Excitatory amino acids in amyotrophic lateral sclerosis: an update.
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Amyotrophic Lateral Sclerosis↗
Biomedical subjects
J T Coyle
Publications and source records attributed to J T Coyle.
Developmental expression of the gene encoding growth-associated protein 43 (Gap43) in the brains of normal and aneuploid mice.
The gene encoding growth-associated protein 43 (Gap43), a neuronal phosphoprotein associated with axonal outgrowth and synaptic plasticity, is located on mouse chromosome 16 (MMU16). We examined the developmental expression of Gap43 in normal, trisomy 16 (Ts16), and trisomy 19 (Ts19) mouse brain using northern blot analysis and in situ hybridization as a first step toward understanding the neurobiologic consequences of increased gene dosage on brain development. Gap43 expression was detected by in situ hybridization throughout the mesencephalon, rhombencephalon, spinal cord, and first branchial arch in whole embryos as early as day 10 of gestation (E10). By E15, Gap43 expression was localized to cells in the retina, the olfactory bulbs, and anterior olfactory structures, the cortical plate, the basal telencephalon, diencephalon, midbrain, hindbrain, and spinal cord. Northern blot analysis detected a three-fold increase in Gap43 mRNA levels in the brains of normal mice between E12-E18. At E15, Gap43 mRNA levels were increased 35-40% in Ts16 mouse brain and decreased 10% in Ts19 mouse brain, relative to euploid littermate controls. Using in situ hybridization we found that overexpression of Gap43 occurred in the diencephalon, medial and lateral basal telencephalon, and cortical plate region in Ts16 mice relative to littermate controls. Thus, the degree of overexpression of Gap43 mRNA in Ts16 mice is consistent with that expected from gene dosage effects.
Aneuploidy↗
Platelet serotonin, a possible marker for familial autism.
Serotonin (5HT) levels in platelet-rich plasma were measured in 5 autistic subjects who had siblings with either autism or pervasive developmental disorder (PDD), 23 autistic subjects without affected siblings, and 10 normal controls. The 5HT levels of autistic subjects with affected siblings were significantly higher than probands without affected siblings, and autistic subjects without affected siblings had 5HT levels significantly higher than controls. Differences in 5HT levels remained significant after adjustment for sex, age, and IQ. These results suggest that 5HT level in autistic subjects may be associated with genetic liability to autism.
Adolescent↗
Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures.
Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.
Animals↗
Immunocytochemical localization of N-acetyl-aspartate with monoclonal antibodies.
N-Acetyl-aspartate is found in high concentrations in all areas of the brain, but is undetectable in non-neuronal tissue. In order to characterize the cellular localization of N-acetyl-aspartate in brain, highly specific monoclonal antibodies against N-acetyl-aspartate were produced by fusing spleen lymphocytes obtained from mice immunized with N-acetyl-aspartate conjugated to thyroglobulin by carbodiimide with P3/x63-Ag8.653 mouse myeloma cells. Clones were selected which secrete IgG2a(k) antibodies highly specific for conjugated N-acetyl-aspartate. Only 3-6% cross-reactivity with conjugated N-acetyl-aspartate-glutamate was observed at high antibody concentrations, whereas no cross-reactivity (less than 1%) was observed with conjugated N-acetyl-glutamate or aspartate. Preincubation of the antibodies with 0.5 mg/ml conjugated N-acetyl-aspartate blocked immunoreactivity more than 90%, while preincubation with conjugated N-acetyl-aspartate-glutamate and free N-acetyl-aspartate had no effect. Immunocytochemical staining has shown that N-acetyl-aspartate-like immunoreactivity is localized in neurons, which are widely distributed throughout the brain. The immunoreactive neurons exhibited intense staining of the perikarya, proximal dendrites and axons. No consistent pattern of distribution of immunoreactivity was observed with regard to primary neurotransmitter characteristics of stained neurons although neurons with long projections or extensive arbors, such as pyramidal cells in cortex, locus coeruleus, motor neurons and Purkinje cells, stained much more intensively than local circuit neurons.
