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Implication of Tryptophan 2,3-Dioxygenase and its Novel Variants in the Hippocampus and Cerebellum During the Developing and Adult Brain

Tryptophan 2,3-dioxygenase (TDO) is a first and rate-limiting enzyme for the kynurenine pathway of tryptophan metabolism. Using Tdo−/− mice, we have recently shown that TDO plays a pivotal role in systemic tryptophan metabolism and brain serotonin synthesis as well as emotional status and adult neurogenesis. However, the expression of TDO in the brain has not yet been well characterized, in contrast to its predominant expression in the liver. To further examine the possible role of local TDO in the brain, we quantified the levels of tdo mRNA in various nervous tissues, using Northern blot and quantitative real-time RT-PCR. Higher levels of tdo mRNA expression were detected in the cerebellum and hippocampus. We also identified two novel variants of the tdo gene, termed tdo variant1 and variant2, in the brain. Similar to the known TDO form (TDO full-form), tetramer formation and enzymatic activity were obtained when these variant forms were expressed in vitro. While quantitative real-time RT-PCR revealed that the tissue distribution of these variants was similar to that of tdo full-form, the expression patterns of these variants during early postnatal development in the hippocampus and cerebellum differed. Our findings indicate that in addition to hepatic TDO, TDO and its variants in the brain might function in the developing and adult nervous system. Given the previously reported associations of tdo gene polymorphisms in the patients with autism and Tourette syndrome, the expression of TDO in the brain suggests the possible influence of TDO on psychiatric status. Potential functions of TDOs in the cerebellum, hippocampus and cerebral cortex under physiological and pathological conditions are discussed.


Introduction
Tryptophan and its metabolites, such as kynurenines and serotonin, are involved in biological functions, such as protein synthesis, energy synthesis, immune regulation, and higher-order brain functioning. [1][2][3][4] The mechanisms of mood modulation and disturbance that contribute to social functioning have become a particularly important issue in the 21st century. Identifying the mechanisms regulating tryptophan and its metabolites in the brain represents a key issue for not only understanding the mechanisms regulating emotion but also alleviating social problems. In contrast to the systemic regulation of tryptophan metabolism, molecular mechanisms and local factors regulating tryptophan (kynurenine) metabolism in the brain are not well understood. There is evidence that, in the brain, indoleamine 2,3-dioxygenase (IDO), [5][6][7][8][9] an initial and rate-limiting enzyme for the kynurenine pathway, is markedly induced by immune signals (including interferon-gamma) and that IDO plays an important role in kynurenine production and immune response under pathological conditions in vivo and in vitro. [6][7][8][9][10][11] In contrast, under physiological conditions, other molecule(s) are likely to play a key role in regulating kynurenine metabolism of the adult brain, as IDO levels in the adult nervous systems are extremely low.
A suitable candidate is tryptophan 2,3-dioxygenase (TDO), which is another initial and rate-limiting enzyme for the kynurenine pathway of tryptophan metabolism. [1][2][3][12][13][14][15] We have previously studied tdo-deficient (Tdo -/-) mice and have demonstrated that TDO plays important roles in systemic tryptophan metabolism and brain serotonin synthesis as well as modulation of emotional status and adult neurogenesis. 16 In support of these findings, some genetic studies have demonstrated an association of human TDO2 single nucleotide polymorphisms (SNPs) with psychiatric diseases, such as Tourette syndrome and autism. 17,18 Moreover, antidepressants have been postulated to act by directly inhibiting the activity of hepatic TDO, thereby enhancing the availability of cerebral tryptophan. 19,20 These findings demonstrate that TDO play an important role not only in systemic tryptophan metabolism but also in mood regulation and psychiatric disease. However, although TDO is predominantly expressed in the liver and a few studies have suggested that tdo is expressed in the rodent or human brain, 11,21,22 the precise distribution and regulation of local TDO in the brain has not yet been well characterized.
Here, we describe the identification, localization and regulation of novel tdo variants, which we termed tdo variant1 and tdo variant2, in addition to tdo fullform, during brain development, and in the brain and liver of adult mice. The potential roles of TDOs in the brain under physiological and pathophysiological conditions are also discussed.

