Altered hippocampal plasticity by prenatal kynurenine administration, kynurenine-3-monoxygenase (KMO) deletion or galantamine.

Glutamate receptors sensitive to N-methyl-D-aspartate (NMDA) are involved in embryonic brain development but their activity may be modulated by the kynurenine pathway of tryptophan metabolism which includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Our previous work has shown that prenatal inhibition of the pathway produces abnormalities of brain development. In the present study kynurenine and probenecid (both 100mg/kg, doses known to increase kynurenic acid levels in the brain) were administered to female Wistar rats on embryonic days E14, E16 and E18 of gestation and the litter was allowed to develop to post-natal day P60. Western blotting revealed no changes in hippocampal expression of several proteins previously found to be altered by inhibition of the kynurenine pathway including the NMDA receptor subunits GluN1, GluN2A and GluN2B, as well as doublecortin, Proliferating Cell Nuclear Antigen (PCNA), sonic hedgehog and unco-ordinated (unc)-5H1 and 5H3. Mice lacking the enzyme kynurenine-3-monoxygenase (KMO) also showed no changes in hippocampal expression of several of these proteins or the 70-kDa and 100-kDa variants of Disrupted in Schizophrenia-1 (DISC1). Electrical excitability of pyramidal neurons in the CA1 region of hippocampal slices was unchanged, as was paired-pulse facilitation and inhibition. Long-term potentiation was decreased in the kynurenine-treated rats and in the KMO(-/-) mice, but galantamine reversed this effect in the presence of nicotinic receptor antagonists, consistent with evidence that it can potentiate glutamate at NMDA receptors. It is concluded that interference with the kynurenine pathway in utero can have lasting effects on brain function of the offspring, implying that the kynurenine pathway is involved in the regulation of early brain development.

Changes in synaptic transmission and protein expression in the brains of adult offspring after prenatal inhibition of the kynurenine pathway.

During early brain development, N-methyl-d-aspartate (NMDA) receptors are involved in cell migration, neuritogenesis, axon guidance and synapse formation, but the mechanisms which regulate NMDA receptor density and function remain unclear. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at NMDA receptors and we have previously shown that inhibition of the pathway using the kynurenine-3-monoxygenase inhibitor Ro61-8048 in late gestation produces rapid changes in protein expression in the embryos and effects on synaptic transmission lasting until postnatal day 21 (P21). The present study sought to determine whether any of these effects are maintained into adulthood. After prenatal injections of Ro61-8048 the litter was allowed to develop to P60 when some offspring were euthanized and the brains removed for examination. Analysis of protein expression by Western blotting revealed significantly reduced expression of the GluN2A subunit (32%) and the morphogenetic protein sonic hedgehog (31%), with a 29% increase in the expression of doublecortin, a protein associated with neurogenesis. No changes were seen in mRNA abundance using quantitative real-time polymerase chain reaction. Neuronal excitability was normal in the CA1 region of hippocampal slices but paired-pulse stimulation revealed less inhibition at short interpulse intervals. The amount of long-term potentiation was decreased by 49% in treated pups and recovery after low-frequency stimulation was delayed. The results not only strengthen the view that basal, constitutive kynurenine metabolism is involved in normal brain development, but also show that changes induced prenatally can affect the brains of adult offspring and those changes are quite different from those seen previously at weaning (P21). Those changes may be mediated by altered expression of NMDAR subunits and sonic hedgehog.

Involvement of the proteasome and caspase activation in hippocampal long-term depression induced by the serine protease subtilisin.

