Ruth Kohen

Cloning, Characterization, and Chromosomal Localization of a Human 5-HT6 Serotonin Receptor

Abstract: We describe the cloning and characterization of a human 5‐HT6 serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5‐HT6 cDNA encodes a 440‐amino‐acid polypeptide whose sequence diverges significantly from that published for the rat 5‐HT6 receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5‐HT6 amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5‐HT6 receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5‐HT1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5‐HT6 receptor also reveals an RsaI restriction fragment length polymorphism within the coding region.

Four 5‐Hydroxytryptamine7 (5‐HT7) Receptor Isoforms in Human and Rat Produced by Alternative Splicing: Species Differences Due to Altered Intron‐Exon Organization

Abstract: The serotonin (5‐HT) 5‐HT7 receptor subtype is thought to mediate a number of physiological effects in mammalian brain and periphery. Previous studies suggested that alternative splicing might contribute to 5‐HT7 receptor diversity as well. We now report that alternative splicing in human and rat tissues produces four 5‐HT7 receptor isoforms that differ in their predicted C‐terminal intracellular tails. Human and rat partial 5‐HT7 cDNAs and intronic sequences were identified and compared. In rat tissues, three 5‐HT7 isoforms, here called 5‐HT7(a), 5‐HT7(b), and 5‐HT7(c), are found. Rat 5‐HT7(a) [448‐amino acid (aa)] and 5‐HT7(b) (435‐aa) forms arise from alternative splice donor sites. A third new isoform found in rat, 5‐HT7(c) (470‐aa), results from a retained exon cassette. Three 5‐HT7 mRNA isoforms were also identified in human tissues, where only one isoform was previously described. Two human isoforms represent 5‐HT7(a) and 5‐HT7(b) forms (445‐ and 432‐aa), but the third form does not correspond to 5‐HT7(c). Instead, it constitutes a distinct isoform, 5‐HT7(d) (479‐aa), resulting from retention of a separate exon cassette. 5‐HT7(d) transcripts are not present in rat because the 5‐HT7(d)‐specifying exon is absent from the rat 5‐HT7 gene. A frame‐shifting homologue of the rat 5‐HT7(c)‐specifying exon is present in the human gene but is not used in the human tissues examined. Tissue‐specific splicing differences are present in human between brain and spleen. These studies suggest that alternative splicing may contribute to diversity of 5‐HT7 receptor action and that the human and rat repertoires of 5‐HT7 splice variants are substantially different.

Function and distribution of three rat 5-hydroxytryptamine7 (5-HT7) receptor isoforms produced by alternative splicing

Serotonin (5-HT7) receptor pre-mRNA is alternatively spliced in rat tissue to produce three isoforms, 5-HT(7a), 5-HT(7b) and 5-HT(7c), which differ in the amino acid sequences of their carboxyl terminal tails. Substantial species differences in structure and expression patterns exist for 5-HT7 isoforms. We have now compared some of the functional characteristics and level of expression for the three rat 5-HT7 receptor isoforms. Recombinant receptor isoforms were expressed in COS-7 cells for examination of [3H]5-HT binding characteristics and in JEG-3 cells to ascertain their ability to stimulate cAMP production. These studies showed that all three isoforms are functionally active and have similar agonist binding characteristics. Distribution of expression of the three rat receptor isoforms were examined in several brain regions and peripheral tissues using RT-PCR and in situ hybridization. The relative proportions of total 5-HT7 receptor message lent by each isoform varied little between these areas. In contrast to what has been observed in human tissue, the 5-HT(7a) isoform predominated in all regions examined, while the 5-HT(7c) isoform revealed a low level of expression (3% of total transcript). In situ hybridization was used to determine if the overall low level of expression of the 5-HT(7c) isoform by RT-PCR could be attributed to a small localized subpopulation of cells expressing high levels 5-HT(7c) message. In situ hybridization results indicate a generalized low level of expression of the 5-HT(7c) isoform throughout the CNS. These data suggest that while all three known 5-HT7 receptor isoforms in the rat are functionally competent, any functionally important differences between the three isoforms are not likely to involve differences in ligand binding or gross differences in adenylate cyclase coupling. However, differences in receptor phosphorylation, regulation or coupling to other effectors or cell trafficking could still exist.

