The role of kynurenine metabolism in the development of the central nervous system

Prenatal exposure to maternal infection has been thought as a major risk factor for neurodevelopmental brain damage and thus contributes to the pathophysiology of neurodegenerative diseases including schizophrenia and autism. The mechanisms of aberrant neurodevelopmental processes on the offspring, in which primary cerebral insults occur during early brain development, are not fully understood. In the present investigation, maternal infection was modelled in timed-pregnant rats at embryonic day (E) 14, 16 and 18 by administering intraperitoneal injections of polyriboinosinic-polyribocytidilic acid,poly(I:C), a viral mimetic double stranded RNA complex which activates Toll-Like-Receptor-3 (TLR-3). The aim was to examine the impact of maternal inflammatory response on the regulation of expression of neurodevelopmental proteins that play important roles in many neurodevelopment aspects, including maintenance of synaptic plasticity, intracellular signalling and neurogenesis which may be relevant in cognitive and behavioural functions. An examination of embryo brains 5 h after maternal poly(I:C) showed significant differences in expression of the NMDA receptor NR2 subunits. The expression of NR2A subunits was reduced, whereas infection induced during pregnancy enhanced NR2B subunit expression. Expression levels of both subunits at postnatal day 21 (P21) were not affected by prenatal poly(I:C) exposure. In utero viral challenge led to significant changes among neurogenesis factor only at P21. In the fetal brain, acute poly(I:C) exposure had no effect on the expression of SHH, PCNA and also SOX2 proteins. However, when poly(I:C) was administered during mid and late gestation in the rodent model, long term effects of prenatal viral challenge on survival and maintenance of cell in the brain as indicated by the expression of SOX2 and SHH was clearly demonstrable. Expression of SOX2 level was increased,while SHH was significantly decreased, suggesting possible increase in the number of cells and changes in the rate of differentiation, respectively. The results demonstrate that poly(I:C) challenge in pregnant dams results in selective molecular changes in the brain, with transient alteration in the levels of NMDA receptor subunit NR2A and NR2B in the foetal brain, and also affecting molecules associated with cell genesis processes at later stages of developmental age of offspring. On the other hand, recent pharmacological interest in kynurenines with respect to CNS diseases has mainly focussed on two neuroactive molecules: quinolinic acid (QUIN) and kynurenic acid (KYNA). Manipulation of the kynurenine pathway and its neuroactive metabolites has been associated with N-methyl-D-aspartate (NMDA) receptor neurotoxicity and dysfunction which linked to the development of various neurological disorders. An early developmental event has been proposed to precipitate alterations in the NMDA receptor function. In this respect, early development during the gestational period of rats is most suitable for investigating the modulating effect of kynurenine pathway inhibition by compound Ro61-8048 (3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulphomide) an inhibitor of kynurenine-3-monooxygenase (KMO) in shifting the balance towards the production of neuroprotective, kynurenic acid. Western blots were generated to indicate the expression of a range of proteins relevant to different aspects of CNS development including neuritogenesis, axon guidance, maintenance of synaptic plasticity, intracellular signalling and cell proliferation and migration. Within 5 h of Ro61-8048, there was a significant decrease in NR2A expression and increased NR2B in the embryo brains, with subsequent changes in SHH and NFB at 24 h post treatment. The litters were left undisturbed until weaning on P21 and other groups were allowed to develop to P60, at which time they euthanized and the brains removed for analysis. At P21, western blot analysis revealed significantly increased protein expression of the NR2A and NR2B subunits and postsynaptic density protein (PSD95). Among several neurodevelopmental proteins, the expression of NFB and proliferating cell nuclear antigen (PCNA) was increased, while reduced level of SHH was detected. We demonstrate here persisting changes in NR2A expression, with reduced level in the hippocampus while a raised level was noted in the cortex suggesting prenatal modulation of kynurenine pathway causes long lasting modifications of NMDA receptor composition and function. It is important to note that kynurenine pathway inhibition can generate a consistent set of long term changes in the SHH in which the levels of this protein remained repressed in some regional areas of the brain including hippocampus, cerebellum and cortex. We show that there are some common pathways that are affected by kynurenine pathway inhibition, and this early modulation tends to disrupt critical molecular processes that are known to be actively occurring at each specific developmental time. Overall, given these selective and differing developmental profile, an early life modulation of the kynurenine pathway might be expected to cause a sufficient disturbance of biological processes that are actively occurring at the time of exposure and also able to leave a series of molecular changes that persist into adulthood. This disruption is likely to influence the resulting physiology of the adolescent and adult brain and subsequently can lead to impairments in social behaviour. It is hoped that this study provides a broad analysis of the long term molecular effects of developmental kynurenine metabolism, and that it allows for a viable opportunity of potential therapeutic targets for disease intervention