Materials and Methods Animal
Male C57BL/6J mice were purchased from Nippon SLC (Hamamatsu, Japan) and maintained as previously described. 23 Acquisition, care, housing, use, and disposition of the animals were in compliance with the institutional laws and regulations of Osaka University Graduate School of Medicine. All efforts were made to minimize both animal discomfort and the number of animals used.
cDnA cloning of mouse tdo variants cDNA cloning of mouse tdo variants was performed as previously decribed. 23 Briefly, cDNAs were synthesized from adult C57BL/6J mice midbrain and liver total RNA using oligo d(T) 20 primer and superscript III reverse transcriptase. Amplification of mouse tdo variants cDNA was done using TaqMan AmpliTaq Gold Master Mix and the following primer set: forward primer, 5′-ATCTCTCTCTCCCTCTACTTC-3′, and reverse primer, 5′-TCAATCCGATTCATCGCT GC-3′. These primer sequences were designed based on the rat tdo minor start site. 24 Amplified cDNA fragments (about 1600 bp and 1560 bp) were subcloned into pGEM-T easy vector (Promega), which was subjected to sequence analyses.

Quantitative real-time RT-pCR
Total RNAs were purified from tissues (liver, cerebral cortex, hippocampus, pons, striatum, midbrain, and cerebellum) of postnatal day 1 to 49. First-strand cDNA was prepared from 5 µg of DNase I-treated total RNA; quantitative real-time RT-PCR and quantitation of the tdo genes were performed as previously described. 23 Briefly, the expression of tdo genes was quantified using universal PCR master mix and TaqMan Gene Expression assays to amplify mouse tdo exons 4-5 (thus amplifying full-length tdo as well as tdo variant1 and variant2) and rodent GAPDH. The expression of tdo variants was quantified using Power SYBR Green PCR master mix and the following primer sets: tdo variant1, 5′-GCACTAAAGTATCTGGGAAGG-3′ and 5′-CTCCTTTGCTGGCTCTGTTT-3′; tdo variant2, 5′-TGTAAGCTGGGTGCTGATTG-3′ and 5′-GTGTATCTTTTATGTATCCTG-3′. Realtime RT-PCR was carried out as described. 23 Since the expression levels of tdo full-form were much higher than those of tdo variant1 and variant2 in all indicated samples examined (for example, the levels of tdo variant1 and varinat2 were about 1% and 0.01% compared to those of tdo full-form in the liver, respectively), calculated values of tdo exon 4-5 (reflecting the sum of tdo full-form, tdo varinat1 and tdo variant2) are nearly identical to the levels of tdo full-form, and thus the values are expressed as the levels of tdo fullform. Results were expressed as the means ± S.E. of duplicate replicates.

TDo enzymatic activity
At 48 h post-Transfection with pCAGGS-tdo, -tdo variant1, or -tdo variant2, COS-7 cells were lysed in 0.02 M phosphate buffer (pH 7.4) containing 2 mM Trp and proteinase inhibitor cocktails (Roche Diagnostics, Germany). 23 TDO enzymatic activities were assessed by the conversion of Trp to Kyn as previously described. 23 TDO specific activities were expressed as units (µmoles of Kyn formed per hour at 37 °C) per milligram of each protein. The amounts of each protein (TDO and its variants) were determined by Western blotting using recombinant TDO protein as a standard (Geneway Biotech, CA). 23 Values represent means ± S.E. (n = 5 in each group).

