The serotonin-2A receptor gene locus does not contain common polymorphism affecting mRNA levels in adult brain.

The serotonin-2A (HTR2A) receptor is a molecule of particular interest in biological psychiatry, as it is an important target for psychotropic drugs, and altered HTR2A expression has been found in several neuropsychiatric conditions, including depression and schizophrenia. Genetic association has been reported between a synonymous 102T/C polymorphism in the gene encoding HTR2A and a number of clinical phenotypes, including schizophrenia, clozapine response, psychotic symptoms in Alzheimer's disease and certain features of depression. Given that there are no known effects of the 102T/C polymorphism on the structure of the receptor, attention has switched to the possibility that the observations of both altered expression and genetic association point to functional sequence variants that alter expression of the HTR2A gene. Moreover, data have been presented recently suggesting that mRNAs containing the 102T- and C-alleles are differentially expressed. This suggests a direct effect of the variant itself on mRNA levels, or the influence of a distinct regulatory variant, such as the -1438A/G promoter polymorphism, with which it is in perfect linkage disequilibrium. The present study tested this hypothesis by employing a highly accurate quantitative allele- specific primer extension assay to measure the relative expression of brain mRNAs carrying each allele in 23 individuals heterozygous for the 102T/C polymorphism. Comparison between allele ratios derived from genomic DNA and mRNA from several cortical regions revealed that the 102C- and T-alleles are expressed identically. Furthermore, the absence of any interindividual variability in relative mRNA allele ratio suggests that the HTR2A locus is unlikely to contain common polymorphisms or epigenetic modification that alter HTR2A mRNA levels in adult brain, and essentially exclude such phenomena as a potential explanation for the altered expression and genetic associations that have been reported to date.

Lack of functional promoter polymorphisms in genes involved in glutamate neurotransmission.

OBJECTIVES: The regulation of genes involved in glutamatergic function is thought to be a critical for many central nervous system processes including memory, learning, synaptic maintenance, and many pathological states. As part of a larger survey into the key regulatory elements in genes of neuro-psychiatric interest, we sought to identify the promoter regions of genes in this broad family, and to identify sequence variants that alter gene expression. METHODS: Mutation analysis was carried out on the promoters of 20 genes encoding 13 glutamate receptor subunits, four transporters and three metabolizing enzymes using denaturing high performance liquid chromatography. Thirty-nine different promoter haplotypes were cloned into a luciferase reporter gene vector and tested for differences in their ability to drive transcription in both HEK293t and TE671 cell lines. RESULTS: We have identified a total of 48 sequence variants in six glutamate receptor subunits, four glutamate transporters and two enzymes. Interestingly, seven promoter sequences gave three or more haplotypes from a single individual, indicating gene duplication. No differences in expression greater than 1.35-fold were found between haplotypes originating from the same or paralogous genes. CONCLUSION: The lack of common functional polymorphisms in any of these promoters indicates that expression of glutamate receptors and transporters is unusually tightly controlled, and suggests the possibility that non-coding polymorphisms in these genes are rare and may be unlikely to contribute in a major way to neuro-psychiatric phenotypes. This study represents the world's largest survey of the any group of promoters yet performed for any gene system.

Streamlined approach to functional analysis of promoter-region polymorphisms.

We have developed a rapid method for identifying functional promoter-region polymorphisms. Using a modified pGL3 luciferase expression T-vector, we can amplify by PCR, clone, identify allelic pairs of a polymorphic gene promoter region, and prepare plasmids for cell culture 10 promoters (20 allele pairs) per week per researcher. By utilizing 96-well plate technology and an internal control plasmid expressing secreted alkaline phosphatase, each of these allele pairs can be tested for relative promoter activity in each of three cell lines (HEK293t, TE671, and JEG3) with similar resources.

Characterisation, mutation detection, and association analysis of alternative promoters and 5' UTRs of the human dopamine D3 receptor gene in schizophrenia.

The dopamine D(3) receptor gene (DRD3) is a candidate for a number of psychiatric conditions including schizophrenia, bipolar disorder and alcohol and drug abuse. Previous studies have reported associations between polymorphisms in DRD3 and these disorders, but these findings may have reflected linkage disequilibrium with pathogenic variants that are further upstream. We have isolated and sequenced approximately 9 kb of genomic sequence upstream of the human DRD3 translational start site. Using 5' RACE, we have identified within this region three additional exons and two putative promoter regions which show promoter activity in three different cell lines. A 5' UTR identified only in lymphoblasts is spread over three exons and is 353 bp long. A second 5' UTR, found in adult and fetal brain, lymphocytes, kidney and placenta is spread over two exons and is 516 bp long. A 260-bp sequence within this 9 kb corresponds to a previously reported EST, but corresponding mRNA could not be found in the tissues above. The EST, 5' UTRs and putative promoter regions have been analysed for polymorphisms, revealing 10 single nucleotide polymorphisms, seven of which were tested for association in a large sample of unrelated patients with schizophrenia and matched controls. No associations were observed with schizophrenia. In addition we failed to replicate previous findings of association with homozygosity of the Ser9Gly variant. The results from this study imply that neither the coding nor the regulatory region of DRD3 plays a major role in predisposition to schizophrenia.

