Increased serotonin-1A (5-HT1A) autoreceptor expression and reduced raphe serotonin levels in deformed epidermal autoregulatory factor-1 (Deaf-1) gene knock-out mice.

Altered regulation of the serotonin-1A (5-HT1A) receptor gene is implicated in major depression and mood disorders. The functional human 5-HT1A C(-1019)G promoter polymorphism (rs6295), which prevents the binding of Deaf-1/NUDR leading to dysregulation of the receptor, has been associated with major depression. In cell models Deaf-1 displays dual activity, repressing 5-HT1A autoreceptor expression in serotonergic raphe cells while enhancing postsynaptic 5-HT1A heteroreceptor expression in nonserotonergic neurons. A functional Deaf-1 binding site on the mouse 5-HT1A promoter was recognized by Deaf-1 in vitro and in vivo and mediated dual activity of Deaf-1 on 5-HT1A gene transcription. To address regulation by Deaf-1 in vivo, Deaf-1 knock-out mice bred to a C57BL/6 background were compared with wild-type siblings for changes in 5-HT1A RNA and protein by quantitative RT-PCR, in situ hybridization, and immunofluorescence. In the dorsal raphe, Deaf-1 knock-out mice displayed increased 5-HT1A mRNA, protein, and 5-HT1A-positive cell counts but reduced 5-HT levels, whereas other serotonergic markers, such as tryptophan hydroxylase (TPH)- or 5-HT-positive cells and TPH2 RNA levels, were unchanged. By contrast, 5-HT1A mRNA and 5-HT1A-positive cells were reduced in the frontal cortex of Deaf-1-null mice, with no significant change in hippocampal 5-HT1A RNA, protein, or cell counts. The region-specific alterations of brain 5-HT1A gene expression and reduced raphe 5-HT content in Deaf-1(-/-) mice indicate the importance of Deaf-1 in regulation of 5-HT1A gene expression and provide insight into the role of the 5-HT1A G(-1019) allele in reducing serotonergic neurotransmission by derepression of 5-HT1A autoreceptors.

Region-specific regulation of 5-HT1A receptor expression by Pet-1-dependent mechanisms in vivo.

Serotonin (5-hydroxytryptamine, 5-HT) neurotransmission is negatively regulated by 5-HT1A autoreceptors on raphe neurons, and is implicated in mood disorders. Pet-1/FEV is an ETS transcription factor expressed exclusively in serotonergic neurons and is essential for serotonergic differentiation, although its regulation of 5-HT receptors has not yet been studied. Here, we show by electrophoretic mobility shift assay that recombinant human Pet-1/FEV binds directly to multiple Pet-1 elements of the human 5-HT1A receptor promoter to enhance its transcriptional activity. In luciferase reporter assays, mutational analysis indicated that while several sites contribute, the Pet-1 site at -1406 bp had the greatest effect on 5-HT1A promoter activity. To address the effect of Pet-1 on 5-HT1A receptor regulation in vivo, we compared the expression of 5-HT1A receptor RNA and protein in Pet-1 null and wild-type littermate mice. In the raphe nuclei of Pet-1-/- mice tryptophan hydroxylase 2 (TPH2) RNA, and 5-HT and TPH immunostaining were greatly reduced, indicating a deficit in 5-HT production. Raphe 5-HT1A RNA and protein levels were also reduced in Pet-1-deficient mice, consistent with an absence of Pet-1-mediated transcriptional enhancement of 5-HT1A autoreceptors in serotonergic neurons. Interestingly, 5-HT1A receptor expression was up-regulated in the hippocampus, but down-regulated in the striatum and cortex. These data indicate that, in addition to transcriptional regulation by Pet-1 in raphe neurons, 5-HT1A receptor expression is regulated indirectly by alterations in 5-HT neurotransmission in a region-specific manner that together may contribute to the aggressive/anxiety phenotype observed in Pet-1 null mice.

Deaf1 isoforms control the expression of genes encoding peripheral tissue antigens in the pancreatic lymph nodes during type 1 diabetes.

Type 1 diabetes may result from a breakdown in peripheral tolerance that is partially controlled by the expression of peripheral tissue antigens (PTAs) in lymph nodes. Here we show that the transcriptional regulator Deaf1 controls the expression of genes encoding PTAs in the pancreatic lymph nodes (PLNs). The expression of canonical Deaf1 was lower, whereas that of an alternatively spliced variant was higher, during the onset of destructive insulitis in the PLNs of nonobese diabetic (NOD) mice. We identified an equivalent variant Deaf1 isoform in the PLNs of patients with type 1 diabetes. Both the NOD mouse and human Deaf1 variant isoforms suppressed PTA expression by inhibiting the transcriptional activity of canonical Deaf1. Lower PTA expression resulting from the alternative splicing of DEAF1 may contribute to the pathogenesis of type 1 diabetes.

