Human 5-HT1A Receptor Gene Research Articles

Transcriptional Regulation of the Human 5-HT1A Receptor Gene by Lithium: Role of Deaf1 and GSK3β.

Serotonin 1A (5-HT1A) autoreceptors located on serotonin neurons inhibit their activity, and their upregulation has been implicated in depression, suicide and resistance to antidepressant treatment. Conversely, post-synaptic 5-HT1A heteroreceptors are important for antidepressant response. The transcription factor deformed epidermal autoregulatory factor 1 (Deaf1) acts as a presynaptic repressor and postsynaptic enhancer of 5-HT1A transcription, but the mechanism is unclear. Because Deaf1 interacts with and is phosphorylated by glycogen synthase kinase 3β (GSK3β)-a constitutively active protein kinase that is inhibited by the mood stabilizer lithium at therapeutic concentrations-we investigated the role of GSK3β in Deaf1 regulation of human 5-HT1A transcription. In 5-HT1A promoter-reporter assays, human HEK293 kidney and 5-HT1A-expressing SKN-SH neuroblastoma cells, transfection of Deaf1 reduced 5-HT1A promoter activity by ~45%. To identify potential GSK3β site(s) on Deaf1, point mutations of known and predicted phosphorylation sites on Deaf1 were tested. Deaf1 repressor function was not affected by any of the mutants tested except the Y300F mutant, which augmented Deaf1 repression. Both lithium and the selective GSK3 inhibitors CHIR-99021 and AR-014418 attenuated and reversed Deaf1 repression compared to vector. This inhibition was at concentrations that maximally inhibit GSK3β activity as detected by the GSK3β-sensitive TCF/LEF reporter construct. Our results support the hypothesis that GSK3β regulates the activity of Deaf1 to repress 5-HT1A transcription and provide a potential mechanism for actions of GSK3 inhibitors on behavior.

Family-based association study of tryptophan hydroxylase 2 and serotonin 1A receptor genes in attention deficit hyperactivity disorder

Attention deficit hyperactivity disorder (ADHD) is a complex genetic disorder that is caused by multiple heritable and environmental factors. Serotonergic system-related genes are likely to be involved in the mechanisms underlying ADHD, because serotonin dysregulation has been related to impulsive and aggressive behavior in children, and has thus been hypothesized to play a causal role in ADHD. The firing rate of the dorsal raphe serotonergic neurons is modulated by somatodendritic 5-hydroxytryptamine 1A (5-HT1A) autoreceptors. The human 5-HT1A receptor gene (HTR1A) transcription is modulated by a functional C-1019G promoter polymorphism (rs6295). In a case–control association study with 78 Korean ADHD patients and 107 normal controls, Shim et al. (2010) found a significant difference in the genotype distributions and allele frequencies of HTR1A C-1019G between the ADHD group and the control group (P = 0.044 and 0.017, respectively). Tryptophan hydroxylase 2 (TPH2) is a rate-limiting enzyme in the biosynthesis of serotonin and is expressed exclusively in the brain. In 103 German families with 225 ADHD children, Walitza et al. (2005) reported a preferential transmission for two polymorphisms in TPH2’s regulatory region (rs4570625, P = 0.049; rs11178997, P = 0.034).

Human Freud-2/CC2D1B: A Novel Repressor of Postsynaptic Serotonin-1A Receptor Expression

Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot. Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

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.

The mental retardation gene CC2D1A/Freud‐1 encodes a long isoform that binds conserved DNA elements to repress gene transcription

