Reconstitution of GABA, Glycine and Glutamate Transporters.

In contrast to water soluble enzymes which can be purified and studied while in solution, studies of solute carrier (transporter) proteins require both that the protein of interest is situated in a phospholipid membrane and that this membrane forms a closed compartment. An additional challenge to the study of transporter proteins has been that the transport depends on the transmembrane electrochemical gradients. Baruch I. Kanner understood this early on and first developed techniques for studying plasma membrane vesicles. This advanced the field in that the experimenter could control the electrochemical gradients. Kanner, however, did not stop there, but started to solubilize the membranes so that the transporter proteins were taken out of their natural environment. In order to study them, Kanner then had to find a way to reconstitute them (reinsert them into phospholipid membranes). The scope of the present review is both to describe the reconstitution method in full detail as that has never been done, and also to reveal the scientific impact that this method has had. Kanner's later work is not reviewed here although that also deserves a review because it too has had a huge impact.

The cystine-glutamate exchanger (xCT, Slc7a11) is expressed in significant concentrations in a subpopulation of astrocytes in the mouse brain.

The cystine-glutamate exchanger (xCT) promotes glutathione synthesis by catalyzing cystine uptake and glutamate release. The released glutamate may modulate normal neural signaling and contribute to excitotoxicity in pathological situations. Uncertainty, however, remains as neither the expression levels nor the distribution of xCT have been unambiguously determined. In fact, xCT has been reported in astrocytes, neurons, oligodendrocytes and microglia, but most of the information derives from cell cultures. Here, we show by immunohistochemistry and by Western blotting that xCT is widely expressed in the central nervous system of both sexes. The labeling specificity was validated using tissue from xCT knockout mice as controls. Astrocytes were selectively labeled, but showed greatly varying labeling intensities. This astroglial heterogeneity resulted in an astrocyte domain-like labeling pattern. Strong xCT labeling was also found in the leptomeninges, along some blood vessels, in selected circumventricular organs and in a subpopulation of tanycytes residing the lateral walls of the ventral third ventricle. Neurons, oligodendrocytes and resting microglia, as well as reactive microglia induced by glutamine synthetase deficiency, were unlabeled. The concentration of xCT protein in hippocampus was compared with that of the EAAT3 glutamate transporter by immunoblotting using a chimeric xCT-EAAT3 protein to normalize xCT and EAAT3 labeling intensities. The immunoblots suggested an xCT/EAAT3 ratio close to one (0.75 ± 0.07; average ± SEM; n = 4) in adult C57BL6 mice. CONCLUSIONS: xCT is present in select blood/brain/CSF interface areas and in an astrocyte subpopulation, in sufficient quantities to support the notion that system xc- provides physiologically relevant transport activity.

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons.

Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045-2064.

Astrocyte membrane properties are altered in a rat model of developmental cortical malformation but single-cell astrocytic glutamate uptake is robust.