The serine protease subtilisin-A produces a long-term depression (LTD) of synaptic potentials in hippocampal slices which differs mechanistically from classical LTD. Since caspases have been implicated in hippocampal plasticity, this study examined a possible role for these enzymes in subtilisin-induced LTD. Subtilisin produced a concentration-dependent decrease in the size of field excitatory synaptic potentials (fEPSPs), which was not prevented or modified by the caspase inhibitors Z-VAD(OMe)-fmk and Z-DEVD-fmk. Similarly Z-VAD(OMe)-fmk did not modify the selective loss of protein expression produced by subtilisin. Subtilisin reduced the expression of procaspase-3 and caspase-9 but, while caspase-9 was converted to its conventionally activated form (39 kDa), caspase-3 was metabolised along a non-canonical pathway to a 29/30 kDa protein rather than the classical 17/19 kDa fragments. Both Z-VAD(OMe)-fmk and Z-DEVD-fmk were unable to prevent the reduced expression of Postsynaptic Density Protein-95, Vesicle-Associated Membrane Protein-1 and Unco-ordinated 5H3 proteins produced by subtilisin, although MG132 did produce partial recovery from subtilisin-induced depression of fEPSPs. When tested on long-term potentiation (LTP) induced by theta stimulation in the stratum radiatum, MG132 inhibited the immediate increase in fEPSP size but generated a higher plateau LTP. Twin LTP stimulation generated a further increase in LTP amplitude in control slices but not in slices exposed to MG132. The results indicate that subtilisin does produce caspase activation but that this does not contribute to its induction of LTD. However, activation of the proteasome does contribute to subtilisin-induced LTD and may also play a modulatory role in electrically induced LTP.

Altered kynurenine metabolism correlates with infarct volume in stroke.

Inflammation and oxidative stress are involved in brain damage following stroke, and tryptophan oxidation along the kynurenine pathway contributes to the modulation of oxidative stress partly via the glutamate receptor agonist quinolinic acid and antagonist kynurenic acid, and via redox-active compounds such as 3-hydroxyanthranilic acid. We have confirmed that following a stroke, patients show early elevations of plasma neopterin, S100B and peroxidation markers, the latter two correlating with infarct volume assessed from computed tomography (CT) scans, and being consistent with a rapid inflammatory response. We now report that the kynurenine pathway of tryptophan metabolism was also activated, with an increased kynurenine : tryptophan ratio, but with a highly significant decrease in the ratio of 3-hydroxyanthranilic acid : anthranilic acid, which was strongly correlated with infarct volume. Levels of kynurenic acid were significantly raised in patients who died within 21 days compared with those who survived. The results suggest that increased tryptophan catabolism is initiated before or immediately after a stroke, and is related to the inflammatory response and oxidative stress, with a major change in 3-hydroxyanthranilic acid levels. Together with previous evidence that inhibiting the kynurenine pathway reduces brain damage in animal models of stroke and cerebral inflammation, and that increased kynurenine metabolism directly promotes oxidative stress, it is proposed that oxidative tryptophan metabolism may contribute to the oxidative stress and brain damage following stroke. Some form of anti-inflammatory intervention between the rise of S100B and the activation of microglia, including inhibition of the kynurenine pathway, may be valuable in modifying patient morbidity and mortality.

Tryptophan, adenosine, neurodegeneration and neuroprotection.

This review summarises the potential contributions of two groups of compounds to cerebral dysfunction and damage in metabolic disease. The kynurenines are oxidised metabolites of tryptophan, the kynurenine pathway being the major route for tryptophan catabolism in most tissues. The pathway includes quinolinic acid -- an agonist at N-methyl-D-aspartate (NMDA) receptors, kynurenic acid -- an antagonist at glutamate and nicotinic receptors, and other redox active compounds that are able to generate free radicals under many physiological and pathological conditions. The pathway is activated in immune-competent cells, including glia in the central nervous system, and may contribute substantially to delayed neuronal damage following an infarct or metabolic insult. Adenosine is an ubiquitous purine that can protect neurons by suppressing excitatory neurotransmitter release, reducing calcium fluxes and inhibiting NMDA receptors. The extent of brain injury is critically dependent on the balance between the two opposing forces of kynurenines and purines.