Results
Identification and expression of tdo variant1 and variant2 mRnAs, in addition to tdo full-form in various nervous tissues We first performed northern blot analyses using a full-length tdo cDNA probe to visualize all tdo transcripts in various brain tissues. Liver total RNA served as a positive control for northern blot analysis. Long exposure film revealed that, in addition to the fullform size band, lower and higher bands were detected in the brainstem, hippocampus, cerebellum, and liver (see Fig. 1A in ref. 23). These results suggested that novel variants of the tdo gene were present.
To further examine brain tdo mRNA expression, we next performed quantitative real-time RT-PCR, using two primer sets, tdo exon 1-2 and exon 4-5, in order to detect different parts of tdo mRNA. Although lower than in the liver, significant levels of tdo mRNAs were detected in various brain regions, including the cerebral cortex, striatum, pons, and midbrain. Consistent with our northern blot findings, high expression levels of tdo were detected in the hippocampus and cerebellum. Interestingly, more than a 10-fold difference was detected in the expression levels of tdo between exon 1-2 and exon 4-5 in the cerebral cortex, striatum, cerebellum, pons, and midbrain (see Fig. 1B in ref. 23). These findings demonstrate that novel variant(s) lacking these regions of exons 1 and 2 of the tdo gene are present in the brain.
After additional analyses, we identified three forms of tdo mRNAs ( Fig. 1; see details in ref. 23). One is a full-length form corresponding to a traditional tdo containing 12 exons. The second is a longer form that contains exon 0a + b of the tdo gene, which we termed tdo variant1. 23 The third is a shorter form that contains exon 0a but lacks exon 1, which we termed tdo variant2. 23 To assess the expression of tdo variants, we performed northern blot analyses using probes covering different parts of the tdo gene. One is a probe covering full-length tdo cDNA, which detects all three forms of tdo genes in covering tdo exon 0 (-400 ∼ -15), and thus detects tdo variant1 and variant2 but does not detect full-length tdo. The last is a probe covering tdo exon 1 (1-70) and thus detects both full-length tdo and tdo variant1. As previously described in the brain tissues and the liver (ref. 23), northern blotting using a probe covering fulllength tdo revealed that at least three tdo mRNAs are present in the liver ( Fig. 2A). Using a probe covering exon 0, we found that tdo mRNA containing exon 0 was detected at low levels and its size was nearly identical to the full-length tdo mRNA in the liver. As both tdo variants contain a part of or whole exon 0, we cannot distinguish which variant (or a new variant) is responsible for the band in Figure 2B. But our findings clearly demonstrate that one of tdo variants is present and its mRNA size is close to that of full-length tdo (Fig. 2B,  arrows). Using a probe covering exon 1, we detected three bands as indicated in Figure 2C. We speculate that additional (new) tdo variant(s) containing exon 1 would be present, as we identified two extra mRNA bands containing exon 1 in addition to the band correspond to the full-length tdo in Figure 2C.
Next, to assess the expression of tdo mRNAs in brain tissues, we performed quantitative RT-PCR. It revealed that both tdo variant1 and variant2 were expressed in various brain regions, and a relatively high expression of these variants was detected in the hippocampus and cerebellum, in addition to the tdo full-form, as previously described (see Fig. 2 in ref. 23). These results suggest that tdo and its two novel variants are expressed in various brain regions, with particularly high levels seen in the hippocampus and cerebellum.

Regulation of tdo mRNA expression in the hippocampus and cerebellum during development
To clarify the regulation of tdo mRNAs in the hippocampus during development, we examined the expression of tdo mRNAs in the hippocampus from postnatal day1 (P1) to P49. 23 Quantitative RT-PCR revealed that the levels of tdo full-form were high from P4, when the organization of neural networks begins, and were maintained at the same levels until P49 (levels of tdo full-form were calculated from the values of exon 4-5; see "Materials and Methods"). 23 While expression levels of tdo variants were lower than the full-form, expression levels of tdo variants gradually increased after P14 in the hippocampus (Fig. 3A). 23 In the cerebellum, levels of tdo full-form were markedly increased at P14, and A. Hippocampus

B. Cerebellum tdo exon 4-5 or variant2
(Relative ratio to P49 liver) tdo exon 4-5 or variant2 (Relative ratio to P49 liver)   maintained at the same level until P49. In comparison, levels of tdo variant2 were low at P1, but were sustained at high levels from P4 to P49 (Fig. 3B). 23 TDo variant1 and variant2 are functional proteins with full-TDo enzymatic activity We synthesized each protein of TDO and its variants in COS-7 cell lines and analyzed its cell lysates by Western blotting using the antibody generated against International Journal of Tryptophan Research 2010:3 rat purified TDO that can also detect mouse TDO. 23 Western blotting of cell lysates revealed that the molecular weight of mouse TDO full-form and that of TDO variant1 was identical. 23 In contrast, the molecular size of variant2 was slightly lower than that of the other forms, which fits well with the notion that translation of tdo variant1 would begin from exon 1, and that of variant2 would begin from exon 2, as there is no theoretical translation start site in exon 0.
We further examined whether recombinant TDO variants form tetramer, since native rat and human TDO full-form proteins form a tetramer with heme, and tetramer formation is required for TDO full enzymatic activity. 25 Western blotting showed that recombinant mouse TDO full-form migrated as a 44 kDa band at the monomer size in SDS-PAGE, while in Native-PAGE it migrated as an approximately 180 kDa band, which was 4 times higher than monomer size, indicating that it forms tetramer. TDO vari-ant1 and variant2 were detected as a large complex in Native-PAGE, and these molecular weights were about 4 times greater than each monomer size, which demonstrates that the two novel variants form tetramer as well (see Fig. 4 in ref. 23). The enzymatic assays for TDOs revealed that all three forms had similar full-enzymatic activity (Table 1). 23 Although further work is required in order to examine the detailed kinetics of these variants, our findings demonstrate that these variants are functional proteins with full-TDO activity.