Developmental cortical malformations (DCMs) are linked with severe epilepsy and are caused by both genetic and environmental insults. DCMs include several neurological diseases, such as focal cortical dysplasia, polymicrogyria, schizencephaly, and others. Human studies have implicated astrocyte reactivity and dysfunction in the pathophysiology of DCMs, but their specific role is unknown. As astrocytes powerfully regulate glutamate neurotransmission, and glutamate levels are known to be increased in human epileptic foci, understanding the role of astrocytes in the pathological sequelae of DCMs is extremely important. Additionally, recent studies examining astrocyte glutamate uptake in DCMs have reported conflicting results, adding confusion to the field. In this study we utilized the freeze lesion (FL) model of DCM, which is known to induce reactive astrocytosis and cause significant changes in astrocyte morphology, proliferation, and distribution. Using whole-cell patch clamp recording from astrocytes, we recorded both UV-uncaging and synaptically evoked glutamate transporter currents (TCs), widely accepted assays of functional glutamate transport by astrocytes. With this approach, we set out to test the hypothesis that astrocyte membrane properties and glutamate transport were disrupted in this model of DCM. Though we found that the developmental maturation of astrocyte membrane resistance was disrupted by FL, glutamate uptake by individual astrocytes was robust throughout FL development. Interestingly, using an immunolabeling approach, we observed spatial and developmental differences in excitatory amino acid transporter (EAAT) expression in FL cortex. Spatially specific differences in EAAT2 (GLT-1) and EAAT1 (GLAST) expression suggest that the relative contribution of each EAAT to astrocytic glutamate uptake may be altered in FL cortex. Lastly, we carefully analyzed the amplitudes and onset times of both synaptically- and UV uncaging-evoked TCs. We found that in the FL cortex, synaptically-evoked, but not UV uncaging-evoked TCs, were larger in amplitude. Additionally, we found that the amount of electrical stimulation required to evoke a synaptic TC was significantly reduced in the FL cortex. Both of these findings are consistent with increased excitatory input to the FL cortex, but not with changes in how individual astrocytes remove glutamate. Taken together, our results demonstrate that the maturation of astrocyte membrane resistance, local distribution of glutamate transporters, and glutamatergic input to the cortex are altered in the FL model, but that single-cell astrocytic glutamate uptake is robust.

Astrocytic glutamate uptake is slow and does not limit neuronal NMDA receptor activation in the neonatal neocortex.

Glutamate uptake by astrocytes controls the time course of glutamate in the extracellular space and affects neurotransmission, synaptogenesis, and circuit development. Astrocytic glutamate uptake has been shown to undergo post-natal maturation in the hippocampus, but has been largely unexplored in other brain regions. Notably, glutamate uptake has never been examined in the developing neocortex. In these studies, we investigated the development of astrocytic glutamate transport, intrinsic membrane properties, and control of neuronal NMDA receptor activation in the developing neocortex. Using astrocytic and neuronal electrophysiology, immunofluorescence, and Western blot analysis we show that: (1) glutamate uptake in the neonatal neocortex is slow relative to neonatal hippocampus; (2) astrocytes in the neonatal neocortex undergo a significant maturation of intrinsic membrane properties; (3) slow glutamate uptake is accompanied by lower expression of both GLT-1 and GLAST; (4) glutamate uptake is less dependent on GLT-1 in neonatal neocortex than in neonatal hippocampus; and (5) the slow glutamate uptake we report in the neonatal neocortex corresponds to minimal astrocytic control of neuronal NMDA receptor activation. Taken together, our results clearly show fundamental differences between astrocytic maturation in the developing neocortex and hippocampus, and corresponding changes in how astrocytes control glutamate signaling.

Network-Related Changes in Neurotransmitters and Seizure Propagation During Rodent Epileptogenesis.

OBJECTIVE: To test the hypothesis that glutamate and GABA are linked to the formation of epilepsy networks and the triggering of spontaneous seizures, we examined seizure initiation/propagation characteristics and neurotransmitter levels during epileptogenesis in a translationally relevant rodent model of mesial temporal lobe epilepsy. METHODS: The glutamine synthetase (GS) inhibitor methionine sulfoximine was infused into one of the hippocampi in laboratory rats to create a seizure focus. Long-term video-intracranial EEG recordings and brain microdialysis combined with mass spectrometry were used to examine seizure initiation, seizure propagation, and extracellular brain levels of glutamate and GABA. RESULTS: All seizures (n = 78 seizures, n = 3 rats) appeared first in the GS-inhibited hippocampus of all animals, followed by propagation to the contralateral hippocampus. Propagation time decreased significantly from 11.65 seconds early in epileptogenesis (weeks 1-2) to 6.82 seconds late in epileptogenesis (weeks 3-4, paired t test, p = 0.025). Baseline extracellular glutamate levels were 11.6-fold higher in the hippocampus of seizure propagation (7.3 µM) vs the hippocampus of seizure onset (0.63 µM, analysis of variance/Fisher least significant difference, p = 0.01), even though the concentrations of the major glutamate transporter proteins excitatory amino acid transporter subtypes 1 and 2 and xCT were unchanged between the brain regions. Finally, extracellular GABA in the seizure focus decreased significantly from baseline several hours before a spontaneous seizure (paired t test/false discovery rate). CONCLUSION: The changes in glutamate and GABA suggest novel and potentially important roles of the amino acids in epilepsy network formation and in the initiation and propagation of spontaneous seizures.

