Synthesis of Serotonin by a Second Tryptophan Hydroxylase Isoform

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Science  03 Jan 2003:
Vol. 299, Issue 5603, pp. 76
DOI: 10.1126/science.1078197

The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] is causally involved in multiple central nervous facets of mood control and in regulating sleep, anxiety, alcoholism, drug abuse, food intake, and sexual behavior (1). In peripheral tissues, 5-HT regulates vascular tone, gut motility, primary hemostasis, and cellmediated immune responses (1). 5-HT is synthesized in two steps, with tryptophan hydroxylase (TPH) as the rate-limiting enzyme (2). TPH belongs to a superfamily of aromatic amino acid hydroxylases, together with phenylalanine (PAH) and tyrosine hydroxylases (TH), and has been detected mainly in the brain stem and gut enterochromaffin cells (2).

To study the physiological impact of the loss of 5-HT synthesis, we generated mice genetically deficient for TPH (Tph−/−). Like wild-type (Tph+/+) siblings and the mouse strains from which the Tph−/− animals were derived,Tph−/− mice still expressed normal amounts of 5-HT in classical serotonergic brain regions. However, Tph−/− mice lacked 5HT in the periphery except for in the duodenum (Fig. 1A), which contained about 4% of normal 5-HT levels, probably because of the serotonergic neurons in this tissue (3). Concordantly, Tph−/− mice exhibited no significant behavioral differences in elevated plus maze and hole board tests (table S1), which are indicative for 5-HT–related behavior.

Figure 1

(A) 5-HT in whole blood, duodenum, hippocampus, and frontal cortex of 129SvJ, C57BL/6, Tph+/+, and Tph−/− mice. n.d., below the detection limit of the assay (<5 pg/μl). *, statistically significant (P < 0.05), as compared with all other investigated mouse lines. +, statistically significant (P < 0.05), as compared withTph+/+ mice, but not with other laboratory mouse lines. (B) RNase protection assays with probes specific for the two isoforms reveal the presence of Tph1 mRNA in duodenum of wild-type animals, but not in Tph−/− mice. Tph2mRNA is present exclusively in brain, irrespective of the genotype. (C) Reversed-phase HPLC-FD chromatograms of TPH activity assays. (Top) Untransfected COS7 cells. (Bottom) COS7 cells transiently transfected with a eukaryotic expression vector containing the mouse Tph2 cDNA. 5-Hydroxylated tryptophan (5-HTP) elutes with a retention time of 5 min.

Despite suggestions of a possible second TPH isoform (4), molecular verification has been lacking. Therefore, we screened the High Throughput Genomic Sequences database of GenBank with short translated sequences of Tph and obtained a human genomic clone on chromosome 12, with an open reading frame similar to Tph exon 4, but different fromPah, which is located on the same chromosome. On the basis of this sequence, we performed 5′- and 3′-RACE experiments with brain RNA of Tph−/− mice and obtained the full-length cDNA (referred to as Tph2; GenBank: AY090565; fig. S1), which was different from the known Tph (referred to here now asTph1), Pah, and Th of the mouse.

We detected Tph1 mRNA in the duodenum, but not in the brain, by ribonuclease (RNase) protection assays (RPAs) specific for each Tph isoform (Fig. 1B). In contrast, Tph2 was detected exclusively in the brain (Fig. 1B). Brain stem total RNA samples from wild-type mice revealed about 150 times moreTph2 than Tph1 mRNA by RPAs (5).

Transfected COS7 cells expressing TPH2 acquired tryptophan-hydroxylating activity, confirming the identity of this enzyme (Fig. 1C). Moreover, commercially available antibodies against TPH1 also detected TPH2 in transfected cells (5), thus explaining previous detection of TPH in the brain.

We also cloned and sequenced the rat and human TPH2 homologs (GenBank: AY098915 and AY098914; fig. S1) and detected TPH2 expression in rat brain (5). By searching GenBank, we found one homologous expressed sequence tag from chicken and several from zebrafish brain, in addition to a sequence from zebrafish ovary with high homology to the mammalian TPH1 (6).

The discovered duality of the serotonin system in vertebrates may open up new avenues for specific therapeutic approaches exclusively affecting central or peripheral 5-HT actions. This is particularly important, because of efforts to find diagnostically useful correlations between peripheral levels of 5-HT and its metabolites and 5-HT function in the central nervous system of human patients suffering from psychiatric disorders (7). Also, numerous attempts linking polymorphisms in the Tph1 gene with such diseases (1) have to be reconsidered now that it is known that this gene is hardly expressed in the brain.

Supporting Online Material

Materials and Methods

Fig. S1

Table S1

  • * To whom correspondence and requests for materials should be addressed. E-mail: dwalther{at} or mbader{at}


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