Discussion
Proper neural network development is important for cognitive development, especially in the hippocampus and cerebellum. Tryptophan is known to be involved in the development of these brain regions and their associated cognitive and behavioral functions (Fig. 4). For example, tryptophan restriction during early postnatal development reduced dendrite arborization, spine density, and cell proliferation in the dentate gyrus of the hippocampus, and caused anxiety-and depressive-like behavior. 26 Furthermore, its metabolite kynurenine, which is produced in primary neurons and glial cells, increases production of nerve growth   18,26,30 TDo may also contribute to physiological mood regulation via hippocampal neurogenesis, maintenance of neural cells, and early cerebellar development. 16,23 factor in astrocytes and potentially promotes survival of new neurons in the adult hippocampus. 2,10,11,27,28 Our previous studies of Tdo -/mice have demonstrated the critical role of TDO in hippocampal neurogenesis and the maintenance of hippocampal neurons as well as in anxiety-related behavior. 16 Moreover, the expression level of tdo mRNA was significantly decreased (over 90%) in the immature dentate gyrus of adult alpha-CaMKII hetero-knockout mice, which impaired working memory and mood regulation. 29 Consistent with previous studies, our quantitative study demonstrated the expression of TDOs in various brain regions, thereby suggesting that TDO and its variants, in addition to IDO, potentially produce N-formylkynurenine locally in the nervous system under physiological condition. 11,21,23 Collectively, the predominant local expression and regulation of fullform tdo among the various forms in the hippocampus suggests that tdo full-form is the dominant TDO form to modulate hippocampal functions including mood and memory, and this activity would be mediated by neurogenesis and maintenance of neural cells during development and adulthood.
In addition to its role in the hippocampus, tryptophan is thought to play an essential role in early cerebellar development. This is based on the finding that a tryptophan deficient corn-based diet begun 5 weeks before mating leads to the retardation of Bergmann glial maturation as well as a concomitant granular cell migration. 30 Furthermore, it is intriguing that human TDO SNPs have been associated with autism, 18 and that pathological changes have been reported in the postmortem cerebellum of the patients with autism (Fig. 4). 31 Since tryptophan is essential for early cerebellar development and tdo SNPs may be associated with autism, local predominant expression and regulation of tdo variant2 in the cerebellum during early postnatal development suggests that the tdo variant2, in addition to tdo full-form, plays an important role in early cerebellar development and may therefore be implicated in the development of autism.
In addition to these roles, dysregulation of the kynurenine pathway has been associated with psychiatric and neurodegenerative diseases, such as depression and Alzheimer's diseases. [2][3][4] There are some reports that local IDO in the brain represents one mechanism of pathology in these disorders, especially when the immune system is activated. 4,7 However, the presence of different molecular sized TDO proteins and altered immunoreactivity against TDO has been reported in the frontal cortex of individuals with schizophrenia and depression. 21 In addition, our findings of local expression of TDOs in the cerebral cortex suggests a local role of TDO and its variants in the specific regional modulation of tryptophan metabolism and subsequent behavioral and immune modulation, which would likely be regulated via a different mechanism from IDO under such pathological conditions.
In summary, in addition to the liver TDO, we present evidence that TDO, as well as newly identified TDO variants, are locally expressed and regulated in the brain, and therefore may play a critical role in the hippocampal and cerebellar development and function (Fig. 5).
Dysregulation of brain or liver TDOs may be involved in psychiatric or neurodegenerative disorders such as anxiety-related disorders, autism, or multiple sclerosis. Further analyses of brain cells expressing newly identified TDO variants in addition to the TDO full-form, the possible identification of new tdo variant(s), and the elucidation of the regulation and roles of these molecules may open a new avenue for understanding the role of tryptophan metabolites in higher-order brain functions in the future.

Liver Brain
• TDO and its novel variants (TDOs) are locally expressed and regulated in the brain.
• TDOs, in addition to IDO, would play a role in converting Trp to N-formylkynurenine under physiologcal conditions.
• TDOs may contribute to physiological hippocampal and cerebellar development and function.

Figure 5.
A working model of TDo function. In addition to the liver, tdo mRNAs are expressed in various brain regions, with high expression in the hippocampus and cerebellum. In the hippocampus, the level of tdo full-form was continuously expressed at a higher level than tdo variants, while tdo variant2 was highly expressed from early development in the cerebellum. Therefore, TDOs, in addition to IDO, potentially contribute to local tryptophan metabolism in the brain, and in turn to physiological hippocampal and cerebellar development and functions. Dysregulation of brain TDos may, at least in part, influence the development of psychiatric disorders, such as anxiety-related disorders, autism, and schizophrenia.
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