Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases in subjects over 65 years of age. Several postulates have been put forward that relate AD neuropathology to intellectual and functional impairment. These range from free-radical-induced damage, through cholinergic dysfunction, to beta-amyloid-induced toxicity. However, therapeutic strategies aimed at improving the cognitive symptoms of patients via choline supplementation, cholinergic stimulation or beta-amyloid vaccination, have largely failed. A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate (L-Glu) may underlie the pathogenic mechanisms of (e.g.) hypoxia-ischemia, epilepsy, and chronic neurodegenerative disorders such as Huntington's disease and AD. Almost all neurons in the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotropic L-glutamate receptors, which can mediate post-synaptic Ca2+ influx. Excitotoxicity resulting from excessive activation of NMDA receptors may enhance the localized vulnerability of neurons in a manner consistent with AD neuropathology, as a consequence of an altered regional distribution of NMDA receptor subtypes. This review discusses mechanisms for the involvement of the NMDA receptor complex and its interaction with polyamines in the pathogenesis of AD. NMDA receptor antagonists have potential for the therapeutic amelioration of AD.     

Differential Regulation of Molecular Subtypes of Muscarinic Receptors in Alzheimer's Disease

Abstract: Molecular subtypes of muscarinic receptors (m1–m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease. However, the status of these receptors in human brain and Alzheimer's disease is incompletely understood. The m1–m5 receptors in brains from control subjects and Alzheimer's disease patients were examined using a panel of specific antisera and radioligand binding. Quantitative immunoprecipitation demonstrated a predominance of the m1, m2, and m4 receptor subtypes in cortical and subcortical regions in control subjects. In Alzheimer's disease, normal levels of m1 receptors measured by radioligand binding contrasted with decreased m1 receptor immunoreactivity, suggesting that the m1 receptor is altered in Alzheimer's disease. The m2 immunoreactivity was decreased, consistent with the loss of m2 binding sites and the location of this receptor subtype on presynaptic cholinergic terminals. The m4 receptor was up-regulated significantly and may offer a target for new memory-enhancing drugs. Differential alterations of molecular subtypes of muscarinic receptors may contribute to the cholinergic component of Alzheimer's disease dementia.     

Muscarinic Cholinergic Receptor Subtypes in Hippocampus in Human Cognitive Disorders

Total muscarinic receptor levels, the levels of the subtypes exhibiting high and low affinity for pirenzepine, and the high- and low-affinity agonist states of the receptor were investigated in hippocampal tissue obtained at autopsy from mentally normal individuals and the following pathological groups: Alzheimer's disease, Parkinson's disease, Down's syndrome, alcoholic dementia, Huntington's chorea, and motor-neurone disease. A moderate decrease in the density of both high-affinity pirenzepine and high-affinity agonist subtypes was found in Alzheimer's disease, whereas a trend towards an increase in the overall muscarinic receptor density was apparent in the parkinsonian patients without dementia, mainly due to an increase in the low-affinity agonist state; the differences between the Alzheimer's disease and nondemented parkinsonian cases were highly significant. As previously reported, the levels of both choline acetyltransferase and acetylcholinesterase were markedly reduced in both Alzheimer's disease and Parkinson's disease--with a greater loss of both enzymes in the demented subgroup of parkinsonian patients. Activities of the cholinergic enzymes were also extensively reduced in Down's syndrome, accompanied by a loss of high-affinity pirenzepine binding. There were no significant receptor or enzyme alterations in the other groups studied. These observations suggest that in the human brain, extensive degeneration of cholinergic axons to the hippocampus, as indicated by a loss of cholinergic enzymes, is not necessarily accompanied by extensive muscarinic receptor abnormalities (as might be expected if a major subpopulation were presynaptic). Moreover, the opposite changes in muscarinic binding in Parkinson's and Alzheimer's diseases may be related to the greater severity of dementia in the latter disease.     

Glutamate-stimulated secretion of amyloid precursor protein from cortical rat brain slices