Purine metabolism and clinical status of patients with rheumatoid arthritis treated with dipyridamole.

The anti-inflammatory activities of methotrexate and sulphasalazine may be mediated by increases in endogenous adenosine levels. Since the vascular protective drug dipyridamole inhibits the uptake and metabolism of adenosine we have now tested this compound in patients with rheumatoid arthritis to assess its effects on their symptoms. Forty patients (aged 18-75 years) received dipyridamole 400 mg/day or placebo. The levels of adenosine and its major metabolites were determined by high performance liquid chromatography (HPLC) in blood samples taken at baseline and at monthly intervals during treatment for 6 months. After three months of treatment there was a significant reduction in the modified Health Assessment Questionnaire (mHAQ) score, but these effects were not maintained, and dipyridamole did not modify disease severity scores or the levels of adenosine and its metabolites. We conclude that the symptoms of rheumatoid arthritis were not modified by treatment with dipyridamole.

Blood 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and melatonin levels in patients with either Huntington's disease or chronic brain injury.

Following a study of oxidative tryptophan metabolism to kynurenines, we have now analysed the blood of patients with either Huntington's disease or traumatic brain injury for levels of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and melatonin. There were no differences in the baseline levels of these compounds between patients and healthy controls. Tryptophan depletion did not reduce 5-HT levels in either the controls or in the patients with Huntington's disease, but it increased 5-HT levels in patients with brain injury and lowered 5-HIAA in the control and Huntington's disease groups. An oral tryptophan load did not modify 5-HT levels in the patients but increased 5-HT in control subjects. The tryptophan load restored 5-HIAA to baseline levels in controls and patients with brain injury, but not in those with Huntington's disease, in whom 5-HIAA remained significantly depressed. Melatonin levels increased on tryptophan loading in all subjects, with levels in patients with brain injury increasing significantly more than in controls. Baseline levels of neopterin and lipid peroxidation products were higher in patients than in controls. It is concluded that both groups of patients exhibit abnormalities in tryptophan metabolism, which may be related to increased inflammatory status and oxidative stress. Interactions between the kynurenine, 5-HT and melatonin pathways should be considered when interpreting changes of tryptophan metabolism.

Tryptophan metabolism and oxidative stress in patients with chronic brain injury.

The kynurenine pathway generates the excitotoxic N-methyl-d-aspartate receptor agonist, quinolinic acid and the glutamate antagonist, kynurenic acid, as well as free-radical generators. We investigated the status of the pathway following severe brain injury sustained at least 1 year previously in 15 patients compared with controls. At baseline, patients with brain injury showed increased levels of neopterin, erythrocyte sedimentation rate, C-reactive protein and peroxidation products in the blood compared with controls, indicating persistent inflammation and oxidative stress. At baseline and following tryptophan depletion, more tryptophan was converted to kynurenine in patients than controls, but less kynurenine was converted into the neuroprotectant, kynurenic acid. This suggests that neuroprotection by kynurenic acid may be inadequate in brain-damaged patients even many years after injury. On tryptophan loading, patients metabolized more kynurenine into kynurenic acid than controls, a process which may be neuroprotective. In addition, lower levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in patients after tryptophan loading should be protective since these compounds generate free radicals. The results suggest that for brain-damaged patients, increased activation of the kynurenine pathway, oxidative stress and raised levels of inflammation continue many years after the original insult, possibly contributing to the continuing cerebral dysfunction in these patients.

Tryptophan metabolism and oxidative stress in patients with Huntington's disease.

