Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)—the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.     

Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)-the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.     

The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted?at?two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders.     

Kynurenine 3-mono-oxygenase inhibitors attenuate post-ischemic neuronal death in organotypic hippocampal slice cultures

Kynurenine 3-mono-oxygenase (KMO) inhibitors reduce 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m-nitrobenzoyl)-alanine (30-100 micro m) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (1-10 micro m) reduced post-ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with alpha7 nicotinic acetylcholine receptors or with the glycineb site of N-methyl-d-aspartate (NMDA) receptors. On the other hand, the addition of either 3-HK or QUIN (1-10 micro m) to OGD-exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3-HK and QUIN.     

Perinatal kynurenine pathway metabolism in the normal and asphyctic rat brain

Summary. The kynurenine pathway of tryptophan degradation contains several metabolites which may influence brain physiology and pathophysiology. The brain content of one of these compounds, kynurenic acid (KYNA), decreases precipitously around the time of birth, possibly to avoid deleterious N-methyl-D-aspartate (NMDA) receptor blockade during the perinatal period. The present study was designed to determine the levels of KYNA, the free radical generator 3-hydroxykynurenine (3-HK), and their common precursor L-kynurenine (L-KYN) between gestational day 16 and adulthood in rat brain and liver. The cerebral activities of the biosynthetic enzymes of KYNA and 3-HK, kynurenine aminotransferases (KATs) I and II and kynurenine 3-hydroxylase, respectively, were measured at the same ages. Additional studies were performed to assess whether and to what extent kynurenines in the immature brain derive from the mother, and to examine the short-term effects of birth asphyxia on brain KYNA and 3-HK levels. The results revealed that 1) the brain and liver content of L-KYN, KYNA and 3-HK is far higher pre-term than postnatally; 2) KAT I and kynurenine 3-hydroxylase activities are quite uniform between E-16 and adulthood, whereas KAT II activity rises sharply after postnatal day 14; 3) during the perinatal period, KYNA, but not L-KYN, may originate in part from the maternal circulation; and 4) oxygen deprivation at birth affects the brain content of both KYNA and 3-HK 1 h but not 24 h later. Received August 31, 1999 Accepted September 20, 1999     

Involvement of the Kynurenine Pathway in Human Glioma Pathophysiology

The kynurenine pathway (KP) is the principal route of L-tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), the excitotoxin, quinolinic acid (QUIN) and the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD⁺). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2,3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. IDO-1 and TDO-2 induction in tumors are crucial mechanisms implicated to play pivotal roles in suppressing anti-tumor immunity. Here, we report the first comprehensive characterisation of the KP in 1) cultured human glioma cells and 2) plasma from patients with glioblastoma (GBM). Our data revealed that interferon-gamma (IFN-γ) stimulation significantly potentiated the expression of the KP enzymes, IDO-1 IDO-2, kynureninase (KYNU), kynurenine hydroxylase (KMO) and significantly down-regulated 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) and kynurenine aminotransferase-I (KAT-I) expression in cultured human glioma cells. This significantly increased KP activity but significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. KP activation (KYN/TRP) was significantly higher, whereas the concentrations of the neuroreactive KP metabolites TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlight a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumors, aimed at restoring anti-tumor immunity and reducing the capacity for malignant cells to produce NAD⁺, which is necessary for energy production and DNA repair.     

Metabolism of [5-3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-³H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-³H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µ M [5-³H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µ M [5-³H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [³H]KYNA (5.0 vs. 1.8%), [³H]3-HK (20.9 vs. 4.5%), [³H]XA (1.5 vs. 0.4%), and [³H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.     

Characterisation of kynurenine pathway metabolism in human astrocytes and implications in neuropathogenesis

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.     

Characterisation of kynurenine pathway metabolism in human astrocytes and implications in neuropathogenesis

Abstract

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.     

In vivo assessment of kynurenate neuroprotective potency and quinolinate excitotoxicity