Genetic association studies of alcoholism--problems with the candidate gene approach.

In recent years, progress has been made in the identification of causative factors in most single gene disorders and those with genes of major effect. In comparison, no genes contributing to a complex disorder have been unambiguously identified. A number of reasons for this have been previously presented in theoretical papers. Alcoholism is such a complex illness and genetic studies into its underlying genetic causes have suffered from lack of power due to small subject numbers, poor selection of control subjects, and over-emphasis on markers with low prior probability of involvement.

The high affinity neurotensin receptor gene (NTSR1): comparative sequencing and association studies in schizophrenia.

Neurotensin and its high affinity receptor (NTSR1) localise within dopaminergic neurones in the mesocortical, mesolimbic and nigrostriatal systems and it is now clear that neurotensin can selectively modulate dopaminergic neurotransmission. This has led to the hypothesis that altered neurotensin function contributes to the pathogenesis of schizophrenia and other psychoses. This hypothesis has been supported circumstantially by a number of lines of evidence. (1) Central administration of neurotensin produces effects similar to those produced by the peripheral administration of atypical antipsychotics. (2) Observations of low levels of neurotensin in the CSF of schizophrenics. (3) Reduced numbers of neurotensin receptors in the brains of schizophrenics. Given the above link between neurotensin and dopamine, and the evidence implicating altered neurotensin function in psychosis, we have postulated that DNA sequence variation in neurotensin or its receptors might be associated with schizophrenia. In keeping with this hypothesis, an association has recently been reported between schizophrenia and the gene encoding the neurotensin high affinity receptor (NTSR1). However, caution is required because the associated marker, a tetranucleotide repeat, is located 3 kb away from the 3' end of the gene and there is no evidence that it is functional. Therefore, as a follow-up to our earlier work on neurotensin, we have now sought to test the hypothesis that DNA sequence variants that alter the structure or expression of the NTSR1 gene (VAPSEs) are associated with schizophrenia. However, while we found 14 novel sequence variants in 28 probands with psychosis, none resulted in an amino acid change, and neither direct nor indirect association studies suggested these are involved in susceptibility to schizophrenia.

Rat liver tryptophan pyrrolase activity and gene expression during alcohol withdrawal.

Rat liver tryptophan (Trp) pyrrolase activity and gene expression were studied in relation to the alcohol-withdrawal syndrome (AWS). Both activity and gene expression were enhanced after withdrawal of ethanol-containing liquid diets and the time-course of these changes mirrored that of development and intensity of the behavioural disturbances of the AWS. By contrast, no correlation was observed between the AWS-induced behaviour and changes in activity of another hepatic glucocorticoid-inducible enzyme, tyrosine aminotransferase, and a negative correlation was noted between behaviour and the gene expression of this latter enzyme and also of that of the hepatic glucocorticoid receptor. We suggest that the metabolic consequences of activation of liver Trp pyrrolase during alcohol withdrawal may play a role in the behavioural features of the AWS.

Association analysis of the proneurotensin gene and bipolar disorder.

Neurotensin (NT) localizes within dopaminergic neurones in the mesocortical, mesolimbic and nigrostriatal systems, and it is now clear that NT can selectively modulate dopaminergic neurotransmission. It has therefore been proposed that altered NT function might contribute to the pathogenesis of neuropsychiatric disorders in which disordered dopaminergic neurotransmission is suspected. We have previously screened the gene encoding NT in a sample of schizophrenic and bipolar subjects, and identified three sequence variants. These have now been tested for association with bipolar disorder using a case-control sample of unrelated bipolar subjects and matched controls. No evidence for association was found, and our data therefore suggest that sequence variation in this gene does not make an important contribution to susceptibility to bipolar disorder.

Comparative sequencing and association studies of aromatic L-amino acid decarboxylase in schizophrenia and bipolar disorder.