The aim of this study was to determine whether L-glutamate, a major excitatory transmitter in the cerebral cortex, modulates the proteolytic cleavage of the amyloid precursor protein (APP) in the brain through specific receptor activation. Native rat brain cerebral cortical slices were stimulated either with L-glutamate or various glutamate receptor agonists, and the soluble APP derivatives released into the incubation medium were assayed by Western blot analysis. Immunoprecipitation with specific antibodies revealed that in the medium only secretory forms of APP lacking intact C-terminus were present, whereas in the brain slices both C- and N-terminal intact APP products were detectable. L-glutamate induced the release of secretory APP from cortical slices in a concentration-dependent but biphasic manner, with the highest release at 50 μM L-glutamate and smaller effects at higher glutamate concentrations. To determine whether the effect of L-glutamate is mediated through distinct glutamate receptor subtypes, brain slices were incubated in the presence of various specific glutamate receptor agonists. Activation of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor with 50 nM (RS)-bromohomoibotenic acid resulted in a reduced release of secretory APP by 17%±3 (P<0.01, one tailed Student's t-test) compared to the incubation without any drug. Stimulation of the metabotropic glutamate receptor with 50 nM (2S,3S,4S)--(carboxycyclopropyl)-glycine (L-CCG-I) led to an enhanced release of secretory APP by 39%±3 (P<0.001), whereas activation of the N-methyl-D-aspartate (NMDA) receptor with 50 nM (1R,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1R,3R)-ACPD) did not significantly change the secretion of APP compared to the incubation without any drug. The data suggest that: (i) cortical glutamatergic neurotransmission is involved in APP metabolism; and (ii) the stimulation of APP cleavage in cerebral cortical brain slices is mainly mediated by the metabotropic but not the NMDA glutamate receptor subtype, whereas the AMPA receptor subtype seems to inhibit the secretory path of APP processing.     

Modulation by L- and D-isoforms of amino acids of the L-glutamate response of N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptor subtypes epsilon 1 and zeta 1 were coexpressed in Xenopus oocytes for the investigation of the magnitude of augmentation of the L-glutamate response by 20 common L-amino acids and their 19 D-isoforms. Simultaneous application of L- and D-alanine, -cysteine, and -serine, or glycine and L-glutamate potentiated the glutamate-induced current. Other amino acids produced only marginal effects. Analysis of the relationship between the response and amino acid size revealed that the critical threshold size is between those of cysteine and aspartate. No amino acid alone induced a current. The effects of L- and D-alanine, -cysteine, and -serine applied with L-glutamate were concentration-dependent. Molecular modeling of these three amino acids revealed a positive relationship between the charge at an atom of the side chain and the receptor sensitivity, which may explain the efficacies of these amino acids.     

Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.     

Cholesterol metabolism, apolipoprotein E, adenosine triphosphate-binding cassette transporters, and Alzheimer's disease

PURPOSE OF REVIEW: Recent evidence suggests that cholesterol metabolism participates in the pathogenesis of Alzheimer's disease. Apolipoprotein E is the main lipid carrier in the brain and the best-established risk factor for late-onset Alzheimer's disease. Intracellular cholesterol levels influence the generation of amyloid-beta peptides, the toxic species thought to be a primary cause of Alzheimer's disease. Finally, compounds that modulate cholesterol metabolism affect amyloid-beta generation. This review summarizes data linking apolipoprotein E and adenosine triphosphate-binding cassette transporters to aspects of cholesterol metabolism and Alzheimer's disease pathogenesis. RECENT FINDINGS: In vivo, the lipidation status of apolipoprotein E affects amyloid-beta burden in mice with Alzheimer's disease, which appears to caused by the modulation of amyloid-beta deposition or clearance rather than amyloid-beta production. State-of-the-art in-vivo assays reveal that amyloid-beta is cleared from the brain by multiple pathways. Members of the adenosine triphosphate-binding cassette superfamily of transporters regulate lipid homeostasis and apolipoprotein metabolism in the brain, and may affect Alzheimer's disease pathogenesis by modulating apolipoprotein E lipidation as well as intracellular sterol homeostasis. SUMMARY: Proteins involved in brain cholesterol metabolism may affect the pathogenesis of Alzheimer's disease. Compounds that modulate the expression of these proteins may be of therapeutic benefit in Alzheimer's disease.     

Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.     

Selective Loss of N-Methyl-d-Aspartate-Sensitive l-[3H]Glutamate Binding Sites in Rat Brain Following Portacaval Anastomosis

Excitatory amino acids have been implicated in the pathogenesis of hepatic encephalopathy. In the present study, kainate, quisqualate and N-methyl-D-aspartate (NMDA) subclasses of L-glutamate receptors were measured in adult rat brain by quantitative receptor autoradiography following surgical construction of an end-to-side portacaval anastomosis (PCA). PCA resulted in sustained hyperammonemia and decreased binding of L-glutamate to the NMDA receptor when compared to sham-operated controls. Decreases in binding ranged from 17 to 39% in several regions of cerebral cortex, hippocampus, striatum, and thalamus. Binding to quisqualate and kainate receptor subtypes was not altered. PCA leads to astrocytic changes in brain but does not result in any measurable loss of neuronal integrity. It is therefore proposed that decreased glutamate binding to the NMDA receptor following PCA results from increased extracellular glutamate caused by decreased reuptake into perineuronal astrocytes and a compensatory down-regulation of these receptors. Such changes could be of pathophysiological significance in hepatic encephalopathy.     

Isolation, localization, and cloning of a kainic acid binding protein from frog brain

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.     