Abnormalities in the kynurenine pathway may play a role in Huntington's disease (HD). In this study, tryptophan depletion and loading were used to investigate changes in blood kynurenine pathway metabolites, as well as markers of inflammation and oxidative stress in HD patients and healthy controls. Results showed that the kynurenine : tryptophan ratio was greater in HD than controls in the baseline state and after tryptophan depletion, indicating increased indoleamine dioxygenase activity in HD. Evidence for persistent inflammation in HD was provided by elevated baseline levels of C-reactive protein, neopterin and lipid peroxidation products compared with controls. The kynurenate : kynurenine ratio suggested lower kynurenine aminotransferase activity in patients and the higher levels of kynurenine in patients at baseline, after depletion and loading, do not result in any differences in kynurenic acid levels, providing no supportive evidence for a compensatory neuroprotective role for kynurenic acid. Quinolinic acid showed wide variations in blood levels. The lipid peroxidation data indicate a high level of oxidative stress in HD patients many years after disease onset. Levels of the free radical generators 3-hydroxykynurenine and 3-hydroxyanthranilic acid were decreased in HD patients, and hence did not appear to contribute to the oxidative stress. It is concluded that patients with HD exhibit abnormal handling of tryptophan metabolism and increased oxidative stress, and that these factors could contribute to ongoing brain dysfunction.

Modulation of cytokine release by purine receptors in patients with rheumatoid arthritis.

OBJECTIVE: Since adenosine receptors are known to modulate the release of some inflammatory mediators in control subjects, we have examined the effects of the mixed A1 and A2 adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) on basal and lipopolysaccharide (LPS)-induced cytokine release in diluted whole blood cultures from rheumatoid arthritis (RA) patients and healthy volunteers. METHODS: Twenty-eight patients with rheumatoid arthritis aged 18-75 years gave their voluntary consent to participate and give a blood sample. Basal levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) were measured by ELISA, and whole blood cultures were prepared to assess the effects of LPS activation. RESULTS: Following a 40-hour incubation, activation of adenosine receptors by NECA, added to the cell cultures from rheumatoid arthritis patients, was found to suppress both the basal and LPS-induced release of TNF-alpha and IL-1beta, while causing an increase in the release of both basal and LPS-induced IL-6. In healthy volunteers basal cytokines were undetectable, but NECA alone induced the release of all three cytokines. Stimulated levels of TNF-alpha were more than double those in patients. In the control blood cultures, NECA suppressed LPS-induced release of TNF-alpha and IL-1beta, but increased IL-6 release. CONCLUSIONS: Adenosine receptor stimulation has a differential effect on the release of pro-inflammatory cytokines, and may induce cytokine release in normal subjects. Stimulated release of TNF-alpha is substantially lower in patients with rheumatoid arthritis than in control subjects, possibly indicating saturation, exhaustion or down-regulation of the release process.

Purine modulation of cytokine release during diuretic therapy of rheumatoid arthritis.

Since free radicals are implicated in rheumatoid arthritis (RA) and since uric acid is a free radical scavenger, we examined the effects of treating RA patients with with the diuretic bumetanide to try to improve their arthritic control. Seventy patients, aged 18-75 years, were randomised to receive bumetanide 4 mg/day or placebo. Uric acid levels increased, but not that of other purines, in the blood of drug-treated patients compared with placebo-treated controls. There were no significant changes in clinical measurements of disease activity or in ESR or CRP levels. There were no over all differences in the blood levels of the cytokines, nor in the basal or stimulated production of cytokines from the blood cultures. The adenosine receptor agonist 5'N-ethylcarboxamido-adenosine (NECA) used to modify cytokine release in cultures of whole blood taken from the patients, depressed the release of tumour necrosis factor-alpha (TNFalpha), but failed to depress the release of interleukin-1b (IL-1b) or interleukin-6 (IL-6), a difference from earlier studies of healthy control subjects and, thus, a difference which may contribute to the disease activity.

Tryptophan loading induces oxidative stress.

