Alan C. Foster

II. Excitotoxic models for neurodegenerative disorders

Abstract In recent years, considerable interest has been shown in the neurotoxic properties of excitatory amino acids and their possible relevance for the study of human neurodegenerative disorders. The term “excitotoxin” has been coined for a family of acidic amino acids which are neuroexcitants and produce a characteristic type of “axon-sparing” neuronal lesion. Intracerebral infusions of kainic and ibotenic acids, the two most commonly used excitotoxins, result in a morphological and biochemical picture in experimental animals which resembles that observed in the brains of Huntingtons disease and epilepsy victims. The emergence of such animal models for neurodegenerative disorders has led to the hypothesis that endogenous excitotoxins may exist which are linked to the pathogenesis of human diseases. The most promising candidate discovered so far is quinolinic acid, a hepatic tryptophan metabolite which has recently also been found to occur in brain tissue. The particular excitotoxic properties of quinolinic acid warrant a thorough investigation of its metabolic and synaptic disposition in normal and abnormal brain function. While little is known about the mechanisms by which excitotoxins cause selective neuronal death, most current speculations propose the participation of specific synaptic receptors for acidic amino acids. The recent development of selective antagonists of such receptors has aided in the elucidation of excitotoxic mechanisms. Although a biochemical link between endogenous excitotoxins and human neurodegenerative disorders remains elusive at present, pharmacological blockade of excitotoxicity may constitute a novel therapeutic strategy for the treatment of these disease states.

Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid

Kynurenic acid (KYNA) was tested as an antagonist of the neurotoxic and epileptogenic effects of the metabolically related brain constituent quinolinic acid (QUIN). In the rat striatum, KYNA blocked the neurotoxic effects of QUIN in preference to those of other excitotoxins. In the hippocampus, KYNA antagonized both the neurodegeneration and seizures caused by the local application of QUIN. These properties of KYNA raise the possibility of a functional link between KYNA and QUIN in the brain which may be of relevance for an understanding of human neurodegenerative disorders.

On the excitotoxic properties of quinolinic acid, 2,3-piperidine dicarboxylic acids and structurally related compounds

To obtain information about the receptors which mediate the neurotoxic actions of quinolinic acid, a series of pyridine dicarboxylates and piperidine dicarboxylates and structurally related compounds were tested for their neurotoxic effects following intrastriatal or intrahippocampal infusion in the rat, and for their activity in assays of binding and uptake sites for acidic amino acids. Of the compounds tested, only cis- and trans-2,3-piperidine dicarboxylates and quinolinic acid showed pronounced neurotoxic effects. At 600 nmol, 2,6- and 3,4-pyridine dicarboxylates were weakly active and the remaining compounds were inactive in both brain regions. After injection into the striatum of the adult rat, trans-2,3-piperidine dicarboxylate, quinolinic acid and cis-2,3-piperidine dicarboxylate caused axon-sparing neuronal degeneration as assessed by light microscopic and neurochemical methods, the threshold doses being 12, 24 and 120 nmol, respectively. In the striatum of the 7-day old rat, 30 nmol quinolinic acid or 600 nmol cis-2,3-piperidine dicarboxylate were inactive. Small doses of cis-2,3-piperidine dicarboxylate (60 nmol) and quinolinic acid (30 nmol) injected into the adult rat hippocampus resulted in a preferential loss of pyramidal neurons. In larger doses granule cells also degenerated. In contrast, trans-2,3-piperidine dicarboxylate was equally toxic to hippocampal neurons, regardless of the dose used. No distant neuronal damage was observed after the intracerebral application of any test compound. Equimolar amounts of (-)-2-amino-7-phosphonoheptanoic acid completely blocked the neurotoxic effects of quinolinic acid, cis- and trans-2,3-piperidine dicarboxylate after injection into the striatum or hippocampus. None of the analogs tested were good inhibitors of Cl--dependent or independent binding of L-[3H]glutamate, [3H]kainate or high-affinity, Na+-dependent uptake of L-glutamate in striatal or hippocampal tissue at 1 mM. The results indicate that the receptors mediating the neurotoxic effects of these compounds have strict structural requirements for activation. Whereas the excitotoxic characteristics of trans-2,3-piperidine dicarboxylate suggest a direct action on N-methyl-D-aspartate receptors, the properties of quinolinic acid and cis-2,3-piperidine dicarboxylate are far more complex and make categorization of their receptor-interactions difficult. Indirect mechanisms may account for the excitotoxicity of quinolinic acid and cis-2,3-piperidine dicarboxylate.

Identification of quinolinic acid in rat and human brain tissue

An analytical technique for the determination of the excitotoxic compound quinolinic acid (2,3-pyridine dicarboxylic acid) in brain tissue has been developed. Following sample prepurification by ion exchange and high pressure liquid chromatography, quinolinic acid is converted to the dihexafluoroisopropyl ester and the derivative is analyzed by mass fragmentography. Using the present technique quinolinic acid has been identified in both rat and human brain tissue.

