Inhibition of STAT-mediated cytokine responses to chemically-induced colitis prevents inflammation-associated neurobehavioral impairments.

Depression can be associated with chronic systemic inflammation, and production of peripheral proinflammatory cytokines and upregulation of the kynurenine pathway have been implicated in pathogenesis of depression. However, the mechanistic bases for these comorbidities are not yet well understood. As tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which convert tryptophan to kynurenine, are rate-limiting enzymes of the kynurenine pathway, we screened TDO or IDO inhibitors for effects on the production of proinflammatory cytokines in a mouse macrophage cell line. The TDO inhibitor 680C91 attenuated LPS-induced pro-inflammatory cytokines including IL-1ß and IL-6. Surprisingly, this effect was TDO-independent, as it occurred even in peritoneal macrophages from TDO knockout mice. Instead, the anti-inflammatory effects of 680C91 were mediated through the suppression of signal transducer and activator of transcription(STAT) signaling. Furthermore, 680C91 suppressed production of proinflammatory cytokines and STAT signaling in an animal model of inflammatory bowel disease. Specifically, 680C91 effectively attenuated acute phase colon cytokine responses in male mice subjected to dextran sulfate sodium (DSS)-induced colitis. Interestingly, this treatment also prevented the development of anxiodepressive-like neurobehaviors in DSS-treated mice during the recovery phase. The ability of 680C91 to prevent anxiodepressive-like behavior in response to chemically-induced colitis appeared to be due to rescue of attenuated dopamine responses in the nucleus accumbens. Thus, inhibition of STAT-mediated, but TDO-independent proinflammatory cytokines in macrophages can prevent inflammation-associated anxiety and depression. Identification of molecular mechanisms involved may facilitate the development of new treatments for gastrointestinal-neuropsychiatric comorbidity.

The kynurenine pathway contributes to long-term neuropsychological changes in experimental pneumococcal meningitis.

Pneumococcal meningitis is a lethal form of bacterial infection in the central nervous system that often causes lifelong neurological sequelae, despite therapeutic advances. The contemporary view is that the inflammatory response to infection contributes to the functional disabilities among survivors of this disease. We previously have established a mouse model of neurobehavioural deficits, using an automated IntelliCage™ system that revealed long-term behavioural and cognitive deficits in C57BL/6J female mice cured of meningitis by ceftriaxone treatment. We now have investigated the roles of two kynurenine pathway enzymes, indoleamine dioxygenase-1 (IDO1) and tryptophan dioxygenase-2 (TDO2), in the pathomechanisms of pneumococcal meningitis. Since tryptophan metabolism has long been implicated in behavioural and cognitive modulation through the production of neuroactive compounds, we hypothesised that preventing the actions of these enzymes through gene knockout would be beneficial in mice subjected to pneumococcal infection. We found no significant effect of IDO1 or TDO2 on mortality. Post-meningitic wild-type mice showed long-term diurnal hypoactivity and nocturnal hyperactivity when they were exposed to an Intellicage adaptation test throughout both the light and dark phases. These changes were not apparent in IDO1(-/-) survivors, but were present in the TDO2(-/-) survivors. Both IDO1(-/-) and TDO2(-/-) survivors were not protected against developing long-term cognitive deficits as measured in IntelliCage-based patrolling or reversal tasks. Collectively, these observations suggest (i) involvement of the kynurenine pathway in causing some behavioural sequelae of pneumococcal meningitis and (ii) that this pathway might operate synergistically with, or independently of, other pathways to cause other aspects of neurological sequelae.

The niacin required for optimum growth can be synthesized from L-tryptophan in growing mice lacking tryptophan-2,3-dioxygenase.

In mammals, nicotinamide (Nam) is biosynthesized from l-tryptophan (l-Trp). The enzymes involved in the initial step of the l-Trp→Nam pathway are l-Trp-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO). We aimed to determine whether tdo-knockout (tdo(-/-)) mice fed a diet without preformed niacin can synthesize enough Nam to sustain optimum growth. Wild-type (WT) and tdo(-/-) mice were fed a chemically defined 20% casein diet with or without preformed niacin (30 mg nicotinic acid/kg) for 28 d. Body weight, food intake, and liver NAD concentrations did not differ among the groups. In the groups of mice fed the niacin-free diet, urinary concentrations of the upstream metabolites kynurenine (320% increase, P < 0.0001), kynurenic acid (270% increase, P < 0.0001), xanthurenic acid (770% increase, P < 0.0001), and 3-hydroxyanthranilic acid (3-HA; 450% increase, P < 0.0001) were higher in the tdo(-/-) mice than in the WT mice, while urinary concentrations of the downstream metabolite quinolinic acid (QA; 50% less, P = 0.0010) and the sum of Nam and its catabolites (10% less, P < 0.0001) were lower in the tdo(-/-) mice than in the WT mice. These findings show that the kynurenine formed in extrahepatic tissues by IDO and subsequent enzymes can be metabolized up to 3-HA, but not into QA. However, the tdo(-/-) mice sustained optimum growth even when fed the niacin-free diet for 1 mo, suggesting they can synthesize the minimum necessary amount of Nam from l-Trp, because the liver can import blood kynurenine formed in extrahepatic tissues and metabolize it into Nam via NAD and the resulting Nam is then distributed back into extrahepatic tissues.