The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction.

The unravelling of the polarized distribution of AQP4 in perivascular astrocytic endfeet has revitalized the interest in the role of astrocytes in controlling water and ion exchange at the brain-blood interface. The importance of the endfeet is based on the premise that they constitute a complete coverage of the vessel wall. Despite a number of studies based on different microscopic techniques this question has yet to be resolved. We have made an electron microscopic 3D reconstruction of perivascular endfeet in CA1 (stratum moleculare) of rat hippocampus. The endfeet interdigitate and overlap, leaving no slits between them. Only in a few sites do processes--tentatively classified as processes of microglia--extend through the perivascular glial sheath to establish direct contact with the endothelial basal lamina. In contrast to the endfoot covering of the endothelial tube, the endfoot covering of the pericyte is incomplete, allowing neuropil elements to touch the basal lamina that enwraps this type of cell. The 3D reconstruction also revealed large bundles of mitochondria in the endfoot processes that came in close apposition to the perivascular endfoot membrane. Our data support the idea that in pathophysiological conditions, the perivascular astrocytic covering may control the exchange of water and solutes between blood and brain and that free diffusion is limited to narrow clefts between overlapping endfeet.

Novel aspects of glutamine synthetase in ammonia homeostasis.

Elevated blood ammonia (hyperammonemia) is believed to be a major contributor to the neurological sequelae following severe liver disease. Ammonia is cleared via two main mechanisms, the urea cycle pathway and the glutamine synthetase reaction. Recent studies of genetically modified animals confirm the importance of the urea cycle, but also suggest that the glutamine synthetase reaction is more important than previously recognized. While the liver clears about two-thirds of the body's ammonia via the combined action of the urea cycle and glutamine synthetase, extrahepatic tissues do not express all the components required for performing a complete urea cycle and therefore depend on the glutamine synthetase reaction for ammonia clearance. The brain is particularly vulnerable to the effects of hyperammonemia, which include impaired extracellular potassium buffering and brain edema. Moreover, the glutamine synthetase reaction is intimately linked to the metabolism of the excitatory and inhibitory neurotransmitters glutamate and gamma aminobutyric acid (GABA), implicating a key role for this enzyme in neurotransmission. This review discusses the emerging roles of glutamine synthetase in brain pathophysiology, particularly aspects related to ammonia homeostasis and hepatic encephalopathy.

Semi-quantitative distribution of excitatory amino acid (glutamate) transporters 1-3 (EAAT1-3) and the cystine-glutamate exchanger (xCT) in the adult murine spinal cord.

