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In Drosophila, one enzyme (Drosophila tryptophan-phenylalanine hydroxylase, DTPHu) hydroxylates both tryptophan to yield 5-hydroxytryptophan, the first step in serotonin synthesis, and phenylalanine, to generate tyrosine. Analysis of the sequenced Drosophila genome identified an additional enzyme with extensive homology to mammalian tryptophan hydroxylase (TPH), which we have termed DTRHn. We have shown that DTRHn can hydroxylate tryptophan in vitro but displays differential activity relative to DTPHu when using tryptophan as a substrate. Recent studies in mice identified the presence of two TPH genes, Tph1 and Tph2, from distinct genetic loci. Tph1 represents the non-neuronal TPH gene, and Tph2 is expressed exclusively in the brain. In this article, we show that DTRHn is neuronal in expression and function and thus represents the Drosophila homologue of Tph2. Using a DTRHn-null mutation, we show that diminished neuronal serotonin affects locomotor, olfactory and feeding behaviors, as well as heart rate. We also show that DTPHu functions in vivo as a phenylalanine hydroxylase in addition to its role as the peripheral TPH in Drosophila, and is critical for non-neuronal developmental events.
Biogenic amines are ancient, evolutionarily conserved molecules, which function in many diverse physiological contexts in both vertebrate and invertebrate species, including modulation of behavior, fertility, reproduction and the development of neuronal and non-neuronal tissues (reviewed by Monastirioti 1999). Perturbation of biogenic amine signaling pathways has been correlated with serious neurological diseases in humans, such as depression, obsessive-compulsive disorder (Mockus & Vrana 1998) and Parkinson’s disease (Cao et al. 1995).
The biogenic amines are formed by the actions of the aromatic amino acid hydroxylases: tryptophan hydroxylase (TPH) (EC 18.104.22.168), tyrosine hydroxylase (TH) (EC 22.214.171.124) and phenylalanine hydroxylase (PAH) (EC 126.96.36.199). TPH catalyzes the synthesis of 5–hydroxytryptophan from tryptophan, the first and rate-limiting step in serotonin (5–hydroxytryptamine, 5-HT) biosynthesis; similarly, TH catalyzes the formation of L-dihydroxyphenylalanine from tyrosine, the first and rate-limiting step in dopamine biosynthesis. PAH is a metabolic enzyme that catalyzes the conversion of phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism, and protein and neurotransmitter biosynthesis. The genes encoding these enzymes have likely derived from an ancestral aromatic amino acid hydroxylase, as there is extensive sequence and biochemical conservation between them.
It has recently been discovered that in mammals, there are two distinct and largely nonoverlapping TPH enzymes. The first, encoded by Tph1, is expressed in the periphery, and the second, Tph2, is expressed within the central nervous system (CNS) (Walther et al. 2003; Zhang et al. 2004). In mice, loss of Tph1 expression results in cardiac dysfunction (Cote et al. 2003). Polymorphisms within the Tph2 locus have been associated with major depressive disorder (Harvey et al. 2004), and a loss of function mutation within Tph2 has been found within a significant proportion of patients with unipolar major depression (Zhang et al. 2005). While these data provide strong evidence for compartmentalization of expression and function of the two 5-HT-synthesizing enzymes, there are limited genetic tools available in mammalian systems to fully assess their individual contributions to 5-HT synthesis.
5–hydroxytryptamine is the final product of the actions of the TPH enzymes. In mammals, 5-HT regulates appetite, sleep, learning and memory, temperature regulation and sexual behavior (Wilkinson & Dourish 1991). There is an overwhelming body of evidence suggesting that 5-HT signaling is a major modulator of emotional behavior including anxiety, impulsivity and aggression, as well as integrated complex brain functions such as cognition, sensory processing and motor activity (Westenberg 1996); serotonin has also been implicated in the actions of numerous drugs of abuse (Lucki & Wieland 1990). Recent studies in mammals show that Tph2 is responsible for the rhythmic fluctuations in 5-HT synthesis (Malek et al. 2005), which modulates entrainment of the circadian clock. 5–hydroxytryptamine also serves as a hormone, cytokine, biological modifier, growth factor and regulator of vascular tone and intestinal activity (Hoyer et al. 2002; Kroeze et al. 2002; Seuwen & Pouyssegur 1990; Talley 2001). It has been suggested that 5-HT orchestrates the activity and interaction of several other transmitter systems, which can account for the diversity of these roles. In Drosophila and other insects, 5-HT also modulates circadian rhythms (Yuan et al. 2005), reproduction (Barreteau et al. 1991), feeding (Novak & Rowley 1994; Novak et al. 1995), heart rate (Dasari & Cooper 2006) and locomotion (Kamyshev et al. 1983), evidence for conservation of these numerous modulatory roles for 5-HT. This is consistent with the observation that there are only ∼100 serotonergic neurons within the Drosophila CNS but, as in higher vertebrates, these neurons send projections to most parts of the nervous system (Valles & White 1988).