Three complementary questions related to the kynurenine pathway and excitotoxicity were addressed in this study: (i) Which extracellular levels of quinolinic acid (QUIN) may be neurotoxic? (ii) Which extracellular levels of kynurenic acid (KYNA) may control excessive NMDA-receptor function? (iii) Can "anti-excitotoxic" levels of KYNA be reached by inhibition of kynurenine-3-hydroxylase (i.e. inhibition of QUIN synthesis and shunts of kynurenine metabolism toward KYNA)? Multifunctional microdialysis probes were used in halothane anaesthetised rats to apply NMDA or QUIN directly to the brain, with or without co-perfusion of KYNA, to record the resulting local depolarisations, and to monitor changes in dialysate KYNA after kynurenine-3-hydroxylase inhibition. QUIN produced concentration-dependent depolarisations with an estimated EC50 (i.e. concentration in the perfusion medium) of 1.22mM. The estimated ED50 for KYNA inhibition of NMDA-responses was 181microM. Kynurenine-3-hydroxylase inhibition (Ro-61-8048, 100mg/kg i.p.) increased dialysate KYNA 11 times (to 33.8nM) but without any reduction of NMDA-responses. These data challenge the notion that extracellular accumulation of endogenous QUIN may contribute to excessive NMDA-receptor activation in some neurological disorders, and the suitability of kynurenine-3-hydroxylase inhibition as an effective anti-excitotoxic strategy.     

Decreased serum and red blood cell kynurenic acid levels in Alzheimer's disease

Kynurenine aminotransferases (KAT I and KAT II) are responsible for the transamination of kynurenine (KYN) to form kynurenic acid (KYNA), an excitatory amino acid receptor antagonist. Since these members of the kynurenine pathway (KP) are proposed to be involved in the pathogenesis of Alzheimer's dementia (AD), the activities of these enzymes and the levels of these metabolites were measured in the plasma and red blood cells (RBCs) of AD and control subjects together with the inheritance of the apolipoprotein (APOE) epsilon4 allele. KYNA levels were significantly decreased both in the plasma and in the RBCs in AD, but the levels of KYN and the activities of KAT I and KAT II remained unchanged. No association has been found with the possession of the epsilon4 allele. These findings indicate an altered peripheral KP in AD regardless of the APOE status of the probands.     

Screening marine natural products for selective inhibitors of key kynurenine pathway enzymes

Abstract

Kynurenine, a metabolite of tryptophan along the ‘kynurenine pathway’, is at a branch point of the pathway which can lead to the synthesis of both quinolinic acid (QUIN) and kynurenic acid (KYNA). KYNA is an antagonist of glutamate receptors; however, QUIN is a selective agonist of NMDA receptors, and has been shown to act as an excitotoxic agent. A high QUIN/KYNA ratio has been implicated in a variety of neurological diseases in which excitotoxic neuronal cell death is found, e.g. AIDS-related dementia, stroke, etc. Inhibiting the key enzymes of this pathway (i.e. kynureninase and kynurenine 3-hydroxylase) would lower the QUIN/KYNA ratio, which may potentially have neuroprotective effects. We have developed high through-put assays for kynurenine pathway enzymes which allow us to screen extracts from marine organisms for selective enzyme inhibitors. Active metabolites are purified, isolated and identified by HPLC, high-field NMR and mass spectral techniques. Extracts from a sponge of the Aka species were found to contain a selective inhibitor of kynureninase. We have recently purified and identified the active principal as being serotonin sulfate. Related indoleamines, serotonin and 5-hydroxyindoleacetic acids are inactive. This finding may be suggestive of a novel interaction between the serotoninergic and excitatory amino acid pathways.     

Kynurenine-3-monooxygenase (KMO) broadly inhibits viral infections via triggering NMDAR/Ca2+ influx and CaMKII/ IRF3-mediated IFN-β production

Tryptophan (Trp) metabolism through the kynurenine pathway (KP) is well known to play a critical function in cancer, autoimmune and neurodegenerative diseases. However, its role in host-pathogen interactions has not been characterized yet. Herein, we identified that kynurenine-3-monooxygenase (KMO), a key rate-limiting enzyme in the KP, and quinolinic acid (QUIN), a key enzymatic product of KMO enzyme, exerted a novel antiviral function against a broad range of viruses. Mechanistically, QUIN induced the production of type I interferon (IFN-I) via activating the N-methyl-d-aspartate receptor (NMDAR) and Ca²⁺ influx to activate Calcium/calmodulin-dependent protein kinase II (CaMKII)/interferon regulatory factor 3 (IRF3). Importantly, QUIN treatment effectively inhibited viral infections and alleviated disease progression in mice. Furthermore, kmo^-/- mice were vulnerable to pathogenic viral challenge with severe clinical symptoms. Collectively, our results demonstrated that KMO and its enzymatic product QUIN were potential therapeutics against emerging pathogenic viruses.     