Aromatic L-amino acid decarboxylase (AADC) is a relatively non specific enzyme involved in the biosynthesis of several classical neurotransmitters including dopamine and 5-hydroxytryptamine (5HT; serotonin). AADC does not catalyse the rate limiting step in either pathway, but is rate limiting in the synthesis of 2-phenylethylamine (2PE) which is a positive modulator of dopaminergic transmission and a candidate natural psychotogenic compound.1 We and others have proposed that polymorphism in AADC resulting in altered 2PE activity might contribute to the pathogenesis of psychosis. In order to test this hypothesis, we have used denaturing high performance liquid chromatography (DHPLC)3 to screen 3943 bases of the AADC gene and its promoter regions for variants that might affect protein structure or expression in 15 unrelated people with schizophrenia, and 15 unrelated people with bipolar disorder. Three polymorphisms were identified by DHPLC: a insertion/deletion polymorphism in the 5' UTR of the neuronal specific mRNA (g.-33-30delAGAG, bases 586-589 of GenBank M77828), a T>A variant in the non-neuronal exon 1 (g. -67T>A, GenBank M88070), and a G>A polymorphism within intron 8 (g. IVS8 +75G>A, GenBank M84598). Case-control analysis did not suggest that genetic polymorphism in the AADC gene is associated with liability for developing schizophrenia or bipolar disorder.

Comparative sequencing of the proneurotensin gene and association studies in schizophrenia.

Neurotensin (NT) is an endogenous tridecapetide1 cleaved from a precursor proneurotensin/ proneuromedin protein. NT localises within dopaminergic neurones in the mesocortical, mesolimbic and nigrostriatal systems1-3 and it is now clear that NT can selectively modulate dopaminergic neurotransmission.2-9 These anatomical and functional connections have led to the hypothesis that NT dysfunction might contribute to the pathogenesis of neuropsychiatric disorders in which disordered dopaminergic neurotransmission is suspected, particularly schizophrenia.3 The latter hypothesis has been supported circumstantially by the observation that central administration of NT produces effects similar to those produced by the peripheral administration of atypical antipsychotics,10,11 and more directly by studies showing levels of NT in cerebral spinal fluid (CSF) is lower in schizophrenics than in controls.12,13 To allow such hypotheses to be tested, we used denaturing high performance liquid chromatography (DHPLC)14 to identify three sequence variants in the neurotensin gene (NTS) that might alter NT structure or expression. However, using a case-control study design and a novel genotyping system based upon a primer extension protocol and HPLC detection,15 we found no evidence to support the hypothesis that variation in the proneurotensin gene contributes to susceptibility to schizophrenia.

Cheap, accurate and rapid allele frequency estimation of single nucleotide polymorphisms by primer extension and DHPLC in DNA pools.

At present, the cost of genotyping single nucleotide polymorphisms (SNPs) in large numbers of subjects poses a formidable problem for molecular genetic approaches to complex diseases. We have tested the possibility of using primer extension and denaturing high performance liquid chromatography to estimate allele frequencies of SNPs in pooled DNA samples. Our data show that this method should allow the accurate estimation of absolute allele frequencies in pooled samples of DNA and also of the difference in allele frequency between different pooled DNA samples. This technique therefore offers an efficient and cheap method for genotyping SNPs in large case-control and family-based association samples.

Inhibition of rat liver tryptophan pyrrolase activity by fluoxetine.

The present study has demonstrated the effectiveness of acute administration of fluoxetine to inhibit rat liver tryptophan pyrrolase activity. The maximum inhibition of basal liver tryptophan pyrrolase activity at 2 h after administration was observed with 1 mg/kg dose for the total enzyme and apoenzyme activities and that significant inhibition of these two activities was evident with a dose of the drug as small as 0.5 mg/kg. Serum free tryptophan concentrations were also increased using 10 mg/kg dose of fluoxetine. In view of the role of tryptophan depletion and thus 5-HT in pathophysiology of depression, it is strongly suggested that the inhibition of liver tryptophan pyrrolase activity may be a major mechanism of antidepressant action.

Tryptophan metabolism in male Sardinian alcohol-preferring (sP) and -non-preferring (sNP) rats.