The CC2D1A/Freud-1 gene has recently been linked to non-syndromic mental retardation and a short isoform of mouse Five prime REpressor Under Dual repression binding protein 1 (Freud-1) can repress the serotonin-1A (5-HT1A) receptor gene in rodent cells. In this study, we addressed the expression, localization and regulation of the human 5-HT1A receptor gene by a long isoform of human Freud-1 protein (Freud-1L). We show that human CC2D1A/Freud-1 RNA is expressed in brain and peripheral tissues and encodes short and long isoforms, which differ by an upstream in-frame translational start site. Whereas previous studies identified the short isoform of Freud-1 as the predominant isoform in rodent cells, we demonstrate that the long isoform is more abundant in human cells, especially in the nuclear fraction. The nuclear localization of Freud-1L was enriched upon inhibition of chromosome region maintenance 1/exportin 1-dependent nuclear export, indicating a dynamic regulation of Freud-1 nuclear localization. Consistent with a functional role in the nucleus, human Freud-1L bound specifically to its dual repressor element in the 5-HT1A receptor gene in vitro and repressed transcription from these sites. Importantly, chromatin immunoprecipitation using antibodies specific for human Freud-1L demonstrated that it is bound to the dual repressor element in chromatin, indicating a functional role in regulating the basal expression of the 5-HT1A receptor gene. Taken together, these results indicate that both the short and long isoforms of Freud-1 are expressed, although Freud-1L is the major isoform that regulates the human 5-HT1A receptor gene. Disruption of transcriptional regulation by mutation of Freud-1 may play a role in abnormal brain function leading to mental retardation.

Functional polymorphisms of the 5-HT1A and 5-HT1B receptor are associated with clinical symptoms in migraineurs

Migraine is regarded as a polygenic disease and serotonergic pathways appear to play a major role in its pathogenesis. In the present study, the role of the 5-HT1A and 5-HT1B receptors in migraine was evaluated. The human 5-HT1A receptor gene transcription is modulated by a functional C-1019G promoter polymorphism. The 5-HT1B receptor is the main effector of vasoconstriction in meningeal and cerebral arteries and its functional G861C promoter polymorphism was investigated. We report a positive association of the GG genotype of the 5-HT1A promoter polymorphism with avoidance of physical activity during a migraine attack in comparison to the CC genotype (p = 0.008). Moreover, a positive association of the CC genotype of the G861C polymorphism of the 5-HT1B receptor with the reported intensity of the headache attack on the visual analogue scale was observed (CC 8.3 +/- 1.5 vs. GG 6.9 +/- 1.8; p < 0.05). An association of either polymorphism with migraine with or without aura could not be found. For the first time, our results indicate a role of allelic variation of the 5-HT1A receptor in motion related discomfort in migraineurs and a role of the 5-HT1B receptor polymorphism in headache intensity.

Human 5-HT1A receptor C(-1019)G polymorphism and psychopathology.

Dysfunction of the serotonin (5-HT1A) receptor (5-HTR1A) has been implicated in mood disorders, anxiety disorders, psychosis and the action of antidepressants. A common C(-1018)G [C(-1019)G] functional polymorphism in the promoter region of the human 5-HT1A receptor gene has been reported, which may be useful in identifying psychopathology associated with altered function of the human 5-HT1A receptor. We studied the relationship of this polymorphism to psychopathology and 5-HT1A binding in prefrontal cortex. The 5-HT1A receptor genotype for the C(-1019)G polymorphism was typed in 696 unrelated psychiatric subjects, 107 unrelated healthy volunteers, and in post-mortem brain samples from 241 cases. 5-HT1A receptor binding was assayed in post-mortem prefrontal cortex using [3H]8-OH-DPAT, and specific binding determined by 1 microM 5-HT. An association of genotype distribution and allele frequency of the 5-HTR1A C(-1019)G locus was observed in schizophrenia (chi2=9.51, d.f.=2, p=0.009; chi2=9.52, d.f.=1, p=0.002; Armitage's trend test: chi2=9.07, d.f.=1, p=0.003), in substance use disorder (chi2=8.41, d.f.=2, p=0.015; chi2=8.35, d.f.=1, p=0.004; Armitage's trend test: chi2=6.27, d.f.=1, p=0.0012), and in panic attack (chi2=6.31, d.f.=2, p=0.043; chi2=6.14, d.f.=1, p=0.013; Armitage's trend test: chi2=6.27, d.f.=1, p=0.012). An association of the 5-HTR1A C(-1019)G locus with schizophrenia, substance use disorder, and panic attack was suggested by our results. In post-mortem brain samples, 5-HT1A receptor binding in prefrontal cortex and suicide were not associated with genotype. The relationship does not appear to be explained by binding differences, although we cannot rule out altered receptor affinity and transduction.

