Chronic Cocaine Administration Increases CNS Tyrosine Hydroxylase Enzyme Activity and mRNA Levels and Tryptophan Hydroxylase Enzyme Activity Levels

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self-administration by rats when given ad libitum access to this dose. This chronic, response-independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striaturn and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response-independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.     

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

Association between Tph2 gene polymorphism, brain tryptophan hydroxylase activity and aggressiveness in mouse strains

The brain neurotransmitter serotonin is involved in the regulation of aggressive behavior. The main factor determining the brain serotonin level is the activity of the rate-limiting enzyme in the biosynthesis of the neurotransmitter--tryptophan hydroxylase isoform (TPH) 2 encoded by the Tph2 gene. Recently the C1473G single-nucleotide polymorphism in the Tph2 gene was reported. Here we study the C1473G polymorphism in 10 inbred mouse strains (C57BL/6J, AKR/J, DD/He, C3H/HeJ, YT/Y, BALB/cJLac, CC57BR/Mv and A/He) and demonstrate the association of the polymorphism with brain TPH activity and intermale aggressiveness. TPH activity in the midbrain of mice homozygous for the 1473C allele was higher than that in mice carrying 1473G alleles. A close association of the 1473C allele with increased number of attacks towards another male was found. The results support a link between the C1473G polymorphism in Tph2 gene, tryptophan hydroxylase activity and intensity of intermale aggression.     

Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington's disease

The pathogenic mechanisms of the mutant huntingtin protein that cause Huntington's disease (HD) are unknown. Previous studies have reported significant decreases in the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brains of the R6/2 transgenic mouse model of HD. In an attempt to elucidate the cause of these neurochemical perturbations in HD, the protein levels and enzymatic activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, were determined. Enzyme activity was measured in brainstem homogenates from 4-, 8-, and 12-week-old R6/2 mice and compared with aged-matched wild-type control mice. We observed a 62% decrease in brainstem TPH activity (p = 0.009) in 4-week-old R6/2 mice, well before the onset of behavioral symptoms. In addition, significant decreases in TPH activity were also observed at 8 and 12 weeks of age (61%, p = 0.02 and 86%, p = 0.005, respectively). In the 12-week-old-mice, no change in immunoreactive TPH was observed. In vitro binding showed that TPH does not bind to exon 1 of huntingtin in a polyglutamine-dependent manner. Specifically, glutathione-S-transferase huntingtin exon 1 proteins with 20, 32 or 53 polyglutamines did not interact with radiolabeled tryptophan hydroxylase. Therefore, the inhibition of TPH activity does not appear to result from a direct huntingtin/TPH interaction. Receptor binding analyses for the 5-HT1A receptor in 12-week-old R6/2 mice revealed significant reductions in 8-OH-[3H]DPAT binding in several hippocampal and cortical regions. These results demonstrate that the serotonergic system in the R6/2 mice is severely disrupted in both presymptomatic and symptomatic mice. The presymptomatic inhibition of TPH activity in the R6/2 mice may help explain the functional consequences of HD and provide insights into new targets for pharmacotherapy.     

Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.     

Functional domains of human tryptophan hydroxylase 2 (hTPH2)

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.     

Tryptophan hydroxylase 2 aggregates through disulfide cross-linking upon oxidation: possible link to serotonin deficits and non-motor symptoms in Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopamine neurons of the nigrostriatal system, resulting in severe motor disturbances. Although much less appreciated, non-motor symptoms are also very common in PD and many can be traced to serotonin neuronal deficits. Tryptophan hydroxylase (TPH) 2, the rate-limiting enzyme in the serotonin biosynthesis, is a phenotypic marker for serotonin neurons and is known to be extremely labile to oxidation. Therefore, the oxidative processes that prevail in PD could cause TPH2 misfolding and modify serotonin neuronal function much as is seen in dopamine neurons. Oxidation of TPH2 inhibits enzyme activity and leads to the formation of high molecular weight aggregates in a dithiothreitol-reversible manner. Cysteine-scanning mutagenesis shows that as long as a single cysteine residue (out of a total of 13 per monomer) remains in TPH2, it cross-links upon oxidation and only cysteine-less mutants are resistant to this effect. The effects of oxidants on TPH2 catalytic function and cross-linking are also observed in intact TPH2-expressing HEK293 cells. Oxidation shifts TPH2 from the soluble compartment into membrane fractions and large inclusion bodies. Sequential non-reducing/reducing 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting confirmed that TPH2 was one of a small number of cytosolic proteins that form disulfide-bonded aggregates. The propensity of TPH2 to misfold upon oxidation of its cysteine residues is responsible for its catalytic lability and may be related to loss of serotonin neuronal function in PD and the emergence of non-motor (psychiatric) symptoms.     

