Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Effects of Kynurenine Pathway Metabolites on Intracellular NAD+ Synthesis and Cell Death in Human Primary Astrocytes and Neurons:

Effects of Kynurenine Pathway Metabolites on Intracellular NAD+ Synthesis and Cell Death in Human... ORIGINAL RESEARCH Effects of Kynurenine Pathway Metabolites on Intracellular NAD Synthesis and Cell Death in Human Primary Astrocytes and Neurons 1 1,2 3,4 1 5 Nady Braidy , Ross Grant , Bruce J Brew , Seray Adams , Tharusha Jayasena 1,4 and Gilles J. Guillemin 1 2 University of New South Wales, Faculty of Medicine, Sydney, Australia. Australasian Research Institute, Sydney Adventist Hospital, Sydney, Australia. St. Vincent’s Centre for Applied Medical Research, Sydney, Australia. Department of Neurology, St. Vincent’s Hospital, Sydney, Australia. Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, Australia. Abstract: The kynurenine pathway (KP) is a major route of L-tryptophan catabolism resulting in the production of the essential pyridine nucleotide nicotinamide adenine dinucleotide, (NAD ). Up-regulation of the KP during infl ammation leads to the release of a number of biologically active metabolites into the brain. We hypothesised that while some of the extracellular KP metabolites may be benefi cial for intracellular NAD synthesis and cell survival at physiological concentra- tions, they may contribute to neuronal and astroglial dysfunction and cell death at pathophysiological concentrations. In this study, we found that treatment of human primary neurons and astrocytes with 3-hydroxyanthranilic acid (3-HAA), 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and picolinic acid (PIC) at concentrations below 100 nM signifi cantly increased intracellular NAD levels compared to non-treated cells. However, a dose dependent decrease in intracellular NAD levels and increased extracellular LDH activity was observed in human astrocytes and neurons treated with 3-HAA, 3-HK, QUIN and PIC at concentrations 100 nM and kynurenine (KYN), at concentrations above 1 μM. Intracellular NAD levels were unchanged in the presence of the neuroprotectant, kynurenic acid (KYNA), and a dose dependent increase + + in intracellular NAD levels was observed for TRP up to 1 mM. While anthranilic acid (AA) increased intracellular NAD levels at concentration below 10 μM in astrocytes. NAD depletion and cell death was observed in AA treated neurons at concentrations above 500 nM. Therefore, the differing responses of astrocytes and neurons to an increase in KP metabolites should be considered when assessing KP toxicity during neuroinfl ammation. Introduction Tryptophan (TRP) catabolism via the kynurenine pathway (KP) represents the major pathway for the + 1 + synthesis of nicotinamide adenine dinucleotide (NAD ). Essential NAD dependent reactions can be 2 + 3 divided into three main categories: (1) NAD is an important contributor to energy (ATP) production; + 4,5 (2) NAD serves as a cofactor for NAD glycohydrolases involved in intracellular calcium regulation; + 6–8 (3) NAD is a substrate for the family of DNA nick sensing poly(ADP-ribose) polymerases (PARP) 9,10 + and the class III histone deacetylases known as sirtuins. NAD levels are extremely volatile and can be signifi cantly reduced under conditions of excessive PARP-1 activation caused by oxidative damage 11 + to DNA, and during mitosis. Thus, continuous biosynthesis of NAD is vital to the maintenance and ongoing cell viability of all cells. The KP is the principal route of L-tryptophan catabolism, resulting in the production of NAD (Fig. 1). Over-activation of the KP has been implicated in the pathogenesis of several neurological disorders including Huntington’s disease (HD), Alzheimer’s disease (AD), and the acquired immunodefi ciency 13–17 syndrome (AIDS)-dementia complex. The pathway is regulated by the immune-factor responsive enzyme indoleamine-2,3-dioxygenase (IDO) in most cells and by tryptophan-2,3 dioxygenase (TDO) 18,19 in the liver which is modulated by tryptophan and glucocorticoids. Several intermediate products of the KP are known to be neurotoxic. Among them, the N-methyl- D-aspartate (NMDA) receptor agonist and neurotoxin, quinolinic acid (QUIN) is likely to be most important in terms of biological activity. Anthranilic acid (AA), 3-hydroxyanthranilic acid (3-HAA), and 3-hydroxykynurenine (3-HK) have been shown to generate free radicals leading to neuronal damage similar to QUIN. The early upstream KP metabolite kynurenic acid (KYNA), has been shown to antagonise the Correspondence: Gilles J. Guillemin, Department of Pharmacology, Faculty of Medicine, University of NSW, Sydney, Australia 2052. Email: g.guillemin@cfi .unsw.edu.au Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/. International Journal of Tryptophan Research 2009:2 61–69 61 Braidy et al neurotoxic effects of QUIN and glutamate-mediated of human neurons and astrocytes treated with 20,21 NMDA receptor activation. The downstream physiological and pathophysiological concentra- metabolite picolinic acid (PIC) is an endogenous tions of TRP, KYN, KYNA, AA, 3-HAA, 3-HK, 22,23 metal chelator within the brain that displays some PIC, and QUIN respectively (0.1–100 μM). Intra- protection against QUIN induced toxicity and cellular NAD levels were measured using the 24,25 posesses immune regulatory activity. thiazolyl blue microcycling assay. The effect of Given the signifi cance of intracellular NAD KP metabolites on cell viability was determined levels for the maintenance of total cell integrity by measuring the release of lactate dehydrogenase and cell viability, we used primary monocultures into the extracellular medium. L-Tryptophan COOH NH A B NH L-Formylkynurenine COOH HN CHO Kynurenic Acid OH H N L-Kynurenine COOH COOH NH Missing in E Astrocytes NH 3-Hydroxykynurenine Anthranilic Acid COOH NH HO COOH Picolinic Acid 3-Hydroxyanthranilic Acid NH OH N COOH COOH 2-Amino-3- CHC Carboxymuconic HOOC NH Glutaryl- 2 Acetyl- Semialdehyde CoA CoA COOH Quinolinic Acid NAD COOH Figure 1. The Kynurenine Pathway of Tryptophan Degradation. A) Indoleamine 2,3-dioxygenase (IDO); B) Tryptophan 2,3 dioxygenase (TDO) C) Kynurenine Formylase; D) Kynurenine-Amino Transferase; E) Kynurenine 3Hydroxylase; F) Kynureninase; G) Non-specifi c hydroxylation; H) 3-Hydroxyanthranilic Acid Oxidase; I) Picolinic Carboxylase J) Non-enzymatic cyclisation; K) Quinolinic Acid Phosphori- bosyltransferase. International Journal of Tryptophan Research 2009:2 62 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis Materials and Methods Extracellular LDH activity as a measurement for cytotoxicity The release of lactate dehydrogenase (LDH) into Reagents and chemicals culture supernatant correlates with the amount of Dulbecco’s phosphate buffer solution (DBPS) and cell death and membrane damage, providing an all other cell culture media and supplements were accurate measure of cellular toxicity. LDH activity from Invitrogen (Melbourne, Australia) unless oth- following 24 hour incubation with the desired erwise stated. Nicotinamide, bicine, β-nicotinamide concentrations of KP metabolites was assayed adenine dinucleotide reduced form (β-NADH), using a standard spectrophotometric technique 3-[-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo- described by Koh and Choi. lium bromide (MTT), alcohol dehydrogenase (ADH), sodium pyruvate, TRIS, γ-globulins, L-tryptophan (TRP), kynurenine (KYN), kynurenic Bradford protein assay acid (KYNA), anthranilic acid (AA), 3-hydroxyan- for the quantifi cation of total protein thranilic acid (3-HAA), 3-hydroxykynurenine NAD concentration and extracellular LDH activity (3-HK), picolinic acid (PIC), and quinolinic acid were adjusted for variations in cell number using the (QUIN) were obtained from Sigma-Aldrich Bradford protein assay described by Bradford. (Castle-Hill, Australia). Phenazine methosulfate (PMS) was obtained from ICN Biochemicals (Ohio, Data analysis U.S.A). Bradford reagent was obtained from BioRad, Results obtained are presented as the means ± the Hercules (CA, U.S.A). standard error of measurement (SEM). Signifi cant differences between results were verifi ed using the Cell cultures two-tailed t-test with equal variance. Differences Human foetal brains were obtained from 16–19 week between treatment groups were considered signifi - old foetuses collected following therapeutic termina- cant if p was less than 0.05 (p  0.05). tion with informed consent. Mixed brain cultures were prepared and maintained using a protocol pre- viously described by Guillemin et al. Astrocytes Results and neurons were prepared from the mixed brain cell cultures, and maintained using a protocol previously 27 Effect of extracellular KP metabolites described by Guillemin et al. on intracellular NAD concentrations in human astrocytes and neurons Primary brain cells and KP metabolite TRP induced a dose-dependent increase in intracel- culture treatments lular NAD levels in both human neurons Human primary astrocytes and neurons were (at 100 nM TRP) and astrocytes (at 500 nM incubated with various concentrations of the