Animals↗
The non-excitatory mechanisms of glutamate induced neurotoxicity.
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Animals↗
The potential for muscarinic receptor subtype-specific pharmacotherapy for Alzheimer's disease.
In several neurodegenerative disorders, including Alzheimer's disease, a loss of the cholinergic projections of the basal forebrain to the cerebral cortex and hippocampus occurs. Studies of the anatomic and physiologic characteristics of these ascending cholinergic systems suggest that they are important in processing information and in memory function. Muscarinic receptors are situated at various critical control points in these pathways. Activation of postsynaptic muscarinic receptors often increases the excitability of neurons; thus, the signal-to-noise ratio for sensory processing is enhanced. In addition, muscarinic receptors negatively control cholinergic tone at presynaptic sites. Molecular biologic methods have disclosed the existence of five muscarinic receptors, which are coupled to different second messenger systems. The evidence reviewed suggests that at least four of the five muscarinic receptor genes are expressed as functional receptor proteins in the neocortex and hippocampal formation. On the basis of the current information about their pharmacologic properties and coupling mechanisms in nervous tissue, drugs that selectively affect subtypes of muscarinic receptors could enhance cortical cholinergic function and thereby ameliorate certain cognitive impairments in Alzheimer's disease.
Alzheimer Disease↗
Titrated scatter treatment in the ETDRS.
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Contraindications↗
Enhanced NAD(P)H:quinone reductase activity prevents glutamate toxicity produced by oxidative stress.
Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.
Antioxidants↗
Glutamate neurotoxicity and the inhibition of protein synthesis in the hippocampal slice.
In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.
Animals↗
Neuroleptic malignant syndrome: life-threatening complication of neuroleptic treatment in adolescents with affective disorder.
Neuroleptic malignant syndrome (NMS) is an uncommon, potentially fatal side effect of neuroleptic treatment characterized by hyperthermia, rigidity, rhabdomyolysis, and delirium. In recent clinical studies of adults it was suggested that affective disorder is a risk factor for the development of neuroleptic malignant syndrome. The cases of two adolescents with neuroleptic malignant syndrome who were treated with neuroleptic therapy because of psychotic symptoms in association with primary affective disorders are reported. The occurrence of these cases, as well as the observations in adults, suggests that attention to the primary psychiatric diagnosis is important in neuroleptic usage and that physicians should be vigilant to the occurrence of neuroleptic malignant syndrome in the pediatric population.
Adolescent↗
Down syndrome and the trisomy 16 mouse: impact of gene imbalance on brain development and aging.
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Aging↗
Developmental time course and ionic dependence of kainate-mediated toxicity in rat cerebellar granule cell cultures.
The mechanisms associated with the neurotoxic response caused by kainate (KA) were examined in cerebellar granule cell cultures. Under the conditions studied, millimolar concentrations of quisqualate, (RS)-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, glutamate and N-methyl-D-aspartate did not cause significant cytolysis. In contrast, KA induced complete cell death, which was antagonized by 6,7-dinitroquinoxaline-2,3-dione, quisqualate, (RS)-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and glutamate. This neurotoxic effect was dependent on the dose of KA and the age of the cultures. Two separate components of KA-induced neurotoxicity were observed and differentiated according to morphological changes, time of onset and ionic dependence. For acute neurotoxicity, release of lactate dehydrogenase measured after 30 min of KA exposure, became apparent between 8 and 11 days in culture and was dependent on both Cl- and Na+. However, vulnerability to acute toxicity did not correlate with [3H]KA receptor expression with receptor-mediated Cl- influx. On the other hand, delayed toxicity, as determined by lactate dehydrogenase release 24 hr after KA exposure, was dependent on Cl-. This delayed neurotoxicity induced by KA shares time course features with N-methyl-D-aspartate-mediated toxicity. Yet in contrast to studies reported for N-methyl-D-aspartate, glutamate was ineffective as an agonist, measured by its ability to elicit a neurotoxic response, and the KA delayed response did not appear to be dependent upon the presence of extracellular Ca++, during the exposure to KA.
