Ursula M. D'Souza

Regulation of striatal dopamine receptors by estrogen

The ability of estrogen to modulate the expression of ventral and dorsal striatal dopamine receptors D1, D2, and D3 was examined in vivo using semi‐quantitative in situ hybridization and ligand binding autoradiography. Two‐week treatment with subcutaneous pellets of 17β‐estradiol (25 mg) downregulated D2 dopamine receptor mRNA in both dorsal and ventral striatum (shell and core regions of nucleus accumbens). No significant changes in D1 or D3 mRNA expression were detected. Ligand binding autoradiography did not reveal changes in D1, D2, or D3 receptor protein expression. We also assessed the ability of 17β‐estradiol to regulate D2 gene promoter activity in NB41A3 neuroblastoma cells that express this gene endogenously using co‐transfections with an estrogen receptor expression vector. While a small fragment of the D2 promoter could be activated 2.5‐fold by estrogen, a larger portion of the D2 gene was not regulated by this treatment. Estrogens do not appear to have a net effect on striatal dopamine receptor expression. The observed downregulation of D2 receptor mRNA in the dorsal and ventral striatum in vivo could be secondary to the increased striatal dopamine release induced by estrogen. Synapse 34:222–227, 1999. Published 1999 Wiley‐Liss, Inc.

Three-amino acid Extension Loop Homeodomain Proteins Meis2 and TGIF Differentially Regulate Transcription

Three-amino acid extension loop (TALE) homeobox proteins are highly conserved transcription regulators. We report that two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in opposite orientations, can function competitively. For example, in theD1A gene, which encodes the predominant dopamine receptor in the striatum, Meis2 and TGIF bind to the activator sequence ACT (−1174 to −1154) and regulate transcription differentially in a cell type-specific manner. Among the five cloned splice variants of Meis2, isoforms Meis2a–d activate theD1A promoter in most cell types tested, whereas TGIF competes with Meis2 binding to DNA and represses Meis2-induced transcription activation. Consequently, Meis2 cannot activate theD1A promoter in a cell that has abundant TGIF expression. The Meis2 message is highly co-localized with the D1A message in adult striatal neurons, whereas TGIF is barely detectable in the adult brain. Our observations provide in vitro and in vivo evidence that Meis2 and TGIF differentially regulate their target genes. Thus, the delicate ratio between Meis2 and TGIF expression in a given cell type determines the cell-specific expression of theD1A gene. We also found that splice variant Meis2e, which has a truncated homeodomain, cannot bind to theD1A ACT sequence or activate transcription. However, Meis2e is an effective dominant negative regulator by blocking Meis2d-induced transcription activation. Thus, truncated homeoproteins with no DNA binding domains can have important regulatory functions.

Hippocampal gene expression profiling across eight mouse inbred strains: towards understanding the molecular basis for behaviour

Mouse inbred strains differ in many aspects of their phenotypes, and it is known that gene expression does so too. This gives us an opportunity to isolate the genetic aspect of variation in expression and compare it to other phenotypic variables. We have investigated these issues using an eight‐strain expression profile comparison with four replicates per strain on Affymetrix MGU74av2 GeneChips focusing on one well‐defined brain tissue (the hippocampus). We identified substantial strain‐specific variation in hippocampal gene expression, with more than two hundred genes showing strain differences by a very conservative criterion. Many such genetically driven differences in gene expression are likely to result in functional differences including differences in behaviour. A large panel of inbred strains could be used to identify genes functionally involved in particular phenotypes, similar to genetic correlation. The genetic correlation between expression profiles and function is potentially very powerful, especially given the current large‐scale generation of phenotypic data on multiple strains (the Mouse Phenome Project). As an example, the strongest genetic correlation between more than 200 probe sets showing significant differences among our eight inbred strains and a ranking of these strains by aggression phenotype was found for Comt, a gene known to be involved in aggression.

Relationship between VNTR polymorphisms of the human dopamine transporter gene and expression in post-mortem midbrain tissue.

