Quinolinate-like neurotoxicity produced by aminooxyacetic acid in rat striatum

Summary The endogenous tryptophan metabolite quinolinic acid elicits in rodent brain a pattern of neuronal degeneration which resembles that caused by L-glutamate. Its qualities as a neurotoxic agent raised the hypothesis that quinolinic acid might be involved in the pathogenesis of human neurodegenerative disorders. Kynurenic acid, another endogenous tryptophan metabolite and preferential N-methyl-D-aspartate (NMDA) antagonist, has been shown to block quinolinic acid neurotoxicity. Here we report that microinjections of aminooxyacetic acid (AOAA), an inhibitor of kynurenine transaminase and of other pyridoxal phosphate-dependent enzymes, into the rat striatum produce neuronal damage resembling that caused by quinolinic acid. AOAA-induced striatal lesions can be prevented by kynurenic acid and the selective NMDA antagonist 2-amino-7-phosphonoheptanoic acid. These results suggest that AOAA produces excitotoxic lesions by depleting brain concentrations of kynurenic acid (inhibition of synthetic enzyme) or due to impairment of intracellular energy metabolism (depletion of cell energy resources). The concept of deficient neuroprotection due to metabolic defects might help to clarify the pathogenesis of human neurodegenerative disorders and to develop strategies that may be useful in their treatment.This work was supported by research grant from the Polish Academy of Sciences.These data have been communicated to the International Congress on Amino Acid Research held in Vienna in August 7–12, 1989.     

Transport of quinolinic acid into rabbit and rat brain

The transport metabolism of [³H]quinolinic acid in the central nervous system of rabbits and rats were studied. In vitro [³H]quinolinic acid was not readily accumulated by isolated choroid plexus. After the intraventricular injection of tracer quantities of [³H]quinolinic acid, the [³H]quinolinic acid did not enter the brain as readily as concurrently injected [¹⁴C]mannitol and was not metabolized, The permeability-surface area constant for [³H]quinolinic acid at the rat blood-brain barrier was 1.5±1.3×10^–5 sec^–1 compared to 2.8±0.4×10^–5 sec^–1 for [³H]mannitol. Our results suggest that: 1) [³H]quinolinic acid is transported in the CNS by passive diffusion and 2) is not metabolized.     

Energetic Dysfunction in Quinolinic Acid-Lesioned Rat Striatum

Abstract: Impairment of mitochondrial energy metabolism may contribute to the selective neuronal degeneration observed in Huntington's disease and other neurodegenerative disorders. Intrastriatal injection of the excitotoxin, quinolinic acid, produces a pattern of neuronal death similar to that seen in Huntington's disease. However, little is known about the effects of quinolinic acid on striatal energetics. In the present work, time-dependent changes in energy metabolism caused by injection of quinolinic acid into rat striatum were examined. Oxygen consumption by free and synaptic mitochondria was quantified and correlated with the concentrations of nucleotides and amino acids at different times after injection. Compared with saline-treated controls, a decrease in ADP-stimulated (state 3) to basal (state 4) oxygen consumption (respiratory control ratio) by free mitochondria was apparent in quinolinic acid-injected striata as early as 6 h after treatment. No significant changes were seen in nucleotide concentrations at this time. By 12 h after injection, the decline in the respiratory control ratio was more pronounced (45%), and reductions in ATP, NAD, aspartate, and glutamate (30–60%) were also observed. These results show that injection of quinolinic acid in vivo produces progressive mitochondrial dysfunction, which may be a common and critical event in the cell death cascade initiated in Huntington's disease and in animal models of this neurodegenerative disorder. The indicators of mitochondrial function examined in this study, therefore, may be useful in evaluating the efficacy of neuroprotective agents.     

II. Excitotoxic models for neurodegenerative disorders

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 Huntington's 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.     

Expression of Tryptophan 2,3-Dioxygenase and Production of Kynurenine Pathway Metabolites in Triple Transgenic Mice and Human Alzheimer's Disease Brain

To assess the role of the kynurenine pathway in the pathology of Alzheimer''s disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.     

