Lourdes Massieu

Accumulation of Extracellular Glutamate by Inhibition of Its Uptake Is Not Sufficient for Inducing Neuronal Damage: An In Vivo Microdialysis Study

Abstract: It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high‐affinity sodium‐dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (l‐trans‐pyrrolidine‐2,4‐dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, dl‐threo‐β‐hydroxyaspartate and l‐aspartate‐β‐hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate or by intrastriatal coinjection of 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used.

Excitotoxic Neuronal Death and the Pathogenesis of Huntington's Disease

Huntingtons disease (HD) is a neurodegenerative hereditary illness originated by the mutation of the gene encoding the huntingtin-protein (htt). Mutated htt (mhtt) is characterized by an increased number of glutamine repeats in the N-terminal end; when 40 or more glutamine residues are present, the disease is manifested. Expression of mhtt leads to the selective death of the medium spiny neurons (MSN) in the neostriatum, resulting in the appearance of generalized involuntary movements, the main phenotypic alteration of HD. The relationship between the expression of mhtt and the death of the MSN is not fully understood. Nonetheless, according to experimental evidence indicating that MSN are selectively vulnerable to the toxicity of glutamate (excitotoxicity) or its analogues, excitotoxic neuronal death is suggested to be involved in neurodegeneration associated with HD. Support for this hypothesis comes from studies in HD postmortem tissue and transgenic mice models, suggesting a correlation between mhtt expression and altered glutamatergic neurotransmission, mainly altered conductance of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype and decreased levels of glutamate transporters. On the other hand, alterations in energy metabolism are well documented in HD patients, which might facilitate excitotoxicity. Throughout this review we will discuss relevant evidence suggesting that altered glutamatergic neurotransmission plays a role in neurodegeneration associated with HD, as well as the possible contribution of deficient energy metabolism to the development of an excitotoxic cell death cascade in MSN. We show data supporting protection by energy substrates against neuronal damage in a rat model combining energy deficit and glutamate toxicity.

Acetoacetate protects hippocampal neurons against glutamate-mediated neuronal damage during glycolysis inhibition.

Glucose is the main substrate that fulfills energy brain demands. However, in some circumstances, such as diabetes, starvation, during the suckling period and the ketogenic diet, brain uses the ketone bodies, acetoacetate and beta-hydroxybutyrate, as energy sources. Ketone body utilization in brain depends directly on its blood concentration, which is normally very low, but increases substantially during the conditions mentioned above. Glutamate neurotoxicity has been implicated in neurodegeneration associated with brain ischemia, hypoglycemia and cerebral trauma, conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. In recent years substantial evidence favoring a close relation between glutamate neurotoxic potentiality and cellular energy levels, has been compiled. We have previously demonstrated that accumulation of extracellular glutamate after inhibition of its transporters, induces neuronal death in vivo during energy impairment induced by glycolysis inhibition. In the present study we have assessed the protective potentiality of the ketone body, acetoacetate, against glutamate-mediated neuronal damage in the hippocampus of rats chronically treated with the glycolysis inhibitor, iodoacetate, and in hippocampal cultured neurons exposed to a toxic concentration of iodoacetate. Results show that acetoacetate efficiently protects against glutamate neurotoxicity both in vivo and in vitro probably by a mechanism involving its role as an energy substrate.

β-Amyloid neurotoxicity is exacerbated during glycolysis inhibition and mitochondrial impairment in the rat hippocampus in vivo and in isolated nerve terminals: Implications for alzheimer's disease

Abstract Senile plaques composed mainly by β-amyloid (Aβ) protein are one of the pathological hallmarks of Alzheimers disease (AD). In vitro, Aβ and its active fragment 25–35 have been shown either to be directly neurotoxic or to exacerbate the damaging effect of other neurotoxic insults. However, the attempts to replicate Aβ neurotoxicity in vivo have yielded conflicting results. One of the most consistent alterations in AD is a reduced resting glucose utilization. Important evidence suggests that impairment of brain energy metabolism can lead to neuronal damage or facilitate the deleterious effects of some neurotoxic agents. In the present study we have investigated the influence of glycolysis inhibition induced by iodoacetate, and mitochondrial impairment induced by 3-nitropropionic acid (3-NP), in the toxicity of Aβ. We have studied Aβ neurotoxicity during energy deficiency both in vivo in the dentate gyrus of the hippocampal formation and in presynaptic terminals isolated from neocortex and hippocampus. Results show that during metabolic inhibition an enhanced vulnerability of hippocampal neurons to Aβ peptide toxicity occurs, probably resulting from decreased glucose metabolism and mitochondrial ATP production. Synaptosomal response to energy impairment and Aβ toxicity was evaluated by the MTT assay. Results suggest that synapses may be particularly sensitive to metabolic perturbation, which in turn exacerbates Aβ toxicity. The present data provide experimental support to the hypothesis that certain risk factors such as metabolic dysfunction and amyloid accumulation may interact to exacerbate AD, and that metabolic substrates such as pyruvate may play a role as a therapeutic tool.

