Kynurenine Pathway Activation in Human African Trypanosomiasis.

Background: The kynurenine pathway of tryptophan oxidation is associated with central nervous system (CNS) inflammatory pathways. Inhibition of this pathway ameliorates CNS inflammation in rodent models of the late (meningoencephalitic) stage of human African trypanosomiasis (HAT). In this study, we evaluate whether the kynurenine pathway is activated in clinical HAT and associated with CNS inflammatory responses. Methods: We measured cerebrospinal fluid (CSF) tryptophan and kynurenine metabolite concentrations in patients infected with Trypanosoma brucei rhodesiense, using liquid chromatography-mass spectrometry. Results: Kynurenine concentration in CSF was increased in both the early and late stages of disease, with a progressive increase in tryptophan oxidation associated with stage progression. Kynurenine pathway activation was associated with increases in neuroinflammatory markers, but there was no clear relationship to neurological symptoms. Conclusions: CNS kynurenine pathway activation occurs during HAT, including cases prior to the current diagnostic cutoff for late-stage infection, providing evidence for early CNS involvement in HAT. Metabolite data demonstrate that the kynurenine-3-monooxygenase and kynurenine aminotransferase branches of the kynurenine pathway are active. The association between tryptophan oxidation and CNS inflammatory responses as measured by CSF interleukin 6 (IL-6) concentration supports a role of kynurenine metabolites in the inflammatory pathogenesis of late-stage HAT.

Quinolinic acid induces neuritogenesis in SH-SY5Y neuroblastoma cells independently of NMDA receptor activation.

Glutamate and nicotinamide adenine dinucleotide (NAD+ ) have been implicated in neuronal development and several types of cancer. The kynurenine pathway of tryptophan metabolism includes quinolinic acid (QA) which is both a selective agonist at N-methyl-D-aspartate (NMDA) receptors and also a precursor for the formation of NAD+ . The effect of QA on cell survival and differentiation has therefore been examined on SH-SY5Y human neuroblastoma cells. Retinoic acid (RA, 10 µm) induced differentiation of SH-SY5Y cells into a neuronal phenotype showing neurite growth. QA (50-150 nm) also caused a concentration-dependent increase in the neurite/soma ratio, indicating differentiation. Both RA and QA increased expression of the neuronal marker ß3-tubulin in whole-cell homogenates and in the neuritic fraction assessed using a neurite outgrowth assay. Expression of the neuronal proliferation marker doublecortin revealed that, unlike RA, QA did not decrease the number of mitotic cells. QA-induced neuritogenesis coincided with an increase in the generation of reactive oxygen species. Neuritogenesis was prevented by diphenylene-iodonium (an inhibitor of NADPH oxidase) and superoxide dismutase, supporting the involvement of reactive oxygen species. NMDA itself did not promote neuritogenesis and the NMDA antagonist dizocilpine (MK-801) did not prevent quinolinate-induced neuritogenesis, indicating that the effects of QA were independent of NMDA receptors. Nicotinamide caused a significant increase in the neurite/soma ratio and the expression of ß3-tubulin in the neuritic fraction. Taken together, these results suggest that QA induces neuritogenesis by promoting oxidizing conditions and affecting the availability of NAD+ , independently of NMDA receptors.

Selective depletion of tumour suppressors Deleted in Colorectal Cancer (DCC) and neogenin by environmental and endogenous serine proteases: linking diet and cancer.

BACKGROUND: The related tumour suppressor proteins Deleted in Colorectal Cancer (DCC) and neogenin are absent or weakly expressed in many cancers, whereas their insertion into cells suppresses oncogenic behaviour. Serine proteases influence the initiation and progression of cancers although the mechanisms are unknown. METHODS: The effects of environmental (bacterial subtilisin) and endogenous mammalian (chymotrypsin) serine proteases were examined on protein expression in fresh, normal tissue and human neuroblastoma and mammary adenocarcinoma lines. Cell proliferation and migration assays (chemoattraction and wound closure) were used to examine cell function. Cells lacking DCC were transfected with an ectopic dcc plasmid. RESULTS: Subtilisin and chymotrypsin selectively depleted DCC and neogenin from cells at nanomolar concentrations without affecting related proteins. Cells showed reduced adherence and increased migration, but after washing they re-attached within 24 h, with recovery of protein expression. These effects are induced by chymotryptic activity as they are prevented by chymostatin and the soybean Bowman-Birk inhibitor typical of many plant protease inhibitors. CONCLUSIONS: Bacillus subtilis, which secretes subtilisin is widely present in soil, the environment and the intestinal contents, while subtilisin itself is used in meat processing, animal feed probiotics and many household cleaning agents. With chymotrypsin present in chyme, blood and tissues, these proteases may contribute to cancer development by depleting DCC and neogenin. Blocking their activity by Bowman-Birk inhibitors may explain the protective effects of a plant diet. Our findings identify a potential non-genetic contribution to cancer cell behaviour which may explain both the association of processed meats and other factors with cancer incidence and the protection afforded by plant-rich diets, with significant implications for cancer prevention.