The non-NMDA receptors common for aspartate and quinolinic acid in some rat cortical neurons

Transmembrane currents elicited by application of N-methyl-D-aspartate (NMDA), aspartate (As), or quinolinic acid (QA) were recorded from isolated unidentified neurons of the rat cortex. It was found that As and QA acted not only on NMDA-receptors: in some neurons they elicited other responses, whose characteristics suggest that As and QA act on their common receptors, different from those of NMDA type.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 264–266, July–August, 1993.     

Quinolinic Acid Responses during Interferon-α-Induced Depressive Symptomatology in Patients with Chronic Hepatitis C Infection - A Novel Aspect for Depression and Inflammatory Hypothesis

Background

The aim of this exploratory study is to gain for the first time a more comprehensive picture of the impact of changes of quinolinic acid concentrations on depressive symptomatology during and after IFN-α therapy.

Methods

The quinolinic acid concentrations of 35 HCV patients are examined in a prospective survey over the entire period of IFN-α treatment as well as three months later at six different times (baseline, one, three, six and nine months after the beginning of IFN-α treatment, and after the end of treatment).

Results

During IFN-α treatment Hamilton Depression Rating Scale scores rise significantly. At the same time there is greater activity of indoleamine 2,3-dioxygenase, with a resulting increase in plasma kynurenine concentrations. Compared to baseline values quinolinic acid concentrations increase significantly during therapy, reflecting an increased neurotoxic challenge. In addition, patients with higher scores in the Hamilton Depression Rating Scale at six and nine months after starting therapy show significantly higher levels of quinolinic acid concentration.

Conclusions

The increase of quinolinic acid during IFN-α therapy might contribute to depressive symptomatology through the neurotoxic challenge caused by quinolinic acid. Subsequently, our exploratory study results support the inflammatory hypothesis of depression. The awareness of relevant risk factors of IFN-α treatment-induced depression is essential to develop preventative treatment strategies.     

Capillary zone electrophoresis separation of tryptophan and its metabolites, including quinolinic acid

Capillary zone electrophoresis with UV absorbance detection was used to separate tryptophan and ten its metabolites. Run buffers of pH 4.0–10.0 were evaluated for their effect on resolution; a pH 9.6 buffer was found to give optimum separation of all components. Ethylenediaminetetraacetic acid (EDTA), which prevents complexation of some analytes with polyvalent cations, was included in the run buffer to insure good peak shape and reproducible mobilities. The resulting method was used to detect the presence of quinolinic acid in a urine sample.     

Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain

Quinolinic acid phosphoribosyltransferase (QPRT) [EC 2.4.2.19] from human liver and brain was purified to homogeneity. Identity of the pure enzymes isolated from the two organs was proven by biochemical, physiocochemical and, following the production and partial purification of anti-liver QPRT antibodies, immunological techniques. Human QPRT has a molecular weight of 170,000 and consists of five identical subunits. Kinetic analyses revealed a Km of 5.6 microM for the substrate (quinolinic acid) and 23 microM for the co-substrate (phosphoribosylpyrophosphate). Enzyme activity was dependent on Mg2+ (optimal concentration: 1 mM) and was inhibited by the enzymatic by-product, inorganic pyrophosphate. Pure QPRT and its antibodies will constitute useful tools in the examination of the possible role of quinolinic acid in the pathogenesis of human neurodegenerative disorders.     

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

In mammalian peripheral organs, 3-hydroxyanthranilic 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 3HAO in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy-14C]3-hydroxyanthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chromatographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed a Km of 3.6 +/- 0.5 microM for 3-hydroxyanthranilic acid and a Vmax of 73.7 +/- 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 less than 25% inhibition of activity at 500 microM. 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 degrees C. Taken together, these data indicate that measurements of brain 3HAO may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.     