A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.     

Genetic mapping of the human tryptophan hydroxylase gene on chromosome 11, using an intronic conformational polymorphism.

The identification of polymorphic alleles at loci coding for functional genes is crucial for genetic association and linkage studies. Since the tryptophan hydroxylase (TPH) gene codes for the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, it would be advantageous to identify a polymorphism in this gene. By examining introns of the human TPH gene by PCR amplification and analysis by the single-strand conformational polymorphism (SSCP) technique, an SSCP was revealed with two alleles that occur with frequencies of .40 and .60 in unrelated Caucasians. DNAs from 24 informative CEPH families were typed for the TPH intron polymorphism and analyzed with respect to 10 linked markers on chromosome 11, between p13 and p15, with the result that TPH was placed between D11S151 and D11S134. This region contains loci for several important genes, including those for Beckwith-Wiedemann syndrome and tyrosine hydroxylase.     

Chronic amphetamine administration decreases brain tryptophan hydroxylase activity in cats

Chronic administration of d-amphetamine sulfate (7.5 mg/kg, i.p. every 12 hrs. for 6 days) to cats produced significant decreases in the Vmax of brain-stem and forebrain tryptophan hydroxylase when measured 1 day (?34 and ?46%) and 10 days (?17 and ?30%) after the final amphetamine injection. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) levels were decreased by a similar magnitude. A single injection of amphetamine (7.5 mg/kg) produced no significant changes in tryptophan hydroxylase activity, serotonin, or 5HIAA when measured 1 day after the injection. Neither acute nor chronic amphetamine treatment produced any significant changes in the Km of tryptophan hydroxylase for either tryptophan or the natural co-factor, tetrahydrobiopterin. These data suggest that chronic amphetamine treatment decreases central serotonergic neurotransmission by an action on the rate-limiting enzyme in serotonin biosynthesis.     

Regulation of pteridine-requiring enzymes by the cofactor tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GCH), 6-pyruvoyltetrahydropterin synthase (PTS), and sepiapterin reductase (SPD). GCH is the rate-limiting enzyme. BH4 is a cofactor for three pteridine-requiring monooxygenases that hydroxylate aromatic L-amino acids, i.e., tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and phenylalanine hydroxylase (PAH), as well as for nitric oxide synthase (NOS). The intracellular concentrations of BH4, which are mainly determined by GCH activity, may regulate the activity of TH (an enzyme-synthesizing catecholamines from tyrosine), TPH (an enzyme-synthesizing serotonin and melatonin from tryptophan), PAH (an enzyme required for complete degradation of phenylalanine to tyrosine, finally to CO2 + H2O), and also the activity of NOS (an enzyme forming NO from arginine), Dominantly inherited hereditary progressive dystonia (HPD), also termed DOPA-responsive dystonia (DRD) or Segawa's disease, is a dopamine deficiency in the nigrostriatal dopamine neurons, and is caused by mutations of one allele of the GCH gene. GCH activity and BH4 concentrations in HPD/DRD are estimated to be 2-20% of the normal value. By contrast, recessively inherited GCH deficiency is caused by mutations of both alleles of the GCH gene, and the GCH activity and BH4 concentrations are undetectable. The phenotypes of recessive GCH deficiency are severe and complex, such as hyperphenylalaninemia, muscle hypotonia, epilepsy, and fever episode, and may be caused by deficiencies of various neurotransmitters, including dopamine, norepinephrine, serotonin, and NO. The biosynthesis of dopamine, norepinephrine, epinephrine, serotonin, melatonin, and probably NO by individual pteridine-requiring enzymes may be differentially regulated by the intracellular concentration of BH4, which is mainly determined by GCH activity. Dopamine biosynthesis in different groups of dopamine neurons may be differentially regulated by TH activity, depending on intracellular BH4 concentrations and GCH activity. The nigrostriatal dopamine neurons may be most susceptible to a partial decrease in BH4, causing dopamine deficiency in the striatum and the HPD/DRD phenotype.     

Developmental role of tryptophan hydroxylase in the nervous system

The serotonin 5-hydroxytryptamine (5-HT) neurotransmitter system contributes to various physiological and pathological conditions. 5-HT is the first neurotransmitter for which a developmental role was suspected. Tryptophan hydroxylase (TPH) catalyzes the rate-limiting reaction in the biosynthesis of 5-HT. Both TPH1 and TPH2 have tryptophan hydroxylating activity. TPH2 is abundant in the brain, whereas TPH1 is mainly expressed in the pineal gland and the periphery. However, TPH1 was found to be expressed predominantly during the late developmental stage in the brain. Recent advances have shed light on the kinetic properties of each TPH isoform. TPH1 showed greater affinity for tryptophan and stronger enzymic activity than TPH2 under conditions reflecting those in the developing brain stem. Transient alterations in 5-HT homeostasis during development modify the fine wiring of brain connections and cause permanent changes to adult behavior. An increasing body of evidence suggests the involvement of developmental brain disturbances in psychiatric disorders. These findings have revived a long-standing interest in the developmental role of 5-HT-related molecules. This article summarizes our understanding of the kinetics and possible neuronal functions of each TPH during development and in the adult.     