Prenatal inhibition of the kynurenine pathway leads to structural changes in the hippocampus of adult rat offspring.

Glutamate receptors for N-methyl-d-aspartate (NMDA) are involved in early brain development. The kynurenine pathway of tryptophan metabolism includes the NMDA receptor agonist quinolinic acid and the antagonist kynurenic acid. We now report that prenatal inhibition of the pathway in rats with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulphonamide (Ro61-8048) produces marked changes in hippocampal neuron morphology, spine density and the immunocytochemical localisation of developmental proteins in the offspring at postnatal day 60. Golgi-Cox silver staining revealed decreased overall numbers and lengths of CA1 basal dendrites and secondary basal dendrites, together with fewer basal dendritic spines and less overall dendritic complexity in the basal arbour. Fewer dendrites and less complexity were also noted in the dentate gyrus granule cells. More neurons containing the nuclear marker NeuN and the developmental protein sonic hedgehog were detected in the CA1 region and dentate gyrus. Staining for doublecortin revealed fewer newly generated granule cells bearing extended dendritic processes. The number of neuron terminals staining for vesicular glutamate transporter (VGLUT)-1 and VGLUT-2 was increased by Ro61-8048, with no change in expression of vesicular GABA transporter or its co-localisation with vesicle-associated membrane protein-1. These data support the view that constitutive kynurenine metabolism normally plays a role in early embryonic brain development, and that interfering with it has profound consequences for neuronal structure and morphology, lasting into adulthood.

Prenatal activation of maternal TLR3 receptors by viral-mimetic poly(I:C) modifies GluN2B expression in embryos and sonic hedgehog in offspring in the absence of kynurenine pathway activation.

Activation of the immune system during pregnancy is believed to lead to psychiatric and neurological disorders in the offspring, but the molecular changes responsible are unknown. Polyinosinic:polycytidylic acid (poly(I:C)) is a viral-mimetic double-stranded RNA complex which activates Toll-Like-Receptor-3 and can activate the metabolism of tryptophan through the oxidative kynurenine pathway to compounds that modulate activity of glutamate receptors. The aim was to determine whether prenatal administration of poly(I:C) affects the expression of neurodevelopmental proteins in the offspring and whether such effects were mediated via the kynurenine pathway. Pregnant rats were treated with poly(I:C) during late gestation and the offspring were allowed to develop to postnatal day 21 (P21). Immunoblotting of the brains at P21 showed decreased expression of sonic hedgehog, a key protein in dopaminergic neuronal maturation. Expression of α-synuclein was decreased, while tyrosine hydroxylase was increased. Disrupted in Schizophrenia-1 (DISC-1) and 5-HT2C receptor levels were unaffected, as were the dependence receptors Unc5H1, Unc5H3 and Deleted in Colorectal Cancer (DCC), the inflammation-related transcription factor NFkB and the inducible oxidative enzyme cyclo-oxygenase-2 (COX-2). An examination of embryo brains 5 h after maternal poly(I:C) showed increased expression of GluN2B, with reduced doublecortin and DCC but no change in NFkB. Despite altered protein expression, there were no changes in the kynurenine pathway. The results show that maternal exposure to poly(I:C) alters the expression of proteins in the embryos and offspring which may affect the development of dopaminergic function. The oxidation of tryptophan along the kynurenine pathway is not involved in these effects.

Involvement of kynurenines in Huntington's disease and stroke-induced brain damage.