Attention deficit hyperactivity disorder (ADHD) is currently one of the most prevalent childhood behavioral disorders. The disorder is found to be highly heritable, suggesting a large genetic component. Association studies have repeatedly implicated the dopamine transporter (DAT1) gene, and in particular the 10‐repeat allele of a variable number tandem repeat (VNTR) polymorphism located in the 3′UTR of the gene. Inconclusive data has been generated from several earlier studies on the functional effects of this polymorphism. Therefore, there is call for further investigation and thus the focus on data described here from TaqMan RT‐PCR assays. The expression levels of the DAT1 gene from post‐mortem midbrain tissue was measured in relation to the polymorphism present at the 3′UTR VNTR, together with a further VNTR marker located within intron 8 of the gene (Int8 VNTR). The findings suggest that the presence of the 10‐repeat allele of the 3′UTR VNTR, the 3‐repeat of the intron 8 VNTR and both VNTR markers are correlated with increased levels of the DAT1 transcript in midbrain post‐mortem tissue. Further work using linear regression (LR) shows agreement with the correlation analysis, and either nominal significance or a trend in that direction. Given the small sample size, bootstrapping‐derived confidence intervals were calculated for the LR. These empirical analyses suggest that the 3′UTR VNTR to show a significant main effect on relative DAT1 expression. Furthermore, a significant effect was found for the combined model (3′UTR and Int8 VNTR markers) on expression. These data provide further evidence on the plausible molecular mechanism underlying the aetiology of the disorder.

Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor

Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain.

Functional genetic polymorphisms in serotonin and dopamine gene systems and their significance in behavioural disorders

Many genes in the monoamine neurotransmitter pathways possess functional variants which have been associated with human behavioural disorders and traits, making them of important clinical relevance. In this chapter, we summarize the most recent literature concerning functional studies on these variants and their possible behavioural consequences. Such studies have adopted a variety of strategies. Key investigations have determined effects on gene expression at the level of transcription in mammalian cell cultures, human lymphoblasts and/or human post-mortem brain tissue employing a range of strategies including allele-specific expression. This has enabled the comparison of in vitro and in vivo data, and furthermore provides an improved perceptive of their respective advantages. Pharmacological studies have focused on the effects of gene variation at the protein level in terms of binding to ligands and drugs. Additionally, molecular biological approaches have identified transcription factors (DNA-binding proteins) that interact with the motifs within the polymorphisms themselves. Various neuroimaging studies have further determined the relationship of genotype with protein availability in the brain, thereby contributing further to an understanding of the in vivo functional significance of gene variants. Finally, there is growing evidence from both human and animal studies on the interaction of functional polymorphisms with the environment in determining behavioural outcomes. Taken together, these findings have contributed to a greater understanding of the plausible molecular mechanisms underpinning the functional significance of polymorphisms in monoamine neurotransmitter pathway genes and how they may influence behavioural phenotypes.

Genes within the serotonergic system are differentially expressed in human brain

BackgroundSerotonin is an important neurotransmitter with wide-ranging functions throughout the central nervous system. There is strong evidence to suggest that regulation of serotonergic gene expression might be related to genetic variability, and several studies have focused on understanding the functional effects of specific polymorphisms within these genes on expression levels. However, the combination of genotype together with gender and brain region could have an overall effect on gene expression. In this study, we report expression patterns of five serotonergic genes (TPH1, TPH2, 5-HT2A, 5-HT2C, 5-HTT) in seven different human post-mortem brain regions (superior frontal gyrus, superior temporal gyrus, striatum, cerebellum, hippocampus, midbrain and thalamus) using TaqMan™ real-time quantitative PCR. In addition, the effect of genotype and gender on their expression levels was determined.ResultsThe data revealed that mRNA from the five genes investigated was detected in all brain regions and showed an overall significant difference in expression levels. Furthermore, the expression of 5-HT2C, 5-HT2A and TPH2 was found to be significantly different between the various brain regions. However, neither gender nor genotype showed significant effects on the expression levels of any of the genes assayed. Interestingly, TPH1 and TPH2 were expressed in all brain regions similarly except for within the striatum and cerebellum, where TPH1 was expressed at a significantly higher level than TPH2.ConclusionThe effect of brain region has a greater influence on serotonergic gene expression than either genotype or gender. These data add to the growing body of evidence that effects of functional polymorphisms on gene expression in vitro are not observed ex vivo, and provide information that will aid in the design of expression studies of the serotonergic gene system within human post-mortem brain.

