Distribution of the C1473G polymorphism in tryptophan hydroxylase 2 gene in laboratory and wild mice

The neurotransmitter serotonin is implicated in the regulation of various forms of behavior, including aggression, sexual behavior and stress response. The rate of brain serotonin synthesis is determined by the activity of neuronal‐specific enzyme tryptophan hydroxylase 2. The missense C1473G substitution in mouse tryptophan hydroxylase 2 gene has been shown to lower the enzyme activity and brain serotonin level. Here, the C1473G polymorphism was investigated in 84 common laboratory inbred strains, 39 inbred and semi‐inbred strains derived from wild ancestors (mostly from Eurasia) and in 75 wild mice trapped in different locations in Russia and Armenia. Among all the classical inbred strains studied, only substrains of BALB/c, A and DBA, as well as the IITES/Nga and NZW/NSlc strains were homozygous for the 1473G allele. In contrast to laboratory strains, the 1473G allele was not present in any of the samples from wild and wild‐derived mice, although the wild mice varied substantially in the C1477T neutral substitution closely linked to the C1473G polymorphism. According to these results, the frequency of the 1473G allele in natural populations does not exceed 0.5%, and the C1473G polymorphism is in fact a rare mutation that is possibly eliminated by the forces of natural selection.     

Knockout mouse points to second form of tryptophan hydroxylase

A second form of tryptophan hydroxylase (TPH) is expressed in the brain by the gene Tph2. The presence of the gene was discovered when Tph 1(-/-)mice were found to express normal amounts of serotonin in brain, but not in the periphery. Additionally, Tph1(-/-) mice showed no observed behavioral differences from wild-type littermates. Veenstra-Vanderweele and Cook discuss the ramifications of these findings and what they might mean for designing drugs that target the expression and activity of TPH in differing tissues.     

The C1473G polymorphism in gene tph2 is the main factor mediating the genetically defined variability of tryptophan hydroxylase-2 activity in the mouse brain

Brain neurotransmitter serotonin is involved in the regulation of many physiological functions and types of behavior. The key enzyme of serotonin synthesis in the brain is tryptophan hydroxylase-2 (TPH-2). An association of the C1473G polymorphism in gene tph2 causing the replacement of Pro447 by Arg447 in TPH-2 molecule with enzyme activity in the mouse brain of 10 inbred strains was found. Association of the polymorphism with the TPH-2 activity in the brain of F2 hybrids between strains C57BL/6 and CC57BR was shown. The results indicate that the C1473G polymorphism in gene tph2 is the main factor determining the genetic defined variability of enzyme activity in the mouse brain.     

From genes to aggressive behavior: the role of serotonergic system

Recent investigations in neurogenomics have opened up new lines of research into a crucial genetic problem-the pathway from genes to behavior. This paper concentrates on the involvement of protein elements in the brain neurotransmitter serotonin (5-HT) system in the genetic control of aggressive behavior. Specifically, it describes: (1) the effect of the knockout of MAO A, the principal enzyme in 5-HT degradation, (2) the association of intermale aggression with the polymorphism in the Tph2 gene encoding the key enzyme in 5-HT synthesis in the brain, tryptophan hydroxylase (TPH), and (3) the effect of selective breeding for nonaggressive behavior on 5-HT metabolism, TPH activity and 5-HT(1A) receptors in the brain. The review provides converging lines of evidence that: (1) brain 5-HT contributes to a critical mechanism underlying genetically defined individual differences in aggressiveness, and (2) genes encoding pivotal enzymes in 5-HT metabolism (TPH and MAO A), 5-HT-transporter, 5-HT(1A) and 5-HT(1B) receptors belong to a group of genes that modulate aggressive behavior.     

Tryptophan hydroxylase 1 knockout and tryptophan hydroxylase 2 polymorphism: effects on hypoxic pulmonary hypertension in mice

Serotonin [5-hydroxytryptamine (5-HT)] biosynthesis depends on two rate-limiting tryptophan hydroxylases (Tph): Tph1, which is expressed in peripheral organs, and Tph2, which is expressed in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used Tph1(-/-) mice characterized by very low 5-HT synthesis rates and contents in the gut and lung and increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P < 0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (100 mgxkg(-1)xday(-1)) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes during hypoxia. Forebrain Tph activity was greater and hypoxic PH was more severe in C57Bl/6 and 129X1/SvJ mice homozygous for the 1473C allele than in DBA/2 and BALB/cJ mice homozygous for the 1473G allele. p-Chlorophenylalanine reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-hydroxytryptophan accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that Tph1 and Tph2 may contribute to PH development.     