KP TRP), (Fig. 2). We found that 3-HAA, 3-HK, QUIN metabolites (0.1–100 μM) for 24 hours. Experiments and PIC signifi cantly increased intracellular NAD were performed with primary cultures derived from levels at a low concentration of 100 nM but sub- three different human foetal brains with each indi- stantially decreased NAD levels at higher con- vidual preparation tested in triplicate. centrations (Fig. 2) in both cell types. Treatment with KYNA had no signifi cant effect on intracel- lular NAD activity in either neuronal or astroglial NAD(H) Microcycling assay cultures (Fig. 2). A dose-dependent decrease in for the measurement of intracellular intracellular NAD levels was observed in KYN NAD concentrations treated astrocytes and neurons at concentra- Intracellular NAD concentration following 24 hour tions above 1 μM. AA increased intracellular incubation with the desired concentrations of KP NAD levels at concentrations below 50 μM in metabolites were measured spectrophotometrically human astrocytes. However in human neurons using the thiazolyl blue microcycling assay estab- NAD depletion was observed at AA concentra- lished by Bernofsky and Swan adapted for 96 well tions 500 nM for neurons and 100 μM for plate format by Grant and Kapoor. astrocytes (Fig. 2). International Journal of Tryptophan Research 2009:2 63 Braidy et al Figure 2. The effect of KP metabolites on intracellular NAD levels in human neurons and astrocytes. (Control = 0 μM treatment for each metabolite and cell type) A) The effect of TRP (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. B) The effect of KYN (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. C) The effect of KYNA (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. D) The effect of 3-HAA (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. E) The effect of 3HK (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. F) The effect of AA (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. G) The effect of QUIN (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared control in human neurons. ¥p  0.05 compared to control in human astrocytes. H) The effect of PIC (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. (n = 3 for each treatment group). International Journal of Tryptophan Research 2009:2 64 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis Effect of extracellular KP metabolites Moreover, under conditions of TRP depletion, supplementation with TRP down-regulates on extracellular LDH activity in human enzymes directing TRP to non-NAD dependent astrocytes and neurons pathways, suggesting a shift of all available TRP Consistent with the results obtained for intracellular + 37 catabolism to NAD synthesis. However, it is NAD levels, no signifi cant change was observed clear that excessive TRP supplementation would in extracellular LDH activity for both astrocyte and aggravate or induce autoimmune disease. neuronal cultures treated with TRP or KYNA up to The primary metabolite of TRP, N-formylkyn- 1 mM (Fig. 3). However treatment with 3-HAA, urenine can be rapidly converted to KYN by the 3-HK, QUIN and PIC increased extracellular LDH enzyme arylamine formamidase. KYN (its activity at concentrations above 100 nM (Fig. 3) in physiological concentration is 1 μM) is converted both cell types. A dose-dependent increase in extra- into several neurotoxic metabolites such as 3-HK cellular LDH activity was observed in astrocytes 39,14 and QUIN in unstimulated human brain cells. and neurons treated with KYN at concentrations Our data indicates that KYN causes intracellular above 1 μM. However the magnitude of LDH NAD depletion and reduced cell viability at greater release following KYN treatment was signifi cantly than physiological concentrations (Figs. 2 and 3). less than that observed for any of the other toxic Human astrocytes and microglial cells demonstrate metabolites. LDH activity was also increased for rapid cellular uptake of KYN. KYN has been shown cells treated with AA at concentrations above to increase QUIN production and KP enzyme 100 nM in astrocytes and at or above 100 nM in expression in human macrophages. The enhanced neurons (Fig. 3). generation of QUIN may account for the reduced NAD levels and increased cytotoxicity observed Discussion in human neurons and astrocytes at pathophysio- Neurodegenerative diseases are often characterised logical concentrations (Figs. 2 and 3). by a loss of neuronal cells in specifi c regions of the KYN and the resulting metabolite, 3-HK can be + 15 brain. Given the importance of intracellular NAD converted to AA and 3-HAA by kynureninase. levels for maintaining overall cellular integrity and These metabolites also provide additional substrate + 40 function, it is conceivable that reduced NAD levels for QUIN formation. The levels of 3-HK, AA and are a potential pathogenic mechanism for neuronal 3-HAA are signifi cantly increased in the CSF of 11,8 41 and astroglial cell death. TRP has been used as a patients with HD. Elevated levels have also been supplement for some years in the United States reported in HIV cases associated with dementia, before being removed due to an outbreak of the lethal infantile spasms, and hepatic encephalopathy. autoimmune disease, eosinophilia-myalgia syn- While 3-HK, AA, and 3-HAA appear less neurotoxic drome (EMS) resulting in 36 deaths. Large doses than QUIN, these KP metabolites have been of TRP can induce the build-up of selected white previously shown to promote neuronal damage blood cells leading to EMS. Since the KP is a major largely through free radical formation but not NMDA 32 15 regulator of the immune response, the toxic effect receptor activation. Under normal conditions, the may be due to inhibition of normal tolerogenic cell serum concentration of 3-HK, 3-HAA and AA has 11 42 35 34 T-cell death following IDO induction. been found to be 383 nM, 24 nM and 21 nM, In this study, we found that TRP supplementa- respectively. In this study, we found that 3-HK, AA tion produced a dose-dependent increase in intra- and 3-HAA supplementation resulted in neuronal + + cellular NAD levels in human astrocytes and and astroglial NAD depletion and cell death in 24 neurons after 24 hours (Fig. 2). The physiological hour cultures at micromolar concentrations (Fig. 1 concentration of TRP is human plasma is estimated and Fig. 2). These results are consistent with a previ- 33,34 to be 40–90 μM. Lower serum concentrations ous study showing that 3-HK, 3-HAA, and AA also have been observed in several disorders including induced a time-and dose-dependent increase in cell 35 36 41 depression and anxiety, rheumatoid arthritis and death at micromolar concentrations (1–100 μM). following infection with HIV. TRP supplementa- These authors reported that the accompanying cell tion has been previously shown to be benefi cial in death was signifi cantly reduced by co-treatment with several neurological conditions, including insom- catalase, suggesting that the neurotoxic effects of nia and depression, since TRP can be used for the 3-HK may be mediated by increased hydrogen per- + 11,19 41 synthesis of serotonin, melatonin and NAD . oxide. 3-HK can be converted to quinoneimines International Journal of Tryptophan Research 2009:2 65 Braidy et al A B 120 120 100 100 80 80 60 60 Neurons Neurons 40 40 Astrocytes Astrocytes * * ¥ ¥ 20 20 * * ¥ ¥ ¥ ¥ * * ¥ ¥ ** ** 0 0 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 1000 TRP conc (μM) KYN conc (μM) C C D D 120 120 ¥ ¥ * * 100 100 * * ¥ ¥ 60 60 * * Neurons Neurons ¥ ¥ Astrocytes Astrocytes * * ¥ ¥ 20 * * ¥ ¥ * * ¥ ¥ 0 0 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 1000 KYNA conc (μM) 3-HAA conc (μM) E E * * ¥ ¥ F F 120 120 * * * * ¥ ¥ 100 100 ¥ ¥ * * 80 80 * * * * ¥ ¥ * * 60 60 Neurons Neurons * * ¥ ¥ ¥ ¥ 40 40 * * Astrocytes Astrocytes * * ¥ ¥ * * 20 20 * * ¥ ¥ * * ¥ ¥ ¥ ¥ 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 3-HK conc (μM) AA conc (μM) G G H H 250 120 * * ¥ ¥ * * ¥ ¥ * * ¥ ¥ * * ¥ ¥ * * * * Neurons Neurons ¥ ¥ * * ¥ ¥ ¥ ¥ 40 * * Astrocytes Astrocytes ¥ ¥ * * 50 * * ¥ ¥ ¥ ¥ * * ¥ ¥ * * ¥ ¥ 0 0 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 1000 QUIN conc (μM) PIC conc (μM) Figure 3. Effect of KP metabolites on extracellular LDH activity in human astrocytes and neurons (Control = 0 μM treatment for each metabolite and cell type). A) The effect of TRP (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. B) The effect of KYN (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. C) The effect of KYNA (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. D) The effect of 3-HAA (1–1000 μM) on extracel- lular LDH activity in human neurons and astrocytes after 24 hours. **p  0.05 compared to control in human neurons (n = 3 for each treatment group). ¥p  0.05 compared to control in human astrocytes. E) The effect of 3-HK (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. F) The effect of AA (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. G) The effect of QUIN (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. H) The effect of PIC (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes (n = 3 for each treatment group). International Journal of Tryptophan Research 2009:2 66 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis that can generate pro-oxidant intermediates, such as undergoes non-specific hydroxylation to form hydrogen peroxide, organic and hydroxyl radicals, 3-HAA and may subsequently be used to form QUIN 43,44 + 27 during processes of autoxidation. Similarly, 3- and NAD . This suggests a greater requirement for HK has been shown to potentiate QUIN toxicity in AA as a substrate for NAD synthesis in human rats, and the cytotoxic effect can be prevented using astrocytes compared to human neurons, and AA may free radical scavengers. become cytotoxic to both cell types in excess con- Although 3-HK and 3-HAA have been shown centrations. These fi ndings provide insight not only to be cytotoxic at high concentrations, physiolog- in the differences between neurons and astrocytes, ical concentrations of 100 nM increased intracel- but also for understanding how different brain cells + + lular NAD levels by 18 and 12 per cent respectively, produce NAD . with no detectable effect on extracellular LDH QUIN is an endogenous NMDA receptor agonist activity (Figs. 1 and Fig. 2). The contribution of involved in neuronal fi ring. The amount of QUIN 3-HK and 3-HAA at these physiological concentra- in the brain and CSF is usually less than 100 nM. tions to neuronal and astroglial cytotoxicity is Increased brain QUIN levels (500–1000 nM) have uncertain as micromolar concentrations appear to been observed in the CSF and serum in several 41,45 49 45 produce neurotoxicity in-vitro. infl ammatory brain diseases, including AD, HD, The observation that addition of these KP traumatic brain injury, AIDS dementia complex + 16 15 metabolites can serve as substrate for NAD synthesis (ADC), and other infections. QUIN has been at low concentrations (100–500 nM) implies that shown to be up-regulated in ageing and in associa- + 50 under normal conditions, NAD levels may be tion with senile plaques in the AD brain. The dependent on substrate availability. We propose results in the study are in line with previously pub- that astroglial and neuronal KP enzymes downstream lished results highlighting the importance of QUIN of QUIN may become saturated in the presence of as a benefi cial substrate for NAD synthesis at low excess extracellular KP metabolites leading to an concentration, but a putative toxin able to induce accumulation of QUIN. Indeed, we have previously oligodendrocyte, neuronal, and astroglial apoptosis 49,51–53 demonstrated that exogenous 3-HAA substantially at pathophysiological concentrations. increases QUIN synthesis in human foetal astrocytes As previously mentioned, PIC is an endogenous to neurotoxic levels implying saturation of down- metal chelator in the brain, and is an effi cient chelator stream enzymes involved in catabolism of QUIN to for minerals such as chromium, zinc, manganese, + 27 23 its essential metabolite, NAD . Similarly, Blight copper, and iron. Unbound (free) redox-active iron et al observed that treatment of 4-chloro-3- and copper significantly increase free radical hydroxyanthranilate, a synthetic inhibitor of generation and are found in abundance in the AD 3-hydroxyanthranilic acid oxidase, was able to reduce brain. Disordered PIC metabolism may yet be found QUIN production and functional defi cits following to play a role in the pathophysiology of AD. The experimental spinal cord injury in guinea pigs. physiological concentration of PIC in human serum On the other hand, our results indicate a clear is thought to be between 100–400 nM. Elevated dichotomy between exogenous AA effects in human PIC levels have been associated with fatal outcome neurons and astrocytes. While AA appears neuro- in Malawian children with cerebral malaria. Our toxic at concentrations as low as 500 nM, AA data shows that PIC can increase intracellular NAD improved intracellular NAD levels in human levels at physiological concentrations (100 nM). This astrocytes by up to 10% in the 0.5 to 10 μM range may be partly due to its intracellular metal chelating (Fig. 2). AA has been previously reported to impair properties reducing the contribution of free redox energy metabolism in the rat cerebral cortex at active metals in free radical generation. micromolar concentrations, possibly through However, PIC appeared to also induce a dose inhibition of complex I–III activities in the mito- dependent decrease in intracellular NAD and chondrial respiratory chain. While the KP is fully increase in extracellular LDH activity in both expressed in human neurons, kynurenine hydrox- human astrocytes and neurons. These results are ylase (KYN-OHase), which converts KYN to 3-HK consistent with one study that observed signfi cant is absent in human foetal astrocytes. Consequently, PIC induced neurotoxicity that was associated with astrocytes cannot produce 3-HK. The KP is divided both prolonged exposure time and increased glu- into two parts in human astrocytes (Fig. 1), and KYN cose concentration in the culture supernatant. may be converted to AA by kynureninase which then While the mechanism leading to PIC toxicity is International Journal of Tryptophan Research 2009:2 67 Braidy et al 3. Berger SJ, Sudar DC, Berger NA. Metabolic consequences of DNA not known, at high concentrations, PIC may be damage: DNA damage induces alterations in glucose metabolism by causing disruption to some of the many trace metal activation of Poly(ADP-ribose) polymerase. Biochem Biophys Res + ++ ++ (Cu , Zn , Fe ) dependent biochemical reactions Commun. 1986;134:227–232. 4. Berger F, Ramirez-Hernandez MH, Ziegler M. The new life of a centenarian: essential for normal cell metabolism. signalling functions of NADP. Trends Biochem Sci. 2004;29:111–118. KYNA is a neuroprotective molecule with 5. Lee HC. Physiologic functions of cyclic ADP-ribose and NAADP as antagonistic properties on both nicotinic-acetylcholine calcium messengers. Ann Rev Pharmacol Toxicol. 2001;41:317–345. 6. Furukawa A, Tada-Oikawa S, Kawanishi S. H O accelerates cellular and glutamatergic receptors at supra-physiological 2 2 senescence by accumulation of accelerates cellular senescence by concentrations. The tissue concentrations of KYNA accumulation of acetylated p53 via decrease in the function of SIRT1 in the human brain have been estimated to be + by NAD depletion. Cell Physiol Biochem. 2007;20:45–54 58,59 between 0.2–1.5 μM. In our study, no signifi cant 7. Ha HC, Snyder SH. Poly(ADP-ribose) polymerase-I in the nervous system. Neurobiol Dis. 2000;7:225–239. changes in intracellular NAD levels and cell 8. Love S, Barber R, Wilcock GK. Increased poly(ADP-ribosyl)ation of viability were observed across micromolar concen- nuclear proteins in Alzheimer’s disease. Brain. 1999;122:247–253. trations in both human primary astrocytes and neu- 9. Sauve AA, Wolberger C, Schramm VL, Boeke JD. The biochemistry of Sirtuins. Annu Rev Biochem. 2006;75:435–465. rons (Figs. 2 and 3). KYNA exhibits a high affi nity 10. Anderson RM, Bitterman KJ, Wood JG, et al. Manipulation of a nuclear for the glycine-binding site of the NMDA receptor NADP salvage pathway delays ageing without altering steady-state at low concentrations, but can act on the glutamate NADP levels. J Biol Chem. 2002;277:18881–18890. 11. Penberthy TW. Pharmacological targeting of IDO-mediated tolerance binding site on the NMDA receptor, and AMPA for treating autoimmune disease. Curr Drug Metab. 2007;8:245–266. receptors, at high micromolar concentrations. 12. Ying W. NAD and NADH in cellular functions and cell death. Front Decrease in KYNA levels below the threshold Biosci. 2006;11:3129–3148. needed for NMDA receptor antagonism can enhance 13. Schwarcz R. The kynurenine pathway of tryptophan degradation as a drug target. Curr Opin Pharm. 2004;4:12–17. the vulnerability of dopaminergic neurons to exci- 14. Heyes MP, Saito K, Crowley JS, et al. Quinolinic acid and kynurenine 60 61 totoxic insult. Moroni proposed that NMDA and pathway metabolism in infl ammatory and non-infl ammatory neurologic glutamate induced excitotoxic neuronal damage can disease. Brain. 1992;115:1249–1273. 15. Stone TW. Endogenous neurotoxins from tryptophan. Toxicon. be ameliorated by diverting KYN metabolism to 2001;39(1):61–73. KYNA synthesis. Likewise, supplementation with 16. Guillemin GJ, Kerr SJ, Brew BJ. Involvement of quinolinic acid in TRP, an endogenous precursor for KYN synthesis, AIDS dementia complex. Neurotox Res. 2005;7(1–2):103–23. 17. Heyes MP, Brew BJ, Martin A, et al. Quinolinic acid in cerebrospinal may increase the levels of KYNA and therefore pos- fl uid and serum in HIV-1 infection: relationship to clinical and neuro- sibly slow down or prevent the formation of cyto- logical status. Ann Neurol. 1991;29(2):202–9. 62,63 toxic concentrations of QUIN. 18. Mackay GM, Forrest CM, Stoy N, et al. Tryptophan metabolism and This study is the fi rst to examine the effects of oxidative stress in patients with chronic brain injury. Eur J Neurol. 2006;13:30–42. extracellular KP metabolites on intracellular NAD 19. Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Lowry CA. synthesis and cell death in human primary astro- Tryptophan metabolism in the central nervous system: medical impli- cytes and neurons. While a large body of literature cations. Expert Rev Mol Med. 2006;8:1–27. 20. Hilmas C, Pereira EF, Alkondon M, Rassoulpour A, Schawrcz R. The is available on the toxic effects of KP metabolites brain metabolite kynureninic acid inhibits alpha7-nicotinic recceptor in regard to neuroinfl ammation, few studies have activity and increases non-alpha7 nicotinic receptor expression. considered the effect of these metabolites on intra- J Neurosci. 2001;21:7463–7473. 21. Perkins MN, Stone TW. An iontophoretic investigation of the actions cellular NAD levels in-vitro. This study demon- of convulsant kynurenines and their interactions with endogenous strates that changes in the brain levels of certain KP excitant quinolinic acid. Brain Res. 1982;247:184–187. metabolites may be important mediators contribut- 22. Jayasena T, Grant RS, Keerthisinghe N, Solaja I, Smythe GA. Membrane ing to brain cell dysfunction in some neurological permeability of redox active metal chelators: An important element in reducing hydroxyl radical induced NAD depletion in neuronal cells. disorders involving increased KP activation. Neurosci Res. 2007;57:454–461. 23. Melillo G, Bosco MC, Musso T, Varesio L. Immunobiology of picolinic acid. Adv Exp Med Biol. 1996;398:135–141. Disclosure 24. Bosco MC, Rapisarda A, Reffo G, Massazza S, Pastorino S, Varesio L. The authors report no confl icts of interest. Macrophage activating properties of the tryptophan catabolite picolinic acid. Adv Exp Med Biol. 2003;527:55–65. 25. Kalisch BE, Jhamandas K, Boegman RJ, Beninger RJ. Picolinic acid References protects against quinolinic acid-induced depletion of NADPH diaphorase 1. Grant RS, Passey R, Matanovic G, Smythe G, Kapoor V. Evidence for containing neurons in the rat striatum. Brain Res. 1994;668(1–2):1–8. increased de-novo synthesis of NAD in immune-activated RAW264.7 26. Guillemin GJ, Smythe G, Takikawa O, Brew BJ. Expression of macrophages: a self-protective mechanism? Arch Biochem Biophys. indoleamine 2,3-dioxygenase and production of quinolinic acid by 1999;372:1–7. human microglia, astrocytes, and neurons. Glia. 2005;49(1):15–23. 2. Braidy N, Guillemin G, Grant R. Promotion of Cellular NAD Anabolism: 27. Guillemin GJ, Kerr SJ, Smythe GA, et al. Kynurenine pathway Therapeutic Potential for Oxidative Stress in Ageing and Alzheimer's metabolism in human astrocytes: a paradox for neuronal protection. Disease. Neurotox Res. 2008;13(3, 4):173–184. J Neurochem. 2001;78:842–853. International Journal of Tryptophan Research 2009:2 68 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis 28. Bernofsky C, Swan M. An improved cycling assay for nicotinamide 45. Guidetti P, Schwarcz R. 3-hydroxykynurenine and quinolinate: adenine dinucleotide. Anal Biochem. 1973;53:452–458. pathological synergism in early Huntington’s disease? Adv Exp Med 29. Grant RS, Kapoor V. Murine glial cells regenerate NAD, after peroxide- Biol. 2003;527:137–45. induced depletion, using either nicotinic acid, nicotinamide, or 46. Blight AR, Cohen TI, Saito K, Heyes MP. Quinolinic acid accumulation quinolinic acid as substrates. J Neurochem. 1998;70:1759–1763. and functional defi cits following experimental spinal cord injury. Brain. 30. Koh JY, Choi DW. Quantitative determination of glutamate mediated 1995;118:735–752. cortical neuronal injury in cell culture by lactate dehydrogenase effl ux 47. Schuck PF, Tonin A, da Costa Ferreira G, et al. Kynurenines impair assay. J Neurosci Methods. 1987;20:83–90. energy metabolism in rat cerebral cortex. Cell Mol Neurobiol. 31. Bradford MM. A rapid and sensitive method for quantitation of 2007;27:147–60. microgram quantities of protein utilising the principle of protein-dye 48. Guillemin GJ, Cullen KM, Lim CK, et al. Characterisation of the binding. Anal Biochem. 1976;53:452–458. kynurenine pathway in human neurons. J Neurosci. 2007;27:1–9. 32. Munn DH, Mellor AL. Indoleamine 2,3-dioxygenase and tumour- 49. Guillemin GJ, Brew BJ. Implications of the kynurenine pathway and induced tolerance. J Clin Invest. 2007;177:1147–54. quinolinic acid in Alzheimer’s disease. Redox Rep. 2002;7(4):199–206. 33. Werner ER, Fuchs D, Hausen A, et al. Tryptophan degradation in 50. Guillemin GJ, Brew BJ, Noonan CE, Takikawa O, Cullen KM. patients infected by human immunodefi ciency virus. Biol Chem Hoppe Indoleamine 2,3 dioxygenase and quinolinic acid immunoreactivity in Seyler. 1988;369:337–340. Alzheimer’s Disease hippocampus. Neuropathol Appl Neurobiol. 34. Forrest CM, Macaky GM, Oxford L, Stoy N, Stone TW, Darlington LG. 2005;31:395–404. Kynurenine pathway metabolism in patients with osteoporosis after 51. Braidy N, Grant R, Adams S, Guillemin G. Mechanism for quinolinic 2 years of drug treatment. Clin Exp Pharmacol Physiol. 2006;33: acid cytotoxicity in human astrocytes and neurons. (submitted). 1078–1087. 52. Cammer W. Apoptosis of oligodendrocytes in secondary cultures from 35. Myint AM, Kim YM, Verkerk R, Scharpe S, Steinbuch H, Leonard B. neonatal rat brains. Neurosci Lett. 2002;327(2):123–7. Kynurenine pathway in major depression: evidence of impaired neu- 53. Guillemin GJ, Wang L, Brew BJ. Quinolinic acid selectively induces roprotection. J Affect Disord. 2007;98:143–51. apoptosis of human astocytes: Potential role in AIDS dementia complex. 36. Schrocksnadel K, Winkler C, Fuith LC, Fuchs D. Tryptophan J Neuroinfl ammation. 2005;2(16):1–6. degradation in patients with gynecological cancer correlates with 54. Bush AI. Metal complexing agents as therapies for Alzheimer’s disease. immune activation. Cancer Lett. 2005;223:323–329. Neurobiol Aging. 2002;23:1031–1038. 37. Shibata K, Motooka K, Murata K. The differences in growth and activity 55. Chen Y, Guillemin GJ. Kynurenine pathway metabolites in humans: of the tryptophan-NAD pathway between Wistar and Sprague-Dawley disease and healthy states. IJTR 2. 2009 strains of rats fed on tryptophan limited diet. J Nutr Sci Vitaminol 56. Medana IM, Day NP, Salahifar-Sabet H, Stocker R, Smythe G, (Tokyo). 1982;28:11–19. Bwanaisa L, Njobvu A, Kayira K, Turner GD, Taylor TE, Hunt NH. 38. Backhaus C, Rahman H, Scheffl er S, Laatsch H, Hardeland R. NO Metabolites of the kynurenine pathway of tryptophan metabolism in scavenging by 3-hydroxyanthranilic acid and 3-hydroxykynurenine: the cerebrospinal fl uid of Malawian children with malaria. J Infect Dis. N-nitrosation leads via oxadiazoles to o-quinone diazides. Nitric Oxide. 2003;188(6): 844–849. 2008;19(3):237–44. 57. Melillo G, Bosco MC, Musso T, Varesio L. Immunobiology of picolinic 39. Sas K, Robotka H, Toldi J, Vecsei L. Mitochondria, metabolic acid. Adv Exp Med Biol. 1996;398:135–141. disturbances, oxidative stress and the kynurenine system, with focus 58. Moroni P, Russi G, Lombardi G, Beni M, Carl V. Presence of kynurenic on neurodegenerative disorders. J Neurol Sci. 2007;257:221–239. acid in the mammalian brain. J Neurochem. 1988;51:177–180. 40. Stone TW, Forrest CM, Mackay GM, Stoy N, Darlington LG. 59. Wu HQ, Guidetti P, Goodman JH, Varasi M, Ceresoli-Borroni G, Tryptophan, adenosine, neurodegeneration and neuroprotection. Metab Speciale C, et al. Kynurenergic manipulations infl uence excitatory Brain Dis. 2007;22(3–4):337–352. synaptic function and excitotoxic vulnerability in the rat hippocampus 41. Smith AJ, Stone TW, Smith RA. Neurotoxicity of tryptophan in vivo. Neuroscience. 2000;97:243–252. metabolites. CNS. 2007;35(5):1287–1289. 60. Poeggeler B, Rassoulpour A, Guidetti P, Wu HQ, Schwarcz R. Dopa- 42. Heyes MP, Saito K, Devinsky O, Nadi NS. Kynurenine pathway minergic control of kynurenate levels and NMDA toxicity in the metabolites in cerebrospinal fl uid and serum in complex partial seizures. developing rat striatum. Dev Neurosci. 1998;20:146–153. Epilepsia. 1994;35:251–257. 61. Moroni P. Tryptophan metabolism and brain function: focus on kynurenine 43. Goldstein LE, Leopold MC, Huang X, et al. 3-Hydroxykynurenine and and other indol metabolites. Eur J Pharmacol. 1999;375:87–100. 3-hydroxyanthranilic acid generate hydrogen peroxide and promote 62. Medana IM, Hien TT, Day NP, et al. The clinical signifi cance of alpha-crystallin cross-linking by metal ion reduction. Biochemistry. cerebrospinal fl uid levels of kynurenine pathway metabolites and 2000;39:7266–7275. lactate in severe malaria. J Biol Chem. 2002;269:8128–8133. 44. Vazquez S, Garner B, Sheil MM, Truscott RJ. Characterisation of the 63. Heyes MP, Lackner A. Increased cerebrospinal fl uid quinolinic acid, major autoxidation products of 3-hydroxykynurenine under kynurenic acid and L-kynurenine in acute septicemia. J Neurochem. physiological conditions. Free Radic Res. 2000;32:11–23. 1990;55:338–341. International Journal of Tryptophan Research 2009:2 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Tryptophan Research SAGE

Effects of Kynurenine Pathway Metabolites on Intracellular NAD+ Synthesis and Cell Death in Human Primary Astrocytes and Neurons:

Download PDF
9 pages

Loading next page...