Proper glutamatergic neurotransmission requires a balance between glutamate release and removal. The removal is mainly catalyzed by the glutamate transporters EAAT1-3, while the glutamate-cystine exchanger (system xc- with specific subunit xCT) represents one of the release mechanisms. Previous studies of the spinal cord have focused on the cellular distribution of EAAT1-3 with special reference to the dorsal horn, but have not provided quantitative data and have not systematically compared multiple segments. Here we have studied the distribution of EAAT1-3 and xCT in sections of multiple spinal cord segments using knockout tissue as negative controls. EAAT2 and EAAT3 were evenly expressed in all gray matter areas at all segmental levels, albeit with slightly higher levels in laminae 1-4 (dorsal horn). Somewhat higher levels of EAAT2 were also seen in lamina 9 (ventral horn), while EAAT3 was also detected in the lateral spinal nucleus. EAAT1 was concentrated in laminae 1-3, lamina 10, the intermediolateral nucleus and the sacral parasympathetic nucleus, while xCT was concentrated in laminae 1-3, lamina 10 and the leptomeninges. The levels of these four transporters were low in white matter, which represents 42% of the spinal cord volume. Quantitative immunoblotting revealed that the average level of EAAT1 in the whole spinal cord was 0.6 ± 0.1% of that in the cerebellum, while the levels of EAAT2, EAAT3 and xCT were, respectively, 41.6 ± 12%, 39.8 ± 7.6%, and 30.8 ± 4.3% of the levels in the hippocampus (mean values ± SEM). Conclusions: Because the hippocampal tissue content of EAAT2 protein is two orders of magnitude higher than the content of the EAAT3, it follows that most of the gray matter in the spinal cord depends almost exclusively on EAAT2 for glutamate removal, while the lamina involved in the processing of autonomic and nociceptive information rely on a complex system of transporters.

Axon-terminals expressing EAAT2 (GLT-1; Slc1a2) are common in the forebrain and not limited to the hippocampus.

The excitatory amino acid transporter type 2 (EAAT2) represents the major mechanism for removal of extracellular glutamate. In the hippocampus, there is some EAAT2 in axon-terminals, whereas most of the protein is found in astroglia. The functional importance of the neuronal EAAT2 is unknown, and it is debated whether EAAT2-expressing nerve terminals are present in other parts of the brain. Here we selectively deleted the EAAT2 gene in neurons (by crossing EAAT2-flox mice with synapsin 1-Cre mice in the C57B6 background). To reduce interference from astroglial EAAT2, we measured glutamate accumulation in crude tissue homogenates. EAAT2 proteins levels were measured by immunoblotting. Although synapsin 1-Cre mediated gene deletion only reduced the forebrain tissue content of EAAT2 protein to 95.5 ±â€¯3.4% of wild-type (littermate) controls, the glutamate accumulation in homogenates of neocortex, hippocampus, striatum and thalamus were nevertheless diminished to, respectively, 54 ±â€¯4, 46 ±â€¯3, 46 ±â€¯2 and 65 ±â€¯7% of controls (average ±â€¯SEM, n = 3 pairs of littermates). GABA uptake was unaffected. After injection of U-13C-glucose, lack of neuronal EAAT2 resulted in higher 13C-labeling of glutamine and GABA in the hippocampus suggesting that neuronal EAAT2 is partly short-circuiting the glutamate-glutamine cycle in wild-type mice. Crossing synapsin 1-Cre mice with Ai9 reporter mice revealed that Cre-mediated excision occurred efficiently in hippocampus CA3, but less efficiently in other regions and hardly at all in the cerebellum. Conclusions: (1) EAAT2 is expressed in nerve terminals in multiple brain regions. (2) The uptake catalyzed by neuronal EAAT2 plays a role in glutamate metabolism, at least in the hippocampus. (3) Synapsin 1-Cre does not delete floxed genes in all neurons, and the contribution of neuronal EAAT2 is therefore likely to be larger than revealed in the present study.

Selective deletion of glutamine synthetase in the mouse cerebral cortex induces glial dysfunction and vascular impairment that precede epilepsy and neurodegeneration.

Glutamate-ammonia ligase (glutamine synthetase; Glul) is enriched in astrocytes and serves as the primary enzyme for ammonia detoxification and glutamate inactivation in the brain. Loss of astroglial Glul is reported in hippocampi of epileptic patients, but the mechanism by which Glul deficiency might cause disease remains elusive. Here we created a novel mouse model by selectively deleting Glul in the hippocampus and neocortex. The Glul deficient mice were born without any apparent malformations and behaved unremarkably until postnatal week three. There were reductions in tissue levels of aspartate, glutamate, glutamine and GABA and in mRNA encoding glutamate receptor subunits GRIA1 and GRIN2A as well as in the glutamate transporter proteins EAAT1 and EAAT2. Adult Glul-deficient mice developed progressive neurodegeneration and spontaneous seizures which increased in frequency with age. Importantly, progressive astrogliosis occurred before neurodegeneration and was first noted in astrocytes along cerebral blood vessels. The responses to CO2-provocation were attenuated at four weeks of age and dilated microvessels were observed histologically in sclerotic areas of cKO. Thus, the abnormal glutamate metabolism observed in this model appeared to cause epilepsy by first inducing gliopathy and disrupting the neurovascular coupling.

