Bicarbonate-dependent peroxidase activity of human Cu,Zn-superoxide dismutase induces covalent aggregation of protein: intermediacy of tryptophan-derived oxidation products

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate (HCO3-)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3-. HCO3--dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion (CO3.). Human SOD1WT has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) that exhibits HCO3--dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,H2O2, and HCO3-did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3.-induced covalent aggregation of hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. Fluorescence experiments revealed that Trp-32 was further oxidized by CO3., forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3-/H2O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3--dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed.     

Mass spectral evidence for carbonate-anion-radical-induced posttranslational modification of tryptophan to kynurenine in human Cu, Zn superoxide dismutase

Previously, we showed that oxidation of tryptophan-32 (Trp-32) residue was crucial for H₂O₂/bicarbonate (HCO₃⁻)-dependent covalent aggregation of human Cu,Zn SOD1 (hSOD1). The carbonate anion radical (CO₃⁻)-induced oxidation of Trp-32 to kynurenine-type oxidation products was proposed to cause the aggregation of hSOD1. Here we used the matrix-assisted laser desorption ionization–time of flight mass spectroscopy, high-performance liquid chromatography–electrospray ionization mass spectroscopy, and liquid chromatography mass spectroscopy methods to characterize products. Results show that a peptide region (31–36) of hSOD1 containing the Trp-32 residue (VWGSIK) is oxidatively modified to the N-formylkynurenine (NFK)- and kynurenine (Kyn)-containing peptides (V(NFK)GSIK) and (V(Kyn)GSIK) during HCO⁻-dependent peroxidase activity of hSOD1. Also, UV photolysis of a cobalt complex that generates authentic CO₃⁻ radical induced a similar product profile from hSOD1. Similar products were obtained using a synthetic peptide with the same amino acid sequence (i.e., VWGSIK). We propose a mechanism involving a tryptophanyl radical for CO₃⁻-induced oxidation of Trp-32 residue (VWGSIK) in hSOD1 to V(NFK)GSIK and V(Kyn)GSIK.     

Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.     

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1^WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp³² residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp³² residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1^WT and hSOD1^G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp³² residue in the process. The results showed that Trp³² residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp³² residue (bovine SOD1 and hSOD1^W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp³² residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.     

Role of superoxide anion in regulating pressor and vascular hypertrophic response to angiotensin II

Our purpose was to address the role of NAPDH oxidase-derived superoxide anion in the vascular response to ANG II. Blood pressure, aortic superoxide anion, 3-nitrotyrosine, and medial cross-sectional area were compared in wild-type mice and in mice that overexpress human superoxide dismutase (hSOD). The pressor response to ANG II was significantly less in hSOD mice. Superoxide anion levels were increased twofold in ANG II-treated wild-type mice but not in hSOD mice. 3-Nitrotyrosine increased in aortic endothelium and adventitia in wild-type but not hSOD mice. In contrast, aortic medial cross-sectional area increased 50% with ANG II in hSOD mice, comparable to wild-type mice. The lower pressor response to ANG II in the mice expressing hSOD is consistent with a pressor role of superoxide anion in wild-type mice, most likely because it reacts with nitric oxide. Despite preventing the increase in superoxide anion and 3-nitrotyrosine, the aortic hypertrophic response to ANG II in vivo was unaffected by hSOD.     

Systemic metabolism of tryptophan and its catabolites,kynurenine and 3-HAA,in mice with inflammatory arthritis

Tryptophan is an essential amino acid. The liver is primary organ involved the oxidative catabolism of tryptophan. However, in the immune system, tryptophan and its catabolites, kynurenine and 3-hydroxyanthranilic acid (3-HAA), play an anti-inflammatory role. Rheumatoid arthritis (RA) is an autoimmune disease. Collagen induced arthritis (CIA) is an animal model of RA. Therefore, it was of interest to measure concentration of tryptophan, kynurenine and 3-HAA in mice with CIA. Concentration of tryptophan and 3-HAA was measured with HPLC methods. Concentration of kynurenine was measured with colorimetric test. mRNA expression for the kynurenine pathway genes was assessed using qRT-PCR. It has been found that in sera from diseased mice concentration of tryptophan was not changed. Concentration of kynurenine and 3-HAA was decreased. Moreover, in the livers from mice with CIA, concentration of tryptophan and kynurenine was decreased. These observations coincided with decreased mRNA expression for Ido2 and Afm and increased mRNA expression for Kynureninase in the liver. It has been also shown that in CIA the concentration of 3-HAA was increased in the kidneys.     