Several components of the kynurenine pathway of tryptophan metabolism are now recognised to have actions of profound biological importance. These include the ability to modulate the activation of glutamate and nicotinic receptors, to modify the responsiveness of the immune system to inflammation and infection, and to modify the generation and removal of reactive oxygen species. As each of these factors is being recognised increasingly as contributing to major disorders of the central nervous system (CNS), so the potentially fundamental role of the kynurenine pathway in those disorders is presenting a valuable target both for understanding the progress of those disorders and for developing potential drug treatments. This review will summarise some of the evidence for an important contribution of the kynurenines to Huntington's disease and to stroke damage in the CNS. Together with preliminary evidence from a study of kynurenine metabolites after major surgery, an important conclusion is that kynurenine pathway activation closely reflects cognitive function, and may play a significant role in cognitive ability.

Kynurenine metabolism predicts cognitive function in patients following cardiac bypass and thoracic surgery.

Cardiac surgery involving extra-corporeal circulation can lead to cognitive dysfunction. As such surgery is associated with signs of inflammation and pro-inflammatory mediators activate tryptophan oxidation to neuroactive kynurenines which modulate NMDA receptor function and oxidative stress, we have measured blood concentrations of kynurenines and inflammatory markers in 28 patients undergoing coronary arterial graft surgery and, for comparison, 28 patients undergoing non-bypass thoracic surgery. A battery of cognitive tests was completed before and after the operations. The results show increased levels of tryptophan with decreased levels of kynurenine, anthranilic acid and 3-hydroxyanthranilic acid associated with bypass, and a later increase in kynurenic acid. Levels of neopterin and lipid peroxidation products rose after surgery in non-bypass patients whereas tumour necrosis factor-α and S100B levels increased after bypass. Changes of neopterin levels were greater after non-bypass surgery. Cognitive testing showed that the levels of tryptophan, kynurenine, kynurenic acid and the kynurenine/tryptophan ratio, correlated with aspects of post-surgery cognitive function, and were significant predictors of cognitive performance in tasks sensitive to frontal executive function and memory. Thus, anaesthesia and major surgery are associated with inflammatory changes and alterations in tryptophan oxidative metabolism which predict, and may play a role in, post-surgical cognitive function.

Molecular changes associated with hippocampal long-lasting depression induced by the serine protease subtilisin-A.

The serine protease subtilisin-A (SubA) induces a form of long-term depression (LTD) of synaptic transmission in the rat hippocampus, and molecular changes associated with SubA-induced LTD (SubA-LTD) were explored by using recordings of evoked postsynaptic potentials and immunoblotting. SubA-LTD was prevented by a selective inhibitor of SubA proteolysis, but the same inhibitor did not affect LTD induced by electrical stimulation or activation of metabotropic glutamate receptors. SubA-LTD was reduced by the protein kinase inhibitors genistein and lavendustin A, although not by inhibitors of p38 mitogen-activated protein kinase, glycogen synthase kinase-3, or protein phosphatases. It was also reduced by (RS)-α-methyl-4-carboxyphenylglycine, a broad-spectrum antagonist at metabotropic glutamate receptors. Inhibition of the Rho kinase enzyme Rho-associated coiled-coil kinase reduced SubA-LTD, although inhibitors of the RhoGTPase-activating enzymes farnesyl transferase and geranylgeranyl transferase did not. In addition, a late phase of SubA-LTD was dependent on new protein synthesis. There was a small, non-significant difference in SubA-LTD between wild-type and RhoB(-/-) mice. Marked decreases were seen in the levels of Unc-5H3, a protein that is intimately involved in the development and plasticity of glutamatergic synapses. Smaller changes were noted, at higher concentrations of SubA, in Unc-5H1, vesicle-associated membrane protein-1 (synaptobrevin), and actin, with no changes in the levels of synaptophysin, synaptotagmin, RhoA, or RhoB. None of these changes was associated with LTD induced electrically or by the metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine. These results indicate that SubA induces molecular changes that overlap with other forms of LTD, but that the overall molecular profile of SubA-LTD is quite different.

Blood levels of kynurenines, interleukin-23 and soluble human leucocyte antigen-G at different stages of Huntington's disease.