Seizure activity and lesions after intrahippocampal quinolinic acid injection

Electroencephalographic, behavioral, and neuropathologic changes were monitored after infusions of the endogenous excitatory amino acid, quinolinic acid (QUIN), into the dorsal hippocampus of unanesthetized, freely moving rats. A dose of 120 nmol QUIN was required to reliably precipitate seizures although EEG changes were observed with doses as small as 3 nmol. Seizure episodes were characterized by repetitive periods of high-voltage spiking typically lasting 20 s but occasional longer multicomponent episodes (60 s) were also observed. The latency of specific QUIN-induced seizures was similar for all doses tested (19 to 32 min); however, the total number of seizures and total time in seizures increased in a dose-dependent fashion from 30 to 300 nmol QUIN. Seizure episodes were often associated with a frozen appearance of the animal and intermittent wet dog shakes. Ataxia was apparent in animals receiving 120 and 300 nmol QUIN. Using light microscopic analyses, pyramidal cell degeneration was observed in the QUIN-injected hippocampus (CA3 and CA1 cells more susceptible than CA2 cells); dentate granule cells showed signs of degeneration only at the largest QUIN dose. No neuropathologic changes were found outside the injected hippocampus. Seizures and neuropathologic changes induced by 120 nmol QUIN were completely blocked by pre- or cotreatment with 12 nmol (-)2-amino-7-phosphonoheptanoic acid. Experiments with [3H]QUIN indicated that only 3% of the injected radioactivity was present in the dorsal hippocampus at the average time of seizure onset (25 min), and consisted entirely of unmetabolized QUIN. The potent convulsant properties of QUIN, an endogenous metabolite, may prove to be of relevance for the etiology of human temporal lobe epilepsy.

Synthesis of Quinolinic Acid by 3-Hydroxyanthranilic Acid Oxygenase in Rat Brain Tissue In Vitro

In mammalian peripheral organs, 3‐hydroxy‐anthranilic acid oxygenase (3HAO), catalyzing the conversion of 3‐hydroxyanthranilic acid to quinolinic acid, constitutes a link in the catabolic pathway of tryptophan to NAD. Because of the possible involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we examined the presence and characteristics of 3HA0 in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy‐14C]3‐hydroxy‐anthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chro‐matographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed a Km of 3.6 ± 0.5 μM for 3‐hydroxyanthranilic acid and a Vmaxof 13.1± 9.5 pmol quinolinic acid/h/mg tissue. The enzyme showed pronounced selectivity for its substrate, since several substances structurally and metabolically related to 3‐hydroxyanthranilic acid caused <25% inhibition of activity at 500 μM. Both the Fe2+ dependency and the distinct subcellular distribution (soluble fraction) of brain 3HAO indicated a close resemblance to 3HAO from peripheral tissues. Examination of the regional distribution in the brain demonstrated a 10‐fold variation between the region of highest (olfactory bulb) and lowest (retina) 3HAO activity. The brain enzyme was present at the earliest age tested (7 days postnatum) and increased to 167% at 15 days before reaching adult levels. Enzyme activity was stable over extended periods of storage at — 80°C. Taken together, these data indicate that measurements of brain 3HA0 may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.

High-affinity CRF1 receptor antagonist NBI-34041: Preclinical and clinical data suggest safety and efficacy in attenuating elevated stress response

There is an extensive evidence that corticotropin releasing factor (CRF) is hypersecreted in depression and anxiety, and blockade of CRF could have therapeutic benefit. We report preclinical data and the results of a clinical Phase I study with the novel nonpeptide CRF1 antagonist NBI-34041/SB723620. Preclinical data conducted with different cell lines expressing human CRF receptors and in Wistar and Sprague–Dawley rats indicate that NBI-34041 is effective in reducing endocrine responses to pharmacological and behavioral challenge mediated by CRF1 receptors. These specific properties and its well-documented safety profile enabled a clinical Phase I study with 24 healthy male subjects receiving NBI-34041 (10, 50, or 100u2009mg) or placebo for 14 days. Regulation of the hypothalamic–pituitary–adrenocortical (HPA) axis was evaluated by intravenous stimulation with 100u2009μg of human CRF. Psychosocial stress response was investigated with the Trier Social Stress Test (TSST). Treatment with NBI-34041 did not impair diurnal adrenocorticotropic hormone (ACTH) and cortisol secretion or CRF evoked ACTH and cortisol responses but attenuated the neuroendocrine response to psychosocial stress. These results suggest that NBI-34041 is safe and does not impair basal regulation of the HPA system but improves resistance against psychosocial stress. NBI-34041 demonstrates that inhibition of the CRF system is a promising target for drug development against depression and anxiety disorders.