Parameters of tryptophan (Trp) and related metabolism were compared in male Sardinian alcohol-preferring (sP) and -non-preferring (sNP) rats. Liver Trp pyrrolase activity was 38-58% higher in sP than in sNP rats, and this was associated with a greater expression of the enzyme mRNA as measured by multiprobe oligonucleotide solution hybridization. Moderately (about 10-19%), but significantly, lower concentrations of free serum, total serum, and brain Trp were also observed in sP compared with sNP rats. Concentrations of whole brain 5-hydroxytryptamine (5-HT) and its major metabolite 5-hydroxyindol-3-yl-acetic acid (5-HIAA) were, however, 14-21% higher in sP rats. Serum corticosterone concentration was 18% higher in sP rats. We conclude that alcohol preference in Sardinian rats is associated with increased liver Trp pyrrolase activity and mRNA expression leading to a decrease in Trp availability to the brain. Although a simple serotonin deficiency could not be demonstrated in the whole brain, the possibility could not be ruled out that a deficiency may be present in discrete areas of the brain of the sP rat.

A family based association study of T102C polymorphism in 5HT2A and schizophrenia plus identification of new polymorphisms in the promoter.

Several studies have shown an association between schizophrenia and the C allele of a T-C polymorphism at nucleotide 102 and the 5HT2A receptor gene. In the present study we observed this association in a sample of 63 parent/offspring trios where the proband received a diagnosis of DSM-III-R schizophrenia using TDT analysis (chi2 = 6.26, P= 0.006, chi2 = 9.00, P=0.001 when one affected offspring was selected at random from each family, suggesting that the results are due to association rather than linkage). There was no significant difference between the transmission of C102 from heterozygous fathers and mothers, which fails to support a role for genomic imprinting in this effect. T102C does not result in an alteration of the amino acid sequence of the protein. We therefore screened the promoter of 5HT2A for polymorphisms using single-strand confirmation polymorphism analysis. An A-G polymorphism at -1438 that creates an HpaII restriction site was identified. This was found to be in complete linkage disequilibrium with T102C and is hence a candidate for the pathogenic variant in schizophrenia. Functional analysis of A-1438G using luciferase assay demonstrated significant basal promoter activity in 5HT2A expressing HeLa cells by both the A and G variants. However, comparison of the A and G variants showed no significant differences in basal activity nor when promoter activity was induced by cAMP and protein kinase C-dependent mechanisms.

Tryptophan pyrrolase gene expression in an alcohol preferring and non-preferring mouse strain.

There is considerable evidence that the level of cerebral serotonin plays a key role in the volitional consumption of ethanol in both man and animals. Naive alcohol-preferring C57BL/6J mice have been shown to have a lower cerebral serotonin content compared to the non-preferring CBA/Ca mouse strain. This has been attributed to the enhancement of hepatic tryptophan pyrrolase activity in C57 mice. Activity and/or expression of tryptophan pyrrolase may be an important biological determinant of alcohol preference. We have investigated the possible mechanism/s underlying this strain difference in tryptophan pyrrolase activity by assaying both mRNA levels encoding for the tryptophan pyrrolase gene and by mutational analysis of tryptophan pyrrolase cDNA. We were unable to demonstrate any difference in tryptophan pyrrolase mRNA levels between naive C57 and CBA mice. Tryptophan pyrrolase mRNA levels were increased following starvation in C57 mice and following glucocorticoid administration in both C57 and CBA mice. Heteroduplex mutational analysis failed to detect any tryptophan pyrrolase cDNA sequence heterogeneity between these mice strains.

Does phenylethylamine have a role in schizophrenia?: LSD and PCP up-regulate aromatic L-amino acid decarboxylase mRNA levels.

Aromatic L-amino acid decarboxylase (AADC) is rate limiting in the production of 2-phenylethylamine (2PE). AADC activity and 2PE serum concentrations have been found to be increased in schizophrenic patients. Both antipsychotic and psychotogenic drugs, including amphetamine, affect the activity and encoding mRNA levels of AADC. Amphetamine is an analogue of 2PE and has a similar physiological effect. We have looked at the effects of chronic (32 day) treatment of rats with LSD (0.12 microg/kg/day) and phencyclidine (PCP; 10 mg/kg/day) on AADC mRNA levels. Both drugs up-regulated AADC mRNA levels in striatum, nucleus accumbens, hippocampus and cerebellum by between 50% and 150%. A splicing variant of AADC, present in human brain, which lacks the 3rd exon does not appear to be present in rat brain. These results are consistent with the hypothesis that over activity of AADC leading to increased production of 2PE is involved in endogenous psychosis such as schizophrenia.

The effects of antipsychotic drugs on the mRNA levels of serotonin 5HT2A and 5HT2C receptors.