Intracerebroventricular Guanine-Based Purines Protect Against Seizures Induced by Quinolinic Acid in Mice

Acute and chronic administration of the nucleoside guanosine have been shown to prevent quinolinic acid (QA) and -dendrotoxin-induced seizures, as well as to impair memory and anxiety in rats and mice. In this study, we investigated the effect of i.c.v. administration of guanine-based purines (GTP, GDP, GMP, and guanosine) against seizures induced by the NMDA agonist and glutamate releaser quinolinic acid in mice. We also aimed to study the effects of the poorly hydrolysable analogs of GTP (GppNHp and GTPS) and GDP (GDPS) in this seizure model. QA produced seizures in 100% of mice, an effect partially prevented by guanine-based purines. In contrast to GTP (480 nmol), GDP (320–640 nmol), GMP (320–480 nmol) and guanosine (300–400 nmol), the poorly hydrolysable analogs of GTP and GDP did not affect QA-induced seizures. Thus, the protective effects of guanine nucleotides seem to be due to their conversion to guanosine. Altogether, these findings suggest a potential role of guanine-based purines for treating diseases involving glutamatergic excitotoxicity.     

Metabolic engineering of Escherichia coli for quinolinic acid production by assembling L-aspartate oxidase and quinolinate synthase as an enzyme complex

Quinolinic acid (QA) is a key intermediate of nicotinic acid (Niacin) which is an essential human nutrient and widely used in food and pharmaceutical industries. In this study, a quinolinic acid producer was constructed by employing comprehensive engineering strategies. Firstly, the quinolinic acid production was improved by deactivation of NadC (to block the consumption pathway), NadR (to eliminate the repression of L-aspartate oxidase and quinolinate synthase), and PtsG (to slow the glucose utilization rate and achieve a more balanced metabolism, and also to increase the availability of the precursor phosphoenolpyruvate). Further modifications to enhance quinolinic acid production were investigated by increasing the oxaloacetate pool through overproduction of phosphoenolpyruvate carboxylase and deactivation of acetate-producing pathway enzymes. Moreover, quinolinic acid production was accelerated by assembling NadB and NadA as an enzyme complex with the help of peptide-peptide interaction peptides RIAD and RIDD, which resulted in up to 3.7 g/L quinolinic acid being produced from 40 g/L glucose in shake-flask cultures. A quinolinic acid producer was constructed in this study, and these results lay a foundation for further engineering of microbial cell factories to efficiently produce quinolinic acid and subsequently convert this product to nicotinic acid for industrial applications.     

A sensitive method for determining quinolinic acid in animal tissues

A sensitive chromatographic method for isolation and measurement of quinolinic acid from rat liver and kidney is described. The method is based on the isolation of quinolinic acid by ion-exchange chromatography. The extraction of quinolinic acid consisted of the freeze clamping of the organ in liquid nitrogen, followed by deproteinization in perchloric acid. The neutralized extract was concentrated by freeze-drying and submitted to the action of concentrated perchloric acid to hydrolyze the nucleotides which interfered in the chromatographic separation of quinolinic acid. The sample was applied to a column of Dowex (HCOO^?) and eluted with a linear gradient of formic acid. The eluted fraction containing quinolinic acid was quantitatively measured by its absorbance at pH 2 and 268 nm in a spectrophotometer.     

Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterisation of the purified enzyme and its in vitro inhibition by Zn2+

3-hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid. Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in Escherichia coli, overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies. The recombinant human protein, obtained in E. coli, retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed. On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn2+, could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the beta type, with a minority of alpha.     

Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain

Quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) was purified 3600-fold from rat liver and 280-fold from rat brain. Kinetic analyses (K_m = 12 μM for the substrate quinolinic acid and K_m 23 μM for the cosubstrate phosphoribosylpyrophosphate), physicochemical properties of the purified enzymes, inhibition by phthalic acid (K_i = 1.4 μM) and molecular weight determination (M_r 160 000 for the holoenzyme, consisting of five identical 32 kDa subunits) indicated the structural identity of quinolinic acid phosphoribosyltransferase from the two rat tissues. This was further confirmed immunologically, using antibodies raised against purified rat liver quinolinic acid phosphoribosyltransferase. Rat quinolinic acid phosphoribosyltransferase differs in several aspects from quinolinic acid phosphoribosyltransferase isolated from other organisms. The purified enzyme will prove a useful tool in the examination of a possible role of quinolinic acid in cellular function and/or dysfunction.     

