Glutamate Efflux at the Blood–Brain Barrier: Cellular Mechanisms and Potential Clinical Relevance

Archives of Medical Research 45 (2014) 639e645

REVIEW ARTICLE

Glutamate Efflux at the BloodeBrain Barrier: Cellular Mechanisms and Potential Clinical Relevance Helms Hans Christian Cederberg, Nielsen Carsten Uhd, and Birger Brodin Department of Pharmacy, The Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark Received for publication October 30, 2014; accepted November 10, 2014 (ARCMED-D-14-00622).

L-Glutamate is considered the most important excitatory amino acid in the mammalian brain. Strict control of its concentration in the brain interstitial fluid is important to maintain neurotransmission and avoid excitotoxicity. The role of astrocytes in handling L-glutamate transport and metabolism is well known, however endothelial cells may also play an important role through mediating brain-to-blood L-glutamate efflux. Expression of excitatory amino acid transporters has been demonstrated in brain endothelial cells of bovine, human, murine, rat and porcine origin. These can account for high affinity concentrative uptake of L-glutamate from the brain interstitial fluid into the capillary endothelial cells. The mechanisms in between L-glutamate uptake in the endothelial cells and L-glutamate appearing in the blood are still unclear and may involve a luminal transporter for L-glutamate, metabolism of L-glutamate and transport of metabolites or a combination of the two. However, both in vitro and in vivo studies demonstrated blood-to-brain transport of L-glutamate, at least during pathological events. This review summarizes the current knowledge on the brain-to-blood L-glutamate efflux hypothesis including possible mechanisms to account for the transport, in vivo studies on blood glutamate scavenging and potential clinical relevance of the phenomenon. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: L-glutamate, Excitotoxicity, Excitatory amino acid transporters, Bloodebrain barrier.

Introduction L-glutamate is generally considered to be the most important excitatory neurotransmitter in the mammalian brain (1) but prolonged elevated concentrations of L-glutamate in the brain interstitial fluid (ISF) are highly cytotoxic, a phenomenon which has been termed excitotoxicity (2,3). Excitotoxicity occurs during a number of pathophysiological conditions such as traumatic brain injury, ischemic stroke, multiple sclerosis, epilepsy and Alzheimer’s disease (4,5). The regulation of L-glutamate in the brain ISF is dependent on glutamate transporters working in concert with intracellular metabolizing enzymes (6). The excitatory

Address reprint requests to: Birger Brodin, Group Leader, Associate Professor, Department of Pharmacy, The Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, DK-2100, Denmark; Phone: þ45 3530 61 69; FAX: þ45 3530 6030; E-mail: [email protected]

amino acid transporters, EAAT-1 (SLC1A3) and EAAT-2 (SLC1A2) are mainly localized in the plasma membrane of astrocytes (7e9) and have been shown to be essential for the control of extracellular L-glutamate levels (10,11). EAAT-3 (SLC1A1) is mainly localized in postsynaptic terminals in neurons (although astrocyte expression has also been demonstrated) and has less significant impact on the regulation of ISF L-glutamate levels (10,12). The uptake of L-glutamate via EAATs is indirectly dependent on the energy status of the cell because the translocation cycle includes co-transport of one molecule of L-glutamate with three sodium ions and one hydrogen ion and an exchange with one potassium ion (13,14). The transport activity and direction is thus coupled to the sodium-potassium ATPase (15). Endothelial cells of the brain capillaries also express EAATs and may thus facilitate significant brain-toblood L-glutamate efflux at least during pathological events (16e22). Previous reviews have focused on the role of EAATs in the brain endothelial cells and possible