In mammals, 5-HT is first detected in the early stages of gastrulation where it appears to regulate cell division and migration (Lauder et al. 1981). It is one of the first biogenic amines to appear in the developing CNS (Lauder & Bloom 1974); high levels of 5-HT are detected within the cells before and during early neurite outgrowth, suggesting that it might serve a trophic role during development prior to assuming its adult functions of neurotransmission and neuromodulation (Azmitia & Whitaker-Azmitia 1991). In Drosophila, 5-HT plays a vital role in early embryonic development, before the appearance of neuroblasts (Colas et al. 1999a,b), and studies in other insects, primarily Manduca sexta, have shown a requirement for 5-HT in the development of olfactory glomeruli (Mercer et al. 1995, 1996; Oland et al. 1995), suggesting the neuronal and non-neuronal developmental roles for 5-HT have also been conserved.
The predicted size of mammalian Tph1 is 51 kDa, which is similar to that of Drosophila tryptophan-phenylalanine hydroxylase (DTPHu) (50 kDa); the predicted size of mammalian Tph2 is 57 kDa, similar to the size of DTRHn (61 kDa); DTRHn shares slightly more sequence homology with Tph2 than with Tph1 (Coleman & Neckameyer 2005). Like mammalian Tph2, a larger N-terminal domain accounts for the increased size of DTRHn relative to Tph1 and DTPHu. Both DTPHu and DTRHn are subject to substrate inhibition by tryptophan, although not by phenylalanine (Coleman & Neckameyer 2004, 2005), which is similar to Tph1 and Tph2 (McKinney et al. 2004, 2005). Both Tph1 and Tph2 are phosphorylated by cyclic adenosine 5′-phosphate-dependent protein kinase; whereas DTPHu TPH activity is increased after phosphorylation by this kinase, that of DTRHn remains unchanged (Coleman & Neckameyer 2004, 2005), which is what has been observed for recombinant Tph2 (McKinney et al. 2005). These data suggest that mechanisms for biochemical regulation of the two enzymes have been conserved.
Given its numerous critical and vital functions, 5-HT levels must be appropriately regulated to correctly modulate these multifunctional roles. A mammalian knockout of Tph2 is not yet available, and assessing behavioral roles for the Tph1 mouse knockout is problematic because these mice die shortly after birth from cardiac malfunction. The genetic tractability of the Drosophila model system, which permits targeted in vivo manipulation of 5-HT synthesis in specific tissues, circumvents these issues. In this article, we show that, like mammals, Drosophila has compartmentalized the neuronal and peripheral regulation of serotonin by having two distinct enzymes, each specific for neuronal or peripheral synthesis, suggesting that this is an ancient and highly conserved mechanism.
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The experiments described above show that compartmentalization of 5-HT synthesis is evolutionarily conserved and may reflect the complex requirements for appropriate regulation of 5-HT as a trophic factor in both neuronal and non-neuronal tissues. Knockdown (Fig. 8) or knockout (Colas et al. 1999a) of DTPHu results in diminished embryonic survival or embryonic lethality, respectively, whereas knockout of DTRHn results in enhanced embryonic survival (Fig. 7a). In the DTRHn mutant, a significant decrease in the number of flies eclosing is observed, attributable to increased larval lethality (Fig. 7b), but knockdown of DTPHu in fat body has no effect at this stage (Fig. 8d). Knockdown of DTPHu in this tissue also results in increased motor activity (Fig. 9b,c), but knockout of DTRHn has no effect on this behavior in larvae (Fig. 5b), and decreases locomotor activity in adults (Fig. 6a). Continued knockdown of DTPHu in fat body increases feeding behavior (Fig. 9c), whereas feeding is significantly depressed in DTRHn-null mutants (Fig. 5a). Clearly, the effects of decreases in DTRHn and DTPHu expression in their target tissues are nonoverlapping, evidence that the two enzymes play different functional roles in 5-HT synthesis. In mammals, knockout of tph1 does not result in overexpression of tph2 to compensate for loss of 5-HT synthesis in the relevant tissues (Cote et al. 2003), suggesting that the two genes are clearly under separate transcriptional and translational controls. Similarly, knockout of DTRHn does not result in synthesis of 5-HT by DTPHu in the CNS (although there is reuptake of peripheral 5-HT synthesized by DTPHu), and DTRHn does not synthesize 5-HT for early developmental events in the absence of DTPHu. The genetic tractability and evolutionary conservation of Drosophila melanogaster thus provide a unique model in which to elucidate the distinct mechanisms governing precise control of 5-HT synthesis.