A common C-1018G polymorphism in the human 5-HT1A receptor gene.

A common C-1018G polymorphism in the human 5-HT1A receptor gene.

Novel mutations in the promoter and coding region of the human 5-HT1A receptor gene and association analysis in schizophrenia.

Dysfunction of serotonin systems has been implicated in schizophrenia. In the present study, the human 5-HT1A receptor gene containing the 5' untranslated region was screened in order to detect genetic variations, through which alteration of protein function or level of expression might contribute to schizophrenia. Genomic DNAs were isolated from whole-blood samples of 61 unrelated schizophrenic patients and 100 healthy controls. Genetic variations were screened systematically by single-strand conformational polymorphism (SSCP) analysis, followed by direct sequencing of polymerase chain reaction (PCR) product as well as restriction fragment-length polymorphism (RFLP). The novel mutations (-51T --> C, -152C --> G, -321G --> C, -480delA, and -581C --> A) were found in the 5' untranslated region. Furthermore, we found a novel missense mutation (Gly272Asp) in the coding region in addition to the mutations (Pro16Leu, 294G --> A, and 549C --> T) reported previously. No significant differences in genotype frequencies as well as allele frequencies were found between patients and controls. Our data provided no evidence of association between schizophrenia and the variants in the 5' untranslated region as well as the coding region of the human 5-HT1A receptor gene.

A 413 bp region upstream the human 5-HT1A receptor gene is sufficient for its in vitro expression

The regulation of a specific pattern of neuronal 5-hydroxytryptamine-1A (5-HT1A) receptor gene expression in the brain is hardly understood. Identification of cis-acting DNA sequences that control expression of the human 5-HT1A receptor gene has been undertaken by fusion of a 413 base pairs (bp)-long DNA segment upstream the human 5-HT1A receptor gene start codon to the 5-HT1A receptor gene. When this fusion product was inserted into the expression vector pcDNA3 and transfected into Cos-7 cells, it was shown to drive expression of specific [3H] (+)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H] 8-OH-DPAT) binding sites with a Kd of 1.18 nM and a Bmax of 105 fmol/mg protein. The yield of expression of [3H] 8-OH-DPAT binding sites by fusion of the 5-HT1A receptor gene to strong promoters, such as the Rous sarcoma virus long terminal repeat and the cytomegalovirus promoter, was 2.5 to 8 times greater. The pharmacological binding profile of these [3H] 8-OH-DPAT binding sites was similar to that of cloned human 5-HT1A receptors stably expressed in HeLa cells. The importance of an Initiator sequence and regulatory Spl sites in the 413 bp region to direct transcription of the human 5-HT1A receptor gene is discussed.

A 413 bp region upstream the human 5-HT1A receptor gene is sufficient for its in vitro expression

The regulation of a specific pattern of neuronal 5-hydroxytryptamine-1A (5-HT1A) receptor gene expression in the brain is hardly understood. Identification of cis-acting DNA sequences that control expression of the human 5-HT1A receptor gene has been undertaken by fusion of a 413 base pairs (bp)-long DNA segment upstream the human 5-HT1A receptor gene start codon to the 5-HT1A receptor gene. When this fusion product was inserted into the expression vector pcDNA3 and transfected into Cos-7 cells, it was shown to drive expression of specific [3H] (+)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H] 8-OH-DPAT) binding sites with a Kd of 1.18 nM and a Bmax of 105 fmol/mg protein. The yield of expression of [3H] 8-OH-DPAT binding sites by fusion of the 5-HT1A receptor gene to strong promoters, such as the Rous sarcoma virus long terminal repeat and the cytomegalovirus promoter, was 2.5 to 8 times greater. The pharmacological binding profile of these [3H] 8-OH-DPAT binding sites was similar to that of cloned human 5-HT1A receptors stably expressed in HeLa cells. The importance of an Initiator sequence and regulatory Spl sites in the 413 bp region to direct transcription of the human 5-HT1A receptor gene is discussed.