Alteration of kynurenic acid concentration in rat plasma following optically pure kynurenine administration: a comparative study between enantiomers

L-Kynurenine (KYN), a tryptophan metabolite, is metabolized to kynurenic acid (KYNA), which is an antagonist of N-methyl-D-aspartate and alpha7 nicotinic acetylcholine receptors, by kynurenine aminotransferase (KAT) I and KAT II. In this study, optically pure KYN, namely L-KYN or D-KYN, was administered intraperitoneally to male Sprague-Dawley rats (16.3 micromol kg(-1)), and the change in plasma KYNA was investigated by using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. Unexpectedly, no remarkable alteration in the plasma KYNA was observed when a natural isomer, L-KYN, was administered, whereas plasma KYNA concentration was unequivocally increased when an unnatural isomer, D-KYN, was administered. Serum protein bindings of L-KYN and D-KYN were also studied, and the protein binding of L-KYN (approximately 65%) in rat serum was larger than that of D-KYN (approximately 12%), suggesting that D-KYN may be easily incorporated and metabolized in tissues during blood circulation to generate KYNA in mammals. In addition, the increase in plasma KYNA by the administration of D-KYN was suppressed in rats pretreated with a selective inhibitor of D-amino acid oxidase (DAAO), 5-methylpyrazole-3-carboxylic acid (80 mg/kg). These results suggest that DAAO might be responsible for the production of KYNA from D-KYN in vivo.     

Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain

The tryptophan metabolite kynurenic acid (KYNA), which is produced enzymatically by the irreversible transamination of l-kynurenine, is an antagonist of alpha7 nicotinic and NMDA receptors and may thus modulate cholinergic and glutamatergic neurotransmission. Two kynurenine aminotransferases (KAT I and II) are currently considered the major biosynthetic enzymes of KYNA in the brain. In this study, we report the existence of a third enzyme displaying KAT activity in the mammalian brain. The novel KAT had a pH optimum of 8.0 and a low capacity to transaminate glutamine or alpha-aminoadipate (the classic substrates of KAT I and KAT II, respectively). The enzyme was inhibited by aspartate, glutamate, and quisqualate but was insensitive to blockade by glutamine or anti-KAT II antibodies. After purification to homogeneity, the protein was sequenced and the enzyme was identified as mitochondrial aspartate aminotransferase (mitAAT). Finally, the relative contributions of KAT I, KAT II, and mitAAT to total KAT activity were determined in mouse, rat, and human brain at physiological pH using anti-mitAAT antibodies. KAT II was most abundant in rat and human brain, while mitAAT played the major role in mouse brain. It remains to be seen if mitAAT participates in cerebral KYNA synthesis under physiological and/or pathological conditions in vivo.     

Screening marine natural products for selective inhibitors of key kynurenine pathway enzymes

Kynurenine, a metabolite of tryptophan along the 'kynurenine pathway', is at a branch point of the pathway which can lead to the synthesis of both quinolinic acid (QUIN) and kynurenic acid (KYNA). KYNA is an antagonist of glutamate receptors; however, QUIN is a selective agonist of NMDA receptors, and has been shown to act as an excitotoxic agent. A high QUIN/KYNA ratio has been implicated in a variety of neurological diseases in which excitotoxic neuronal cell death is found, e.g. AIDS-related dementia, stroke, etc. Inhibiting the key enzymes of this pathway (i.e. kynureninase and kynurenine 3-hydroxylase) would lower the QUIN/KYNA ratio, which may potentially have neuroprotective effects. We have developed high through-put assays for kynurenine pathway enzymes which allow us to screen extracts from marine organisms for selective enzyme inhibitors. Active metabolites are purified, isolated and identified by HPLC, high-field NMR and mass spectral techniques. Extracts from a sponge of the Aka species were found to contain a selective inhibitor of kynureninase. We have recently purified and identified the active principal as being serotonin sulfate. Related indoleamines, serotonin and 5-hydroxyindoleacetic acids are inactive. This finding may be suggestive of a novel interaction between the serotoninergic and excitatory amino acid pathways.     

Biochemical and phenotypic abnormalities in kynurenine aminotransferase II-deficient mice

Kynurenic acid (KYNA) can act as an endogenous modulator of excitatory neurotransmission and has been implicated in the pathogenesis of several neurological and psychiatric diseases. To evaluate its role in the brain, we disrupted the murine gene for kynurenine aminotransferase II (KAT II), the principal enzyme responsible for the synthesis of KYNA in the rat brain. mKat-2(-/-) mice showed no detectable KAT II mRNA or protein. Total brain KAT activity and KYNA levels were reduced during the first month but returned to normal levels thereafter. In contrast, liver KAT activity and KYNA levels in mKat-2(-/-) mice were decreased by >90% throughout life, though no hepatic abnormalities were observed histologically. KYNA-associated metabolites kynurenine, 3-hydroxykynurenine, and quinolinic acid were unchanged in the brain and liver of knockout mice. mKat-2(-/-) mice began to manifest hyperactivity and abnormal motor coordination at 2 weeks of age but were indistinguishable from wild type after 1 month of age. Golgi staining of cortical and striatal neurons revealed enlarged dendritic spines and a significant increase in spine density in 3-week-old mKat-2(-/-) mice but not in 2-month-old animals. Our results show that gene targeting of mKat-2 in mice leads to early and transitory decreases in brain KAT activity and KYNA levels with commensurate behavioral and neuropathological changes and suggest that compensatory changes or ontogenic expression of another isoform may account for the normalization of KYNA levels in the adult mKat-2(-/-) brain.     

