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.

The serine protease subtilisin suppresses epileptiform activity in rat hippocampal slices and neocortex in vivo.

Serine proteases of the S8A family and those belonging to the subtilase group generate a long-lasting inhibition of hippocampal evoked potentials, which shows little recovery and resembles long-term depression. The present work investigates the effects of subtilisin A on epileptiform activity induced in hippocampal slices. Interictal bursts were generated by perfusion with 4-aminopyridine in magnesium-free medium, whereas ictal bursts were produced by the addition of baclofen. Subtilisin A superfused for 10 min at concentrations of 50 nM and above reduced the duration of ictal bursts, whereas higher concentrations reduced the frequency of interictal activity with little or no recovery, indicating similarity with the long-term depression reported previously. The anti-epileptiform activity was not prevented by inhibitors of phosphatases or several kinases, but the inhibition of ictal activity was selectively reduced by the tyrosine kinase inhibitor genistein. The rho-activated coiled-coil kinase (ROCK) inhibitor Y-27632 had no effect on the suppression of ictal or interictal bursts. Subtilisin applied at nanomolar concentrations to the surface of the cerebral cortex in vivo also suppressed epileptiform spikes induced by bicuculline. It is concluded that serine proteases of the subtilase group are highly potent inhibitors of epileptiform activity, especially ictal bursts, and that tyrosine kinases may be involved in that inhibition. The mechanism of inhibition is different from the long-lasting depression of evoked potentials, which is partly mediated via ROCK.

Neurotoxicity of tryptophan metabolites.

The metabolism of tryptophan by the kynurenine pathway leads to the production of several neurotoxic compounds, some of which have been associated with neurological disorders. Recent investigation of some relevant compounds in this pathway has provided further evidence of their neurotoxicity.

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.

Differences in the neurochemical characteristics of the cortex and striatum of mice with cerebral malaria.

Fatal murine cerebral malaria is an encephalitis and not simply a local manifestation in the brain of a systemic process. Histopathologically, murine cerebral malaria has been characterized by monocyte adherence to the endothelium of the microvasculature, activation of microglial cells, swelling of endothelial cell nuclei, microvasculature damage, and breakdown of the blood-brain barrier with cerebral oedema. Brain parenchymal cells have been proposed to be actively involved in the pathogenesis of murine cerebral malaria. We, therefore, compared the neurochemical characteristics of Plasmodium berghei ANKA-infected mice with controls to determine whether cerebral malarial infection significantly impairs specific neuronal populations. Between 6 and 7 days after infection, we found a significant loss of neurones containing substance P, with preservation of cells containing somatostatin, neuropeptide Y and calbindin in the striatum of infected mice compared with controls. In the cortex of infected mice, we found a significant reduction in the number of cells containing substance P, somatostatin and neuropeptide Y. The number of calbindin-containing neurones was unchanged. This study found significant changes in the neurochemical characteristics of the cortex and striatum of mice infected with P. berghei ANKA, which may contribute to their cerebral symptoms.

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.

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.

Long-term follow-up study with repetitive transcranial magnetic stimulation (rTMS) in Parkinson's disease.