We have looked for both global and regional changes in rat brain mRNA levels encoding serotonin 5HT2A and 5HT2C receptors following acute and chronic administration of antipsychotic drugs. In whole brain clozapine (30 mg/kg/day) elicited a transient rise in 5HT2C mRNA levels after 4 days and chlorpromazine (15 mg/kg/day) elicited a decrease of 20% in 5HT2A mRNA levels after 32 days. Regionally, 32 days treatment with haloperidol (3 mg/kg/day), sulpiride (100 mg/kg/day) or clozapine (10 mg/kg/day) resulted in a drop of approximately 30-40% in 5HT2C mRNA levels in both cortex and cerebellum, and decreases (or non-significant trends) of 15-40% in 5HT2A mRNA levels in hippocampus, brainstem and mid brain. 4 days treatment with clozapine resulted in a 40% rise of 5HT2C mRNA in the mid brain and a 24% rise of 5HT2A mRNA in the nucleus accumbens which were not found after 32 days of treatment. These results demonstrate common chronic effects of typical and atypical drugs but unique short term effects of clozapine.

Antipsychotic regulation of dopamine D1, D2 and D3 receptor mRNA.

A range of antipsychotic drugs, both "typical" and "atypical", was administered to rats over a time course and at several different dosages. The mRNA levels of dopamine D1, D2 and D3 receptor were measured in either whole brain or dissected brain regions. D3 receptor mRNA was up-regulated in whole brain by clozapine (10 and 30 but not 3 mg/kg/day), sulpiride (50 and 100 but not 20 mg/kg/day). haloperidol (3 but not 1 or 0.3 mg/kg/day), flupenthixol (3 but not 1 or 0.3 mg/kg/day), pimozide (4.5 but not 1.5 or 0.5 mg/kg/day) and loxapine (1.2 and 4 mg/kg/day but not 0.4 mg/kg/day). Sulpiride (100 mg/kg/day), clozapine (30 mg/kg/ day) and haloperidol (3 mg/kg/day) all up-regulated the D3 receptor mRNA in nucleus accumbens and olfactory tubercles but not striatum. D1 and D2 receptor mRNA was up-regulated in whole brain by haloperidol and loxapine only, and in the case of haloperidol this was localized to striatum and prefrontal cortex. Haloperidol, clozapine and sulpiride all down-regulated D1 mRNA in hippocampus and additionally haloperidol and sulpiride down-regulated it in the cerebellum. This work shows that all the drugs tested up-regulated D3 receptor, but effects on D1 and D2 receptors were less general.

Prevention by cycloheximide of the audiogenic seizures and tryptophan metabolic disturbances of ethanol withdrawal in rats.

Cycloheximide (20 mg/kg body wt, given intraperitoneally at-1 and 3 h after withdrawal of an ethanol-containing liquid diet) prevents the activation of liver tryptophan pyrrolase, the consequent inhibition of synthesis of brain 5-hydroxytryptamine, and the audiogenic seizures observed at 7 h after alcohol withdrawal. We suggest that a rapidly-turning-over protein mediates the alcohol withdrawal syndrome and discuss the possible role of liver tryptophan pyrrolase.

Effects of chronic administration and subsequent withdrawal of ethanol-containing liquid diet on rat liver tryptophan pyrrolase and tryptophan metabolism.

An investigation of the effects of chronic administration of ethanol by the liquid diet procedure and its subsequent withdrawal on tryptophan (Trp) metabolism and disposition was performed in rats. Treatment with the control liquid diet caused an enhancement of liver Trp pyrrolase activity and mRNA abundance. These effects are not due to the starvation associated with this feeding procedure, because they occur in rats maintained on the liquid diet ad libitum. Chronic ethanol administration in the liquid diet did not further influence the above increased expression of Trp pyrrolase mRNA but caused inhibition of pyrrolase activity in competition with the effects of the diet. The control liquid diet decreased liver Trp concentration, but exerted no significant effects on other aspects of Trp disposition. The most striking and robust finding was a highly significant elevation in both Trp pyrrolase activity and mRNA expression at 7 h following discontinuation of ethanol availability, at which time there were demonstrable behavioural signs of ethanol withdrawal. The increase in Trp pyrrolase mRNA during alcohol withdrawal may be caused by corticosterone, whose circulating concentration was also increased. The changes in Trp pyrrolase activity during ethanol withdrawal were associated with significant alterations in Trp disposition including decreased brain Trp concentration and 5-hydroxytryptamine synthesis and turnover. These alterations may play a pivotal role in the behavioural manifestations of ethanol withdrawal including the hyperexcitement underlying audiogenic seizures. We suggest that rat Trp pyrrolase gene regulation may be an important biological determinant of the ethanol withdrawal syndrome and requires further study, and that the use of the liquid diet procedure in Trp metabolic studies requires inclusion of adequate controls and special attention to the effects of the liquid diet itself.