J. Timothy Greenamyre

Excitatory amino acids and Alzheimer's disease.

Excitatory amino acids (EAA) such as glutamate and aspartate are major transmitters of the cerebral cortex and hippocampus, and EAA mechanisms appear to play a role in learning and memory. Anatomical and biochemical evidence suggests that there is both pre- and postsynaptic disruption of EAA pathways in Alzheimers disease. Dysfunction of EAA pathways could play a role in the clinical manifestations of Alzheimers disease, such as memory loss and signs of cortical disconnection. Furthermore, EAA might be involved in the pathogenesis of Alzheimers disease, by virtue of their neurotoxic (excitotoxic) properties. Circumstantial evidence raises the possibility that the EAA system may partially determine the distribution of pathology in Alzheimers disease and may be important in producing the neurofibrillary tangles, RNA reductions and dendritic changes which characterize this devastating disorder. In this article, we will review the evidence suggesting a role for EAA in the clinical manifestations and pathogenesis of Alzheimers disease.

Glutamate dysfunction in Alzheimer's disease: an hypothesis

Abstract Glutamate is a major excitatory neurotransmitter that has been implicated in memory formation and learning. This acidic amino acid also has neurotoxic properties, and in animals produces lesions reminiscent of human neurodegenerative diseases. Here we present evidence that supports the hypothesis that glutamate dysfunction is involved in the pathophysiology of Alzheimers disease and can account for many of the neurochemical and behavioral deficits observed in this disease.

Dementia of the Alzheimer's Type: Changes in Hippocampal L‐[3H]Glutamate Binding

Abstract: Glutamate or a related excitatory amino acid is thought to be the major excitatory neurotransmitter of hippocampal afferents, intrinsic neurons, and efferents. We have used an autoradiographic technique to investigate the status of excitatory amino acid receptors in the hippocampal formation of patients dying with dementia of the Alzheimer type (DAT). We examined l‐[3H]glutamate binding to sections from the hippocampal formation of six patients dying of DAT and six patients without DAT and found marked reductions in total [3H]glutamate binding in all regions of hippocampus and adjacent parahippocampal cortex in DAT brains as compared to controls. When subtypes of excitatory amino acid receptors were assayed, it was found that binding to the N‐methyl‐d‐aspartate (NMDA)‐sensitive receptor was reduced by 75–87%, with the greatest loss found in stratum moleculare and stratum pyramidale of CA1. Binding to quisqualate (QA)‐sensitive receptors was reduced by 45–69%. There were smaller reductions (21–46%) in GABAA receptors in DAT cases. Muscarinic cholinergic receptors assayed in adjacent sections of hippocampal formation were unchanged in DAT. Benzodiazepine receptors were reduced significantly only in parahippocampal cortex by 44%. These results suggest that glutamatergic neurotransmission within the hippocampal formation is likely to be severely impaired in Alzheimers disease. Such impairment may account for some of the cognitive decline and memory deficits that characterize DAT.

GLUTAMATE TRANSMISSION AND TOXICITY IN ALZHEIMER'S DISEASE

1. Despite intensive research, the cause of Alzheimers disease is unknown. 2. Glutamate is the major excitatory transmitter of the cerebral cortex and hippocampus and it appears to have an important role in learning and memory. In addition to its transmitter function, glutamate is a neurotoxin which has been implicated in the pathogenesis of a variety of neurodegenerative disorders. 3. Glutamate toxicity may play a role in the pathogenesis of Alzheimers disease. 4. Disruption of glutamatergic neurotransmission may account, in part, for the learning and memory deficits of Alzheimers disease. 5. Labeling of the glutamate receptor complex may allow in vivo diagnosis by positron emission tomography. 6. Glutamate receptor ligands may provide a means of therapeutic intervention in Alzheimers disease.