Modulation of mutant superoxide dismutase 1 aggregation by co-expression of wild-type enzyme

Mutations in superoxide dismutase 1 (SOD1, EC 1.15.1.1) cause familial amyotrophic lateral sclerosis; with aggregated forms of mutant protein accumulating in spinal cord tissues of transgenic mouse models and human patients. Mice over-expressing wild-type human SOD1 (WT hSOD1) do not develop amyotrophic lateral sclerosis-like disease, but co-expression of WT enzyme at high levels with mutant SOD1 accelerates the onset of motor neuron disease compared with mice expressing mutant hSOD1 alone. Spinal cords of mice expressing both proteins contain aggregated forms of mutant protein and, in some cases, evidence of co-aggregation of WT hSOD1 enzyme. In the present study, we used a cell culture model of mutant SOD1 aggregation to examine how the presence of WT SOD1 affects mutant protein aggregation, finding that co-expression of WT SOD1, hSOD1 or mouse SOD1, delayed the formation of mutant hSOD1 aggregates; in essence appearing to slow the aggregation rate. In some combinations of WT and mutant hSOD1 co-expression, the aggregates that did eventually form appeared to contain WT hSOD1 protein. However, WT mouse SOD1 did not co-aggregate with mutant hSOD1 despite displaying a similar ability to slow mutant hSOD1 aggregation. Together, these studies indicate that WT SOD1 (human or mouse), when expressed at levels equivalent to the mutant protein, modulates the aggregation of mutant SOD1.     

An antibody-based affinity chromatography tool to assess Cu,Zn superoxide dismutase (SOD) G93A structural complexity in vivo

‘Conformational diseases’ are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1^G93A mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSOD^G93A rats provided evidence about heterodimers rSOD–hSOD^G93A formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSOD^G93A showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues.     

Cu,Zn-superoxide dismutase-driven free radical modifications: copper- and carbonate radical anion-initiated protein radical chemistry

The understanding of the mechanism, oxidant(s) involved and how and what protein radicals are produced during the reaction of wild-type SOD1 (Cu,Zn-superoxide dismutase) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We have used immuno-spin trapping and MS analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using HSA (human serum albumin) and mBH (mouse brain homogenate) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and CO3(*-) (carbonate radical anion)-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO(*-) from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (diethylenetriaminepenta-acetic acid) (to suppress copper chemistry), CO(*-) produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO(*-) produced by the reaction of hSOD1 with H2O2 also produced distinctive DMPO (5,5-dimethylpyrroline-N-oxide) nitrone adduct-positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO(*-) production from CO2, enhanced protein radical formation and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our present study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with H2O2; these results are the first step in analysing the critical targets of SOD1-driven oxidation during pathological processes such as neuroinflammation.     

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.     

Depressive and anxiety symptoms in the early puerperium are related to increased degradation of tryptophan into kynurenine,a phenomenon which is related to immune activation

There is now some evidence that i) the availability of plasma tryptophan, the precursor of serotonin, is significantly lower in pregnant women at the end of term and the first few days after delivery than in nonpregnant women; and ii) both pregnancy and the early puerperium are accompanied by activation of the inflammatory response system. The aims of the present study were to examine the effects of pregnancy and delivery on plasma kynurenine, a major tryptophan catabolite synthesized after induction of indoleamine-2, 3 dioxygenase (IDO) by pro-inflammatory cytokines. We measured plasma kynurenine and tryptophan and immune markers, such as serum interleukin-6 (IL-6), IL-8 and the leukemia inhibitory factor-receptor (LIF-R) in healthy, nonpregnant and pregnant women at the end of term and one and three days after delivery. Plasma kynurenine was significantly lower in pregnant women at the end of term than in nonpregnant women, findings which may be attributed to lower plasma tryptophan at the end of term. The kynurenine/tryptophan (K/T) quotient was significantly higher in the pregnant women at the end of term and in the early puerperium than in nonpregnant women. In the early puerperium there was a significant increase in plasma kynurenine and the K/T quotient. The increases in plasma kynurenine and the K/T quotient were significantly more pronounced in women whose anxiety and depression scores significantly increased in the puerperium. The changes from the end of term to the early puerperium in plasma kynurenine and the K/T quotient were significantly related to those in the immune markers. It is concluded that 1) lower plasma kynurenine at the end of term is the consequence of lower plasma tryptophan; 2) the increased K/T quotient at the end of term and in the early puerperium indicates inflammation-induced degradation of tryptophan along the kynurenine pathway; and 3) that depressive and anxiety symptoms in the early puerperium are (causally) related to an increased catabolism of tryptophan into kynurenine, a phenomenon which probably results from immune activation.     