Pharmacokinetic simulations of SPECT quantitation of the M2 muscarinic neuroreceptor subtype in disease states using radioiodinated (R,R)-4IQNB.

Alzheimer's disease (AD) involves selective loss of muscarinic M2, but not M1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of M2 receptors in AD is limited by the fact that there is currently no available M2-selective radioligand which can penetrate the blood-brain barrier. However, by taking advantage of the different pharmacokinetic properties of (R,R)-[123I]IQNB for the M1 and M2 subtypes, it may be possible to estimate losses in M2. It has previously been hypothesized that the difference between an early study and a late study should provide information on the M2 receptor population. In order to test this hypothesis, we present here the results of pharmacokinetic simulations of the in vivo localization of (R,R)-[123I]IQNB in brain regions containing various proportions of M1 and M2 subtypes. These results permit us to conclude that SPECT imaging of (R,R)-[123I]IQNB localization can potentially be used to quantitate changes in the M2 subtype in a disease state within a brain region for which the ratio M2/M1 is sufficiently high in normal individuals.     

GABAA receptor subtypes: from pharmacology to molecular biology.

GABAA receptors are GABA (gamma-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. Although previous pharmacological and biochemical data had suggested a degree of heterogeneity, recent cloning of at least 15 different receptor subunits, thought to be combined in groups of five, indicates that the brain may contain a truly astonishing variety of GABAA receptor subtypes. This review describes the little that is known about these subtypes, emphasizing possible molecular bases of receptor heterogeneity. We also discuss approaches to establishing the subunit composition of subtypes.     

Ionotropic glutamate receptors mediate juvenile hormone synthesis in the cockroach,Diploptera punctata

By monitoring changes in the cytosolic [Ca2+](i) and rates of juvenile hormone (JH) synthesis in response to L-glutamate agonists and antagonists, we identified and characterized glutamate receptor subtypes in corpus allatum (CA) cells of the cockroach, Diploptera punctata. During the first ovarian cycle, corpora allata exhibited a cycle of changes in sensitivity to L-glutamate correlated to cyclic changes in rates of JH synthesis. When exposed to 60 microM L-glutamate in vitro, the active corpora allata of day-4 mated females produced 60% more JH, while inactive corpora allata at other ages showed 10-20% stimulatory response. Pharmacological characterization using various L-glutamate receptor agonists and antagonists indicated that several ionotropic subtypes of L-glutamate receptors were present in the CA. The CA showed an increase in rates of JH synthesis in response to NMDA, kainate, and quisqualate, but not to AMPA in both L-15 medium and minimum incubation medium. In contrast, applications of the metabotropic receptor-specific agonist trans-ACPD failed to elicit a change in the cytosolic [Ca2+](i) and JH production.An elevation of cytosolic calcium concentration, followed by 20-30% rise in JH production, was observed when active CA cells were exposed to 10-40 microM kainate. Kainate had no stimulatory effect on JH synthesis in calcium-free medium. The kainate-induced JH synthesis was blocked by 20 microM CNQX but was not affected by 20 microM NBQX. Kainate-stimulated JH production was not suppressed by MK-801 (a specific blocker of NMDA-receptor channel), nor was NMDA-stimulated JH production affected by CNQX (a specific antagonist of kainate receptor). These data suggest that active CA cells are stimulated to synthesize more JH by a glutamate-induced calcium rise via NMDA-, kainate- and/or quisqualate-sensitive subtypes of ionotropic L-glutamate receptors. The metabotropic-subtype and ionotropic AMPA-subtype L-glutamate receptors are unlikely to be present on active CA cells.     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

Fusion antibody for Alzheimer's disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation

Delivery of monoclonal antibody therapeutics across the blood-brain barrier is an obstacle to the diagnosis or therapy of CNS disease with antibody drugs. The immune therapy of Alzheimer's disease attempts to disaggregate the amyloid plaque of Alzheimer's disease with an anti-Abeta monoclonal antibody. The present work is based on a three-step model of immune therapy of Alzheimer's disease: (1) influx of the anti-Abeta monoclonal antibody across the blood-brain barrier in the blood to brain direction, (2) binding and disaggregation of Abeta fibrils in brain, and (3) efflux of the anti-Abeta monoclonal antibody across the blood-brain barrier in the brain to blood direction. This is accomplished with the genetic engineering of a trifunctional fusion antibody that binds (1) the human insulin receptor, which mediates the influx from blood to brain across the blood-brain barrier, (2) the Abeta fibril to disaggregate amyloid plaque, and (3) the Fc receptor, which mediates the efflux from brain to blood across the blood-brain barrier. This fusion protein is a new antibody-based therapeutic for Alzheimer's disease that is specifically engineered to cross the human blood-brain barrier in both directions.     