Gender-specific decrease in NUDR and 5-HT1A receptor proteins in the prefrontal cortex of subjects with major depressive disorder.

A variety of studies have documented alterations in 5-HT1A receptor binding sites in the brain of subjects with major depressive disorder (MDD). The recently identified transcription factor, nuclear deformed epidermal autoregulatory factor (NUDR/Deaf-1) has been shown to function as a transcriptional modulator of the human 5-HT1A receptor gene. The present study was undertaken to document the regional and cellular localization of NUDR in the human prefrontal cortex and to examine the levels of NUDR and 5-HT1A receptor protein in prefrontal cortex of female and male depressed and control subjects. NUDR immunoreactivity was present in neurons and glia across cortical layers and was co-localized with 5-HT1A receptor immunoreactive neurons. NUDR immunoreactivity as measured by Western blot was significantly decreased in the prefrontal cortex of female depressed subjects (42%, p=0.02) and unchanged in male depressed subjects relative to gender-matched control subjects. Similarly, 5-HT1A receptor protein level was significantly reduced in the prefrontal cortex of female depressed subjects (46%, p=0.03) and unchanged in male depressed subjects compared to gender-matched control subjects. Reduced protein expression of NUDR in the prefrontal cortex of female subjects with MDD may reflect a functional alteration in this transcription factor, which may contribute to the decrease in 5-HT1A receptors observed in the same female subjects with MDD. In addition, the gender-specific alterations in cortical NUDR and 5-HT1A receptor proteins could represent an underlying biological mechanism associated with the higher incidence of depression in women.

Transcriptional regulation at a HTR1A polymorphism associated with mental illness.

The serotonin-1A (5-HT1A) receptor serves as a hub to regulate the activity and actions of the serotonin system, and is expressed both as a presynaptic autoreceptor on raphe neurons, and as a major postsynaptic receptor in hippocampal, cortical, and hypothalamic regions involved in mood, emotion and stress response. As such, the level of expression of 5-HT1A receptors is implicated in the development of anxiety and depression phenotypes. This review focuses on the C(-1019)G (rs6295) promoter polymorphism of the 5-HT1A receptor gene (HTR1A) and its effect on the activity of transcription factors that recognize the C-allele, including Deaf-1, Hes1 and Hes5; its effects on 5-HT1A receptor expression in pre- and postsynaptic areas; as well as its implication in early postnatal development and adult neurogenesis in the hippocampus and cortex. Although several studies have now replicated the association of the G-allele with depression, panic disorder, neuroticism, and reduced response to antidepressant or antipsychotic treatment, ethnic, disease and genetic heterogeneity among subjects in different studies may obscure such associations. Gene-gene interaction studies suggest that the 5-HT1A receptor G(-1019) allele is a risk allele which could be used as a marker for depression and related mood disorders. Finally, association of the G(-1019) allele with increased raphe 5-HT1A binding potential, increased amygdala reactivity to emotional stimuli, and reduced amygdala volume, particularly in disease states, suggests a functional role for the C(-1019)G site in 5-HT1A receptor dys-regulation and predisposition to mental illness.

Cell-type specific induction of tryptophan hydroxylase-2 transcription by calcium mobilization.

Alterations in brain serotonin levels are implicated in major depression and are regulated by tryptophan hydroxylase-2 (TPH2). To study its regulation, we measured TPH2 RNA by quantitative RT-PCR in differentiated serotonergic rat raphe RN46A and GH4C1 pituitary cells, which express TPH2. Upon calcium mobilization using KCl (40 mmol/L), TPH2 RNA was rapidly (1 h) and strongly (> 10-fold) induced in differentiated RN46A cells, but not in GH4C1 cells. This effect was blocked by actinomycin D, implicating transcriptional activation. Similarly, calcium ionophore ionomycin induced TPH2 RNA by threefold in RN46A cells. To address the promoter sites involved, the transcription start site was identified and a series of TATA-containing TPH2 promoter-luciferase constructs were analyzed. In differentiated RN46A cells, the TPH2 promoter was induced 2.5-fold by ionomycin, similar to its action on TPH2 RNA. By contrast, ionomycin had no effect on TPH2 promoter activity in GH4C1 cells or TPH2-negative L6 myoblasts. Ionomycin sensitivity was localized to within 88 bp of the start site, containing putative CCATT-enhancer binding protein element, activator protein-1 and -2 (AP-1, AP-2) elements. These results are the first to identify calcium-mediated regulation of the proximal TPH2 promoter as critical for cell-specific TPH2 expression.

Differential signaling of dopamine-D2S and -D2L receptors to inhibit ERK1/2 phosphorylation.