 
/lp/sage/effects-of-kynurenine-pathway-metabolites-on-intracellular-nad-wv6Pqp9Ert

References (72)

Publisher
SAGE
Copyright
Copyright © 2022 by SAGE Publications Ltd unless otherwise noted. Manuscript content on this site is licensed under Creative Commons Licenses
eISSN
1178-6469
DOI
10.4137/ijtr.s2318
Publisher site
See Article on Publisher Site

Abstract

ORIGINAL RESEARCH Effects of Kynurenine Pathway Metabolites on Intracellular NAD Synthesis and Cell Death in Human Primary Astrocytes and Neurons 1 1,2 3,4 1 5 Nady Braidy , Ross Grant , Bruce J Brew , Seray Adams , Tharusha Jayasena 1,4 and Gilles J. Guillemin 1 2 University of New South Wales, Faculty of Medicine, Sydney, Australia. Australasian Research Institute, Sydney Adventist Hospital, Sydney, Australia. St. Vincent’s Centre for Applied Medical Research, Sydney, Australia. Department of Neurology, St. Vincent’s Hospital, Sydney, Australia. Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, Australia. Abstract: The kynurenine pathway (KP) is a major route of L-tryptophan catabolism resulting in the production of the essential pyridine nucleotide nicotinamide adenine dinucleotide, (NAD ). Up-regulation of the KP during infl ammation leads to the release of a number of biologically active metabolites into the brain. We hypothesised that while some of the extracellular KP metabolites may be benefi cial for intracellular NAD synthesis and cell survival at physiological concentra- tions, they may contribute to neuronal and astroglial dysfunction and cell death at pathophysiological concentrations. In this study, we found that treatment of human primary neurons and astrocytes with 3-hydroxyanthranilic acid (3-HAA), 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and picolinic acid (PIC) at concentrations below 100 nM signifi cantly increased intracellular NAD levels compared to non-treated cells. However, a dose dependent decrease in intracellular NAD levels and increased extracellular LDH activity was observed in human astrocytes and neurons treated with 3-HAA, 3-HK, QUIN and PIC at concentrations 100 nM and kynurenine (KYN), at concentrations above 1 μM. Intracellular NAD levels were unchanged in the presence of the neuroprotectant, kynurenic acid (KYNA), and a dose dependent increase + + in intracellular NAD levels was observed for TRP up to 1 mM. While anthranilic acid (AA) increased intracellular NAD levels at concentration below 10 μM in astrocytes. NAD depletion and cell death was observed in AA treated neurons at concentrations above 500 nM. Therefore, the differing responses of astrocytes and neurons to an increase in KP metabolites should be considered when assessing KP toxicity during neuroinfl ammation. Introduction Tryptophan (TRP) catabolism via the kynurenine pathway (KP) represents the major pathway for the + 1 + synthesis of nicotinamide adenine dinucleotide (NAD ). Essential NAD dependent reactions can be 2 + 3 divided into three main categories: (1) NAD is an important contributor to energy (ATP) production; + 4,5 (2) NAD serves as a cofactor for NAD glycohydrolases involved in intracellular calcium regulation; + 6–8 (3) NAD is a substrate for the family of DNA nick sensing poly(ADP-ribose) polymerases (PARP) 9,10 + and the class III histone deacetylases known as sirtuins. NAD levels are extremely volatile and can be signifi cantly reduced under conditions of excessive PARP-1 activation caused by oxidative damage 11 + to DNA, and during mitosis. Thus, continuous biosynthesis of NAD is vital to the maintenance and ongoing cell viability of all cells. The KP is the principal route of L-tryptophan catabolism, resulting in the production of NAD (Fig. 1). Over-activation of the KP has been implicated in the pathogenesis of several neurological disorders including Huntington’s disease (HD), Alzheimer’s disease (AD), and the acquired immunodefi ciency 13–17 syndrome (AIDS)-dementia complex. The pathway is regulated by the immune-factor responsive enzyme indoleamine-2,3-dioxygenase (IDO) in most cells and by tryptophan-2,3 dioxygenase (TDO) 18,19 in the liver which is modulated by tryptophan and glucocorticoids. Several intermediate products of the KP are known to be neurotoxic. Among them, the N-methyl- D-aspartate (NMDA) receptor agonist and neurotoxin, quinolinic acid (QUIN) is likely to be most important in terms of biological activity. Anthranilic acid (AA), 3-hydroxyanthranilic acid (3-HAA), and 3-hydroxykynurenine (3-HK) have been shown to generate free radicals leading to neuronal damage similar to QUIN. The early upstream KP metabolite kynurenic acid (KYNA), has been shown to antagonise the Correspondence: Gilles J. Guillemin, Department of Pharmacology, Faculty of Medicine, University of NSW, Sydney, Australia 2052. Email: g.guillemin@cfi .unsw.edu.au Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/. International Journal of Tryptophan Research 2009:2 61–69 61 Braidy et al neurotoxic effects of QUIN and glutamate-mediated of human neurons and astrocytes treated with 20,21 NMDA receptor activation. The downstream physiological and pathophysiological concentra- metabolite picolinic acid (PIC) is an endogenous tions of TRP, KYN, KYNA, AA, 3-HAA, 3-HK, 22,23 metal chelator within the brain that displays some PIC, and QUIN respectively (0.1–100 μM). Intra- protection against QUIN induced toxicity and cellular NAD levels were measured using the 24,25 posesses immune regulatory activity. thiazolyl blue microcycling assay. The effect of Given the signifi cance of intracellular NAD KP metabolites on cell viability was determined levels for the maintenance of total cell integrity by measuring the release of lactate dehydrogenase and cell viability, we used primary monocultures into the extracellular medium. L-Tryptophan COOH NH A B NH L-Formylkynurenine COOH HN CHO Kynurenic Acid OH H N L-Kynurenine COOH COOH NH Missing in E Astrocytes NH 3-Hydroxykynurenine Anthranilic Acid COOH NH HO COOH Picolinic Acid 3-Hydroxyanthranilic Acid NH OH N COOH COOH 2-Amino-3- CHC Carboxymuconic HOOC NH Glutaryl- 2 Acetyl- Semialdehyde CoA CoA COOH Quinolinic Acid NAD COOH Figure 1. The Kynurenine Pathway of Tryptophan Degradation. A) Indoleamine 2,3-dioxygenase (IDO); B) Tryptophan 2,3 dioxygenase (TDO) C) Kynurenine Formylase; D) Kynurenine-Amino Transferase; E) Kynurenine 3Hydroxylase; F) Kynureninase; G) Non-specifi c hydroxylation; H) 3-Hydroxyanthranilic Acid Oxidase; I) Picolinic Carboxylase J) Non-enzymatic cyclisation; K) Quinolinic Acid Phosphori- bosyltransferase. International Journal of Tryptophan Research 2009:2 62 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis Materials and Methods Extracellular LDH activity as a measurement for cytotoxicity The release of lactate dehydrogenase (LDH) into Reagents and chemicals culture supernatant correlates with the amount of Dulbecco’s phosphate buffer solution (DBPS) and cell death and membrane damage, providing an all other cell culture media and supplements were accurate measure of cellular toxicity. LDH activity from Invitrogen (Melbourne, Australia) unless oth- following 24 hour incubation with the desired erwise stated. Nicotinamide, bicine, β-nicotinamide concentrations of KP metabolites was assayed adenine dinucleotide reduced form (β-NADH), using a standard spectrophotometric technique 3-[-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo- described by Koh and Choi. lium bromide (MTT), alcohol dehydrogenase (ADH), sodium pyruvate, TRIS, γ-globulins, L-tryptophan (TRP), kynurenine (KYN), kynurenic Bradford protein assay acid (KYNA), anthranilic acid (AA), 3-hydroxyan- for the quantifi cation of total protein thranilic acid (3-HAA), 3-hydroxykynurenine NAD concentration and extracellular LDH activity (3-HK), picolinic acid (PIC), and quinolinic acid were adjusted for variations in cell number using the (QUIN) were obtained from Sigma-Aldrich Bradford protein assay described by Bradford. (Castle-Hill, Australia). Phenazine methosulfate (PMS) was obtained from ICN Biochemicals (Ohio, Data analysis U.S.A). Bradford reagent was obtained from BioRad, Results obtained are presented as the means ± the Hercules (CA, U.S.A). standard error of measurement (SEM). Signifi cant differences between results were verifi ed using the Cell cultures two-tailed t-test with equal variance. Differences Human foetal brains were obtained from 16–19 week between treatment groups were considered signifi - old foetuses collected following therapeutic termina- cant if p was less than 0.05 (p  0.05). tion with informed consent. Mixed brain cultures were prepared and maintained using a protocol pre- viously described by Guillemin et al. Astrocytes Results and neurons were prepared from the mixed brain cell cultures, and maintained using a protocol previously 27 Effect of extracellular KP metabolites described by Guillemin et al. on intracellular NAD concentrations in human astrocytes and neurons Primary brain cells and KP metabolite TRP induced a dose-dependent increase in intracel- culture treatments lular NAD levels in both human neurons Human primary astrocytes and neurons were (at 100 nM TRP) and astrocytes (at 500 