Hepatocyte growth factor overexpression in the nervous system enhances learning and memory performance in mice.

Hepatocyte growth factor (HGF) and its receptor, c-Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron-specific manner (HGF-Tg) to deliver HGF into the brain without injury. HGF-Tg mice displayed increased alternation rates in the Y-maze test compared with age-matched wild-type (WT) controls. In the Morris water maze (MWM) test, HGF-Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short- and long-term memory. Western blot analyses revealed that the levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF-Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

Overexpression of HGF attenuates the degeneration of Purkinje cells and Bergmann glia in a knockin mouse model of spinocerebellar ataxia type 7.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder associated with cerebellar neurodegeneration caused by expansion of a CAG repeat in the ataxin-7 gene. Hepatocyte growth factor (HGF), a pleiotrophic growth factor, displays highly potent neurotrophic activities on cerebellar neurons. A mutant c-met/HGF receptor knockin mouse model has revealed a role for HGF in the postnatal development of the cerebellum. The present study was designed to elucidate the effect of HGF on cerebellar neurodegeneration in a knockin mouse model of SCA7 (SCA7-KI mouse). SCA7-KI mice were crossed with transgenic mice overexpressing HGF (HGF-Tg mice) to produce SCA7-KI/HGF-Tg mice that were used to examine the phenotypic differences following HGF overexpression. The Purkinje cellular degeneration is thought to occur via cell-autonomous and non-cell autonomous mechanisms mediated by a reduction of glutamate transporter levels in Bergmann glia. The Purkinje cellular degeneration and reduced expression of glutamate transporters in the cerebellum of SCA7-KI mice were largely attenuated in the SCA7-KI/HGF-Tg mice. Moreover, phenotypic impairments exhibited by SCA7-KI mice during rotarod tests were alleviated in SCA7-KI/HGF-Tg mice. The bifunctional nature of HGF on both Purkinje cells and Bergmann glia highlight the potential therapeutic utility of this molecule for the treatment of SCA7 and related disorders.

Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus.

New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO), whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation.

Identification and characterization of novel variants of the tryptophan 2,3-dioxygenase gene: differential regulation in the mouse nervous system during development.

Tryptophan 2,3-dioxygenase (TDO), an initial and rate-limiting enzyme for the kynurenine pathway of tryptophan (Trp) metabolism, is thought to play an important role in systemic Trp metabolism as well as in emotional and psychiatric status. In contrast to its predominant expression in the liver, expression of TDO in the brain is poorly understood. Here, we show that tdo mRNA is expressed in various nervous tissues, including the hippocampus, cerebellum, striatum and brainstem. During development, tdo mRNA was differentially regulated in brain tissues. Further, we identified two novel variants of the tdo gene, termed tdo variant1 and variant2. Similar tetramer formation and enzymatic activity were obtained when these forms were expressed in wheat germ and COS-7 cells, respectively. Quantitative real-time RT-PCR revealed that tdo variants were expressed in various nervous tissues, with high expression in the cerebellum and hippocampus, followed by the midbrain. tdo variant2 was the only variant expressed in the cerebellum from postnatal day 4 (P4) to P7, suggesting a unique role for this variant during early postnatal development. Our findings indicate that tdo and its novel variants may play an important role in not only the liver but also in local areas in developing and adult brain.

Tryptophan 2,3-dioxygenase is a key modulator of physiological neurogenesis and anxiety-related behavior in mice.

Although nutrients, including amino acids and their metabolites such as serotonin (5-HT), are strong modulators of anxiety-related behavior, the metabolic pathway(s) responsible for this physiological modulation is not fully understood. Regarding tryptophan (Trp), the initial rate-limiting enzymes for the kynurenine pathway of tryptophan metabolism are tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Here, we generated mice deficient for tdo (Tdo(-/-)). Compared with wild-type littermates, Tdo(-/-) mice showed increased plasma levels of Trp and its metabolites 5-hydroxyindoleacetic acid (5-HIAA) and kynurenine, as well as increased levels of Trp, 5-HT and 5-HIAA in the hippocampus and midbrain. These mice also showed anxiolytic modulation in the elevated plus maze and open field tests, and increased adult neurogenesis, as evidenced by double staining of BrdU and neural progenitor/neuronal markers. These findings demonstrate a direct molecular link between Trp metabolism and neurogenesis and anxiety-related behavior under physiological conditions.