutathione (GSH) (Murphy et al., 1990), the major intracellular antioxidant in brain cells. This inhibition of precursor uptake decreases neuronal GSH levels (Murphy et al., 1990) and thus increases neuronal susceptibility to pro-oxidant events, such as those seen following exposure to the neurotoxic heavy metal, methylmercury (MeHg) (Aschner et al., 1994). Glutamate transporters have been identified on neuronal and astrocytic membranes for removal of extracellular glutamate (Pines et al., 1992; Storck et al., 1992; Rothstein et al., 1994). One of these transporters, commonly referred to as glutamate/aspartate transporter (GLAST), was shown to be the predominant one in cultured astrocytes (Gegelashvili et al., 1996). Disruption in glutamate homeostasis is thought to be a factor in the pathogenesis of certain neurological and psychiatric diseases. Glutamate uptake decreases with normal brain aging and can thus contribute to age-related neurodegenerative disorders (Danbolt, 2001). During ischemia, glutamate accumulates in the extracellular space, which may contribute to cell death. The main cause of such accumulation is presumably the reversal or failure of glutamate uptake transporters, rather than synaptic release (Attwell et al., 1993). The present review underscores the role of glutamate transporters in maintaining optimal CNS function, with specific emphasis on the cycling of glutamate between astrocytes and neurons. Neuropathologic conditions that might be associated with aberrant glutamate transporter function are highlighted. Finally, the roles of astrocytes and neurons in glutamate cycling are discussed within the context of anesthetic treatment with pentobarbital and thiopental. Because of the broad nature of this subject and the limited scope of this review, we have been unable to cite many relevant articles. The curious reader will find additional information in a scholarly review by Danbolt (2001). Glutamate Transporters in Neurotoxicology To prevent initiation of glutamate-induced neurotoxic cascades, a family of high-affinity Na -dependent transporters has evolved to keep extracellular levels of glutamate below toxic concentrations. These transporters maintain a 10,000fold gradient of astrocytic intracellular glutamate (3–10 mM) to extracellular glutamate (0.3–1 M) (Schousboe and Divac, 1979) that is driven by the ionic gradients generated by ion-exchanging pumps such as Na /K -ATPase. Originally termed system XAG , at present there are five distinct Na and K -dependent EAA transporters that have been identified and cloned. They are referred to as EAAT1 (also designated as GLAST in rodents) (Storck et al., 1992), EAAT2 (also designated as GLT1 in rodents) (Pines et al., 1992), EAAT3 (also designated as EAAC1 in rodents) (Kanai et al., 1995b), EAAT4 (Fairman et al., 1995), and EAAT5 (Attwell and Mobbs, 1994). These transporters display heterogeneous regional and cellular expression. EAAT1 and EAAT2 are localized to astrocytes, with EAAT1 predominating in the cerebellum and EAAT2 predominating in the cortex and forebrain (Furuta et al., 1997). EAAT3 is localized to neurons throughout the CNS (Kanai et al., 1995a), whereas EAAT4 localization is largely restricted to cerebellar Purkinje cells (Fairman et al., 1995). EAAT5 has been localized exclusively in the retina (Arriza et al., 1997) and has been hypothesized to function as a photoreceptor on bipolar rod and cone cells in the rat (Pow and Barnett, 2000). Studies with antisense knockdown of specific transporter subtypes have revealed that the astrocytic transporters (EAAT1 and EAAT2) are responsible for removing the majority of the extracellular glutamate (Rothstein et al., 1996). Interaction between astrocytes and neurons is a prerequisite for the expression and maintenance of glutamate transporter function. Astroglial cultures express only GLAST, whereas astrocytes, which are cocultured with neurons, express both GLAST and GLT1 (Gegelashvili et al., 1997). Soluble factors derived from neurons are important for the induction of GLT1 protein and its mRNA in astrocytes, given that pure cortical astroglial cultures supplemented with conditioned medium from cortical neuronal cultures or from mixed neuron-glia cultures (Gegelashvili et al., 1997; Swanson et al., 1997) express this protein (GLT1). Treatment of astrocyte cultures with dibutyryl cyclic AMP (dB-cAMP) led to expression of GLT1 and increased expression of GLAST (Swanson et al., 1997) with an increase in glutamate uptake Vmax but no change in the glutamate Km and no increased sensitivity to inhibition by dihydrokainate. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia (Gegelashvili et al., 1997) and that dB-cAMP can partially mimic this influence (Swanson et al., 1997). In addition to transporting L-glutamate, these proteins also have an approximately equal affinity for D-aspartate and L-aspartate, although D-glutamate is a poor substrate (Fairman and Amara, 1999). For each EAA molecule that is transported, there is also cotransport of two or three Na ions, countertransport of one K ion (Wadiche et al., 1995), and either the cotransport of a proton (Zerangue and Kavanaugh, 1996) or the counter-transport of a hydroxyl ion (or HCO3 ) (Attwell and Mobbs, 1994). In addition, there is a glutamate-activated Cl flux distinct from EAA transport (Wadiche et al., 1995; Arriza et al., 1997; Wadiche and Kavanaugh, 1998) that is not blocked by compounds that inhibit endogenous Cl channels (Wadiche et al., 1995). The existence of channel-like, substrate-mediated ion flux is not limited to EAA transporters but has been described for numerous neurotransmitter transporters (Wadiche et al., 1995). The function of this Cl flux in EAA transporters has been hypothesized to counteract Na -induced cellular depolarization that would otherwise decrease EAA transport (Eliasof and Jahr, 1996). The importance of astrocytic EAA transporters in controlling extracellular levels of glutamate has been demonstrated by anti-sense knockdown (Rothstein et al., 1996) and genomic disruption (Tanaka et al., 1997; Watase et al., 1998). Inactivation of the astrocytic transporters EAAT1 (GLAST) or EAAT2 (GLT1) in rats by chronic antisense oligonucleotide infusion produced increased extracellular glutamate and excitotoxic neurodegeneration (Rothstein et al., 1996). In transgenic mice, knockout of EAAT1 (Watase et al., 1998) or EAAT2 (Tanaka et al., 1997) produced increased susceptibility to traumatic injury and increased brain edema. Antisense knockdown of the neuronal EAA transporter EAAT3 2 Sonnewald et al. at A PE T Jornals on M ay 1, 2017 jpet.asjournals.org D ow nladed from (EAAC1) did not elevate glutamate levels and produced only mild neurotoxicity (Storck et al., 1992). Loss of EAAT3 by genomic disruption in mice produced no neurodegeneration (Peghiniet al., 1997). These data clearly attest to the vital role of astrocytic EAA transporters in preventing glutamatemediated neurotoxicity. The potential involvement of altered glutamate homeostasis in both acute and chronic neurodegenerative diseases has been reviewed in a seminal paper by Danbolt (2001). Given the comprehensive nature of the review, we will only briefly address the role of glutamate transporters in the etiology of neurodegenerative disorders, encouraging those who are interested in the topic to seek additional details in Danbolt’s review (2001). In patients with amyotrophic lateral sclerosis (ALS), abnormality in the glutamate transport system has been implicated as potentially playing an etiologic role. Synaptic preparations from the motor and sensory cortex of these patients demonstrate decreased glutamate transporter activity (Rothstein et al., 1992), reflecting reduced activity of the glutamate transporter isoform, GLT1 (Rothstein et al., 1995). An abnormal RNA editing process was invoked as a probable cause for loss of GLT1 in the sporadic form of ALS. More recent studies, in which the functional impact of a naturally occurring mutation of human GLT1 in a patient with sporadic ALS suggests that the mutation involves a substitution of the putative N-linked glycosylation site, asparagine 206, by a serine residue (N206S), resulting in reduced glycosylation of the transporter and attenuated uptake activity (Trotti et al., 2001). Selective, focal loss of GLT1 and GLAST transporter proteins was also invoked as a potential explanation for the increase in interstitial glutamate levels and the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death (Hazell et al., 2001). Given evidence that in patients with various epilepsies the level