Glutamate uptake impairment and neuronal damage in young and aged rats in vivo

Abstract: The extracellular concentration of glutamate increases during hypoxia/ischemia probably due to deficient uptake. Glutamate might contribute to neuronal damage associated with this disorder and to neurodegeneration during aging. In the present study, we have tested the effect of two inhibitors of glutamate transport, l‐trans‐pyrrolidine‐2,4‐dicarboxylate and dihydrokainate, on the extracellular levels of glutamate and on neuronal damage, which was quantitatively studied by image analysis of histological brain sections. Drugs were administered by microdialysis and glutamate concentration was determined by HPLC in the striatum and the hippocampus of 3‐month‐old and 22–24‐month‐old rats. In both regions studied, the basal concentration of extracellular glutamate was higher in aged than in young rats. Pyrrolidine dicarboxylate induced a substantial elevation of extracellular glutamate in both regions, and although this increase was almost twofold higher in old than in young animals, no neuronal damage was observed. In contrast, dihydrokainate had a poor effect on glutamate levels, but induced clear neuronal damage in the striatum and the hippocampus in both groups of rats. The present results suggest that age appears not to be a significant factor in the sensitivity of neurons to the toxic effect of extracellular glutamate increase via blockade of its transport system.

Transient Inhibition of Glutamate Uptake In Vivo Induces Neurodegeneration when Energy Metabolism Is Impaired

Abstract : Impairment of glutamate transport during ischemia might be related to the elevation of the extracellular concentration of glutamate and ischemic neuronal damage. Additionally, impairment of energy metabolism in vivo leads to neurodegeneration apparently mediated by a secondary excitotoxic mechanism. In vitro observations show that glucose deprivation and inhibition of energy metabolism exacerbate the toxic effects of glutamate. We have previously shown that glutamate uptake inhibition in vivo by l‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC) leads to a substantial elevation in the extracellular concentration of excitatory amino acids that is not associated with cell death. These observations suggest that energy depletion during ischemia might be determinant of ischemic neuronal damage. To investigate whether impairment of energy metabolism in vivo increases neuronal susceptibility to glutamate uptake inhibition, we studied the effect of glutamate accumulation induced by the intrahippocampal or intrastriatal administration of PDC in energy‐deficient rats chronically treated with 3‐nitropropionic acid (3‐NP), which irreversibly inhibits the tricarboxylic acid cycle and electron transport chain. Extracellular glutamate levels were monitored by HPLC from fractions collected from microdialysis probes, and neuronal damage was evaluated by histological analysis. Our results show that glutamate uptake inhibition leads to marked neuronal damage in energy‐deficient rats but not in intact animals, which apparently is not related to an additional elevation of glutamate levels induced by 3‐NP.

Glutamate toxicity in the striatum of the R6/2 Huntington's disease transgenic mice is age-dependent and correlates with decreased levels of glutamate transporters

Glutamate excitotoxicity has been implicated in the neuropathology of Huntingtons disease (HD), due to the toxicity of glutamate receptor agonists on striatal medium spiny neurons (MSN), the most affected neuronal population in HD. Previous studies showed functional alterations of NMDA glutamate receptors and decreased expression of glutamate transporters in transgenic models and HD patients, suggesting the presence of excitotoxic damage. We have studied the vulnerability of the striatum to glutamate toxicity in R6/2 mice at 10 and 14 weeks of age. At 10 weeks R6/2 and wild-type mice are equally vulnerable to glutamate toxicity, while at 14 weeks transgenic mice show increased damage, as assessed by Nissl and Fluoro Jade staining. In addition, increased electrical brain activity is observed after glutamate administration in transgenic mice, as monitored electroencephalographically. According to western blot analysis, increased vulnerability to glutamate toxicity correlates with decreased levels of GLT-1 and GLAST glutamate transporters in the striatum.