There is substantial evidence that abnormal concentrations of oxidised tryptophan metabolites, produced via the kynurenine pathway, contribute to progressive neurodegeneration in Huntington's disease. We have now examined the blood levels of these metabolites in patients at different stages of Huntington's disease, assessed both in terms of clinical disease severity and numbers of CAG repeats. Close relatives of the patients were included in the study as well as unrelated healthy controls. Levels of lipid peroxidation products, the pro-inflammatory cytokine interleukin (IL)-23 and the soluble human leucocyte antigen-G (sHLA-G) were also measured. There were lower levels of tryptophan and a higher kynurenine : tryptophan ratio, indicating activation of indoleamine-2,3-dioxygenase, in the most severely affected group of patients, with increased levels of IL-23 and sHLA-G. Marked correlations were noted between IL-23 and the patient severity group, anthranilic acid levels and the number of CAG repeats, and between anthranilic acid and IL-23, supporting our previous evidence of a relationship between anthranilic acid and inflammatory status. Tryptophan was negatively correlated with symptom severity and number of CAG repeats, and positively correlated with sHLA-G. The results support the proposal that tryptophan metabolism along the kynurenine pathway in Huntington's disease is related to the degree of genetic abnormality, to clinical disease severity and to aspects of immunopathogenesis.

Kynurenine metabolites and inflammation markers in depressed patients treated with fluoxetine or counselling.

1. Depression could result from changes in tryptophan availability caused by activation of the kynurenine pathway as a result of inflammation. In the present study, we examined patients newly diagnosed with depression to determine whether kynurenines and related factors change in parallel with improvements in mood. 2. Concentrations of 5-hydroxytryptamine (5-HT; serotonin), 5-hydroxyindoleacetic acid (5-HIAA), oxidized tryptophan metabolites, brain-derived neurotrophic factor (BDNF) and inflammatory mediators (interleukin (IL)-2, C-reactive protein (CRP), neopterin) were measured in peripheral blood during an 18 week period of treatment with fluoxetine, fluoxetine plus tri-iodothyronine (T(3)) or psychiatric counselling. 3. The results showed significant improvements in mood, with reduced 5-HT concentrations in patients given fluoxetine and a rise in plasma tryptophan in patients given counselling or fluoxetine and T(3). The addition of T(3) to the fluoxetine regimen appeared to slow recovery from depression, although the use of T(3) was associated with a fall in thyroxine concentrations. Changes in 5-HT concentrations did not correlate with psychiatric scores and were seen only in drug-treated groups, not those given counselling. There were no associated changes in absolute concentrations of kynurenines, BDNF, CRP, neopterin or IL-2. With fluoxetine treatment, there were correlations between the concentrations of kynurenine metabolites and the psychiatric rating scores, whereas no correlations were found with BDNF or inflammatory markers. 4. It is concluded that depression scores are largely independent of inflammatory status, but kynurenine metabolism may be related to the degree of depression after fluoxetine treatment.

Serine protease modulation of Dependence Receptors and EMT protein expression.

Expression of the tumour suppressor Deleted in Colorectal Cancer (DCC) and the related protein neogenin is reduced by the mammalian serine protease chymotrypsin or the bacterial serine protease subtilisin, with increased cell migration. The present work examines whether these actions are associated with changes in the expression of cadherins, ß-catenin and vimentin, established markers of the Epithelial-Mesenchymal Transition (EMT) which has been linked with cell migration and tumour metastasis. The results confirm the depletion of DCC and neogenin and show that chymotrypsin and subtilisin also reduce expression of ß-catenin in acutely prepared tissue sections but not in human mammary adenocarcinoma MCF-7 or MDA-MB-231 cells cultured in normal media, or primary normal human breast cells. A loss of ß-catenin was also seen in low serum media but transfecting cells with a dcc-containing plasmid induced resistance. E-cadherin was not consistently affected but vimentin was induced by low serum-containing media and was increased by serine proteases in MCF-7 and MDA-MB-231 cells in parallel with increased wound closure. Vimentin might contribute to the promotion of cell migration. The results suggest that changes in EMT proteins depend on the cells or tissues concerned and do not parallel the expression of DCC and neogenin. The increased cell migration induced by serine proteases is not consistently associated with the expression of the EMT proteins implying either that the increased migration may be independent of EMT or supporting the view that EMT is not itself consistently related to migration. (241).

Dependence receptor involvement in subtilisin-induced long-term depression and in long-term potentiation.