Low-affinity excitatory amino acid uptake in hippocampal astrocytes: a possible role of Na+/dicarboxylate cotransporters.

The excitatory amino acid transporters (EAATs) underlie the so-called "high affinity" uptake of glutamate, which is well characterized. In contrast, the "low-affinity" uptake of glutamate remains poorly defined, and it has been discussed whether it may represent a mere in vitro artifact. Here we have visualized "low-affinity" excitatory amino acid uptake sites by incubating rat hippocampal slices with the glutamate analogue D-aspartate in the presence of PMB-TBOA, which blocks the EAATs. After fixation of the slices, D-aspartate taken up into the tissue was localized with the use of light microscopic immunoperoxidase and electron microscopic immunogold methods, exploiting highly specific antibodies against D-aspartate. PMB-TBOA blocked uptake of both low and high exogenous D-aspartate concentrations (0.01-1.0 mM) into nerve terminals, as well as the uptake of 0.01 mM D-aspartate into astrocytes. Interestingly, there was a residual PMB-TBOA insensitive uptake of D-aspartate in astrocytes at higher exogenous D-aspartate concentrations (0.05-1.0 mM), strongly suggesting that astrocytes have "low-affinity" uptake sites for excitatory amino acid. The PMB-TBOA insensitive D-aspartate uptake in astrocytes was sodium dependent and inhibited by succinate and to certain extent by homocysteate, but not by cystine or DIDS. We suggest that excitatory amino acid is transported into astrocytes in a "low-affinity" fashion by sodium/dicarboxylate transporters.

Expression of Glutamate Transporters in Mouse Liver, Kidney, and Intestine.

Glutamate transport activities have been identified not only in the brain, but also in the liver, kidney, and intestine. Although glutamate transporter distributions in the central nervous system are fairly well known, there are still uncertainties with respect to the distribution of these transporters in peripheral organs. Quantitative information is mostly lacking, and few of the studies have included genetically modified animals as specificity controls. The present study provides validated qualitative and semi-quantitative data on the excitatory amino acid transporter (EAAT)1-3 subtypes in the mouse liver, kidney, and intestine. In agreement with the current view, we found high EAAT3 protein levels in the brush borders of both the distal small intestine and the renal proximal tubules. Neither EAAT1 nor EAAT2 was detected at significant levels in murine kidney or intestine. In contrast, the liver only expressed EAAT2 (but 2 C-terminal splice variants). EAAT2 was detected in the plasma membranes of perivenous hepatocytes. These cells also expressed glutamine synthetase. Conditional deletion of hepatic EAAT2 did neither lead to overt neurological disturbances nor development of fatty liver.

The Glutamate-Glutamine Cycle in Epilepsy.

Epilepsy is a complex, multifactorial disease characterized by spontaneous recurrent seizures and an increased incidence of comorbid conditions such as anxiety, depression, cognitive dysfunction, and sudden unexpected death. About 70 million people worldwide are estimated to suffer from epilepsy, and up to one-third of all people with epilepsy are expected to be refractory to current medications. Development of more effective and specific antiepileptic interventions is therefore requisite. Perturbations in the brain's glutamate-glutamine cycle, such as increased extracellular levels of glutamate, loss of astroglial glutamine synthetase, and changes in glutaminase and glutamate dehydrogenase, are frequently encountered in patients with epilepsy. Hence, manipulations of discrete glutamate-glutamine cycle components may represent novel approaches to treat the disease. The goal of his review is to discuss some of the glutamate-glutamine cycle components that are altered in epilepsy, particularly neurotransmitters and metabolites, enzymes, amino acid transporters, and glutamate receptors. We will also review approaches that potentially could be used in humans to target the glutamate-glutamine cycle. Examples of such approaches are treatment with glutamate receptor blockers, glutamate scavenging, dietary intervention, and hypothermia.