In previous studies tryptophan loads have been administered to human subjects in order to raise central levels of 5-hydroxytryptamine (5HT) and assess the effects of 5HT on behaviour and mood. However, tryptophan is metabolised primarily along the oxidative kynurenine pathway. In this study a 6 g oral tryptophan load was administered to 15 healthy volunteers and the levels of kynurenines and lipid peroxidation products (indicative of oxidative stress) were measured. The results demonstrate that tryptophan loading produces a highly significant increase in lipid peroxidation products in parallel with increased kynurenines. The oxidative stress may result from the generation of quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, all of which are known to have the ability to generate free radicals. The results may have implications for the use of tryptophan loading in psychiatric practice, and for the chronic use of diets high in tryptophan.

Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders.

The generation of reactive oxygen species (free radicals) is an important factor in the development and maintenance of rheumatoid arthritis in humans and animal models. One source of free radicals is nitric oxide produced within the synoviocytes and chondrocytes and giving rise to the highly toxic radical peroxynitrite. Several cytokines, including tumour necrosis factor-alpha (TNFalpha) are involved in the formation of free radicals, partly by increasing the activity of nitric oxide synthase. Indeed, nitric oxide may mediate some of the deleterious effects of cytokines on bone resorption. Aspirin, tetracyclines, steroids and methotrexate can suppress nitric oxide synthase. Dietary antioxidants include ascorbate and the tocopherols and beneficial effects of high doses have been reported especially in osteoarthritis. There is also evidence for beneficial effects of beta-carotene and selenium, the latter being a component of the antioxidant enzyme glutathione peroxidase. The polyunsaturated fatty acids (PUFA) include the n-3 compounds, some of which are precursors of eicosanoid synthesis, and the n-6 group which can increase formation of the pro-inflammatory cytokines TNFalpha and interleukin-6, and of reactive oxygen species. Some prostaglandins, however, suppress cytokine formation, so that n-3 PUFA often oppose the inflammatory effects of some n-6-PUFA. gamma-linolenic acid (GLA) is a precursor of prostaglandin E1, a fact which may account for its reported ability to ameliorate arthritic symptoms. Fish oil supplements, rich in n-3 PUFA such as eicosapentaenoic acid have been claimed as beneficial in rheumatoid arthritis, possibly by suppression of the immune system and its cytokine repertoire. Some other oils of marine origin (e.g. from the green-lipped mussel) and a range of vegetable oils (e.g. olive oil and evening primrose oil) have indirect anti-inflammatory actions, probably mediated via prostaglandin E1. Overall, there is a growing scientific rationale for the use of dietary supplements as adjuncts in the treatment of inflammatory disorders such as rheumatoid arthritis and osteoarthritis.

The role of kynurenines in the production of neuronal death, and the neuroprotective effect of purines.

The kynurenine metabolic pathway from tryptophan accounts for a large proportion of the metabolism of this amino acid in the brain. Although a major route for the generation of the essential co-factor nicotinamide adenine dinucleotide (NAD), two components of the pathway have marked effects on neurons. Quinolinic acid is an agonist at N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors, while kynurenic acid is an antagonist and, thus, a potential neuroprotectant. The levels of quinolinic acid are known to increase substantially following cerebral insults or infection, and has been most clearly implicated in the AIDS-dementia complex. The actions of quinolinic acid and NMDA show subtle differences, however, which suggest other factors contributing to cell damage. In this article we review the evidence that free radicals may be involved in the neurotoxic effects of quinolinic acid and consider the possibility that quinolinic acid might be involved in Alzheimer's disease. Finally, adenosine receptor ligands can modulate neuronal damage, supporting the view that they may represent suitable targets for the development of novel neuroprotectant drugs for the treatment of Alzheimer's and other neurodegenerative disorders.

Levels of lipid peroxidation products in a chronic inflammatory disorder.

We have examined the plasma levels of the lipid peroxidation products 4-hydroxy-nonenal and malondialdehyde in a carefully controlled study of age and sex-matched subjects with rheumatoid arthritis in whom potentially confounding influences such as disease modifying anti-rheumatic drugs (DMARDs), self-medication and vitamin supplements were eliminated. The plasma concentrations of the antioxidants uric acid and vitamin E were also measured. The results reveal a strong and consistent inverse correlation between the levels of lipid peroxidation products in the plasma and erythrocyte sedimentation rate (ESR). There is no indication that vitamin E or urate function as the major antioxidant agent in arthritis, as has been suggested in more seriously affected patients. It is concluded that there is no evidence that vitamin E is more important, and urate less important as an antioxidant in mild arthritis. The correlation between lipid peroxidation and ESR suggests a more complex relationship than has been assumed.

Possible mediation of quinolinic acid-induced hippocampal damage by reactive oxygen species.

Several differences exist between quinolinic acid and N-methyl-D-aspartate (NMDA) in the potency and pharmacology of their neurotoxic actions in the brain, suggesting that quinolinic acid may act by mechanisms additional to the activation of NMDA receptors, possibly involving lipid peroxidation. In the present review, studies are considered which have attempted to determine whether free radicals might contribute to the neuronal damage induced by quinolinic acid. Following Injections into the hippocampus of anaesthetised rats, quinolinic acid induced damage is prevented by melatonin, by an action not blocked by the melatonin receptor blocker luzindole. Deprenyl, but not the non-selective monoamine oxidase inhibitor nialamide, also prevent quinolinic acid-induced damage. In vitro, several groups have shown that quinolinic acid can induce lipid peroxidation of brain tissue The results suggest that free radical formation contributes significantly to quinolinic acid-induced damage in vivo.

Oxidative stress as a mechanism for quinolinic acid-induced hippocampal damage: protection by melatonin and deprenyl.

1. There are differences between the excitotoxic actions of quinolinic acid and N-methyl-D-aspartate (NMDA) which suggest that quinolinic acid may act by mechanisms additional to the activation of NMDA receptors. The present study was designed to examine the effect of a potent antioxidant, melatonin, and the potential neuroprotectant, deprenyl, as inhibitors of quinolinic acid-induced brain damage. Injections were made into the hippocampus of anaesthetized rats, which were allowed to recover before the brains were taken for histology and the counting of surviving neurones. 2. Quinolinic acid (120 nmols) induced damage to the pyramidal cell layer, which was prevented by the co-administration of melatonin (5 nmols locally plus 2x20 mg kg(-1) i.p.). This protective effect was not prevented by the melatonin receptor blocker luzindole. Neuronal damage produced by NMDA (120 nmols) was not prevented by melatonin. 3. Quinolinic acid increased the formation of lipid peroxidation products from hippocampal tissue and this effect was prevented by melatonin. 4. Deprenyl also prevented quinolinic acid-induced damage at a dose of 50 nmols but not 10 nmols plus 2x1.0 mg kg(-1) i.p. The non-selective monoamine oxidase inhibitor nialamide (10 and 50 nmols plus 2x25 mg kg(-1)) did not afford protection. 5. The results suggest that quinolinic acid-induced neuronal damage can be prevented by a receptor-independent action of melatonin and deprenyl, agents which can act as a potent free radical scavenger and can increase the activity of endogenous antioxidant enzymes respectively. This suggests that free radical formation contributes significantly to quinolinic acid-induced damage in vivo.

Review of dietary therapy for rheumatoid arthritis.

There are now sufficient good scientific studies, from the UK and abroad, to suggest that, at least in some patients with RA, dietary therapy may influence at least the symptoms and possibly the progression of the disease. Since dietary treatment is safe and may reduce or avoid the need for drugs, it is appealing to patients, who are increasingly anxious about potential drug toxicity. It must, however, be medically supervised to avoid misinterpretation of results, to avoid patients taking diets to extremes, with resultant malnutrition, particularly in children, and to prevent patients from persisting with ineffective diets when they should be receiving drug treatment. Medical interest in dietary treatment also ensures that patients discuss their diets with orthodox practitioners rather than being driven by our scepticism into the hands of unqualified people who may exploit patients' interest in the subject.