The serine protease subtilisin induces a form of long-term depression (LTD) which is accompanied by a reduced expression of the axo-dendritic guidance molecule Unco-ordinated-5C (Unc-5C). One objective of the present work was to determine whether a loss of Unc-5C function contributed to subtilisin-induced LTD by using Unc-5C antibodies in combination with the pore-forming agents Triton X-100 (0.005%) or streptolysin O in rat hippocampal slices. In addition we have assessed the effect of subtilisin on the related dependence receptor Deleted in Colorectal Cancer (DCC) and used antibodies to this protein for functional studies. Field excitatory postsynaptic potentials (fEPSPs) were analyzed in rat hippocampal slices and protein extracts were used for Western blotting. Subtilisin produced a greater loss of DCC than of Unc-5C, but the antibodies had no effect on resting excitability or fEPSPs and did not modify subtilisin-induced LTD. However, antibodies to DCC but not Unc-5C did reduce the amplitude of theta-burst long-term potentiation (LTP). In addition, two inhibitors of endocytosis - dynasore and tat-gluR2(3Y) - were tested and, although the former compound had no effect on neurophysiological responses, tat-gluR2(3Y) did reduce the amplitude of subtilisin-induced LTD without affecting the expression of DCC or Unc-5C but with some loss of PostSynaptic Density Protein-95. The results support the view that the dependence receptor DCC may be involved in LTP and suggest that the endocytotic removal of a membrane protein or proteins may contribute to subtilisin-induced LTD, although it appears that neither Unc-5C nor DCC are involved in this process.

Kynurenine pathway metabolism following prenatal KMO inhibition and in Mecp2+/- mice, using liquid chromatography-tandem mass spectrometry.

To quantify the full range of tryptophan metabolites along the kynurenine pathway, a liquid chromatography - tandem mass spectrometry method was developed and used to analyse brain extracts of rodents treated with the kynurenine-3-mono-oxygenase (KMO) inhibitor Ro61-8048 during pregnancy. There were significant increases in the levels of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (3-HK) in the maternal brain after 5 h but not 24 h, while the embryos exhibited high levels of kynurenine, kynurenic acid and anthranilic acid after 5 h which were maintained at 24 h post-treatment. At 24 h there was also a strong trend to an increase in quinolinic acid levels (P = 0.055). No significant changes were observed in any of the other kynurenine metabolites. The results confirm the marked increase in the accumulation of some neuroactive kynurenines when KMO is inhibited, and re-emphasise the potential importance of changes in anthranilic acid. The prolonged duration of metabolite accumulation in the embryo brains indicates a trapping of compounds within the embryonic CNS independently of maternal levels. When brains were examined from young mice heterozygous for the meCP2 gene - a potential model for Rett syndrome - no differences were noted from control mice, suggesting that the proposed roles for kynurenines in autism spectrum disorder are not relevant to Rett syndrome, supporting its recognition as a distinct, independent, condition.

Kynurenine and neopterin levels in patients with rheumatoid arthritis and osteoporosis during drug treatment.

The kynurenine pathway from tryptophan generates compounds which can act on glutamate receptors in peripheral tissues or modulate free radical activity. We have measured the concentrations of several of these compounds in the plasma of patients with rheumatoid arthritis (RA) and osteoporosis (OP) before treatment with drugs and then at monthly intervals for 6 months during treatment. Kynurenine analysis was performed by HPLC. Compared with healthy controls, RA patients showed significantly decreased baseline levels of tryptophan, 3-hydroxykynurenine and 3-hydroxyanthranilic acid and increased levels of kynurenine and xanthurenic acid, while kynurenic acid concentrations were normal. Different results were recorded from patients with OP with only a significant reduction in tryptophan and 3-hydroxyanthranilic acid when compared with healthy controls. During 6 months of treating the RA patients with prednisolone or methotrexate, and the OP patients with raloxifene or etidronate and calcium there were significant therapeutic responses and a significant trend towards a reduction in levels of neopterin in RA patients receiving methotrexate but no changes in the profiles of tryptophan metabolites. The results are consistent with the induction of indoleamine-2,3-dioxygenase (IDO) in both RA and OP but with far greater activation of the pathway in the much more inflammatory condition, i.e. RA. It is concluded that there are changes in the kynurenine pathway, which may modify the activation of tissue glutamate receptors, in RA and OP, but that these are not affected by the drug treatments studied.

Levels of purine, kynurenine and lipid peroxidation products in patients with inflammatory bowel disease.