3-hydroxykynurenine-mediated modification of human lens proteins: structure determination of a major modification using a monoclonal antibody

Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.     

Kynurenine metabolism in vitamin-B-6-deficient rat liver after tryptophan injection.

Tryptophan contents of liver, serum and kidney were determined in normal and vitamin-B-6-deficient rats after tryptophan injection. Tryptophan contents of normal and B-6-deficient liver were different, but not those in serum and kidney. Both kynurenine and 3-hydroxykynurenine accumulated in B-6-deficient liver more than in the normal. The 3-hydroxykynurenine contents after tryptophan injection (30 mg/100 g body wt.) increased to 1380 nmol/g of liver at 1-1.5 h, a value sufficient to produce xanthurenate, in view of the Km value of kynurenine aminotransferase. The enzymes metabolizing kynurenine were assayed at various times after tryptophan injection. The activity of kynureninase holoenzyme in B-6-deficient liver was much decreased, but the activity of total enzyme was not changed. It appeared that a high dose of tryptophan in B-6-deficient rats could cause a greater deficiency of pyridoxal 5-phosphate. Tryptophan metabolism in B-6-deficient rat liver after tryptophan administration is discussed.     

Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain

We describe here a gas chromatography-tandem mass spectrometry (GC/MS/MS) method for the sensitive and concurrent determination of extracellular tryptophan and the kynurenine pathway metabolites kynurenine, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN) in rat brain. This metabolic cascade is increasingly linked to the pathophysiology of several neurological and psychiatric diseases. Methodological refinements, including optimization of MS conditions and the addition of deuterated standards, resulted in assay linearity to the low nanomolar range. Measured in samples obtained by striatal microdialysis in vivo, basal levels of tryptophan, kynurenine, and QUIN were 415, 89, and 8 nM, respectively, but 3-HK levels were below the limit of detection (<2 nM). Systemic injection of kynurenine (100 mg/kg, i.p.) did not affect extracellular tryptophan but produced detectable levels of extracellular 3-HK (peak after 2-3 h: ~50 nM) and raised extracellular QUIN levels (peak after 2h: ~105 nM). The effect of this treatment on QUIN, but not on 3-HK, was potentiated in the N-methyl-D-aspartate (NMDA)-lesioned striatum. Our results indicate that the novel methodology, which allowed the measurement of extracellular kynurenine and 3-HK in the brain in vivo, will facilitate studies of brain kynurenines and of the interplay between peripheral and central kynurenine pathway functions under physiological and pathological conditions.     

The metabolism of L-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism.

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5--6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.     

Investigation of the kynurenine pathway in Indoleamine 2, 3 dioxygenase deficient mice with inflammatory arthritis

Tryptophan is an essential amino acid involved in the protein synthesis, cognition, and immunity. Oxidative catabolism of tryptophan is executed by the sets of biochemical reactions collectively referred to as the kynurenine pathway. In the immune system, two distinct enzymes, Indoleamne 2,3 dioxygenase 1 (IDO1) and Indoleamine 2, 3 dioxygenase 2 (IDO2) can initiate metabolic flux through the kynurenine pathway. Rheumatoid arthritis is an autoimmune disease driven by the exacerbated immune response towards self antigens and characterized by the chronic inflammatory reaction of the diarthrodial joints. Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis. Using CIA in wild type (WT) and mice deficient with Indoleamine 2,3 dioxygenase (Ido1KO), it was of interest to test the impact of Ido1 deletion on the concentration of tryptophan and its catabolites as well as on mRNA expression for other genes on the kynurenine pathway. Here, when compared with samples taken from naïve WT animals and those with CIA, it was found that only in the inguinal lymph nodes (iLN) taken from Ido1KO mice with CIA tryptophan concentration was significantly increased. In contrast, mRNA expression for Ido2 was decreased in naïve as well as in the diseased iLN taken from Ido1KO mice. Deletion of Ido1 and reduced mRNA expression for Ido2 neither affected the concentration of the downstream metabolites of tryptophan nor mRNA expression for downstream genes on the kynurenine pathway in iLN. Moreover, the concentration of kynurenine in sera of mice with CIA was significantly decreased in Ido1KO mice with arthritis.     