Although they have distinct functions, the signaling of dopamine-D(2) receptor short and long isoforms (D(2)S and D(2)L) is virtually identical. We compared inhibitory regulation of extracellular signal-regulated kinases (ERK1/2) in GH4 pituitary cells separately transfected with these isoforms. Activation of rat or human dopamine-D(2)S, muscarinic or somatostatin receptors inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation, while the D(2)L receptor failed to inhibit this response. In order to address the structural basis for the differential signaling of D(2)S and D(2)L receptors, we examined the D(2)L-SS mutant, in which a protein kinase C (PKC) pseudosubstrate site that is present in the D(2)L but not D(2)S receptor was converted to a consensus PKC site. In transfected GH4 cells, the D(2)L-SS mutant inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation almost as strongly as the D(2)S receptor. A D(2)S-triple mutant that eliminates PKC sites involved in D(2)S receptor desensitization also inhibited ERK1/2 activation. Similarly, in striatal cultures, the D(2)-selective agonist quinpirole inhibited potassium-stimulated ERK1/2 phosphorylation, indicating the presence of this pathway in neurons. In conclusion, the D(2)S and D(2)L receptors differ in inhibitory signaling to ERK1/2 due to specific residues in the D(2)L receptor alternatively spliced domain, which may account for differences in their function in vivo.

Characterization of rat rostral raphe primary cultures: multiplex quantification of serotonergic markers.

Previous reports establishing raphe cultures typically yield less than 1% serotonin (5-HT)-positive neurons and are impractical for transcriptional studies. In this study, we have established primary cultures enriched in 5-HT neurons and quantified the proportion of cells expressing serotonergic and non-serotonergic markers. We have also shown the feasibility of using the multiplex real-time PCR technique to measure the relative amounts of RNA for some of these markers. Rostral raphe cells derived from E13-15 rat embryos were cultured for 7 days and analyzed by quantitative immunofluorescence and western blot analysis. In these cultures, approximately 8% of neurons were immunopositive for serotonergic markers (5-HT or tryptophan hydroxylase (TPH)). The percentage of cells labeled for GFAP (glial marker), tyrosine hydroxylase (catecholaminergic), and GAD65/67 (GABAergic) was 5, 1, and 54%, respectively. Transcription factors REST/NRSF and Deaf-1 were present in 9 and 98% of cells, respectively. Multiplex quantitative RT-PCR (Q-PCR) analysis was done for TPH2, 5-HT1A receptor or Deaf-1 RNAs paired with GAPDH RNA as control. Using this approach, standard curves for each RNA were obtained over 200-fold concentration range of dilution with r2 values >0.99. The relative abundances determined by Q-PCR are consistent with the expression of TPH2>Deaf-1>5-HT1A receptor RNA in serotonergic raphe cells. The standard error of TPH2 RNA levels between cultures was <20%, indicating a consistent purity of 5-HT neurons. Thus, we have generated a highly consistent and reproducible model system that is enriched in 5-HT neurons and that will be valuable in future investigation of serotonergic regulation.

Cell-specific repressor or enhancer activities of Deaf-1 at a serotonin 1A receptor gene polymorphism.

The serotonin-1A (5-HT1A) receptor is the primary somatodendritic autoreceptor that inhibits the activity of serotonergic raphe neurons and is also expressed in nonserotonergic cortical and limbic neurons. Alterations in 5-HT1A receptor levels are implicated in mood disorders, and a functional C(-1019)G 5-HT1A promoter polymorphism has been associated with depression, suicide, and panic disorder. We examined the cell-specific activity of identified transcription factors, human nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR)/Deaf-1 and Hes5, at the 5-HT1A C(-1019) site. In serotonergic raphe RN46A cells, Deaf-1 and Hes5 repressed the 5-HT1A receptor gene at the C(-1019)-allele but not the G(-1019)-allele. However, in nonserotonergic cells that express 5-HT1A receptors (septal SN48, neuroblastoma SKN-SH, and neuroblastoma/glioma NG108-15 cells), Deaf-1 enhanced 5-HT1A promoter activity at the C(-1019)-allele but not the G-allele, whereas Hes5 repressed in all cell types. The enhancer activity of Deaf-1 was orientation independent and competed out Hes5 repression. To test whether Deaf-1 activity is intrinsic, the activity of a Gal4DBD (DNA binding domain)-Deaf-1 fusion protein at a heterologous Gal4 DNA element was examined. Gal4DBD-Deaf-1 repressed transcription in RN46A cells but enhanced transcription in SN48 cells, indicating that these opposite activities are intrinsic to Deaf-1. Repressor or enhancer activities of Deaf-1 or Gal4DBD-Deaf-1 were blocked by histone deacetylase inhibitor trichostatin A. Thus, the intrinsic activity of Deaf-1 at the 5-HT1A promoter is opposite in presynaptic versus postsynaptic neuronal cells and requires deacetylation. Cell-specific regulation by Deaf-1 could underlie region-specific alterations in 5-HT1A receptor expression in different mood disorders.