The factors affecting gut activity in inflammatory bowel disease are unclear, but purines and kynurenines may be involved in the regulation of neuronal activity and therefore gut motility and secretion. We have measured the serum levels of these compounds in patients and in sex- and age-matched controls. Purines and kynurenines were analysed using HPLC. The levels of tryptophan and its metabolites 3-hydroxykynurenine, 3-hydroxyanthranilic acid and xanthurenic acid were unchanged in all patients. However, the levels of kynurenine and kynurenic acid were significantly elevated in patients with inflammatory bowel disease when compared to control subjects. There were no significant differences between patients and controls for any of the purines analysed or for neopterin. In the inflammatory bowel disease patients serum lipid peroxidation products were significantly elevated when compared to control subjects, suggesting the presence of increased oxidative stress consistent with inflammatory activity. The elevated level of kynurenic acid may represent either a compensatory response to elevated activation of enteric neurones, or a primary abnormality, which induces a compensatory increase in gut activity, but may indicate a role for kynurenine modulation of glutamate receptors in the symptoms of inflammatory bowel disease.

Modified neocortical and cerebellar protein expression and morphology in adult rats following prenatal inhibition of the kynurenine pathway.

Inhibition of the kynurenine pathway of tryptophan metabolism during gestation can lead to changes in synaptic transmission, neuronal morphology and plasticity in the rat hippocampus. This suggests a role for the kynurenine pathway in early brain development, probably caused by kynurenine modulation of N-methyl-d-aspartate (NMDA) glutamate receptors which are activated by the tryptophan metabolite quinolinic acid and blocked by kynurenic acid. We have now examined samples of neocortex and cerebellum of adult animals to assess the effects of a prenatally administered kynurenine-3-monoxygenase inhibitor (Ro61-8048) on protein and mRNA expression, dendritic structure and immuno-histochemistry. No changes were seen in mRNA expression using quantitative real-time polymerase chain reaction. Changes were detected in the expression of several proteins including the GluN2A subunit, unco-ordinated-5H3 (unc5H3), doublecortin, cyclo-oxygenase, sonic hedgehog and Disrupted in schizophrenia-1 (DISC1), although no differences in immunoreactive cell numbers were observed. In the midbrain, dependence receptor expression was also changed. The numbers and lengths of individual dendritic regions were not changed but there were significant increases in the overall complexity values of apical and basal dendritic trees. The data support the hypothesis that constitutive kynurenine metabolism plays a critical role in early, embryonic brain development, although fewer effects are produced in the neocortex and cerebellum than in the hippocampus and the nature of the changes seen are qualitatively different. The significant changes in DISC1 and unc5H3 may be relevant to cerebellar dysfunction and schizophrenia respectively, in which these proteins have been previously implicated.

Prenatal inhibition of the tryptophan-kynurenine pathway alters synaptic plasticity and protein expression in the rat hippocampus.

Glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) are important in early brain development, influencing cell proliferation and migration, neuritogenesis, axon guidance and synapse formation. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Rats were treated in late gestation with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]-benzene-sulphonamide (Ro61-8048), an inhibitor of kynurenine-3-monoxygenase which diverts kynurenine metabolism to kynurenic acid. Within 5h of drug administration, there was a significant decrease in GluN2A expression and increased GluN2B in the embryo brains, with changes in sonic hedgehog at 24h. When injected dams were allowed to litter normally, the brains of offspring were removed at postnatal day 21 (P21). Recordings of hippocampal field excitatory synaptic potentials (fEPSPs) showed that prenatal exposure to Ro61-8048 increased neuronal excitability and paired-pulse facilitation. Long-term potentiation was also increased, with no change in long-term depression. At this time, levels of GluN2A, GluN2B and postsynaptic density protein PSD-95 were all increased. Among several neurodevelopmental proteins, the expression of sonic hedgehog was increased, but DISC1 and dependence receptors were unaffected, while raised levels of doublecortin and Proliferating Cell Nuclear Antigen (PCNA) suggested increased neurogenesis. The results reveal that inhibiting the kynurenine pathway in utero leads to molecular and functional synaptic changes in the embryos and offspring, indicating that the pathway is active during gestation and plays a significant role in the normal early development of the embryonic and neonatal nervous system.

Kynurenine pathway inhibition as a therapeutic strategy for neuroprotection.