A novel glutamate transporter blocker, LL-TBOA, attenuates ischaemic injury in the isolated, perfused rat heart despite low transporter levels.

OBJECTIVES: Loss of glutamate from cardiomyocytes during ischaemia may aggravate ischaemia-reperfusion injury in open heart surgery. This may be due to reversal of excitatory amino acid transporters (EAATs). However, the expression of such transporters in cardiomyocytes is ambiguous and quantitative data are lacking. Our objective was to study whether EAATs were expressed in the rat heart and to study whether blocking of transporter operation during cardiac ischaemia could be beneficial. METHODS: We used TaqMan real-time PCR and immunoisolation followed by western blotting to unequivocally identify EAAT subtypes in rat hearts. We used a novel high-affinity non-transportable competitive inhibitor, named LL-TBOA [(2S,3S)-3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethoxy)ethoxy) acetamido)hexanamido)- hexanamido)-5-(4-(trifluoromethyl)benzamido)benzyloxy) aspartic acid], to block EAAT-mediated transport during global ischaemia and reperfusion of isolated rat hearts. RESULTS: Rat hearts expressed EAAT subtypes 1 and 3, while subtypes 2 and 4 were not detected. Hearts were isolated and perfused with 1.6 µM LL-TBOA for 5 min before 30 min of induced global ischaemia and 60 min of reperfusion (n = 8). Control hearts were perfused either with the solvent dimethylsulfoxide 3.5 mM (n = 7) or with no pretreatment (n = 8). Infarct size was evaluated by triphenyl tetrazolium chloride (TTC) staining. LL-TBOA reduced infarct size from 33 ± 14 to 20 ± 5% (mean ± SD) (P = 0.015). Dimethylsulfoxide alone had no effect (35 ± 2%). Reperfusion arrhythmias were reduced by LL-TBOA (P = 0.009), but not by dimethylsulfoxide alone. CONCLUSION: Rat hearts express EAAT1 and EAAT3, but the mRNA levels are, respectively, ∼ 25 and 200 times lower than in the brain. Addition of LL-TBOA has a beneficial effect against ischaemia-reperfusion injury.

GABA and Glutamate Transporters in Brain.

The mammalian genome contains four genes encoding GABA transporters (GAT1, slc6a1; GAT2, slc6a13; GAT3, slc6a11; BGT1, slc6a12) and five glutamate transporter genes (EAAT1, slc1a3; EAAT2, slc1a2; EAAT3, slc1a1; EAAT4, slc1a6; EAAT5, slc1a7). These transporters keep the extracellular levels of GABA and excitatory amino acids low and provide amino acids for metabolic purposes. The various transporters have different properties both with respect to their transport functions and with respect to their ability to act as ion channels. Further, they are differentially regulated. To understand the physiological roles of the individual transporter subtypes, it is necessary to obtain information on their distributions and expression levels. Quantitative data are important as the functional capacity is limited by the number of transporter molecules. The most important and most abundant transporters for removal of transmitter glutamate in the brain are EAAT2 (GLT-1) and EAAT1 (GLAST), while GAT1 and GAT3 are the major GABA transporters in the brain. EAAT3 (EAAC1) does not appear to play a role in signal transduction, but plays other roles. Due to their high uncoupled anion conductance, EAAT4 and EAAT5 seem to be acting more like inhibitory glutamate receptors than as glutamate transporters. GAT2 and BGT1 are primarily expressed in the liver and kidney, but are also found in the leptomeninges, while the levels in brain tissue proper are too low to have any impact on GABA removal, at least in normal young adult mice. The present review will provide summary of what is currently known and will also discuss some methodological pitfalls.

The rates of postmortem proteolysis of glutamate transporters differ dramatically between cells and between transporter subtypes.

Glutamate transporters (GLT-1, GLAST, EAAC1) limit the actions of excitatory amino acids. Because a disturbed transporter operation can cause or aggravate neurological diseases, transporters are of considerable neuropathological interest. Human samples, however, are seldom obtained fresh. Here, we used mice brains to study how fast glutamate transporters are degraded after death. Immunoblots showed that terminal GLT-1 epitopes (within residues 1-26 and 518-573) had mostly disappeared after 24 hr. GLAST termini (1-25 and 522-543) degraded slightly slower. In contrast, epitopes within central parts of GLT-1 (493-508) and the EAAC1 C-terminus (510-523) were readily detectable after 72 hr. The decline in immunoreactivity of the GLT-1 and GLAST termini was also seen in tissue sections, but proteolysis did not happen synchronously in all cells. At 24 hr, scattered cells remained strongly immunopositive, while the majority of cells were completely immunonegative. GLAST and GLT-1 co-localized in neocortical tissue, but at 12 hr, many GLAST-positive cells had lost the GLT-1 termini. The uneven disappearance of labeling was not observed with the antibodies to GLT-1 residues 493-508. The immunoreactivity to this epitope correlated better with the reported glutamate uptake activity. Thus, postmortem delay may affect epitopes differently, possibly causing erroneous conclusions about relative expression levels.

Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity.

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.

The sodium-dependent inorganic phosphate transporter SLC34A1 (NaPi-IIa) is not localized in the mouse brain: a case of tissue-specific antigenic cross-reactivity.

The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, the authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa immunoreactive band (~90 kDa) was only present in wild-type kidney membranes and not in kidney knockout or wild-type brain membranes. In the water-soluble fraction of wild-type and knockout brains, another band (~50 kDa) was observed; this band was not detected in the kidney. Light and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed immunolabeling of kidney tubules in wild-type but not knockout mice. In the brain, labeling of presynaptic nerve terminals was present also in NaPi-IIa-deficient mice. This labeling pattern was also produced by the NaPi-IIa preimmune serum. The authors conclude that the polyclonal antiserum is specific toward NaPi-IIa in the kidney, but in the brain, immunolabeling is caused by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest and underlines the utility of specificity testing using preimmune sera.

Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom.

Previous studies have shown that a compound purified from the spider Parawixia bistriata venom stimulates the activity of glial glutamate transporters and can protect retinal tissue from ischemic damage. To understand the mechanism by which this compound enhances transport, we examined its effects on the functional properties of glutamate transporters after solubilization and reconstitution in liposomes and in transfected COS-7 cells. Here, we demonstrate in both systems that Parawixin1 promotes a direct and selective enhancement of glutamate influx by the EAAT2 transporter subtype through a mechanism that does not alter the apparent affinities for the cosubstrates glutamate or sodium. In liposomes, we observed maximal enhancement by Parawixin1 when extracellular sodium and intracellular potassium concentrations are within physiological ranges. Moreover, the compound does not enhance the reverse transport of glutamate under ionic conditions that favor efflux, when extracellular potassium is elevated and the sodium gradient is reduced, nor does it alter the exchange of glutamate in the absence of internal potassium. These observations suggest that Parawixin1 facilitates the reorientation of the potassium-bound transporter, the rate-limiting step in the transport cycle, a conclusion further supported by experiments showing that Parawixin1 does not stimulate uptake by an EAAT2 transport mutant (E405D) defective in the potassium-dependent reorientation step. Thus, Parawixin1 enhances transport through a novel mechanism targeting a step in the transport cycle distinct from substrate influx or efflux and provides a basis for the design of new drugs that act allosterically on transporters to increase glutamate clearance.