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.

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.

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.

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.

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.

Amphetamine and vigabatrin down regulate aromatic L-amino acid decarboxylase mRNA levels.

Aromatic L-amino acid decarboxylase (AADC) has previously been shown to be up-regulated at the level of its protein activity and its mRNA abundance by antipsychotic drugs. Its activity has also been shown to be down-regulated by dopamine agonists including amphetamine. In this study we have injected rats for up to 32 days with amphetamine and the anti-epileptic drug vigabatrin, both of which can cause psychosis with similarities to schizophrenia. We have shown that AADC mRNA levels are reduced in most brain regions by both drugs. Cocaine and other non-psychotogenic anti-epileptic drugs had no effect in this paradigm. Two products of this enzyme are implicated in psychotogenesis.

Lack of effect of antipsychotic and antidepressant drugs on glutamate receptor mRNA levels in rat brains.

By employing multiprobe oligonucleotide solution hybridisation (MOSH) we have measured the levels of mRNA encoding the NMDA receptor subtypes (R1, R2A, R2B and R2C) and the non-NMDA glutamate receptor subtypes (GluR1, 2, 3, and 4) within rat brain following, 1-32 days of antipsychotic or antidepressant drug administration. The results suggest that the drugs studied do not significantly alter rat glutamatergic system mRNA levels when compared to controls.

Clozapine and sulpiride up-regulate dopamine D3 receptor mRNA levels.

Chronic treatment (32 days) with sulpiride (100 mg/kg/day) up-regulated rat brain dopamine D3 receptor mRNA levels by 4-fold but had no effect on the mRNA levels encoding the dopamine D1A, D1B or D2 receptors or the enzymes tyrosine hydroxylase and aromatic amino acid decarboxylase as measured by multiprobe oligonucleotide solution hybridisation. Clozapine (30 mg/kg/day) increased D3 receptor mRNA levels by 5-fold after 4 days, the level dropping to basal after 32 days and also increased D1B mRNA levels by 0.5-fold in a similar pattern. Clozapine did not affect any other dopamine receptors or the synthesising enzyme mRNA levels. We have previously shown that the typical antipsychotics haloperidol and loxapine also increased the mRNA levels of the dopamine D3 receptor and these results suggest that up-regulation of dopamine D3 receptor mRNA may be associated with the therapeutic action of antipsychotic drugs.

Both splicing variants of the dopamine D2 receptor mRNA are up-regulated by antipsychotic drugs.

We have investigated the effects of antipsychotic drugs on the expression of the two dopamine D2 receptor splicing variants using multiprobe oligonucleotide solution hybridisation. Both long and short mRNA variants were increased in abundance by approximately 2-fold by both haloperidol and loxapine after 32 days drug administration.

Lack of effect of chronic antipsychotic treatment on dopamine D5 receptor mRNA level.

The effects of administration of antipsychotic drugs (haloperidol, loxapine, sulpiride; 1-32 day time course) on the rat brain mRNA levels of the dopamine D5 receptor has been assessed using solution hybridisation with oligonucleotides. In contrast with the previously reported increases of D1, D2 and D3 receptor mRNA levels in identical experiments, no changes were found in dopamine D5 receptor mRNA levels, suggesting that the mechanism of regulation of D5 receptor mRNA is different to the other cloned dopamine receptors. We also conclude that up-regulation of the D5 receptor is not likely to be involved in the mechanism of action of antipsychotic drugs.

Bi-directional changes in the levels of messenger RNAs encoding gamma-aminobutyric acidA receptor alpha subunits after flurazepam treatment.

Changes in gamma-aminobutyric acidA (GABAA) receptor function have been observed following chronic benzodiazepine administration. The molecular mechanisms responsible are unknown, but one possibility is that benzodiazepines induce alterations in the expression of genes which encode subunits of the GABAA receptor complex, resulting in changes in the receptor structure and function. We have investigated this hypothesis by evaluating the effect of flurazepam 40 mg/kg i.p. on brain levels of the mRNAs which encode the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA receptor complex. Rats were treated with flurazepam or vehicle for up to 32 days. No changes were found in the levels of alpha 1 and alpha 2 mRNA. A rapid decrease was found in the level of alpha 5 mRNA; alpha 3 mRNA was increased by 4 days of treatment and this was followed by an increase in alpha 6 levels. These results support the hypothesis that the alteration in GABAA receptor function after benzodiazepine administration results from changes in subunit gene expression. Furthermore, the predicted consequences of the pattern of mRNA changes we have observed suggest that altered gene expression may be important in the genesis of benzodiazepine tolerance.