Animals↗
Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization.
N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.
Animals↗
Selective release of N-acetylaspartylglutamate from rat optic nerve terminals in vivo.
Glutamate (Glu) and aspartate (Asp) are considered to be the neurotransmitters of the optic pathway in submammalian species, but their roles in mammals is uncertain. Recently, N-acetylaspartylglutamate (NAAG) has been proposed as a neurotransmitter in mammalian optic pathway; however, the release of endogenous NAAG on stimulation of the optic pathway has not been demonstrated. Using an in vivo microdialysis technique, we now report that electrical stimulation of rat optic nerve markedly increased the extracellular concentration of NAAG but not Glu/Asp in superficial superior colliculus where retinal afferents terminate, whereas non-specific stimulation of neurotransmitter release by high potassium or veratridine increased both extracellular Glu/Asp and NAAG concentration in the perfusate. The release of NAAG was dependent on Ca2+ and the presence of optic terminals. We conclude that NAAG is a better candidate as a neurotransmitter of rat optic nerve terminals than Glu/Asp.
Animals↗
Developmental expression of somatostatin in mouse brain. I. Immunocytochemical studies.
The postnatal development of the distribution of somatostatin immunoreactive (SOMLI) neurons and fibers in the forebrain of the Balb/C mouse and their relationship to cholinergic afferents have been examined. SOMLI was first discernable in the hypothalamus on postnatal day (PND) 3 and increased gradually to reach adult levels by PND 30. In the limbic system, SOMLI is detectable at birth. In all other structures of the forebrain, SOMLI could be observed by PND 3 but the distribution, density and morphology of the immunoreactive neurons evolved over the following 2-3 weeks. In general, SOMLI cells and fibers increased for 1-3 weeks after their initial appearance and subsequently declined to achieve adult levels. The distribution pattern of SOMLI elements in adult mouse brain was similar to previous reports in rat with a few notable differences in thalamus, olfactory structures and, to a lesser degree, cortex and hippocampus. The temporal pattern of SOMLI expression in extrahypothalamus forebrain regions, during development, suggests a role of this peptide in differentiation and synapse formation. Such an hypothesis receives further support from neonatal lesions of the basal forebrain which resulted in transient cortical cholinergic deafferentation, a delay of cortical differentiation and a transient increase in the number of SOMLI cells in cortex.
Aging↗
Developmental expression of somatostatin in mouse brain. II. In situ hybridization.
The distribution and the levels of expression of preprosomatostatin (PPSOM) mRNA were examined during pre- and postnatal development of the mouse brain using the in situ hybridization technique. The signal obtained by in situ hybridization of embryonic tissues at day 14 and day 17 of gestation was highest over the neurons of the pyriform cortex, amygdala, and entopeduncular nucleus. The signal was very low over cells of the neocortex and the developing hippocampal formation. The density of grains overlying the neurons of the amygdala and pyriform cortex continued to be high during early postnatal life, but decreased as the animals became adults. A progressive increase of PPSOM mRNA expression was observed in postnatal animals in the stratum oriens and dentate gyrus of the hippocampal formation. In the cerebral cortex and striatum, the number of these neurons became maximal between postnatal weeks 1 and 3. In the diencephalon, the highest densities of grains were found over neurons in the nucleus reticularis thalami and zona incerta at postnatal day 21; these levels declined slightly thereafter. The cells of the periventricular nucleus of the hypothalamus had high densities of grains as early as postnatal week 1 and continued to have high densities of grains in adult animals. These patterns of hybridization density parallelled the distribution of SOM-like immunoreactivity in the mouse brain. When PPSOM mRNA expression was examined in the cerebral cortices of mice that received lesions of the nucleus basalis of Meynert as neonates, a transient increase in the number of cells expressing PPSOM mRNA was observed in the frontoparietal cortex ipsilateral to the lesion at postnatal day 10, but not at postnatal day 30. Importantly, the density of grains over the individual cells was not altered in lesioned animals at these two ages.
Aging↗
Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake.
Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.
Animals↗