Compartmentalization of neuronal and peripheral serotonin synthesis in Drosophila melanogaster

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.     

Effect of the C1473G Polymorphic Variant of the Tryptophan Hydroxylase 2 Gene and Photoperiod Length on the Dopamine System of the Mouse Brain

A decrease in the light in autumn and winter causes depression like seasonal affective disorders (SAD) in sensitive patients, in which the serotonin (5-HT) and dopamine (DA) brain mediator systems are involved. We studied the interaction of the 5-HT and DA brain systems in an experimental SAD model in sexually mature male mice of the congenic B6-1473C and B6-1473G lines with high and low activity of tryptophan hydroxylase 2, a key enzyme of 5-HT synthesis in the brain. Mice of each line (divided into two groups of eight individuals) were kept for 30 days in standard (14 h light/10 h dark) and short (4 h light/20 h dark) daylight. The presence of the C1473G variant in the tryptophan hydroxylase 2 gene did not affect the expression of key genes of DA system: Drd1, Drd2, Scl6a3, Th, and Comt, that encode the D1 and D2 receptors, dopamine transporter, tyrosine hydroxylase, and catechol-o-methyltransferase, respectively. A decrease in the level of DA in the midbrain, as well as of its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, was detected in B6-1473G mice. Keeping mice in short daylight did not affect expression of the Drd1 gene in all brain structures nor the expression of the Slc6a3 and Th genes in the midbrain. Drd2 expression increased in the midbrain and decreased in the hippocampus, where Comt expression increased. An increase in DA level in the midbrain and DOPAC in the striatum was detected in mice kept in short daylight. This indicates the involvement of the brain’s DA system in the reaction to a decrease in daylight duration. No statistically significant effect of the interaction between the presence of the C1473G variant and daylight length on indicators of the activity of DA system was detected. No reasons were found to assert that this polymorphism determines the observed reaction of the brain DA system in keeping of animals under short daylight conditions.     

Impact of the tryptophan hydroxylase 1 gene A218C polymorphism on amygdala activity in response to affective facial stimuli in patients with major depressive disorder

Tryptophan hydroxylase-1 (TPH1) is the rate-limiting enzyme in serotonin biosynthesis, and allelic variations at the TPH1 locus have been implicated in the pathophysiology of depression. Using 1.5-Tesla functional magnetic resonance imaging, we investigated the possible relationship between TPH1 A218C polymorphism and amygdala response to negative facial stimuli in 26 right-handed female subjects with major depressive disorder (MDD). Genotyping was performed with the polymerase chain reaction. We found a significant association between A allele of the TPH1 A218C polymorphism and neural activations in response to negative facial stimuli. Subjects with the A allele of the TPH1 A218C polymorphism showed greater brain activity in the bilateral amygdala under the sad vs. the neutral condition compared with subjects homozygous for the C allele. Our results suggest that the A218C polymorphism of the TPH1 gene serves as a modulator of amygdala activity in patients with MDD.     

Strain differences in basal and post-citalopram extracellular 5-HT in the mouse medial prefrontal cortex and dorsal hippocampus: relation with tryptophan hydroxylase-2 activity

