Vimal Kapoor

Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN), play a role in the pathogenesis of neurological diseases, in particular AIDS dementia complex. Although QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the role of astrocytes in QUIN production is controversial. Using cytokine‐stimulated cultures of human astrocytes, we assayed key enzymes and products of the KP. We found that human astrocytes lack kynurenine hydroxylase so that large amounts of kynurenine and the QUIN antagonist kynurenic acid were produced. However, the amounts of QUIN that were synthesized were subsequently completely degraded. We then showed that kynurenine in concentrations comparable with those produced by astrocytes led to significant production of QUIN by macrophages. These results suggest that astrocytes alone are neuroprotective by minimizing QUIN production and maximizing synthesis of kynurenic acid. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes become indirectly neurotoxic by the production of large concentrations of kynurenine that can be secondarily metabolized by neighbouring or infiltrating monocytic cells to form the neurotoxin QUIN.

Characterization of the Kynurenine Pathway in Human Neurons

The kynurenine pathway is a major route of l-tryptophan catabolism producing neuroactive metabolites implicated in neurodegeneration and immune tolerance. We characterized the kynurenine pathway in human neurons and the human SK-N-SH neuroblastoma cell line and found that the kynurenine pathway enzymes were variably expressed. Picolinic carboxylase was expressed only in primary and some adult neurons but not in SK-N-SH cells. Because of this difference, SK-N-SH cells were able to produce the excitotoxin quinolinic acid, whereas human neurons produced the neuroprotectant picolinic acid. The net result of kynurenine pathway induction in human neurons is therefore predicted to result in neuroprotection, immune regulation, and tumor inhibition, whereas in SK-N-SH cells, it may result in neurotoxicity, immune tolerance, and tumor promotion. This study represents the first comprehensive characterization of the kynurenine pathway in neurons and the first description of the involvement of the kynurenine pathway as a mechanism for controlling both tumor cell neurotoxicity and persistence.

Murine Glial Cells Regenerate NAD, After Peroxide-Induced Depletion, Using Either Nicotinic Acid, Nicotinamide, or Quinolinic Acid as Substrates

Abstract: The potential for regeneration of intracellular pyridine nucleotide levels from different precursors, after peroxide‐induced NAD depletion, in cultured glial cells was investigated. Cultured murine glial cells showed a decrease in intracellular NAD levels of >40% after treatment with H2O2 (100 µM). Removal of the H2O2 followed by a 2‐h incubation did not result in NAD recovery in the absence of precursors. However, NAD levels increased significantly in these cells after the following substrate additions, at minimum effective concentrations of 1 mM for quinolinic acid (QUIN), 500 µM for nicotinamide, and 2 µM for nicotinic acid. The regeneration of significant amounts of NAD from nicotinic acid at doses 250 and 500 times lower than either nicotinamide or QUIN indicates a preferred route for NAD biosynthesis in glial cells in vitro, probably via nicotinic acid phosphoribosylation.

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.

Induction of Indolamine 2,3-Dioxygenase in Primary Human Macrophages by Human Immunodeficiency Virus Type 1 Is Strain Dependent

ABSTRACT Increased kynurenine pathway metabolism has been implicated in the etiology of AIDS dementia complex (ADC). The rate-limiting enzyme for this pathway is indolamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of human immunodeficiency virus type 1 (HIV1-BaL, HIV1-JRFL, and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain-derived HIV-1 isolates, laboratory-adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the highly replicating macrophage-tropic LA strain HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day 8 postinfection. Abundant HIV-1 replication did not reduce the ability of exogenous gamma interferon (IFN-γ) to induce IDO and kynurenine synthesis in HIV-infected MDM. The addition of anti-IFN-γ antibody to MDM infected with HIV1-JRFL resulted in an absence of detectable IDO protein after 48 h and a decrease of 64% ± 1% in supernatant kynurenine concentration. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent kynurenine metabolism in MDM. The induction of IDO, while apparently independent of replication capacity, appears to be mediated by a transient production of IFN-γ in MDM responding to the initial infection with selected strains of HIV-1.

Preliminary evidence for a link between schizophrenia and NMDA–glycine site receptor ligand metabolic enzymes, d-amino acid oxidase (DAAO) and kynurenine aminotransferase-1 (KAT-1)

Preliminary investigations, studying gene expression and biochemical activities of enzymes d-amino acid oxidase (DAAO) and kynurenine aminotransferase-1 (KAT-1), revealed elevated cerebellar KAT-1 and DAAO activities in post-mortem brain samples from schizophrenic versus normal individuals. In addition, we have identified a transcript of DAAO, which was expressed in significantly higher quantities in the diseased cerebellum but not detected in the parietal cortex where DAAO activity is absent.