Ciliary Neurotrophic Factor Protects Striatal Neurons against Excitotoxicity by Enhancing Glial Glutamate Uptake

Ciliary neurotrophic factor (CNTF) is a potent neuroprotective cytokine in different animal models of glutamate-induced excitotoxicity, although its action mechanisms are still poorly characterized. We tested the hypothesis that an increased function of glial glutamate transporters (GTs) could underlie CNTF-mediated neuroprotection. We show that neuronal loss induced by in vivo striatal injection of the excitotoxin quinolinic acid (QA) was significantly reduced (by ∼75%) in CNTF-treated animals. In striatal slices, acute QA application dramatically inhibited corticostriatal field potentials (FPs), whose recovery was significantly higher in CNTF rats compared to controls (∼40% vs. ∼7%), confirming an enhanced resistance to excitotoxicity. The GT inhibitor dl-threo-β-benzyloxyaspartate greatly reduced FP recovery in CNTF rats, supporting the role of GT in CNTF-mediated neuroprotection. Whole-cell patch-clamp recordings from striatal medium spiny neurons showed no alteration of basic properties of striatal glutamatergic transmission in CNTF animals, but the increased effect of a low-affinity competitive glutamate receptor antagonist (γ-d-glutamylglycine) also suggested an enhanced GT function. These data strongly support our hypothesis that CNTF is neuroprotective via an increased function of glial GTs, and further confirms the therapeutic potential of CNTF for the clinical treatment of progressive neurodegenerative diseases involving glutamate overflow.     

De Novo Biosynthesis of Nicotinamide Adenine Dinucleotide in Escherichia coli: Excretion of Quinolinic Acid by Mutants Lacking Quinolinate Phosphoribosyl Transferase

The excretion of quinolinic acid was studied in growing and resting cells of Escherichia coli K-12 nadC(13). Under optimal conditions, this organism could synthesize quinolinic acid in several-fold excess of the amount which would be required for normal growth. The excretion of quinolinic acid was controlled by the concentration of nicotinamide adenine dinucleotide (NAD) precursors available to the organism either during growth or during incubation in dense cell suspensions. These observations suggest that biosynthesis of NAD de novo is regulated by both repression and feedback inhibition. Analogues of niacin which inhibit bacterial growth also inhibited and repressed the synthesis (excretion) of quinolinic acid. The pH optimum for quinolinic acid excretion agreed favorably with the optimum observed for its synthesis in vitro. The rate of quinolinic acid excretion was strongly influenced by the concentration of ribose or glycerol in the medium.     

Neuronal damage in the albino rat: excitotoxin, microtubule inhibitor synergism

Separate lines of investigation implicate excitotoxins or disruption of cytoskeletal architecture in the pathogenesis of human neurodegenerative disease. To assess the hypothesis that these neurotoxic mechanisms may have a synergistic effect, albino rats were given intrastriatal administrations of either the excitotoxin, kainic acid, or the microtubule inhibitor, colchicine, or both. Assessment of neuronal damage, either by loss of neurotransmitter metabolic enzyme activities or by histologic analysis, demonstrated synergism between the agents. Further, the synergism was still apparent with substitution of quinolinic acid, but not dimethyl kainate, for kainic acid, and Vinca alkaloids, but not lumicolchicine, for colchicine.     

Role of zinc in blockade of excitotoxic action of quinolinic acid by picolinic acid

Summary This study examined whether picolinic acid (PIC) inhibits quinolinic acid (QUIN) — induced excitotoxicity through zinc chelation. Injection of QUIN into the nucleus basalis magnocellularis significantly depleted cortical choline acetyltransferase activity 7 days post injection and PIC inhibited this response. Zinc augmented the QUIN- but not NMDA-induced response. When PIC was co-administered with zinc, PIC failed to attenuate the QUIN-induced response. The inhibition of QUIN — induced cholinergic toxicity by PIC may involve chelation of zinc.     

A radioenzymatic assay for quinolinic acid

A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.