0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2014.11.004

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blood-to-brain transport of L-glutamate originating from dietary monosodium-glutamate (23), on EAATs and blood L-glutamate scavenging (18) and on possible therapeutic applications of blood glutamate scavenging (16). The present review aims to give an updated overview of mechanistic evidence on the molecular mechanisms of the bloodebrain barrier (BBB) in the handling of brain L-glutamate, as well as a brief overview of animal studies related to lowering blood L-glutamate levels and brain ISF L-glutamate levels. Finally we will discuss the possible clinical relevance of brain glutamate efflux, and point to future directions of research. Early In Vivo Evidence for Efflux of L-glutamate from Brain Parenchyma to the Blood L-glutamate concentration in plasma is in the range of 30e90 mM and varies depending on nutritional status and exercise (24,25). L-glutamate is accumulated in erythrocytes, where the concentration has been estimated to range from 280 mM (24) to 450 mM (26). The mean blood concentration of L-glutamate will thus be an average of plasma and erythrocyte L-glutamate concentrations, corrected for their volume fractions, estimated to be |140 mM (24). The concentration of L-glutamate in the ISF of the resting, undisturbed brain parenchyma is not readily measured but estimates point at concentrations in the lower micromolar/submicromolar range. Values from 0.1e3 mM have been recorded, using microdialysis techniques (27). The large concentration difference between plasma and brain extracellular fluid indicates that the BBB has a very low permeability for L-glutamate, an active efflux of Lglutamate, a rapid brain metabolism of L-glutamate or a combination of these. The uptake of L-glutamate from blood to brain is low (28,29) and has been hypothesized to take place via a saturable carrier mechanism (30). However, brain microvessel concentrations of L-glutamate has been estimated to |750 nmol g1 (31), equivalent to |785 mM assuming that endothelial cells have a density 1.048 g/mL (32) and that the L-glutamate is exclusively present in endothelial cells and not astrocyte or neuron remnants contaminating the microvessels. If this estimate is correct, then the L-glutamate concentration gradient will be unfavorable for uptake from the blood and into the endothelial cells. However, there would be a measurable unidirectional isotope uptake flux as observed by Oldendorf and Szabo, but not a net uptake (30). Efflux of L-glutamate from the brain was demonstrated by Drewes and colleagues (33) in studies on perfused dog brains. Concentrations of amino acids were measured in the arterial and venous perfusates and net movements of amino acids were calculated from the differences, indicating a net efflux of L-glutamate from the brain in the order of |1 mmol 100 g brain1 min1 under basal conditions (33). Based on these data, Pardrigde suggested that an active L-glutamate efflux system must be

present at the BBB (34). This suggestion was supported by Hutchison and colleagues who investigated uptake kinetics of L-glutamate into isolated rat brain capillaries (35). They demonstrated that a high affinity (KM |2 mM), temperature dependent and ouabain sensitive L-glutamate uptake system was present in the capillaries, which could account for the previous observed brain efflux. This was further supported by studies by Hosoya et al. who performed intracerebral microinjections of radioactive L-glutamate, L-aspartate and D-aspartate in rats (36). They demonstrated rapid clearance from the brain of L-glutamate and Laspartate, which correlated with the appearance of the two isotopes in jugular vein samples, whereas D-aspartate stayed in the brain compartment. Hosoya et al. used thin layer chromatography to verify that at least the main part of the appearing radioactivity in the blood originated from intact L-aspartate/L-glutamate (|70 and 84% respectively) (36). Initially, this efflux was not believed to be associated with EAAT transporters, partly because of lack of Daspartate efflux and partly because immunolabeling studies had not shown EAAT-1 and -2 expression in rat brain endothelial cells (7,8,36). However, in recent decades, evidence has accumulated that EAATs are present in brain capillary endothelial cells and may take part in the brain L-glutamate efflux (see sections below). Studies of EAAT Expression and Activity Table 1 provides an overview of reported EAAT subtype expression patterns in different preparations from different species. Expression in Intact Brain Capillaries Brain capillaries from mice have been shown to have a high expression of EAAT-3 mRNA (22,37,38) as well as protein expression of EAAT-1, -2 and -3 (although mainly subtypes -1 and -2) (39). Freshly isolated bovine brain capillaries express EAAT-1, -2 and -3 mRNA, whereas only EAAT-1 has been detected at the protein level (21,40). Large quantities of EAAT-1 mRNA (41) and protein (42) were found in human brain capillaries. These studies indicate that EAAT’s are present in endothelial cells. However, contamination by glial tissue or remnants of astrocyte endfeet may have Table 1. Overview of EAAT subtype expression patterns in different species EAAT-1 Capillaries or intact tissue mRNA B,H, M Protein B, H, M Cell culture mRNA B Protein B, M, P, R

EAAT-2

EAAT-3

preparations B, M M M M

M, P

B, M B, M, P, R

B, bovine; H, human; M, mouse; P, porcine; R, rat.