TPH gene may be associated with suicidal behavior, but not with schizophrenia in the Korean population

Tryptophan hydroxylase (TPH) is a rate-limiting enzyme in the biosynthesis of serotonin. This study was designed to examine whether A218C polymorphism, which has been identified in intron 7 of the TPH gene, may be associated with schizophrenia or the suicidal behavior of schizophrenics in the Korean population. TPH genotypes were determined in DNA samples from 217 schizophrenics and 236 healthy volunteers. Among the schizophrenic group, 27 patients had a history of suicidal behavior. Genomic DNA was amplified by a polymerase-chain-reaction-based method and restricted by Nhel. A218C polymorphism was associated with a history of suicidal behavior in schizophrenics. This finding suggests that the TPH gene or a gene in its vicinity may influence suicidal behavior in schizophrenics. However, genotypic and allelic distrubutions of this polymorphism did not significantly differ between schizophrenics and controls.     

Amino-Terminal Analysis of Tryptophan Hydroxylase: Protein Kinase Phosphorylation Occurs at Serine-58

Abstract: Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion muta-genesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., NΔ50, NΔ60, NΔ90, NΔ106, and NΔ116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (NΔ116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and NΔ50 enzymes were phosphorylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.     

Cerebral serotonin and the hypothalamo-hypophyseal-adrenal system of the rat during aging]

The role of brain serotonin (5HT) on the hypothalamus-pituitary-adrenal system (HPAs) under basal condition and after injections of p-chlorophenylalanine (pCPA) and L-5-hydroxytryptophan (L-5HTP) has been studied in 6, 12 and 28 month old male Wistar rats. Four experimental groups were made for each age: control, saline, injected with pCPA (250 mg/kg i.p.) and L-5HTP (200 mg/kg i.p.), the effects being valued 2 hours after L-5HTP administration and 24 hours after pCPA injection. In all groups the plasmatic ACTH, the corticosterone levels as well as the simultaneous changes of the 5TH content tryptophan hydroxylase activity in whole brain were estimated two hours after the L-5HTP injection and 24 hours after that of pCPA. Significant changes are not found in the plasmatic ACTH and corticosterone values with respect to age under basal condition. Nevertheless, the response of HPAs differs with the age after pCPA or L-5HTP injection. The ACTH and corticosterone levels augment by L-5HTP and decrease by pCPA in all age groups, but this corresponding increase or decrease was less marked in the older rats. The 5HT content as tryptophan hydroxylase activity in brain decreased in old animals. pCPA and L-5HTP determine, respectively, high falls and rise of 5TH values, these changes being more intense for pCPA in old rats and for L-5HTP in young and mature animals. The tryptophan hydroxylase activity is decreased by pCPA as L-5HTP injections.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Phosphorylation and activation of tryptophan hydroxylase 2: identification of serine-19 as the substrate site for calcium, calmodulin-dependent protein kinase II

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. TPH was once thought to be a single-gene product but it is now known to exist in two isoforms. TPH1 is found in the periphery and pineal gland whereas TPH2 is expressed specifically in the CNS. Both TPH isoforms are known to be regulated by protein kinase-dependent phosphorylation and the sites of modification of TPH1 by protein kinase A have been identified. While TPH2 is activated by calcium, calmodulin-dependent protein kinase II (CaMKII), the sites at which this isoform is modified are not known. Treatment of wild-type TPH2 with CaMKII followed by mass spectrometry analysis revealed that the enzyme was activated and phosphorylated at a single site, serine-19. Mutagenesis of serine-19 to alanine did not alter the catalytic function of TPH2 but this mutant enzyme was neither activated nor phosphorylated by CaMKII. A phosphopeptide bracketing phosphoserine-19 in TPH2 was used as an antigen to generate polyclonal antibodies against phosphoserine-19. The antibodies are highly specific for phosphoserine-19 in TPH2. The antibodies do not react with wild-type TPH2 or TPH1 and they do not recognize phophoserine-58 or phosphoserine-260 in TPH1. These results establish that activation of TPH2 by CaMKII is mediated by phosphorylation of serine-19 within the regulatory domain of the enzyme. Production of a specific antibody against the CaMKII phosphorylation site in TPH2 represents a valuable tool to advance the study of the mechanisms regulating the function of this important enzyme.