of extracellular glutamate is increased (Ferrie et al., 1999), recent studies have also examined whether changes in glutamate transporter function and expression in the medial temporal cortex and hippocampus could be invoked in the development or maintenance of seizures. Studies by Tessler et al. (1999) have demonstrated that there is no reduction in the level of GLT1 encoding messenger RNA in the temporal lobe of epilepsy patients compared with controls, suggesting that major changes in the level of expression of GLT1 do not play an important role in the development of human temporal lobe epilepsy. It has yet to be determined whether GLT1 plays a role in the etiology of other types of epilepsy (Danbolt, 2001). Similarly, a definitive role for altered expression or activity of GLAST (EAAT1) and GLT1 (EAAT2) in Alzheimer’s disease has, so far, not been established (Danbolt, 2001). Studies by Beckstrom et al. (1999) showed a negative correlation between Alzheimer’s disease and glutamate transporters with both normal and reduced levels of GLAST and GLT1. Although reports (Danbolt, 2001) suggest that glutamate uptake inhibition might aggravate the progression of Alzheimer’s disease, direct evidence for altered GLAST and GLT1 expression and activity has yet to be established. Glutamate and Glucose Metabolism The metabolic fate of glutamate has been studied in cultured cortical astrocytes (Sonnewald et al., 1993), cerebellar astrocytes (Qu et al., 2001b), and cerebellar granule neurons (Sonnewald et al., 1996) using [U-C]glutamate and magnetic resonance spectroscopy. In cortical and cerebellar astrocytes, it could be shown that glutamate was not only converted to glutamine but, to a large extent, entered the tricarboxylic acid (TCA) cycle. The carbon skeleton was found in aspartate and in newly synthesized glutamate and glutamine (Fig. 1, Table 1). Surprisingly, labeled lactate signifying glutamate metabolism in the TCA cycle was detected in the medium from these cells (Table 1; Sonnewald et al., 1993, Qu et al., 2001b). In cerebellar neurons [U-C]glutamate was converted to aspartate and glutathione (Sonnewald et al., 1996). It should be noted that [U-C]glutamate is handled differently in astrocytes and cerebellar granule neurons (Table 1). Pyruvate recycling as indicated by the labeling pattern of aspartate and lactate in astrocytes (Håberg et al., 1998) was not observed in these neurons (Sonnewald et al., 1996). The physiological role of pyruvate recycling is at present not clear. Astrocytic uptake of glutamate by specific transporters has also been invoked as potentially regulating signal-transducing properties that are distinct from their transporter activity (Pellerin and Magistretti, 1994). Astrocytic uptake of glutamate has been proposed to stimulate glycolysis (glucose utilization and lactate production) via the activation of a Na -dependent uptake system that involves the Na /K ATPase (resulting from increased intracellular Na concentration that is cotransported with glutamate by the electrogenic uptake system). It was suggested (Pellerin and Magistretti, 1994) that when glutamate is released from active synapses and taken up by astrocytes, this signaling pathway provides a simple and direct mechanism to tightly couple neuronal activity to glucose utilization. Glutamatestimulated glycolysis and lactate production is also consistent with data obtained from functional brain imaging studies, which indicate that the mammalian CNS normally shifts to local nonoxidative glucose utilization during physiological activation (Pellerin and Magistretti, 1994). These observations point to a critical role of the astrocyte in coupling neuronal activity to glucose utilization. Indeed, it appears that in response to glutamate released by active neurons, glucose is predominantly taken up by specialized astrocytic processes, the end-feet. Subsequently, glucose is metabolized to lactate, which provides a preferred energy substrate for neurons (Pellerin et al., 1996). However there are conflicting Fig. 1. Schematic representation of possible isotopomers arising from [U-C]glutamate. represents C; lac, lactate; Mal, malate; OAA, oxaloacetate. Pharmacological Modification of Glutamate Metabolism 3 at A PE T Jornals on M ay 1, 2017 jpet.asjournals.org D ow nladed from