Calcium-dependent production of reactive oxygen species is involved in neuronal damage induced during glycolysis inhibition in cultured hippocampal neurons

Neuronal damage associated with in vivo hypoglycemia has been suggested to be excitotoxic due to the release of excitatory amino acids and the protective effect of glutamate receptor antagonists. The production of reactive oxygen species (ROS) has been also implicated in hypoglycemic damage. Excitotoxicity involves oxidative stress, insofar as the influx of calcium through N‐methyl‐D‐aspartate (NMDA) receptors stimulates ROS production. We have studied the participation of NMDA receptors and intracellular calcium in ROS production and cell death triggered during moderate and severe glycolysis inhibition in cultured hippocampal neurons. Iodoacetate (IOA), an inhibitor of the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), dose dependently reduces ATP levels and cell survival and increases the intracellular concentration of calcium. During mild glycolysis inhibition, the increases in intracellular calcium, ROS production, and cell death are dependent on NMDA receptor activation. In contrast, during severe glycolysis impairment, these processes are not inhibited by NMDA receptor blockade. BAPTA‐AM and vitamin E efficiently reduce ROS generation and cell death under both conditions. Results suggest that calcium influx through NMDA receptors is involved in ROS production and neuronal damage resulting from moderate energy depletion, whereas intracellular calcium increase and ROS generation during severe glycolysis inhibition are more related to energy depletion.

Differential effects of the substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC) and the non-substrate inhibitor dl-threo-β-benzyloxyaspartate (dl-TBOA) of glutamate transporters on neuronal damage and extracellular amino acid levels in rat brain in vivo

The extracellular concentration of glutamate is highly regulated by transporter proteins, due to its neurotoxic properties. Dysfunction or reverse activation of these transporters is related to the extracellular accumulation of excitatory amino acids and neuronal damage associated with ischemia and hypoglycemia. We have investigated by microdialysis the effects of the substrate and the non-substrate inhibitors of glutamate transporters, l-trans-2,4-pyrrolidine dicarboxylate (PDC) and DL-threo-beta-benzyloxyaspartate (DL-TBOA), respectively, on the extracellular levels of amino acids in the rat hippocampus in vivo. In addition, we have studied the effect of both inhibitors on neuronal damage after direct administration into the hippocampus and striatum. Electroencephalographic activity was recorded after the intrahippocampal infusion of DL-TBOA or PDC. Microdialysis administration of 500 microM DL-TBOA into the hippocampus increased 3.4- and nine-fold the extracellular levels of aspartate and glutamate, respectively. Upon stereotaxic administration it induced neuronal damage dose-dependently in CA1 and dentate gyrus, and convulsive behavior. Electroencephalographic recording showed the appearance of limbic seizures in the hippocampus after DL-TBOA infusion. In the striatum it also induced dose-dependent neuronal damage. These effects were prevented by the i.p. administration of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-iminemaleate and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline. In contrast to dl-TBOA, PDC (500 microM) induced a more discrete elevation of excitatory amino acids levels (2.6- and three-fold in aspartate and glutamate, respectively), no neuronal damage or behavioral changes, and no alterations in electroencephalographic activity. The differential results obtained with DL-TBOA and PDC might be attributed to their distinct effects on the extracellular concentration of amino acids. Results are relevant to the understanding of the role of glutamate transporters in amino acid removal or release and the induction of excitotoxic cell death.

INHIBITION OF GLUTAMATE UPTAKE INDUCES PROGRESSIVE ACCUMULATION OF EXTRACELLULAR GLUTAMATE AND NEURONAL DAMAGE IN RAT CORTICAL CULTURES

It is known that neurons exposed to high concentrations of glutamate degenerate and die. The clearance of this amino acid from the extracellular space depends on their active transport by Na+‐dependent high‐affinity carriers. In the present study we tested whether inhibition of glutamate transport in mixed glial/neuronal cortical cultures induces accumulation of extracellular glutamate and whether such increase results in cell damage. Three inhibitors of glutamate transport were used: L‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC), DL‐threo‐β‐hydroxyaspartate (THA), and dihydrokainate (DHK). Cell damage was assessed by light microscopy observations, reduction of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, and leakage of lactate dehydrogenase. PDC induced a significant concentration‐ and time‐dependent neuronal damage, whereas pure glial cultures were not affected. A good correlation was found between this damage and elevations of glutamate concentration in the medium. These effects of PDC were similar in glutamine‐free medium and in medium supplemented with glutamine. THA induced identical cell damage and elevations of extracellular glutamate to those produced by PDC, while DHK did not affect at all any of these parameters. PDC‐ and THA‐induced toxicity was protected by the N‐methyl‐D‐aspartate receptor antagonist (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo‐(a,d)cyclohepten‐5,10‐imine maleate but not by the non‐N‐methyl‐D‐aspartate receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo(f)quinoxaline.