References

(21,22,37,38,40,41) (17,21,39,40,42) (21,22) (19e21,39)

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caused false positive results. A study on rat capillaries revealed little or no EAAT staining on the abluminal surface of brain capillaries (7). However, a recent electron microscopy study by Roberts et al. showed a clear EAAT-1 expression in brain capillary endothelial cells from human postmortem cortex samples (17). Expression and Activity in Cultured Endothelial Cells and Vesicle Preparations Abluminal sodium dependent saturable uptake was initially demonstrated in membrane vesicles from bovine brain endothelial cells (43). Subsequently, the uptake was attributed to EAATs through protein expression and uptake kinetics studies showing expression of EAAT-1, -2 and -3 in abluminal membrane vesicles as well as high affinity uptake matching that of EAATs in other cell types (40). In cell cultures, EAAT-3 seemed to be the main transporter in mouse brain endothelial cells (22,39), whereas EAAT-1 displayed the highest expression level in bovine endothelial cells (21). EAAT-1, -2 and -3 were all present in endothelial cells from porcine brains (20), whereas only EAAT-1 and -3 were found in rat brain endothelial cells (19). Polarized transport of L-glutamate favoring the brain-to-blood direction, inhibitable by the general EAAT inhibitor DL-TBOA, has been demonstrated in primary cells from bovine and porcine endothelium in both studies in co-culture with rat astrocytes

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(20,21). Helms et al. further showed a similar polarized transport of L-aspartate and lack of polarized D-aspartate transport. D-aspartate was instead accumulated intracellularly when applied from the abluminal side, which indicated that the passage of the luminal membrane depended on either intracellular metabolism or a stereoselective transporter accepting only L-isomers of glutamate and aspartate (21). Kinetic measurements revealed a high capacity for intracellular transport, although the KM value for L-glutamate efflux was |140 mM, which is higher than expected for purely EAAT-mediated transport. Cellular Mechanisms for Brain Glutamate Efflux Different cellular mechanisms for the observed L-glutamate efflux can be hypothesized based on the present in vivo and in vitro evidence. It seems evident that uptake from the brain ISF into capillary endothelial cells via EAATs is the initial step in the brain efflux. EAATs are able to take up L-glutamate against a large concentration gradient because of the coupling to sodium gradient (14). Thus, brain endothelial cells are able to accumulate high concentrations of L-glutamate. The following fate of accumulated L-glutamate is unclear and may involve both transport of intact L-glutamate, metabolism of L-glutamate and transport of resulting metabolites or a combination of the two. Possible pathways are shown in Figure 1.

Figure 1. Glutamate transport and metabolism pathways in the neurovascular unit and how metabolites may appear in the blood after intracellular transport. a-KG, a-ketoglutarate; AAT, amino acid transferase; AGC1, mitochondrial aspartate/glutamate carrier-1; EAAT, excitatory amino acid transporter; GDH, glutamate dehydrogenase; GLN, glutamine; GLNase, glutaminase; GLU, L-glutamate; GS, glutamine synthetase; ISF, interstitial fluid; LAC, lactate; LDH, lactate dehydrogenase; MCT-1, monocarboxylic acid transporter-1; MPC1/2, mitochondrial pyruvate carrier-1/2; Pyr, pyruvate; SNAT, small neutral amino acid transporter; TCA, tri-carboxylic acid cycle; VGLUT, vesicular glutamate transporter; X?, unknown luminal glutamate transporter.

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Transport Early in vivo studies indicate the presence of a facilitative L-glutamate transporter at the luminal membrane of the endothelial cells. L-Glutamate uptake from blood-to-brain was demonstrated in rats after carotid bolus injections containing radioactive L-glutamate (30). The transporter was named xGe (44) and has been shown to be sodium independent and inhibited by L-glutamate and L-aspartate (30,43,45). The transporter has not been cloned, and it is possible that an already known amino acid transporter facilitates the passage of the luminal membrane. The glutamate-cystine exchanger (XCe) would be an ideal candidate to efflux intracellular L-glutamate from the endothelial cells to the blood in exchange for cystine uptake. However, previous studies have shown no effects of cystine on luminal endothelial L-glutamate uptake (40,45). A lowaffinity L-glutamate transporter has previously been discovered in rat brain slices (46,47). This transporter was inhibited by L-glutamate and L-aspartate as well as L-homocysteate (48), which matches the inhibition pattern observed by Benrabh et al. (45). Low affinity L-glutamate uptake has been characterized with a KM of 1e2 mM (46,47), which matches the KM value for L-glutamate in luminal membrane vesicles determined by Lee et al. (43). Metabolism L-Glutamate is extensively metabolized in astrocytes via glutamine synthetase to glutamine or via aminotransferases or glutamate dehydrogenase to a-ketoglutarate (6). The observed efflux across the bloodebrain barrier obtained using radiolabeled L-glutamate could therefore be explained by uptake and metabolism in astrocytes and transport of labeled metabolites across the endothelial cells. Two studies tested the effect of glutamine synthetase inhibition in endothelial/astrocyte co-cultures (via methionine sulfoximine inhibition) and found no effects on the intracellular Lglutamate transport (20,21). However, conversion to a-ketoglutarate may still be involved in the transport process. Alternatively, EAATs may mediate uptake into brain endothelial cells followed by intraendothelial a metobolism. Oldendorf et al. demonstrated a high mitochondria density in brain endothelial cells (49). It has been suggested that endothelial cells may utilize L-glutamate as an energy substrate to fuel the ABC-transporters (50). Furthermore, endothelial cells express branched chain aminotransferases, which may catalyze the conversion of L-glutamate to a-ketoglutarate (51). Labeled L-glutamate can thus enter endothelial cells (or astrocytes), be metabolized to a-ketoglutarate and go through the Krebs cycle in the mitochondria. From there, the radiolabel can appear on pyruvate, which may be converted to lactate in the cytosol and transported through MCT-1 in the luminal membrane to the blood, or the label may simply end up on CO2 or water (depending on the radiolabel). As mentioned in the previous section, intact labeled L-glutamate