Regional variations in the pharmacology of NMDA receptor channel blockers : implications for therapeutic potential

Abstract: Quantitative receptor autoradiography was used to examine the regional binding characteristics of a diverse group of N‐methyl‐d‐aspartate (NMDA)‐receptor channel blockers that varied in potency 105‐fold. Full competition curves were generated in each of six brain regions for 11 different compounds. MK‐801 was the most potent compound studied, with an IC50 of ∼10 nM in the forebrain regions and 24 nM in the cerebellar granule cell layer (p < 0.05). The binding affinities of nine of the 11 compounds examined were significantly different in cerebellar granule cell layer than in forebrain regions. In addition, the apparent Hill slopes of five of the compounds were significantly different in cerebellum compared with forebrain. That the rank order of drug potencies in cerebellum diverges from that in forebrain suggests that cerebellar NMDA‐receptor ion channels differ pharmacologically from those in forebrain. There was a general trend that drugs known to be well tolerated in humans (remacemide hydrochloride and its metabolites, amantadine, budipine, and memantine) had lower affinities than compounds with severe neurobehavioral or psychotomimetic effects. Moreover, all of the compounds known to be well tolerated in humans had a significantly higher affinity in the cerebellum than in forebrain regions, in contrast to MK‐801, 1‐[1‐(2‐thienyl)cyclohexyl]‐piperidine hydrochloride (TCP), phencyclidine (PCP), and ketamine, which had lower affinities in cerebellum. Our results are consistent with the notion that low affinity (rapid kinetics) and, possibly, subunit specificity (as indicated by distinct regional pharmacologies) may be important determinants of the clinical tolerability of NMDA‐receptor channel blockers.

HIGH CORRELATION BETWEEN THE LOCALIZATION OF (3H)TCP BINDING AND NMDA RECEPTORS

Tritiated N-(1-[2-thienylcyclohexyl)-3,4-piperidine ([3H]TCP), a derivative of the dissociative anesthetic phencyclidine (PCP), has recently been shown to bind specifically to a o-opiate receptor within the central nervous system of the rat (Sircar and Zukin, 1985). Drugs of this class noncompetitively inhibit the excitatory properties of Nmethyl-D-aspartate (NMDA), a glutamate analogue, in the spinal cord (Martin and Lodge, 1985), and inhibit long-term potentiation in the hippocampus (Stringer et al., 1983). Both of these effects are thought to be mediated by NMDA-sensitive glutamate receptors. The NMDA receptor is coupled to a voltage-sensitive cation channel that is gated by Mg 2÷. It is at this.channel site that PCP and related compounds are believed to act. In order to further delineate the relationship between the dissociative anesthetics and NMDA receptors, we have compared the regional binding distribution of the o-specific compound [3H]TCP and NMDA-sensitive [3H]glutamate binding sites within the rat CNS using quantitative autoradiography. Radiolabelling of each receptor class was carried out on alternate 20 /~m thick slide mounted sections taken at various levels of rat forebrain. For [3H]TCP binding, sections were preincubated for 30 min in cold 50 mM Tris-acetate, pH 7.4 and then dried. Sections were then incubated for 45 min in the same buffer with 1 mM magnesium acetate and 20 nM [3H]TCP (52.9 Ci/mmol, New England Nuclear Corp.) in the presence or ab

Characterization of the excitotoxic potential of the reversible succinate dehydrogenase inhibitor malonate.

Abstract: Although the mechanism of neuronal death in neurodegenerative diseases remains unknown, it has been hypothesized that relatively minor metabolic defects may predispose neurons to N‐methyl‐d‐aspartate (NMDA) receptor‐mediated excitotoxic damage in these disorders. To further investigate this possibility, we have characterized the excitotoxic potential of the reversible succinate dehydrogenase (SDH) inhibitor malonate. After its intrastriatal stereotaxic injection into male Sprague‐Dawley rats, malonate produced a dose‐dependent lesion when assessed 3 days after surgery using cytochrome oxidase histochemistry. This lesion was attenuated by coadministration of excess succinate, indicating that it was caused by specific inhibition of SDH. The lesion was also prevented by administration of the noncompetitive NMDA antagonist MK‐801. MK‐801 did not induce hypothermia, and hypothermia itself was not neuroprotective, suggesting that the neuroprotective effect of MK‐801 was due to blockade of the NMDA receptor ion channel and not to any nonspecific effect. The competitive NMDA antagonist LY274614 and the glycine site antagonist 7‐chlorokynurenate also profoundly attenuated malonate neurotoxicity, further indicating an NMDA receptor‐mediated event. Finally, the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) antagonist NBQX (2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)‐quinoxaline) was ineffective at preventing malonate toxicity at a dose that effectively reduced S‐AMPA toxicity, indicating that non‐NMDA receptors are involved minimally, if at all, in the production of the malonate lesion. We conclude that inhibition of SDH by malonate results in NMDA receptor‐mediated excitotoxic neuronal death. If this mechanism of “secondary” or “weak” excitotoxicity plays a role in neurodegenerative disease, NMDA antagonists and other “antiexcitotoxic” strategies may have therapeutic potential for these diseases.