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L-kynurenine (KYN) and by determining the irreversible conversion of [3H]KYN to [3H]KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2-oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT-rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate-specific. Thus, of the amino acids tested, only alpha-aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3-hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 microM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 microM for KYN with 1 mM 2-oxoglutarate, but a much higher Km (910 microM) with 1 mM pyruvate. Km values for 2-oxoglutarate and pyruvate were 150 and 160 microM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related increase of kynurenic acid in human cerebrospinal fluid - IgG and beta2-microglobulin changes

Kynurenic acid (KYNA) is an endogenous metabolite in the kynurenine pathway of tryptophan degradation and is an antagonist at the glycine site of the N-methyl-D-aspartate as well as at the alpha 7 nicotinic cholinergic receptors. In the brain tissue KYNA is synthesised from L-kynurenine by kynurenine aminotransferases (KAT) I and II. A host of immune mediators influence tryptophan degradation. In the present study, the levels of KYNA in cerebrospinal fluid (CSF) and serum in a group of human subjects aged between 25 and 74 years were determined by using a high performance liquid chromatography method. In CSF and serum KAT I and II activities were investigated by radioenzymatic assay, and the levels of beta(2)-microglobulin, a marker for cellular immune activation, were determined by ELISA. The correlations between neurochemical and biological parameters were evaluated. Two subject groups with significantly different ages, i.e. <50 years and >50 years, p < 0.001, showed statistically significantly different CSF KYNA levels, i.e. 2.84 +/- 0.16 fmol/microl vs. 4.09 +/- 0.14 fmol/microl, p < 0.001, respectively; but this difference was not seen in serum samples. Interestingly, KYNA is synthesised in CSF principally by KAT I and not KAT II, however no relationship was found between enzyme activity and ageing. A positive relationship between CSF KYNA levels and age of subjects indicates a 95% probability of elevated CSF KYNA with ageing (R = 0.6639, p = 0.0001). KYNA levels significantly correlated with IgG and beta(2)-microglobulin levels (R = 0.5244, p = 0.0049; R = 0.4253, p = 0.043, respectively). No correlation was found between other biological parameters in CSF or serum. In summary, a positive relationship between the CSF KYNA level and ageing was found, and the data would suggest age-dependent increase of kynurenine metabolism in the CNS. An enhancement of CSF IgG and beta(2)-microglobulin levels would suggest an activation of the immune system during ageing. Increased KYNA metabolism may be involved in the hypofunction of the glutamatergic and/or nicotinic cholinergic neurotransmission in the ageing CNS.     

Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain

We describe here a gas chromatography-tandem mass spectrometry (GC/MS/MS) method for the sensitive and concurrent determination of extracellular tryptophan and the kynurenine pathway metabolites kynurenine, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN) in rat brain. This metabolic cascade is increasingly linked to the pathophysiology of several neurological and psychiatric diseases. Methodological refinements, including optimization of MS conditions and the addition of deuterated standards, resulted in assay linearity to the low nanomolar range. Measured in samples obtained by striatal microdialysis in vivo, basal levels of tryptophan, kynurenine, and QUIN were 415, 89, and 8 nM, respectively, but 3-HK levels were below the limit of detection (<2 nM). Systemic injection of kynurenine (100 mg/kg, i.p.) did not affect extracellular tryptophan but produced detectable levels of extracellular 3-HK (peak after 2-3 h: ~50 nM) and raised extracellular QUIN levels (peak after 2h: ~105 nM). The effect of this treatment on QUIN, but not on 3-HK, was potentiated in the N-methyl-D-aspartate (NMDA)-lesioned striatum. Our results indicate that the novel methodology, which allowed the measurement of extracellular kynurenine and 3-HK in the brain in vivo, will facilitate studies of brain kynurenines and of the interplay between peripheral and central kynurenine pathway functions under physiological and pathological conditions.