Studies on the disposition of quinolinic acid after intracerebral or systemic administration in the rat

Quinolinic acid (QUIN) is an endogenous, excitotoxic amino acid which is currently under investigation as a possible etiological factor in human neurodegenerative disorders such as Huntingtons disease and epilepsy. We explored certain aspects of this hypothesis, using the adult rat as an experimental animal. After intrastriatal infusions of [3H]QUIN, radioactivity was cleared from the injected region with an apparent half-life of 22 min. To 2 h after injection, all radioactivity recovered from the striatum corresponded to unmetabolized QUIN. Consistent with these data was the lack of significant uptake of [3H]QUIN by slices or crude synaptosomes prepared from rat hippocampus or striatum. When applied intravenously, a high dose of QUIN (450 mg/kg) caused relatively minor seizure-related EEG changes and no signs of neuronal degeneration. Direct measurements indicated negligible penetration of the blood-brain barrier by QUIN. The lack of an effective inactivation mechanism for extracellular QUIN in the brain negates QUINs proposed role as a classical neurotransmitter substance, but may be of significance for the postulated effects of this compound in neurodegenerative diseases. An important role of blood-borne QUIN or QUIN precursors in human disorders cannot be ruled out at present; although the brain appears to be well protected by the blood-brain barrier from an acute elevation of blood QUIN, a possible breakdown of the barrier under pathologic conditions and the effects of chronic elevations of blood QUIN remain to be examined.

Delayed Treatment With an Adenosine Kinase Inhibitor, GP683, Attenuates Infarct Size in Rats With Temporary Middle Cerebral Artery Occlusion

BACKGROUND AND PURPOSEnBrain ischemia is associated with a marked increase in extracellular adenosine levels. This results in activation of cell surface adenosine receptors and some degree of neuroprotection. Adenosine kinase is a key enzyme controlling adenosine metabolism. Inhibition of this enzyme enhances the levels of endogenous brain adenosine already elevated as a result of the ischemic episode. We studied a novel adenosine kinase inhibitor (AKI), GP683, in a rat focal ischemia model.nnnMETHODSnFour groups of 10 adult Sprague-Dawley rats were exposed to 90 minutes of temporary middle cerebral artery (MCA) occlusion. Animals were injected intraperitoneally with vehicle, 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg of GP683 30, 150, and 270 minutes after the induction of ischemia by a researcher blinded to treatment group. The animals were euthanatized 24 hours after MCA occlusion, and brains were stained with 2,3,5-triphenyltetrazolium chloride. We measured brain temperatures in a separate group of 6 rats before and after administration of 1.0 mg/kg GP683.nnnRESULTSnAll treated groups showed a reduction in infarct volumes, but a significant effect was observed only in the 1.0 mg/kg-dose group (44% reduction, P=0.0077). Body weight, physiological parameters, neurological scores, and mortality did not differ among the 4 groups. No apparent behavioral side effects were observed. Brain temperatures did not change after drug injection.nnnCONCLUSIONSnOur results indicate that the use of AKIs offers therapeutic potential and may represent a novel approach to the treatment of acute brain ischemia. The therapeutic effect observed was not caused by a decrease in brain temperature.

Quinolinic acid phosphoribosyltransferase in human and rat brain: Activity in Huntington's disease and in quinolinate-lesioned rat striatum

Quinolinic acid (QUIN) is an excitotoxic compound which is present in rat and human brain and has been hypothetically linked to neurodegenerative disorders including Huntingtons disease (HD). We have examined the biochemistry of QUIN by measuring the activity of its degradative enzyme QUIN phosphoribosyltransferase (QPRT) in post-mortem samples of human brain from normal and HD subjects, and in the striata of rats injected intrastriatally with QUIN. In normal human brain, QPRT activity was highest in the caudate nucleus and substantia nigra, less in the thalamus, hypothalamus, frontal cortex and hippocampus and lowest in the spinal cord and cerebellum. QPRT activity in HD caudate tended to be higher than control, the respective values (mean +/- S.E.M., n = 9 for each group) being 365.7 +/- 52.5 and 242.0 +/- 50.8 fmol/h/mg protein (0.1 greater than P greater than 0.05, t-test); values of enzyme activity in the putamen were similar between normal and HD groups. Kinetic analyses indicated that the Km values for QUIN and its co-substrate phosphoribosylpyrophosphate (PRPP) were similar in normal and HD caudate, but Vmax values were elevated in HD caudate. Rat striatal QPRT activity was increased in QUIN-injected striata, and when expressed as a percentage of the contralateral side it was 163.6% at 2 days, 344.4% at 14 days and 198.8% at 7 months post-injection. Kinetic analyses in the 7-month QUIN-injected group showed an increase of Vmax but no change of Km values for QUIN or PRPP. The results indicate that QPRT activity increases in response to specific neurodegenerative events.(ABSTRACT TRUNCATED AT 250 WORDS)