has been recovered in the blood after intracerebral injections in rats (36), which indicated that at least some L-glutamate is transported through the cells. This may also be explained by paracellular leakage of radiolabeled L-glutamate following the injection, whereas the true balance between metabolism and transport of intact L-glutamate has not been established so far. The possible transport mechanisms are summarized in Figure 1. Studies of Glutamate Scavengers and Brain Glutamate Concentration in Animal Models The presence of EAATs in the bloodebrain barrier and their ability to accumulate large intracellular L-glutamate concentrations started the hypothesis that lowering bloodglutamate levels could increase the concentration gradient from endothelium-to-blood and thereby increase elimination of L-glutamate from the brain, the blood glutamate scavenging hypothesis (18,52). Gottlieb et al. demonstrated that L-glutamate injected in the cerebral ventricles of healthy rats appeared partly unchanged in the blood and that inducing glutamate oxaloacetate transaminase (GOT) and/or glutamate pyruvate transaminase (GPT), caused decreased glutamate concentrations both in the blood and the cerebrospinal fluid (52). This study represents cerebrospinal fluid-to-blood transport and cannot be directly related to glutamate efflux across the BBB. However, the study inspired several investigations of the effects of blood-glutamate scavenging during pathological conditions such as ischemia (19,53e55), subarachnoid hemorrhage (56), closed head injury (57,58), traumatic brain injury (59) and paraoxon intoxication (60). The studies utilized different approaches to lower blood L-glutamate levels, all based on increasing L-glutamate metabolism either by inducing GOT or GPT and/or by direct intravenous injection of the same enzymes. All these studies came to the same general conclusion that lowering blood-L-glutamate levels by metabolism decreases the morbidity of the disease states, for instance through better recovery, better neuron survival or smaller stroke volumes. Together these studies present seemingly convincing evidence for the brain Lglutamate efflux hypothesis. However, it cannot be excluded that oxaloacetate/pyruvate or GOT/GPT may diffuse from the blood to the CNS compartment. GOT and GPT are both large molecules (GOT 5 92 kDA, GPT 5 54.5 kDA), and an in vivo pharmacokinetic study demonstrated that they do not distribute into the CNS in healthy rats (61). However, BBB breakdown with consequent albumin leakage to the brain parenchyma has been shown during stroke, traumatic brain injury and subarachnoid hemorrhage (62e65). Hence the studies performed so far do not rule out the possibility that the injected compounds distribute to the brain parenchyma to exert their effect or that L-glutamate diffuses from the brain ISF via an opened paracellular space between the endothelial cells and