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.     

CCK receptors and human neurological disease

Cholecystokinin (CCK) receptor binding was measured in postmortem brain tissue of patients with Alzheimer's dementia, Huntington's chorea, and neurologically healthy matched controls. CCK binding was significantly reduced inthe basal ganglia and cerebral cortex of Huntington's patients, but was normal in the temporal and cingulate cortex of patients with Alzheimer's disease. These findings indicate that CCK receptor loss is unique to specific neurodegenerative disease(s), and that CCK may be involved in the symptoms of Huntington's disease but is not implicated in the neuropathology of Alzheimer's dementia.     

Plasticity of Glutamate and GABAA Receptors in the Hippocampus of Patients with Alzheimer's Disease

1. Aim: In Alzheimer's disease (AD) it is well known that specific regions of the brain are particularly vulnerable to the pathologic insults of the disease. In particular, the hippocampus is affected very early in the disease and by end stage AD is ravaged by neurofibrillary tangles and senile plaques (i.e., the pathologic hallmarks of AD). Throughout the past several years our laboratory has sought to determine the molecular mechanisms underlying the selective vulnerability of neurons in AD.2. Methods: To this end, we employed immunohistochemical, biochemical, and in situ hybrization methods to examine glutamate and -aminobutyric acid (GABA_A) receptor subtypes in the hippocampus of patients displaying the full spectrum of AD pathology.3. Results: Despite the fact that the hippocampus is characterized by a marked loss of neurons in the late stages of the disease, our data demonstrate a rather remarkable preservation among some glutamate and GABA_A receptor subtypes.4. Conclusions: Collectively, our data support the view that the relatively constant levels of selected receptor subtypes represent a compensatory up-regulation of these receptors subunits in surviving neurons. The demonstration that glutamate and GABA receptor subunits are comparably unaffected implies that even in the terminal stages of the disease the brain is attempting to maintain a balance in excitatory and inhibitory tone. Our data also support the concept that receptor subunits are differentially affected in AD with some subunits displaying no change while others display alterations in protein and mRNA levels within selected regions of the hippocampus. Although many of these changes are modest, they do suggest that the subunit composition of these receptors may be altered and hence affect the pharmacokinetic and physiological properties of the receptor. The latter findings stress the importance of understanding the subunit composition of individual glutamate/GABA receptors in the diseased brain prior to the development of drugs targeted towards those receptors.     

Characterisation, Density, and Distribution of Kainate Receptors in Normal and Alzheimer''s Diseased Human Brain

The specific binding of [3H]kainic acid was investigated in membrane preparations from human parietal cortex obtained postmortem. Saturation studies revealed that binding occurred to a single population of sites with a KD of 15 nM and a Bmax of 110 fmol/mg of protein. The kinetically determined dissociation constant for these sites agreed well with that obtained from saturation analyses. Pharmacological characterisation of these sites gave a profile consistent with those reported for kainate receptor sites in animal brain. The integrity of kainate receptors was studied in several brain regions from six patients who had died of Alzheimer's disease and from six closely matched control subjects. No change in either the affinity or the number of kainate receptors was seen in any of the regions studied, despite the loss of neocortical and hippocampal glutamatergic terminals in the Alzheimer's diseased brains, as previously reported.     

Effects of Anti-Glutamate-Binding Protein Antibodies on Synaptic Membrane Ion Flux, Glutamate Transport and Release, and l-Glutamate Binding Activities

Antibodies (Abs) raised against the L-glutamate-binding protein (GBP) purified from bovine brain were used to define the possible physiologic activity of GBP in synaptic membranes. Three processes were examined for their sensitivity to the Abs: the excitatory amino acid stimulation of thiocyanate (SCN-) flux, the transport of L-glutamic acid across the synaptic membrane, and the depolarization-induced release of L-glutamate. Only the amino acid-induced changes in ion flux were inhibited by the anti-GBP Abs. The change in membrane potential produced by exposure of synaptic membranes to excitatory amino acids was measured as the increase in the uptake of the lipophilic anion SCN-. The L-glutamate-induced SCN- influx was 40 times more sensitive to inhibition by the anti-GBP Abs than the stimulation of ion flux by kainate, and 60 times more sensitive than that produced by quisqualate. The anti-GBP Abs did not inhibit the activation of ion flux produced by N-methyl-D-aspartate. The inhibition of glutamate-stimulated ion fluxes by the Abs was complete, whereas the inhibition of L-glutamate binding to either the rat or bovine brain GBP was not. The results obtained indicated that although the majority of the anti-GBP Abs were not directed against the glutamate recognition site of the GBP and of presumed synaptic membrane receptors, they were effective in blocking the activation of receptor-associated ion channels. Thus, the GBP may be considered a component of some excitatory amino acid receptor complexes.