nM incubated with various concentrations of the KP TRP), (Fig. 2). We found that 3-HAA, 3-HK, QUIN metabolites (0.1–100 μM) for 24 hours. Experiments and PIC signifi cantly increased intracellular NAD were performed with primary cultures derived from levels at a low concentration of 100 nM but sub- three different human foetal brains with each indi- stantially decreased NAD levels at higher con- vidual preparation tested in triplicate. centrations (Fig. 2) in both cell types. Treatment with KYNA had no signifi cant effect on intracel- lular NAD activity in either neuronal or astroglial NAD(H) Microcycling assay cultures (Fig. 2). A dose-dependent decrease in for the measurement of intracellular intracellular NAD levels was observed in KYN NAD concentrations treated astrocytes and neurons at concentra- Intracellular NAD concentration following 24 hour tions above 1 μM. AA increased intracellular incubation with the desired concentrations of KP NAD levels at concentrations below 50 μM in metabolites were measured spectrophotometrically human astrocytes. However in human neurons using the thiazolyl blue microcycling assay estab- NAD depletion was observed at AA concentra- lished by Bernofsky and Swan adapted for 96 well tions 500 nM for neurons and 100 μM for plate format by Grant and Kapoor. astrocytes (Fig. 2). International Journal of Tryptophan Research 2009:2 63 Braidy et al Figure 2. The effect of KP metabolites on intracellular NAD levels in human neurons and astrocytes. (Control = 0 μM treatment for each metabolite and cell type) A) The effect of TRP (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. B) The effect of KYN (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. C) The effect of KYNA (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. D) The effect of 3-HAA (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. E) The effect of 3HK (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. F) The effect of AA (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. G) The effect of QUIN (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared control in human neurons. ¥p  0.05 compared to control in human astrocytes. H) The effect of PIC (1–1000 μM) on intracellular NAD in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. (n = 3 for each treatment group). International Journal of Tryptophan Research 2009:2 64 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis Effect of extracellular KP metabolites Moreover, under conditions of TRP depletion, supplementation with TRP down-regulates on extracellular LDH activity in human enzymes directing TRP to non-NAD dependent astrocytes and neurons pathways, suggesting a shift of all available TRP Consistent with the results obtained for intracellular + 37 catabolism to NAD synthesis. However, it is NAD levels, no signifi cant change was observed clear that excessive TRP supplementation would in extracellular LDH activity for both astrocyte and aggravate or induce autoimmune disease. neuronal cultures treated with TRP or KYNA up to The primary metabolite of TRP, N-formylkyn- 1 mM (Fig. 3). However treatment with 3-HAA, urenine can be rapidly converted to KYN by the 3-HK, QUIN and PIC increased extracellular LDH enzyme arylamine formamidase. KYN (its activity at concentrations above 100 nM (Fig. 3) in physiological concentration is 1 μM) is converted both cell types. A dose-dependent increase in extra- into several neurotoxic metabolites such as 3-HK cellular LDH activity was observed in astrocytes 39,14 and QUIN in unstimulated human brain cells. and neurons treated with KYN at concentrations Our data indicates that KYN causes intracellular above 1 μM. However the magnitude of LDH NAD depletion and reduced cell viability at greater release following KYN treatment was signifi cantly than physiological concentrations (Figs. 2 and 3). less than that observed for any of the other toxic Human astrocytes and microglial cells demonstrate metabolites. LDH activity was also increased for rapid cellular uptake of KYN. KYN has been shown cells treated with AA at concentrations above to increase QUIN production and KP enzyme 100 nM in astrocytes and at or above 100 nM in expression in human macrophages. The enhanced neurons (Fig. 3). generation of QUIN may account for the reduced NAD levels and increased cytotoxicity observed Discussion in human neurons and astrocytes at pathophysio- Neurodegenerative diseases are often characterised logical concentrations (Figs. 2 and 3). by a loss of neuronal cells in specifi c regions of the KYN and the resulting metabolite, 3-HK can be + 15 brain. Given the importance of intracellular NAD converted to AA and 3-HAA by kynureninase. levels for maintaining overall cellular integrity and These metabolites also provide additional substrate + 40 function, it is conceivable that reduced NAD levels for QUIN formation. The levels of 3-HK, AA and are a potential pathogenic mechanism for neuronal 3-HAA are signifi cantly increased in the CSF of 11,8 41 and astroglial cell death. TRP has been used as a patients with HD. Elevated levels have also been supplement for some years in the United States reported in HIV cases associated with dementia, before being removed due to an outbreak of the lethal infantile spasms, and hepatic encephalopathy. autoimmune disease, eosinophilia-myalgia syn- While 3-HK, AA, and 3-HAA appear less neurotoxic drome (EMS) resulting in 36 deaths. Large doses than QUIN, these KP metabolites have been of TRP can induce the build-up of selected white previously shown to promote neuronal damage blood cells leading to EMS. Since the KP is a major largely through free radical formation but not NMDA 32 15 regulator of the immune response, the toxic effect receptor activation. Under normal conditions, the may be due to inhibition of normal tolerogenic cell serum concentration of 3-HK, 3-HAA and AA has 11 42 35 34 T-cell death following IDO induction. been found to be 383 nM, 24 nM and 21 nM, In this study, we found that TRP supplementa- respectively. In this study, we found that 3-HK, AA tion produced a dose-dependent increase in intra- and 3-HAA supplementation resulted in neuronal + + cellular NAD levels in human astrocytes and and astroglial NAD depletion and cell death in 24 neurons after 24 hours (Fig. 2). The physiological hour cultures at micromolar concentrations (Fig. 1 concentration of TRP is human plasma is estimated and Fig. 2). These results are consistent with a previ- 33,34 to be 40–90 μM. Lower serum concentrations ous study showing that 3-HK, 3-HAA, and AA also have been observed in several disorders including induced a time-and dose-dependent increase in cell 35 36 41 depression and anxiety, rheumatoid arthritis and death at micromolar concentrations (1–100 μM). following infection with HIV. TRP supplementa- These authors reported that the accompanying cell tion has been previously shown to be benefi cial in death was signifi cantly reduced by co-treatment with several neurological conditions, including insom- catalase, suggesting that the neurotoxic effects of nia and depression, since TRP can be used for the 3-HK may be mediated by increased hydrogen per- + 11,19 41 synthesis of serotonin, melatonin and NAD . oxide. 3-HK can be converted to quinoneimines International Journal of Tryptophan Research 2009:2 65 Braidy et al A B 120 120 100 100 80 80 60 60 Neurons Neurons 40 40 Astrocytes Astrocytes * * ¥ ¥ 20 20 * * ¥ ¥ ¥ ¥ * * ¥ ¥ ** ** 0 0 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 1000 TRP conc (μM) KYN conc (μM) C C D D 120 120 ¥ ¥ * * 100 100 * * ¥ ¥ 60 60 * * Neurons Neurons ¥ ¥ Astrocytes Astrocytes * * ¥ ¥ 20 * * ¥ ¥ * * ¥ ¥ 0 0 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 1000 KYNA conc (μM) 3-HAA conc (μM) E E * * ¥ ¥ F F 120 120 * * * * ¥ ¥ 100 100 ¥ ¥ * * 80 80 * * * * ¥ ¥ * * 60 60 Neurons Neurons * * ¥ ¥ ¥ ¥ 40 40 * * Astrocytes Astrocytes * * ¥ ¥ * * 20 20 * * ¥ ¥ * * ¥ ¥ ¥ ¥ 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 3-HK conc (μM) AA conc (μM) G G H H 250 120 * * ¥ ¥ * * ¥ ¥ * * ¥ ¥ * * ¥ ¥ * * * * Neurons Neurons ¥ ¥ * * ¥ ¥ ¥ ¥ 40 * * Astrocytes Astrocytes ¥ ¥ * * 50 * * ¥ ¥ ¥ ¥ * * ¥ ¥ * * ¥ ¥ 0 0 0 0.1 0.5 1 10 50 100 1000 0 0.1 0.5 1 10 50 100 1000 QUIN conc (μM) PIC conc (μM) Figure 3. Effect of KP metabolites on extracellular LDH activity in human astrocytes and neurons (Control = 0 μM treatment for each metabolite and cell type). A) The effect of TRP (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. B) The effect of KYN (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. C) The effect of KYNA (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. D) The effect of 3-HAA (1–1000 μM) on extracel- lular LDH activity in human neurons and astrocytes after 24 hours. **p  0.05 compared to control in human neurons (n = 3 for each treatment group). ¥p  0.05 compared to control in human astrocytes. E) The effect of 3-HK (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. F) The effect of AA (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. G) The effect of QUIN (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes. H) The effect of PIC (1–1000 