CommentaryA unique central tryptophan hydroxylase isoform
Introduction
Serotonin is a monoaminergic neurotransmitter involved in a wide variety of brain functions such as mood control, the regulation of sleep and body temperature, anxiety, drug abuse, food intake, and sexual behavior [1], [2], [3], [4], [5], [6] with tryptophan hydroxylase (TPH; EC 1.14.16.4) as the first-step and rate-limiting enzyme in its biosynthesis [7], [8], [9]. TPH uses Fe2+ as cofactor and O2 and tetrahydrobiopterin (BH4) as co-substrates to hydroxylate tryptophan generating 5-hydroxytryptophan (5-HT) (Fig. 1). This metabolite is decarboxylated by aromatic amino acid decarboxylase (AADC) to 5-HT or serotonin.
The serotonergic projection system is the most extensive monoaminergic system in the brain of vertebrates, but it is also the most difficult to study. The roots of this system are confined to a handful of selectively 5-HT-synthesizing neurons within the midbrain, pons, and medulla oblongata, which altogether constitute the several groups of raphe nuclei B1–B9 [10]. Furthermore, serotonin represents an intermediate product in melatonin synthesis and, therefore, the pineal gland expresses highest amounts of TPH under circadian control with maximal activity in the dark period [11]. Projections from the pineal gland to several brain regions have been described [12], [13], in which the rate-limiting enzyme in the biosynthesis of melatonin, serotonin N-acetyltransferase (AANAT) is present [14], suggesting local conversion of 5-HT to melatonin in the projection areas.
Besides the brain and the pineal gland, TPH has been found in enteric neurons [15], in preimplantation embryos [16], mast cells [17], and most prominently in enterochromaffin cells of the gastrointestinal tract [18]. These cells are supposed to be the source of 5-HT in the blood where it is almost exclusively located in the dense core storage vesicles of thrombocytes [19]. 5-HT has been implicated in different processes in peripheral tissues, such as regulation of vascular tone (“serotonin” [20], [21]) and intestinal motility [22], primary haemostasis [23], and T cell-mediated immune responses [24].
The enzyme TPH belongs to a superfamily of aromatic amino acid hydroxylases (AAAH), together with phenylalanine (PAH) and tyrosine hydroxylase (TH) [25], [26]. While the other family members have been studied in great detail concerning structure, characteristics, and regulation [27], TPH has been left behind, due to the extremely low abundance of TPH mRNA in the CNS and the difficulties to purify the protein [28], as well as to the lack of serotonin-producing neuronal cell lines suitable for in vitro studies [29].
TPH-cDNAs have been cloned from different mammalian species (rabbit: [30]; mouse: [31]; rat: [32]; human: [33]). The first TPH gene to be characterized contained 11 exons and was located on human chromosome 11 and mouse chromosome 7 [31], [34], [35], [36], [37]. For more than a decade this gene has been thought to be the only TPH gene in the vertebrate genome [38]. By targeted ablation of this TPH gene (now called Tph1) in mice, we recently discovered the existence of a second TPH isoform, TPH2, encoded by an additional gene on human chromosome 12 and mouse chromosome 10 [39]. TPH1 is mainly present in pineal gland, thymus, spleen, and gut while TPH2 predominates in brain stem. This commentary will summarize the history and the relevance of this discovery and the characteristics of TPH2.
Section snippets
Historical evidence for two TPH isoforms
Since more than thirty years there is evidence in the literature for the existence of isoforms of TPH [28], [29]. In purification procedures, two peaks of activity with different isoelectric points were detected in total brain protein preparations probably including brain stem and pineal gland [40]. Moreover, partially purified TPH enzymes with different biochemical properties were described, depending on the analyzed tissues [41], [42], [43], [44], [45]. Furthermore, the first generation of
Mice deficient in TPH1
To elucidate the physiological impact of the loss of 5-HT synthesis, we generated mice genetically deficient for TPH1 [39]. Although we had expected a lethal phenotype of this genetic manipulation, as has been found for TH knockout mice [56], surprisingly, we obtained viable homozygous Tph1-deficient (Tph1−/−) mice. These mice lack 5-HT in the periphery, in particular in the gut, in the blood and in the pineal gland (Fig. 2), and, therefore, they allow to uncover the actions of 5-HT in
Predicted characteristics of TPH2
TPH1 and TPH2 are highly homologous proteins exhibiting 71% of amino acid identity in humans (Fig. 3). All residues which have been detected to be important for the structural and functional properties of TPH1 are conserved in TPH2 [58], [59], [60], [61], [62]. Therefore, most of the features of TPH1 should also be present in TPH2. In fact, by using TPH preparations from brain stem, previous reports have already unwittingly supported this notion. It has been shown that brain stem TPH (TPH2) can
Clinical implications
As expected from the biochemical and molecular biological findings in Tph1−/− mice showing normal serotonin levels in the brain, no behavioral alterations were detectable, although the peripheral 5-HT pools were almost depleted [39]. Therefore, the behavioral effects of 5-HT are fully uncoupled from 5-HT and its metabolites in peripheral tissues. This fact is particularly important, since many efforts have been undertaken to find diagnostically useful correlations between peripheral levels of
Conclusions
The discovery of TPH2 explains the previously puzzling data compiled in the past thirty years about divergent protein/mRNA ratios and biochemical characteristics of TPH from peripheral sources and from the CNS. More importantly, it justifies a change in the concept of the serotonin system. In fact, there are two serotonin systems in vertebrates with independent regulation and distinct functions defined by the two TPH isoforms, TPH1 and TPH2 (Fig. 4). Consequently, both systems can be targeted
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