We used the microdialysis technique to compare basal extracellular serotonin (5-HT) and the response to citalopram in different strains of mice with functionally different allelic forms of tryptophan hydroxylase-2 (TPH-2), the rate-limiting enzyme in brain 5-HT synthesis. DBA/2J, DBA/2N and BALB/c mice carrying the 1473G allele of TPH-2 had less dialysate 5-HT in the medial prefrontal cortex and dorsal hippocampus (DH) (20-40% reduction) than C57BL/6J and C57BL/6N mice carrying the 1473C allele. Extracellular 5-HT estimated by the zero-net flux method confirmed the result of conventional microdialysis. Citalopram, 1.25, 5 and 20 mg/kg, dose-dependently raised extracellular 5-HT in the medial prefrontal cortex of C57BL/6J mice, with maximum effect at 5 mg/kg, but had significantly less effect in DBA/2J and BALB/c mice and in the DH of DBA/2J mice. A tryptophan (TRP) load enhanced basal extracellular 5-HT in the medial prefrontal cortex of DBA/2J mice but did not affect citalopram's ability to raise cortical and hippocampal extracellular 5-HT. The impairment of 5-HT synthesis quite likely accounts for the reduction of basal 5-HT and the citalopram-induced rise in mice carrying the mutated enzyme. These findings might explain why DBA/2 and BALB/c mice do not respond to citalopram in the forced swimming test. Although TRP could be a useful strategy to improve the antidepressant effect of citalopram (Cervo et al. 2005), particularly in subjects with low 5-HT synthesis, the contribution of serotonergic and non-serotonergic mechanisms to TRP's effect remains to be elucidated.     

The <Emphasis Type="Italic">C1473G</Emphasis> polymorphism in gene <Emphasis Type="Italic">tph2</Emphasis> is the main factor mediating the genetically defined variability of tryptophan hydroxylase-2 activity in the mouse brain

Brain neurotransmitter serotonin is involved in the regulation of many physiological functions and types of behavior. The key enzyme of serotonin synthesis in the brain is tryptophan hydroxylase-2 (TPH-2). Linkage between the C1473G polymorphism in gene tph2 causing the replacement of Pro⁴⁴⁷ by Arg⁴⁴⁷ in TPH-2 molecule and enzyme activity in the mouse brain of 10 inbred strains was found. Association of the polymorphism with the TPH-2 activity in the brain of F₂ hybrids between strains C57BL/6 and CC57BR was shown. The results indicate that the C1473G polymorphism in gene tph2 is the main factor determining the genetically defined variability of enzyme activity in the mouse brain.     

The interaction between TPH2 promoter haplotypes and clinical-demographic risk factors in suicide victims with major psychoses

Tryptophan hydroxylase isoform 2 (TPH2) is a rate-limiting enzyme in the biosynthesis of serotonin (5-HT) and is predominantly localized in the brain. Previous studies have suggested that there is an association between serotonergic dysfunction in the brain and suicidality. This study was designed to examine whether the -473T > A and -8396G > C polymorphisms of the TPH2 gene may be associated with completed suicide in subjects with major psychoses from the Stanley Foundation Brain Bank sample. TPH2 genotypes were determined in 69 subjects with a diagnosis of schizophrenia or bipolar disorder, among which 22 died by suicide. Genomic DNA was amplified by polymerase chain reaction and typed by automated methods. Both markers were found to be in Hardy-Weinberg equilibrium and in strong linkage disequilibrium. No association with history of suicide was found for either polymorphism. Haplotype analysis with EHAP showed no association between completed suicide and haplotype distribution (chi2 = 1.877; 3 df; P = 0.598). Nor was there any association between suicide and these genetic markers even when clinical-demographic factors were considered as covariates in the haplotype analysis. These findings suggest that these 5' marker haplotypes in the TPH2 gene do not influence suicidal behaviour.     

Serotonergic genes and amygdala activity in response to negative affective facial stimuli in Korean women

Serotonergic genes have been implicated in mood disorders, alcoholism and certain personality traits. We investigated the possible relationship between several polymorphisms in the serotonin (5-HT) system and amygdala responses to negative facial stimuli in Korean women using functional magnetic resonance imaging. All participants were genotyped with regard to the following polymorphisms: the serotonin transporter-gene-linked polymorphic region (5-HTTLPR), tryptophan hydroxylase 2 (TPH2) G(-703)T, 5-HT(1A) C(-1019)G and 5-HT(2A) single nucleotide polymorphism (SNP) rs6311. We found increased activations in response to angry facial stimuli in the bilateral amygdala of subjects with the long allele of 5-HTTLPR compared with those with two copies of the short allele. Higher activations in response to sad facial stimuli were found in the bilateral amygdala of subjects with the T/T genotype of 5-HT(2A) SNP rs6311, compared with C allele carriers, and in subjects with the G/G genotype of TPH2 G(-703)T, compared with those with T/T and G/T genotypes. Our results for individuals from an Asian population countered a previous finding for a Caucasian population and identified the moderating role of genetic background in the relationships between these serotonergic gene polymorphisms and amygdala function elicited by negative emotional stimuli.     