μM) on extracellular LDH activity in human neurons and astrocytes after 24 hours. *p  0.05 compared to control in human neurons. ¥p  0.05 compared to control in human astrocytes (n = 3 for each treatment group). International Journal of Tryptophan Research 2009:2 66 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis that can generate pro-oxidant intermediates, such as undergoes non-specific hydroxylation to form hydrogen peroxide, organic and hydroxyl radicals, 3-HAA and may subsequently be used to form QUIN 43,44 + 27 during processes of autoxidation. Similarly, 3- and NAD . This suggests a greater requirement for HK has been shown to potentiate QUIN toxicity in AA as a substrate for NAD synthesis in human rats, and the cytotoxic effect can be prevented using astrocytes compared to human neurons, and AA may free radical scavengers. become cytotoxic to both cell types in excess con- Although 3-HK and 3-HAA have been shown centrations. These fi ndings provide insight not only to be cytotoxic at high concentrations, physiolog- in the differences between neurons and astrocytes, ical concentrations of 100 nM increased intracel- but also for understanding how different brain cells + + lular NAD levels by 18 and 12 per cent respectively, produce NAD . with no detectable effect on extracellular LDH QUIN is an endogenous NMDA receptor agonist activity (Figs. 1 and Fig. 2). The contribution of involved in neuronal fi ring. The amount of QUIN 3-HK and 3-HAA at these physiological concentra- in the brain and CSF is usually less than 100 nM. tions to neuronal and astroglial cytotoxicity is Increased brain QUIN levels (500–1000 nM) have uncertain as micromolar concentrations appear to been observed in the CSF and serum in several 41,45 49 45 produce neurotoxicity in-vitro. infl ammatory brain diseases, including AD, HD, The observation that addition of these KP traumatic brain injury, AIDS dementia complex + 16 15 metabolites can serve as substrate for NAD synthesis (ADC), and other infections. QUIN has been at low concentrations (100–500 nM) implies that shown to be up-regulated in ageing and in associa- + 50 under normal conditions, NAD levels may be tion with senile plaques in the AD brain. The dependent on substrate availability. We propose results in the study are in line with previously pub- that astroglial and neuronal KP enzymes downstream lished results highlighting the importance of QUIN of QUIN may become saturated in the presence of as a benefi cial substrate for NAD synthesis at low excess extracellular KP metabolites leading to an concentration, but a putative toxin able to induce accumulation of QUIN. Indeed, we have previously oligodendrocyte, neuronal, and astroglial apoptosis 49,51–53 demonstrated that exogenous 3-HAA substantially at pathophysiological concentrations. increases QUIN synthesis in human foetal astrocytes As previously mentioned, PIC is an endogenous to neurotoxic levels implying saturation of down- metal chelator in the brain, and is an effi cient chelator stream enzymes involved in catabolism of QUIN to for minerals such as chromium, zinc, manganese, + 27 23 its essential metabolite, NAD . Similarly, Blight copper, and iron. Unbound (free) redox-active iron et al observed that treatment of 4-chloro-3- and copper significantly increase free radical hydroxyanthranilate, a synthetic inhibitor of generation and are found in abundance in the AD 3-hydroxyanthranilic acid oxidase, was able to reduce brain. Disordered PIC metabolism may yet be found QUIN production and functional defi cits following to play a role in the pathophysiology of AD. The experimental spinal cord injury in guinea pigs. physiological concentration of PIC in human serum On the other hand, our results indicate a clear is thought to be between 100–400 nM. Elevated dichotomy between exogenous AA effects in human PIC levels have been associated with fatal outcome neurons and astrocytes. While AA appears neuro- in Malawian children with cerebral malaria. Our toxic at concentrations as low as 500 nM, AA data shows that PIC can increase intracellular NAD improved intracellular NAD levels in human levels at physiological concentrations (100 nM). This astrocytes by up to 10% in the 0.5 to 10 μM range may be partly due to its intracellular metal chelating (Fig. 2). AA has been previously reported to impair properties reducing the contribution of free redox energy metabolism in the rat cerebral cortex at active metals in free radical generation. micromolar concentrations, possibly through However, PIC appeared to also induce a dose inhibition of complex I–III activities in the mito- dependent decrease in intracellular NAD and chondrial respiratory chain. While the KP is fully increase in extracellular LDH activity in both expressed in human neurons, kynurenine hydrox- human astrocytes and neurons. These results are ylase (KYN-OHase), which converts KYN to 3-HK consistent with one study that observed signfi cant is absent in human foetal astrocytes. Consequently, PIC induced neurotoxicity that was associated with astrocytes cannot produce 3-HK. The KP is divided both prolonged exposure time and increased glu- into two parts in human astrocytes (Fig. 1), and KYN cose concentration in the culture supernatant. may be converted to AA by kynureninase which then While the mechanism leading to PIC toxicity is International Journal of Tryptophan Research 2009:2 67 Braidy et al 3. Berger SJ, Sudar DC, Berger NA. Metabolic consequences of DNA not known, at high concentrations, PIC may be damage: DNA damage induces alterations in glucose metabolism by causing disruption to some of the many trace metal activation of Poly(ADP-ribose) polymerase. Biochem Biophys Res + ++ ++ (Cu , Zn , Fe ) dependent biochemical reactions Commun. 1986;134:227–232. 4. Berger F, Ramirez-Hernandez MH, Ziegler M. The new life of a centenarian: essential for normal cell metabolism. signalling functions of NADP. Trends Biochem Sci. 2004;29:111–118. KYNA is a neuroprotective molecule with 5. Lee HC. Physiologic functions of cyclic ADP-ribose and NAADP as antagonistic properties on both nicotinic-acetylcholine calcium messengers. Ann Rev Pharmacol Toxicol. 2001;41:317–345. 6. Furukawa A, Tada-Oikawa S, Kawanishi S. H O accelerates cellular and glutamatergic receptors at supra-physiological 2 2 senescence by accumulation of accelerates cellular senescence by concentrations. The tissue concentrations of KYNA accumulation of acetylated p53 via decrease in the function of SIRT1 in the human brain have been estimated to be + by NAD depletion. Cell Physiol Biochem. 2007;20:45–54 58,59 between 0.2–1.5 μM. In our study, no signifi cant 7. Ha HC, Snyder SH. Poly(ADP-ribose) polymerase-I in the nervous system. Neurobiol Dis. 2000;7:225–239. changes in intracellular NAD levels and cell 8. Love S, Barber R, Wilcock GK. Increased poly(ADP-ribosyl)ation of viability were observed across micromolar concen- nuclear proteins in Alzheimer’s disease. Brain. 1999;122:247–253. trations in both human primary astrocytes and neu- 9. Sauve AA, Wolberger C, Schramm VL, Boeke JD. The biochemistry of Sirtuins. Annu Rev Biochem. 2006;75:435–465. rons (Figs. 2 and 3). KYNA exhibits a high affi nity 10. Anderson RM, Bitterman KJ, Wood JG, et al. Manipulation of a nuclear for the glycine-binding site of the NMDA receptor NADP salvage pathway delays ageing without altering steady-state at low concentrations, but can act on the glutamate NADP levels. J Biol Chem. 2002;277:18881–18890. 11. Penberthy TW. Pharmacological targeting of IDO-mediated tolerance binding site on the NMDA receptor, and AMPA for treating autoimmune disease. Curr Drug Metab. 2007;8:245–266. receptors, at high micromolar concentrations. 12. Ying W. NAD and NADH in cellular functions and cell death. Front Decrease in KYNA levels below the threshold Biosci. 2006;11:3129–3148. needed for NMDA receptor antagonism can enhance 13. Schwarcz R. The kynurenine pathway of tryptophan degradation as a drug target. Curr Opin Pharm. 2004;4:12–17. the vulnerability of dopaminergic neurons to exci- 14. Heyes MP, Saito K, Crowley JS, et al. Quinolinic acid and kynurenine 60 61 totoxic insult. Moroni proposed that NMDA and pathway metabolism in infl ammatory and non-infl ammatory neurologic glutamate induced excitotoxic neuronal damage can disease. Brain. 1992;115:1249–1273. 15. Stone TW. Endogenous neurotoxins from tryptophan. Toxicon. be ameliorated by diverting KYN metabolism to 2001;39(1):61–73. KYNA synthesis. Likewise, supplementation with 16. Guillemin GJ, Kerr SJ, Brew BJ. Involvement of quinolinic acid in TRP, an endogenous precursor for KYN synthesis, AIDS dementia complex. Neurotox Res. 2005;7(1–2):103–23. 17. Heyes MP, Brew BJ, Martin A, et al. Quinolinic acid in cerebrospinal may increase the levels of KYNA and therefore pos- fl uid and serum in HIV-1 infection: relationship to clinical and neuro- sibly slow down or prevent the formation of cyto- logical status. Ann Neurol. 1991;29(2):202–9. 62,63 toxic concentrations of QUIN. 18. Mackay GM, Forrest CM, Stoy N, et al. Tryptophan metabolism and This study is the fi rst to examine the effects of oxidative stress in patients with chronic brain injury. Eur J Neurol. 