Glutamate Transport at the BloodeBrain Barrier

thus appear in the blood without involvement of EAATs. Thus, although the experiments strongly support the brain L-glutamate efflux hypothesis, the results can be explained by alternative mechanisms. Potential Clinical Relevance of the Brain L-glutamate Efflux Phenomenon L-glutamate excitotoxicity has been shown to occur during different pathological conditions, including traumatic brain injury, ischemic stroke, status epilepticus, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis and Parkinson’s disease. It can thus be hypothesized that decreasing blood glutamate levels by therapeutic intervention would dampen excitotoxic events following brain injury, stroke and neurodegenerative disease. Clinical trials have not yet been performed to investigate the hypothesis. There are, however, a few studies on humans, which can be interpreted so as to indicate a connection between low blood glutamate levels and increased brain L-glutamate efflux. Castillo et al. showed from clinical data that L-glutamate levels increase in both CSF and blood following ischemic stroke, and that the two levels correlate (66). Furthermore, correlations between blood levels of especially GOT and clinical outcome in stroke and migraine patients have been shown (67e69). This indicates that brain efflux of L-glutamate may take place in humans following ischemic stroke. Furthermore, the observations can be taken as support for the hypothesis that individuals with a large capacity for L-glutamate metabolism in the blood have a faster L-glutamate clearance from the brain than individuals with a lower L-glutamate metabolism capacity. It has thus been suggested that GOT/oxaloacetate treatments could pose an alternative treatment strategy to limit excitotoxic insults. There are some practical challenges involved in implementing such a therapy for human use. As pointed out by Zlotnik and coworkers, there is a limited time window for the treatment in animal studies, which may also apply to humans (58,70). In rats, treatment should be applied within a short timeframe, no later than 60 min after the onset of the excitotoxic event. In humans there is a longer time course of the ischemic insult following traumatic brain injury, and it has been suggested that a therapeutic treatment thus might be beneficial also at later stages (56). Introducing oxaloacetate as a therapeutic agent may also pose some challenges as its safety profile has not been investigated (16). Summary and Perspectives Since Pardridge formulated the brain glutamate efflux hypothesis (34), numerous studies have demonstrated presence of EAATs in brain endothelial cells in vitro and in vivo in different species, although the subtype expression pattern varies. EAAT mediated uptake into the endothelial cells constitute the first step of the brain glutamate efflux;

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however, the following steps remain unclear. Saturable, low-affinity luminal uptake has been shown, which could account for facilitated transport through the luminal membrane from endothelium-to-blood once sufficient glutamate concentrations accumulate in the endothelium. However, it is also likely that metabolism takes place in the endothelial cells (and/or the astrocytes) followed by efflux of the metabolism products to the blood. The glutamate-glutamine cycle does not seem to play a role in the brain efflux hypothesis, but metabolism to a-ketoglutarate via aminotransferases or glutamate dehydrogenase could take place both in endothelial cells and astrocytes. The effects of these pathways in the overall scheme have not been elucidated and they may account for a large part of the apparent L-glutamate efflux. Several in vivo studies have demonstrated the effect of blood-glutamate scavenging in improving the outcome after stroke, subarachnoid hemorrhage, traumatic brain injury and paraoxon intoxication. These studies support the transporter mediated brain glutamate efflux hypothesis and its possible clinical relevance, but as yet no firm in vivo evidence exists. Future studies should thus focus on verifying the presence of EAAT’s on the endothelial cells in vivo, determining EAAT subtype expression profile, identifying the possible transporter responsible for the passage of the luminal membrane, investigating to what extent metabolism takes part in the apparent L-glutamate efflux and clarifying the mechanisms behind the apparently beneficial effects of blood L-glutamate scavengers. References 1. Zhou Y, Danbolt NC. Glutamate as a neurotransmitter in the healthy brain. J Neural Transm 2014;121:799e817. 2. Nakanishi S. Molecular diversity of glutamate receptors and implications for brain function. Science 1992;258:597e603. 3. Rothman S. Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J Neurosci 1984;4: 1884e1891. 4. Gillessen T, Budd SL, Lipton SA. Excitatory amino acid neurotoxicity. Adv Exp Med Biol 2002;513:3e40. 5. Leibowitz A, Boyko M, Shapira Y, et al. Blood glutamate scavenging: insight into neuroprotection. Int J Mol Sci 2012;13: 10041e10066. 6. Danbolt NC. Glutamate uptake. Prog Neurobiol 2001;65:1e105. 7. Chaudhry FA, Lehre KP, van Lookeren Campagne M, et al. Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron 1995;15:711e720. 8. Lehre KP, Levy LM, Ottersen OP, et al. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci 1995;15:1835e1853. 9. Rothstein JD, Martin L, Levey AI, et al. Localization of neuronal and glial glutamate transporters. Neuron 1994;13:713e725. 10. Rothstein JD, Dykes-Hoberg M, Pardo CA, et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996;16:675e686. 11. Tanaka K, Watase K, Manabe T, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 1997;276:1699e1702.

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