Regulation of striatal dopamine receptors by corticosterone: an in vivo and in vitro study

The effects of chronic corticosterone administration and adrenalectomy on the expression of brain dopamine receptors were studied in rats. In situ hybridization and receptor binding autoradiography were carried out to determine D1, D2 and D3 receptor expression in dorsal and ventral striata. Except for down-regulation of D2 mRNA in dorsal striatum after 2 week corticosterone treatment, no other significant changes were detected. In addition, the transcriptional regulation of D1 and D2 gene promoters by glucocorticoids was studied in neuroblastoma cell lines using transient transfections. While a small segment of the D2-promoter could be activated three-fold by dexamethasone, large fragments of neither D1 or D2 promoters were regulated by this treatment. Glucocorticoids do not appear to have direct overall effects on striatal dopamine receptor expression. The observed down-regulation of D2 receptor mRNA in the dorsal striatum in vivo is likely secondary to increased striatal dopamine release induced by corticosterone.

Putative transcriptomic biomarkers in the inflammatory cytokine pathway differentiate major depressive disorder patients from control subjects and bipolar disorder patients

Mood disorders consist of two etiologically related, but distinctly treated illnesses, major depressive disorder (MDD) and bipolar disorder (BPD). These disorders share similarities in their clinical presentation, and thus show high rates of misdiagnosis. Recent research has revealed significant transcriptional differences within the inflammatory cytokine pathway between MDD patients and controls, and between BPD patients and controls, suggesting this pathway may possess important biomarker properties. This exploratory study attempts to identify disorder-specific transcriptional biomarkers within the inflammatory cytokine pathway, which can distinguish between control subjects, MDD patients and BPD patients. This is achieved using RNA extracted from subject blood and applying synthesized complementary DNA to quantitative PCR arrays containing primers for 87 inflammation-related genes. Initially, we use ANOVA to test for transcriptional differences in a ‘discovery cohort’ (total n = 90) and then we use t-tests to assess the reliability of any identified transcriptional differences in a ‘validation cohort’ (total n = 35). The two most robust and reliable biomarkers identified across both the discovery and validation cohort were Chemokine (C-C motif) ligand 24 (CCL24) which was consistently transcribed higher amongst MDD patients relative to controls and BPD patients, and C-C chemokine receptor type 6 (CCR6) which was consistently more lowly transcribed amongst MDD patients relative to controls. Results detailed here provide preliminary evidence that transcriptional measures within inflammation-related genes might be useful in aiding clinical diagnostic decision-making processes. Future research should aim to replicate findings detailed in this exploratory study in a larger medication-free sample and examine whether identified biomarkers could be used prospectively to aid clinical diagnosis.

Characterization of the 5′ flanking region of the rat D3 dopamine receptor gene

The D3 dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D3 gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D3 gene. In this study, the 5′ flanking region of the rat D3 gene was characterized by isolating the 5′ end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196‐bp and 120‐bp long, separated by an 855‐bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D3 gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5′ RACE and was found to localize at a pyrimidine‐rich consensus ‘initiator’ sequence, similar to the rat D2 gene. The D3 promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D3 promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases −37 and −537. The D3 promoter is inactive in C6 and COS7 cells. We conclude that the D3 gene, similar to the closely related D2 gene, is transcribed from a tissue specific promoter which is under intense negative control.