2006;13:30–42. extracellular KP metabolites on intracellular NAD 19. Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Lowry CA. synthesis and cell death in human primary astro- Tryptophan metabolism in the central nervous system: medical impli- cytes and neurons. While a large body of literature cations. Expert Rev Mol Med. 2006;8:1–27. 20. Hilmas C, Pereira EF, Alkondon M, Rassoulpour A, Schawrcz R. The is available on the toxic effects of KP metabolites brain metabolite kynureninic acid inhibits alpha7-nicotinic recceptor in regard to neuroinfl ammation, few studies have activity and increases non-alpha7 nicotinic receptor expression. considered the effect of these metabolites on intra- J Neurosci. 2001;21:7463–7473. 21. Perkins MN, Stone TW. An iontophoretic investigation of the actions cellular NAD levels in-vitro. This study demon- of convulsant kynurenines and their interactions with endogenous strates that changes in the brain levels of certain KP excitant quinolinic acid. Brain Res. 1982;247:184–187. metabolites may be important mediators contribut- 22. Jayasena T, Grant RS, Keerthisinghe N, Solaja I, Smythe GA. Membrane ing to brain cell dysfunction in some neurological permeability of redox active metal chelators: An important element in reducing hydroxyl radical induced NAD depletion in neuronal cells. disorders involving increased KP activation. Neurosci Res. 2007;57:454–461. 23. Melillo G, Bosco MC, Musso T, Varesio L. Immunobiology of picolinic acid. Adv Exp Med Biol. 1996;398:135–141. Disclosure 24. Bosco MC, Rapisarda A, Reffo G, Massazza S, Pastorino S, Varesio L. The authors report no confl icts of interest. Macrophage activating properties of the tryptophan catabolite picolinic acid. Adv Exp Med Biol. 2003;527:55–65. 25. Kalisch BE, Jhamandas K, Boegman RJ, Beninger RJ. Picolinic acid References protects against quinolinic acid-induced depletion of NADPH diaphorase 1. Grant RS, Passey R, Matanovic G, Smythe G, Kapoor V. Evidence for containing neurons in the rat striatum. Brain Res. 1994;668(1–2):1–8. increased de-novo synthesis of NAD in immune-activated RAW264.7 26. Guillemin GJ, Smythe G, Takikawa O, Brew BJ. Expression of macrophages: a self-protective mechanism? Arch Biochem Biophys. indoleamine 2,3-dioxygenase and production of quinolinic acid by 1999;372:1–7. human microglia, astrocytes, and neurons. Glia. 2005;49(1):15–23. 2. Braidy N, Guillemin G, Grant R. Promotion of Cellular NAD Anabolism: 27. Guillemin GJ, Kerr SJ, Smythe GA, et al. Kynurenine pathway Therapeutic Potential for Oxidative Stress in Ageing and Alzheimer's metabolism in human astrocytes: a paradox for neuronal protection. Disease. Neurotox Res. 2008;13(3, 4):173–184. J Neurochem. 2001;78:842–853. International Journal of Tryptophan Research 2009:2 68 + Effects of kynurenine pathway metabolites on intracellular NAD synthesis 28. Bernofsky C, Swan M. An improved cycling assay for nicotinamide 45. Guidetti P, Schwarcz R. 3-hydroxykynurenine and quinolinate: adenine dinucleotide. Anal Biochem. 1973;53:452–458. pathological synergism in early Huntington’s disease? Adv Exp Med 29. Grant RS, Kapoor V. Murine glial cells regenerate NAD, after peroxide- Biol. 2003;527:137–45. induced depletion, using either nicotinic acid, nicotinamide, or 46. Blight AR, Cohen TI, Saito K, Heyes MP. Quinolinic acid accumulation quinolinic acid as substrates. J Neurochem. 1998;70:1759–1763. and functional defi cits following experimental spinal cord injury. Brain. 30. Koh JY, Choi DW. Quantitative determination of glutamate mediated 1995;118:735–752. cortical neuronal injury in cell culture by lactate dehydrogenase effl ux 47. Schuck PF, Tonin A, da Costa Ferreira G, et al. Kynurenines impair assay. J Neurosci Methods. 1987;20:83–90. energy metabolism in rat cerebral cortex. Cell Mol Neurobiol. 31. Bradford MM. A rapid and sensitive method for quantitation of 2007;27:147–60. microgram quantities of protein utilising the principle of protein-dye 48. Guillemin GJ, Cullen KM, Lim CK, et al. Characterisation of the binding. Anal Biochem. 1976;53:452–458. kynurenine pathway in human neurons. J Neurosci. 2007;27:1–9. 32. Munn DH, Mellor AL. Indoleamine 2,3-dioxygenase and tumour- 49. Guillemin GJ, Brew BJ. Implications of the kynurenine pathway and induced tolerance. J Clin Invest. 2007;177:1147–54. quinolinic acid in Alzheimer’s disease. Redox Rep. 2002;7(4):199–206. 33. Werner ER, Fuchs D, Hausen A, et al. Tryptophan degradation in 50. Guillemin GJ, Brew BJ, Noonan CE, Takikawa O, Cullen KM. patients infected by human immunodefi ciency virus. Biol Chem Hoppe Indoleamine 2,3 dioxygenase and quinolinic acid immunoreactivity in Seyler. 1988;369:337–340. Alzheimer’s Disease hippocampus. Neuropathol Appl Neurobiol. 34. Forrest CM, Macaky GM, Oxford L, Stoy N, Stone TW, Darlington LG. 2005;31:395–404. Kynurenine pathway metabolism in patients with osteoporosis after 51. Braidy N, Grant R, Adams S, Guillemin G. Mechanism for quinolinic 2 years of drug treatment. Clin Exp Pharmacol Physiol. 2006;33: acid cytotoxicity in human astrocytes and neurons. (submitted). 1078–1087. 52. Cammer W. Apoptosis of oligodendrocytes in secondary cultures from 35. Myint AM, Kim YM, Verkerk R, Scharpe S, Steinbuch H, Leonard B. neonatal rat brains. Neurosci Lett. 2002;327(2):123–7. Kynurenine pathway in major depression: evidence of impaired neu- 53. Guillemin GJ, Wang L, Brew BJ. Quinolinic acid selectively induces roprotection. J Affect Disord. 2007;98:143–51. apoptosis of human astocytes: Potential role in AIDS dementia complex. 36. Schrocksnadel K, Winkler C, Fuith LC, Fuchs D. Tryptophan J Neuroinfl ammation. 2005;2(16):1–6. degradation in patients with gynecological cancer correlates with 54. Bush AI. Metal complexing agents as therapies for Alzheimer’s disease. immune activation. Cancer Lett. 2005;223:323–329. Neurobiol Aging. 2002;23:1031–1038. 37. Shibata K, Motooka K, Murata K. The differences in growth and activity 55. Chen Y, Guillemin GJ. Kynurenine pathway metabolites in humans: of the tryptophan-NAD pathway between Wistar and Sprague-Dawley disease and healthy states. IJTR 2. 2009 strains of rats fed on tryptophan limited diet. J Nutr Sci Vitaminol 56. Medana IM, Day NP, Salahifar-Sabet H, Stocker R, Smythe G, (Tokyo). 1982;28:11–19. Bwanaisa L, Njobvu A, Kayira K, Turner GD, Taylor TE, Hunt NH. 38. Backhaus C, Rahman H, Scheffl er S, Laatsch H, Hardeland R. NO Metabolites of the kynurenine pathway of tryptophan metabolism in scavenging by 3-hydroxyanthranilic acid and 3-hydroxykynurenine: the cerebrospinal fl uid of Malawian children with malaria. J Infect Dis. N-nitrosation leads via oxadiazoles to o-quinone diazides. Nitric Oxide. 2003;188(6): 844–849. 2008;19(3):237–44. 57. Melillo G, Bosco MC, Musso T, Varesio L. Immunobiology of picolinic 39. Sas K, Robotka H, Toldi J, Vecsei L. Mitochondria, metabolic acid. Adv Exp Med Biol. 1996;398:135–141. disturbances, oxidative stress and the kynurenine system, with focus 58. Moroni P, Russi G, Lombardi G, Beni M, Carl V. Presence of kynurenic on neurodegenerative disorders. J Neurol Sci. 2007;257:221–239. acid in the mammalian brain. J Neurochem. 1988;51:177–180. 40. Stone TW, Forrest CM, Mackay GM, Stoy N, Darlington LG. 59. Wu HQ, Guidetti P, Goodman JH, Varasi M, Ceresoli-Borroni G, Tryptophan, adenosine, neurodegeneration and neuroprotection. Metab Speciale C, et al. Kynurenergic manipulations infl uence excitatory Brain Dis. 2007;22(3–4):337–352. synaptic function and excitotoxic vulnerability in the rat hippocampus 41. Smith AJ, Stone TW, Smith RA. Neurotoxicity of tryptophan in vivo. Neuroscience. 2000;97:243–252. metabolites. CNS. 2007;35(5):1287–1289. 60. Poeggeler B, Rassoulpour A, Guidetti P, Wu HQ, Schwarcz R. Dopa- 42. Heyes MP, Saito K, Devinsky O, Nadi NS. Kynurenine pathway minergic control of kynurenate levels and NMDA toxicity in the metabolites in cerebrospinal fl uid and serum in complex partial seizures. developing rat striatum. Dev Neurosci. 1998;20:146–153. Epilepsia. 1994;35:251–257. 61. Moroni P. Tryptophan metabolism and brain function: focus on kynurenine 43. Goldstein LE, Leopold MC, Huang X, et al. 3-Hydroxykynurenine and and other indol metabolites. Eur J Pharmacol. 1999;375:87–100. 3-hydroxyanthranilic acid generate hydrogen peroxide and promote 62. Medana IM, Hien TT, Day NP, et al. The clinical signifi cance of alpha-crystallin cross-linking by metal ion reduction. Biochemistry. cerebrospinal fl uid levels of kynurenine pathway metabolites and 2000;39:7266–7275. lactate in severe malaria. J Biol Chem. 2002;269:8128–8133. 44. Vazquez S, Garner B, Sheil MM, Truscott RJ. Characterisation of the 63. Heyes MP, Lackner A. Increased cerebrospinal fl uid quinolinic acid, major autoxidation products of 3-hydroxykynurenine under kynurenic acid and L-kynurenine in acute septicemia. J Neurochem. physiological conditions. Free Radic Res. 2000;32:11–23. 1990;55:338–341. International Journal of Tryptophan Research 2009:2

Journal

International Journal of Tryptophan Research – SAGE

Published: Apr 3, 2009

There are no references for this article.