The kynurenine pathway of tryptophan metabolism detected in the nervous system of Drosophila melanogaster

Isolated thoracic ganglia were incubated in physiological solution containing 14C-tryptophan. After this procedure, they were homogenized in a 1 per cent solution of HCL in methanol, and supernatant was subjected to two-dimensional thin layer chromatography in the presence of tryptophan, kynurenine, 3-hydroxy kynurenine, as well as kynurenic, anthranilic and xanthurenic acids. The spots were cut out and counted by liquid scintillation technique. Except tryptophan, only kynurenine and 3-hydroxy kynurenine spots contained notable radioactivity. Therefore, at least the initial stages of kynurenine pathway operate in the nervous system of Drosophila melanogaster. This finding is in accordance with observations of the effects of kynurenines on insect behaviour.     

Alterations in kynurenine precursor and product levels in schizophrenia and bipolar disorder

Increased concentrations of kynurenine pathway metabolites have been reported by several groups for disorders involving psychosis, including schizophrenia and bipolar disorder. To identify components of the pathway that may be relevant as biomarkers or may underlie the etiology of psychosis, it is essential to characterize the extent of kynurenine pathway activation and to investigate known regulators of one of the key kynurenine-producing enzymes, tryptophan 2,3-dioxygenase (TDO2), previously shown in this laboratory to be increased commensurate with kynurenine in postmortem anterior cingulate brain tissue from individuals with schizophrenia. Using this same anterior cingulate sample set from individuals with schizophrenia, bipolar disorder, depression and controls (N=12-14 per group), we measured the precursor of kynurenine and two downstream products. The precursor, tryptophan, was significantly increased only in the schizophrenia group (1.54-fold the mean control value, p=0.02), and through substrate-induced activation, may be one cause of the increased kynurenine and kynurenine metabolites. This finding for tryptophan differs from some, but not all, previous reports and methodological reasons for the discrepancies are discussed. A product of kynurenine metabolism, 3-OH-anthranilic acid was also significantly increased only in the schizophrenia group (1.68-fold the mean control value, p=0.03). 3-OH-anthranilic acid is a reactive species with cytotoxic properties, although the threshold for such effects is not known for neurons. Analysis of major pre- and post-mortem variables showed that none were confounding for these between-group experimental comparisons. Nicotinamide, a pathway end product, did not differ between groups but was associated with cause of death (suicide) within the bipolar group (p=0.03).     

SYNTHESIS AND METABOLISM OF l-KYNURENINE IN RAT BRAIN

Abstract— A method for the quantitative analysis of femtomole amounts of kynurenine (along with tryptophan, 3-hydroxykynurenine and kynuramine) in rat brain using high pressure liquid chroma-tography and electron-capture GLC is described. Endogenous concentrations of these substances in rat brain regions were measured, and their formation after the injection of radioactive tryptophan or kynurenine was determined. Kynurenine was formed from tryptophan in brain and was also taken up from the periphery. Extracerebral kynurenine was calculated to account for 60% of the cerebral pool of kynurenine. The cerebral rates of synthesis of kynurenine and 3-hydroxykynurenine were 0.29 and 0.17nmol/g/h. The turnover rate of kynurenine in the brain was 1.02 nmol/g/h measured from [¹⁴C]tryptophan or 1.14 nmol/g/h from [³H]kynurenine injected intraperitoneally. Kynuramine levels in different areas of the brain were similar to those of tryptamine. Following intraperitoneal injection of [¹⁴C]tryptophan, the presence of anthranilic, 3-hydroxyanthranilic, xanthurenic, kynurenic and quinaldic acids was demonstrated in the brain.     

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

Background

By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures.

Methodology/Principal Findings

In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures.

Conclusions/Significance

Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells.