Serotonin synthesis by two distinct enzymes inDrosophila melanogaster

Abstract

Annotation of the sequenced Drosophila genome suggested the presence of an additional enzyme with extensive homology to mammalian tryptophan hydroxylase, which we have termed DTRH. In this work, we show that enzymatic analyses of the putative DTRH enzyme expressed in Escherichia coli confirm that it acts as a tryptophan hydroxylase but can also hydroxylate phenylalanine, in vitro. Building upon the knowledge gained from the work in mice and zebrafish, it is possible to hypothesize that DTRH may be primarily neuronal in function and expression, and DTPH, which has been previously shown to have phenylalanine hydroxylation as its primary role, may be the peripheral tryptophan hydroxylase in Drosophila. The experiments presented in this report also show that DTRH is similar to DTPH in that it exhibits differential hydroxylase activity based on substrate. When DTRH uses tryptophan as a substrate, substrate inhibition, catecholamine inhibition, and decreased tryptophan hydroxylase activity in the presence of serotonin synthesis inhibitors are observed. When DTRH uses phenylalanine as a substrate, end product inhibition, increased phenylalanine hydroxylase activity after phosphorylation by cAMP-dependent protein kinase, and a decrease in phenylalanine hydroxylase activity in the presence of the serotonin synthesis inhibitor, alpha-methyl-(DL)-tryptophan are observed. These experiments suggest that the presence of distinct tryptophan hydroxylase enzymes may be evolutionarily conserved and serve as an ancient mechanism to appropriately regulate the production of serotonin in its target tissues.

Tryptophan Hydroxylase as Novel Target for the Treatment of Depressive Disorders

“…Therefore, for a long time Henna has been considered to be the PAH and the TPH of insects until the main TPH, CG9122, was discovered [158] .…”

A Trophic Role for Serotonin in the Development of a Simple Feeding Circuit

“…In Drosophila , as in mammals, there are two discrete Tph enzymes: Drosophila tryptophan-phenylalanine hydroxylase (DTPH) is largely nonneuronal, expressed in the periphery, and corresponds to mammalian Tph1; Drosophila tryptophan hydroxylase (DTRH) is exclusively neuronal and corresponds to mammalian Tph2 [Walther et al, 2003;Zhang et al, 2004;Coleman and Neckameyer, 2005;Neckameyer et al, 2007]. Biochemical regulation of these enzymes is strikingly similar to that of mammals Neckameyer, 2004, 2005].…”

“…DTRH is expressed solely within 5-HT neurons; the only homologous genes are DTPH and Drosophila tyrosine hydroxylase, both expressed in DA neurons. However, there is considerable third base wobble, greatly limiting homology at the DNA level, so it is not likely that the dsRNAi DTRH transgene will affect the expression of these genes [Coleman and Neckameyer, 2005]. Although both the GSelav and elav C155 promoters drive ectopic DTRH expression, by immunohistochemical analyses, 5-HT expression is limited to 5-HT neurons (data not shown).…”

“…Larval brains were dissected from each genotype (elav C155 /w 1118 , n = 9; elav C155 /DTRHE, n = 10, and elav C155 / DTRHA, n = 14). The 5-HT cell pattern in each brain was visualized with a polyclonal antibody raised against DTRH [Coleman and Neckameyer, 2005] and viewed under fluorescence. a The pixel intensity of the DTRH immunofluorescence of the two unpaired neurons in the 8th abdominal neuromere was determined to ascertain the extent of knockdown of DTRH.…”

“…Induction and knockdown of DTRH expression and 5-HT levels were assessed by quantitation of changes in fluorescent intensity of specific neurons after visualization with an antibody raised against DTRH [Coleman and Neckameyer, 2005] or against 5-HT (Spring Biosciences, Fremont, Calif., USA). The GSelav driver [Osterwalder et al, 2001] was used to induce temporal expression of the TRH RNAi transgene or the TRH UAS transgene using RU486 (Mifepristone, Sigma).…”