Brain serotonin deficiency leads to social communication deficits in mice

A deficit in brain serotonin is thought to be associated with deteriorated stress coping behaviour, affective disorders and exaggerated violence. We challenged this hypothesis in mice with a brain-specific serotonin depletion caused by a tryptophan hydroxylase 2 (TPH2) deficiency. We tested TPH2-deficient (Tph2^−/–) animals in two social situations. As juveniles, Tph2^−/− mice displayed reduced social contacts, whereas ultrasonic vocalizations (USVs) were unchanged within same-sex same-genotype pairings. Interestingly, juvenile females vocalized more than males across genotypes. Sexually naive adult males were exposed to fresh male or female urine, followed by an interaction with a conspecific, and re-exposed to urine. Although Tph2^−/− mice showed normal sexual preference, they were hyper-aggressive towards their interaction partners and did not vocalize in response to sexual cues. These results highlight that central serotonin is essential for prosocial behaviour, especially USV production in adulthood, but not for sexual preference.     

Implication of 5-HT2A receptors in the genetic mechanisms of the brain 5-HT system autoregulation

Brain serotonin (5-HT) system has been implicated in pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT2A receptor is involved in the mechanisms of stress-induced psychopathology and impulsive behavior. Here, we investigated the role of 5-HT2A receptor in the autoregulation of the brain 5-HT system. The chronic treatment with agonist of 5-HT2A receptor DOI (1.0 mg/kg, i.p./14 days) produced considerable decrease of 5-HT2A receptor-mediated "head-twitches" in AKR/J mice indicating desensitization of 5-HT2A receptors. Chronic DOI treatment failed to alter 5-HT2A receptor gene expression in the midbrain, hippocampus and frontal cortex. At the same time, the increase in the expression of the gene encoding key enzyme of 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), the increase in TPH2 activity and 5-HT levels and decreased expression of serotonin transporter (5-HTT) gene was found in the midbrain of DOI-treated mice. The results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and the implication of 5-HT2A receptor in the autoregulation of the brain 5-HT system.     

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2 ^flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2 ^null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2 ^null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2 ^null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.     

The role of the glycoprotein gp130 in serotonin mediator system in mouse brain

Glycoprotein gp130 is involved in signaling out of significant cytokine receptors as interleukin-6 (IL-6), leukemia inhibitory factor and ciliary neurotrophic factor, which play critical role in immunity, inflammation and neurogenesis. IL-6 and brain neurotransmitter serotonin are involved in the mechanism of depression. The aim of this work was to investigat the role of protein gp130 in the regulation of expression of genes, coding the key enzyme of serotonin synthesis--tryptophan hydroxylase 2 (TPH2), 5-HT-transporter, 5-HT(1A)- and 5-HT(2A)-receptors of serotonin. The study was carried out on adult mouse males of AKR and congenic AKR.CBA-D13Mit76 strains, created by transfer of the fragment of chromosome 13 containing the gene coding gp130 protein from CBA/Lac strain to the genome of AKR/J strain. Decreased expression of 5-HT(1A) - 5-HT(2A)-receptor genes in hippocampus midbrain and TPH2 gene in midbrain in AKR.CBA-D13Mit76 mice compared with AKR mice were shown. Activation of nonspecific immunity by bacterial endotoxin lipopolysaccharide (LPS) administration did not affect the genes expression in AKR mice, but increased 5-HT(2A)-receptor expression in midbrain and decreased 5-HT(1A)-receptor expression in cortex in AKR.CBA-D13Mit76 mice. The results indicate: 1) the participation of gp130 in the regulation of TPH2, 5-HT(1A)- and 5-HT(2A)-receptor genes and 2) association of this protein in the genetically determined sensitivity to LPS.