IDO induction in IFN-γ activated astroglia: A role in improving cell viability during oxidative stress

Abstract In the central nervous system (CNS), astrocytes play an integral role in the maintenance of neuronal viability and function. Inflammation within the CNS increases the concentration of oxidative metabolites and, therefore, the potential for NAD depletion through increased poly-(ADP-ribose) polymerase (PARP) activity. However, the activity of indoleamine 2,3-dioxygenase (IDO), the rate limiting enzyme for de novo NAD synthesis, is also markedly increased in astrocytes during inflammation. This study investigated the role of IDO induction in the maintenance of intracellular NAD and its relationship to improved cell viability under conditions of increased oxidative stress in the human astroglioma cell line, HTB-138. Treatment with the pro-inflammatory cytokine IFN-γ increased IDO activity in these cells. Intracellular NAD levels also increased significantly after treatment with IFN-γ in the presence of a PARP inhibitor. Pretreatment of astroglial cells with IFN-γ significantly moderated both the drop in intracellular NAD concentration and cell death following exposure to hydrogen peroxide. These results suggest that induction of IDO and subsequent de novo NAD synthesis may contribute to the maintenance of intracellular NAD levels and cell viability under conditions of increased oxidative stress.

Effects of acute postoperative pain on catecholamine plasma levels, hemodynamic parameters, and cardiac autonomic control.

Summary Measurements of hemodynamic, endocrine, and autonomic parameters are not useful as surrogates to estimate the severity of acute postoperative pain in the clinical setting. Abstract Postoperative pain is often stated to be a significant contributor to a sympathetic stress response after surgery. However, hardly any evidence has been published to support this assumption. Hence it was the aim of this trial to investigate the relationship between postoperative pain and hemodynamic, endocrine, and autonomic parameters. A total of 85 postoperative patients in the recovery room were repeatedly asked to rate their pain on a numeric rating scale (NRS). Concurrently, the parameters of heart rate variability (HRV) were analysed, and mean arterial pressure (MAP), heart rate (HR) and respiration rate (RR) were recorded. Pain was categorized into no, mild, moderate, and severe. Blood samples were taken for epinephrine (EPI) and norepinephrine (NE) plasma level assessment at the time of recovery room admission and discharge, and each time pain was found decreased in categorized severity. A total of 239 pain readings were obtained. None of the investigated parameters correlated with NRS scores. NE was higher at NRS 5 to 10 vs. NRS 0 to 4 (mean [SEM]: 1009 [73] pg/mL vs. 872 [65] pg/mL; P < 0.01). This was also found for MAP, but not for EPI or the parameters of HRV, HR, and RR. In contrast to common belief, the severity of postoperative pain does not appear to be associated with the degree of sympathetic stress response after surgery, and other factors such as surgical trauma may be more important. Importantly, the absence of signs of sympathetic stimulation cannot be seen as a guarantee for the absence of significant pain.

Inhibition of indoleamine 2,3-dioxygenase activity in IFN-γ stimulated astroglioma cells decreases intracellular NAD levels

Astroglia provide essential metabolic and neurotropic support to cells within the CNS and participate in the cellular immune response with microglia/macrophages following activation by the pro-inflammatory cytokine IFN-gamma. Activation of glial cells results in local oxidative stress and induction of a number of proteins including the enzyme indoleamine 2,3-dioxygenase (IDO). As a rate-limiting enzyme, IDO regulates tryptophan catabolism via the kynurenine pathway producing a series of metabolic precursors (some of which are neurotoxic) before complete oxidation to the essential pyridine nucleotide NAD. Inhibition of this pathway may therefore prove therapeutic in neuroinflammatory disease by reducing production of cell toxins. However, kynurenine metabolism may also be cytoprotective through de novo synthesis of cellular NAD levels. We investigated the hypothesis that IDO activity is directly involved in maintenance of intracellular [NAD] in activated astroglial cells through control of de novo synthesis. Exposure to IFN-gamma increased IDO activity from 7+/-1 nmol to 129+/-11 nmol kynurenine/hr/mg protein. Inhibition of IDO activity with either 6-chloro-D-tryptophan (competitive inhibition), or 3-ethoxy beta-carboline (non-competitive inhibition) resulted in a dose-dependent decrease in IDO activity that correlated directly with decreasing [NAD] (R(2)=0.92 and 0.81, respectively). These results support the hypothesis that one important consequence of increasing IDO activity in astroglial cells during inflammation is to maintain NAD levels through de novo synthesis from tryptophan. Inhibition of kynurenine pathway metabolism under these conditions may significantly decrease cell viability and CNS functions unless alternate precursors for NAD synthesis are available.

Induction of indoleamine 2,3-dioxygenase in primary human macrophages by HIV-1

Abstract Increased kynurenine pathway metabolism has been implicated in the aetiology of the AIDS dementia complex (ADC). The rate limiting enzyme for this pathway is indoleamine 2,3- dioxygenase (IDO). We tested the efficacy of different strains of HIV-1 (HIV1-BaL, HIV1-JRFL and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain derived HIV-1 isolates, laboratory adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the high replicating macrophage tropic LA strain, HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day-8 post-infection. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent oxidative tryptophan catabolism in MDM.