Synaptic localization of striatal NMDA, quisqualate and kainate receptors

Striatal binding of labeled glutamate to N-methyl-D-aspartate (NMDA) receptors, D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) to quisqualate receptors and kainate to kainate receptors was examined in rats which had received unilateral decortications or unilateral striatal quinolinic acid lesions. One week after decortication, there were no significant changes in NMDA, quisqualate or kainate receptors in the striatum ipsilateral to the lesion, when compared to the striatum contralateral to the lesion. In contrast, binding to NMDA receptors was reduced by 92%, to quisqualate receptors by 80% and to kainate receptors by 81% in striatum 3 months after quinolinic acid lesions. The reduction in NMDA receptor binding was significantly greater than the loss of quisqualate or kainate receptors. These results suggest that NMDA, quisqualate and kainate receptor recognition sites are located postsynaptically in the striatum. These results also have implications for the quinolinic acid model of Huntingtons disease.

[3H]Dihydrorotenone Binding to NADH: Ubiquinone Reductase (Complex I) of the Electron Transport Chain: An Autoradiographic Study

Abnormalities of mitochondrial energy metabolism may play a role in normal aging and certain neurodegenerative disorders. In this regard, complex I of the electron transport chain has received substantial attention, especially in Parkinson’s disease. The conventional method for studying complex I has been quantitation of enzyme activity in homogenized tissue samples. To enhance the anatomic precision with which complex I can be examined, we developed an autoradiographic assay for the rotenone site of this enzyme. [3H]dihydrorotenone ([3H]DHR) binding is saturable (KD = 15–55 nm) and specific, and Hill slopes of 1 suggest a single population of binding sites. Nicotinamide adenine dinucleotide (NADH) enhances binding 4- to 80-fold in different brain regions (EC50 = 20–40 μm) by increasing the density of recognition sites (Bmax). Nicotinamide adenine dinucleotide phosphate also increases binding, but NAD+ does not. In skeletal muscle, heart, and kidney, binding was less affected by NADH. [3H]DHR binding is inhibited by rotenone (IC50 = 8–20 nm), meperidine (IC50 = 34–57 μm), amobarbitol (IC50 = 375–425 μm), and MPP+(IC50 = 4–5 mm), consistent with the potencies of these compounds in inhibiting complex I activity. Binding is heterogeneously distributed in brain with the density in gray matter structures varying more than 10-fold. Lesion studies suggest that a substantial portion of binding is associated with nerve terminals. [3H]DHR autoradiography is the first quantitative method to examine complex I with a high degree of anatomic precision. This technique may help to clarify the potential role of complex I dysfunction in normal aging and disease.

Exacerbation of NMDA, AMPA, and l‐Glutamate Excitotoxicity by the Succinate Dehydrogenase Inhibitor Malonate

Abstract: We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N‐methyl‐d‐aspartate (NMDA), S‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (S‐AMPA), and l‐glutamate. In male Sprague‐Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 µmol), NMDA alone (15 nmol), S‐AMPA alone (1 nmol), or glutamate alone (0.6 µmol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 ± 1.4 mm3), as did coinjection of malonate with S‐AMPA (11.0 ± 1.0 mm3) or glutamate (12.8 ± 0.7 mm3). Administration of the noncompetitive NMDA antagonist MK‐801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 ± 0.3 mm3). This dose of MK‐801 had little effect on the lesion produced by malonate plus S‐AMPA (9.0 ± 0.7 mm3), but it attenuated the toxicity of malonate plus glutamate by ∼40% (7.5 ± 0.9 mm3). Coinjection of the AMPA antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)‐quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 ± 1.8 and 14.0 ± 0.9 mm3, respectively) but greatly attenuated malonate plus S‐AMPA toxicity (1.5 ± 0.9 mm3). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor‐ and non‐NMDA receptor‐mediated excitotoxicity. They also suggest that the NMDA receptor may play a major role in situations of metabolic compromise in vivo, where glutamate is the endogenous agonist. Furthermore, glutamate toxicity under conditions of metabolic compromise may not be mediated entirely by ionotropic glutamate receptors.