The oxidative pathway for the metabolism of tryptophan along the kynurenine pathway generates quinolinic acid, an agonist at N-methyl-D-aspartate receptors, as well as kynurenic acid which is an antagonist at glutamate and nicotinic receptors. The pathway has become recognized as a key player in the mechanisms of neuronal damage and neurodegenerative disorders. As a result, manipulation of the pathway, so that the balance between the levels of components of the pathway can be modified, has become an attractive target for the development of pharmacological agents with the potential to treat those disorders. This review summarizes some of the relevant background information on the pathway itself before identifying some of the chemical strategies for its modification, with examples of their successful application in animal models of infection, stroke, traumatic brain damage, cerebral malaria and cerebral trypanosomiasis.

Prenatal activation of Toll-like receptors-3 by administration of the viral mimetic poly(I:C) changes synaptic proteins, N-methyl-D-aspartate receptors and neurogenesis markers in offspring.

BACKGROUND: There is mounting evidence for a neurodevelopmental basis for disorders such as autism and schizophrenia, in which prenatal or early postnatal events may influence brain development and predispose the young to develop these and related disorders. We have now investigated the effect of a prenatal immune challenge on brain development in the offspring. Pregnant rats were treated with the double-stranded RNA polyinosinic:polycytidylic acid (poly(I:C); 10 mg/kg) which mimics immune activation occurring after activation of Toll-like receptors-3 (TLR3) by viral infection. Injections were made in late gestation (embryonic days E14, E16 and E18), after which parturition proceeded naturally and the young were allowed to develop up to the time of weaning at postnatal day 21 (P21). The brains of these animals were then removed to assess the expression of 13 different neurodevelopmental molecules by immunoblotting. RESULTS: Measurement of cytokine levels in the maternal blood 5 hours after an injection of poly(I:C) showed significantly increased levels of monocyte chemoattractant protein-1 (MCP-1), confirming immune activation. In the P21 offspring, significant changes were detected in the expression of GluN1 subunits of NMDA receptors, with no difference in GluN2A or GluN2B subunits or the postsynaptic density protein PSD-95 and no change in the levels of the related small GTPases RhoA or RhoB, or the NMDA receptor modulator EphA4. Among presynaptic molecules, a significant increase in Vesicle Associated Membrane Protein-1 (VAMP-1; synaptobrevin) was seen, with no change in synaptophysin or synaptotagmin. Proliferating Cell Nuclear Antigen (PCNA), as well as the neurogenesis marker doublecortin were unchanged, although Sox-2 levels were increased, suggesting possible changes in the rate of new cell differentiation. CONCLUSIONS: The results reveal the induction by prenatal poly(I:C) of selective molecular changes in the brains of P21 offspring, affecting primarily molecules associated with neuronal development and synaptic transmission. These changes may contribute to the behavioural abnormalities that have been reported in adult animals after exposure to poly(I:C) and which resemble symptoms seen in schizophrenia and related disorders.

On the Biological Importance of the 3-hydroxyanthranilic Acid: Anthranilic Acid Ratio.

Of the major components of the kynurenine pathway for the oxidative metabolism of tryptophan, most attention has focussed on the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid, and the glutamate receptor blocker kynurenic acid. However, there is increasing evidence that the redox-active compound 3-hydroxyanthranilic acid may also have potent actions on cell function in the nervous and immune systems, and recent clinical data show marked changes in the levels of this compound, associated with changes in anthranilic acid levels, in patients with a range of neurological and other disorders including osteoporosis, chronic brain injury, Huntington's disease, coronary heart disease, thoracic disease, stroke and depression. In most cases, there is a decrease in 3-hydroxyanthranilic acid levels and an increase in anthranilic acid levels. In this paper, we summarise the range of data obtained to date, and hypothesise that the levels of 3-hydroxyanthranilic acid or the ratio of 3-hydroxyanthranilic acid to anthranilic acid levels, may contribute to disorders with an inflammatory component, and may represent a novel marker for the assessment of inflammation and its progression. Data are presented which suggest that the ratio between these two compounds is not a simple determinant of neuronal viability. Finally, a hypothesis is presented to account for the development of the observed changes in 3-hydroxyanthranilic acid and anthranilate levels in inflammation and it is suggested that the change of the 3HAA:AA ratio, particularly in the brain, could possibly be a protective response to limit primary and secondary damage.