Several studies have claimed the effectiveness of repetitive transcranial magnetic stimulation (rTMS) in Parkinson's disease (PD). The rTMS therapy has to be repeated regularly to achieve a permanent effect but the side effects of long-term administration of low frequency rTMS are not known. Further, there is no information about its influence on the development of Parkinson's disease. Two different groups of patients with PD were compared in a retrospective study for 3 years. The first group (A) was treated with drugs, the second group (B) was treated with drugs + rTMS (1 Hz, 0.6 T, 100 stimuli per day for 7 days using a round coil). rTMS was repeated at least twice each year for 3 years. Symptoms of PD were assessed using the Graded Rating Scale. Although at the onset of the study group B patients had greater disease severity and were receiving higher doses of levodopa, this group (receiving rTMS) showed no deterioration in these parameters, whereas those in group A receiving drugs alone showed a marked deterioration. Hoehn-Yahr (H-Y) stages at the onset of the study and 3 years later were: group A: 1.93 +/- 0.75, 3.03 +/- 1.01; group B: 2.50 +/- 0.83, 2.45 +/- 0.62. The dose of levodopa (mg/day) was at the onset of trial and 3 years later was: group A: 124.4 +/- 144.0, 555.5 +/- 247.2; group B: 287.7 +/- 217.1, 333.4 +/- 181.0. The yearly increment in the scores was: group A: 1.308 +/- 0.307 (P < 0.001), group B: 0.642 +/- 0.389 (P < 0.1). Accordingly, this retrospective study using regularly repeated rTMS with 1 Hz for 7 days, at least twice yearly for 3 years, significantly slowed the development of Parkinson's disease. Unwanted side effects were not observed during the 3 years.

Increased long-term potentiation in the CA1 region of rat hippocampus via modulation of GTPase signalling or inhibition of Rho kinase.

There is accumulating evidence that Ras, and Ras-related GTPases of the Rho family, such as RhoA, RhoB and Rac1, are involved in synaptic plasticity in brain regions such as the hippocampus. We have recently shown that Rho family GTPases are activated by synaptic transmission in the CA1 region of the hippocampus. Since the function of these GTPases is dependent on post-translational isoprenylation by either farnesyl or geranylgeranyl transferases, we tested the hypothesis that inhibition of isoprenylation would modify long-term potentiation (LTP). Farnesyl transferase inhibition, which suppressed activation of RhoB and Ras but not RhoA or Rac1, reduced the magnitude of LTP, while geranylgeranyl transferase inhibition, which inhibited RhoA and Rac1 but not RhoB, increased the magnitude of LTP. In addition, Y-27632, a specific inhibitor of a downstream effector of Rho GTPases-Rho-kinase-also increased the magnitude of LTP. This provides strong evidence that GTPases are important mediators of synaptic plasticity, and demonstrates that Rho-kinase acts to reduce the degree of plasticity at hippocampal synapses during LTP. Rho-kinase inhibitors have the unusual property of increasing the magnitude of LTP, and so may be potential cognitive enhancers.

Activation of Rho GTPases by synaptic transmission in the hippocampus.

Ras-related GTPases of the Rho family, such as RhoA and RhoB, are well-characterised mediators of morphological change in peripheral tissues via their effects on the actin cytoskeleton. We tested the hypothesis that Rho family GTPases are involved in synaptic transmission in the CA1 region of the hippocampus. We show that GTPases are activated by synaptic transmission. RhoA and RhoB were activated by low frequency stimulation, while the induction of long-term potentiation (LTP) by high frequency stimulation was associated with specific activation of RhoB via NMDA receptor stimulation. This illustrates that these GTPases are potential mediators of synaptic transmission in the hippocampus, and raises the possibility that RhoB may play a role in plasticity at hippocampal synapses during LTP.

Distribution of Rho family GTPases in the adult rat hippocampus and cerebellum.

Small GTPases are monomeric guanine nucleotide binding proteins of 20-25 kDa mass. Rho GTPases belong to the Ras superfamily of small GTPases. The small GTPases of the Rho family have been shown to participate in the organisation of the actin cytoskeleton and signal transduction pathways leading to gene transcription. Recent evidence suggests that Rho family GTPases may play an important role in synaptic communication in the brain, and particularly in synatic plasticity. In this study the distribution of RhoA, RhoB, RhoG, Cdc42, and Rac1 was investigated in hippocampal and cerebellar tissue of adult rat brain using immunohistochemical techniques. Previous studies suggest that distribution of Rho family mRNA is uniform throughout these structures. Here we provide evidence for differences in expression of these proteins between different regions of the hippocampus, and between the molecular and granular layers in the cerebellum. These differences may prove important with regard to the physiological functions of Rho family GTPases.