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 5 h of drug administration, there was a significant decrease in GluN2A expression and increased GluN2B in the embryo brains, with changes in sonic hedgehog at 24 h. 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

Antioxidant and anti-inflammatory activities of extracts of betel leaves (Piper betle) from solvents with different polarities

The influence of solvents with different polarities on the antioxidant and anti-inflammatory properties of betel leaf extracts (Piper betle) was investigated. The solvents used were water, ethanol, ethyl acetate and hexane. High performance liquid chromatography (HPLC) was used to determine the chemical profiles and concentrations of the active compounds, namely, hydroxychavicol (HC) and eugenol (EU). The antioxidant potential of the extracts was evaluated using two in vitro assays—xanthine/xanthine oxidase superoxide scavenging assay (SOD assay) and 1,2-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay (DPPH assay). The anti-inflammatory assays used were hyaluronidase (HYA), xanthine oxidase (XOD) and lipoxygenase (LOX) inhibition assays. The HPLC results revealed that HC and EU were detected in all types of extracts and the concentrations were highest in the water extract. The highest extraction yield was obtained using water. All the extracts were highly active in both antioxidant assays with water extract showing the strongest inhibition. The extracts also exhibited significant inhibition in XOD and LOX assays. The results indicated that the bioactivity of the extracts was related to HC and EU

Anti-inflammatory action of components from Melastoma malabathricum

Ethnic folklore or empirical therapeutic uses of plant parts have often provided the early indication of the possibility of discovering some pharmacologically active substance from a plant. Melastoma malabathricum L. (Melastomaceae), locally known as sendudok putih, is a small shrub. Traditional medicinal uses include diarrhea, dysentery, ulcers, wound care, and piles. In the search for natural compounds useful against anti-inflammatory activity, a-amyrin, betulinic acid, and isolated flavonoids, including quercetin and quercitrin, were assessed in vitro by determining their inhibitory effects on platelet activating factor (PAF) binding to rabbit platelets using 3H-PAF as a ligand. The results indicated that quercetin, quercitrin, a-amyrin, and betulinic acid showed inhibition of PAF receptor binding with IC50 values of 33.0, 45.4, 20.0, and 22.2 mM, respectively. The IC50 values of these compounds were comparable to Cedrol (13.1 mM), which is a known PAF receptor antagonist. These results suggest that natural flavonoid and pentacyclic triterpenes from M. malabathricum possess selective antagonistic activity toward PAF and could be an attractive candidate as a natural anti-inflammatory compound

In vitro inhibitory effect of Rubraxanthone isolated from Garcinia psrvifolia on platelet- Activating factor receptor binding

Rubraxanthone and isocowanol isolated from Garcinia parvifolia Miq. were investigated for their inhibitory effects on platelet-activating factor (PAF) binding to rabbit platelets using 3H-PAF as a ligand. Rubraxanthone showed a strong inhibition with IC 50 value of 18.2 microM. The IC 50 values of macluraxanthone, 6-deoxyjacareubin, 2-(3-methylbut-2-enyl)-1,3,5-trihydroxyxanthone, 2-(3-methylbut-2-enyl)-1,3,5,6-tetrahydroxyxanthone and 1,3,5-trihydroxy-6,6'-dimethylpyrano(2',3':6,7)-4-(1,1-dimethylprop-2-enyl)-xanthone were also determined for comparison. In the course of our study on structure-activity relationship of xanthones, the results revealed that a geranyl group substituted at C-8 was beneficial to the binding while a hydroxylated prenyl group at C-4 resulted in a significant loss in binding to the PAF receptor

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

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

Abstract 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.</p

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