Cloning of the mouse 5-HT6 serotonin receptor and mutagenesis studies of the third cytoplasmic loop

We have cloned the mouse 5-HT6 serotonin receptor and examined structure-function relationships in the C-terminal end of the third cytoplasmic (CIII) loop, introducing point mutations by site-directed mutagenesis at positions 264 to 268. We examined the ability of 5-HT6 wild type and receptor mutants to activate a cAMP responsive reporter gene when transiently expressed in JEG-3 or COS-7 cells. The wild type 5-HT6 receptor showed strong constitutive activity even when expressed at very low levels and which increased in proportion to the amount of receptor cDNA transfected. Three of the five mutants investigated (K264I, K267A and A268R) showed reduction in constitutive activity compared to wild type. These data suggest that constitutive activity may be important to 5-HT6 receptor activity in vivo and that, unlike some other G-protein coupled receptors, alteration in the BBXXB CIII-loop motif reduces rather than further activates basal activity of the murine 5-HT6 receptor.

Association of Serotonin Transporter Gene Polymorphisms With Poststroke Depression

CONTEXT Polymorphisms of the serotonin transporter gene (SERT) have been associated with mental illness. In people with long-term medical conditions, variants of the 5-HTTLPR and STin2 VNTR polymorphisms of SERT have been shown to confer a heightened vulnerability to comorbid depression. OBJECTIVE To determine whether the 5-HTTLPR, STin2 VNTR, and rs25531 polymorphisms of SERT are associated with poststroke depression (PSD) in stroke survivors. DESIGN A case-control study in which stroke survivors were screened for depressive symptoms and assigned to either a depressed group or a nondepressed group. SETTING Outpatient clinic. PARTICIPANTS Seventy-five stroke survivors with PSD and 75 nondepressed stroke survivors. INTERVENTIONS Blood or saliva samples were collected from each participant for DNA extraction and genotyping. MAIN OUTCOME MEASURES The associations between the 5-HTTLPR, STin2 VNTR, and rs25531 polymorphisms and PSD. RESULTS Individuals with the 5-HTTLPR s/s genotype had 3-fold higher odds of PSD compared with l/l or l/xl genotype carriers (odds ratio, 3.1; 95% confidence interval, 1.2-8.3). Participants with the STin2 9/12 or 12/12 genotype had 4-fold higher odds of PSD compared with STin2 10/10 genotype carriers (odds ratio, 4.1; 95% confidence interval, 1.2-13.6). An association of rs25531 with PSD was not shown. CONCLUSIONS The 5-HTTLPR and the STin2 VNTR, but not the rs25531, polymorphisms of SERT are associated with PSD in stroke survivors. This gives further evidence of a role of SERT polymorphisms in mediating resilience to biopsychosocial stress.

Relationship of SERT polymorphisms to depressive and anxiety symptoms in irritable bowel syndrome

This study investigates the association of psychological symptoms with the distribution of two serotonin transporter gene (SERT) polymorphisms, located in the promoter region (5-HTTLPR) and in intron 2 (STin2 VNTR), in patients with irritable bowel syndrome (IBS). Participants, 21 men and 117 women, were assessed for mental health history and current psychological distress. A blood sample was used for genotyping. Participants who were homozygous for the short allele of 5-HTTLPR or carried a STin2.9 VNTR allele were significantly more likely to have a history of depression. Participants did not differ by genotype in their history of anxiety or suicidal ideation nor in their current levels of depression, anxiety, or general psychological distress. The results support a biopsychosocial model of IBS in which SERT genotype modifies the risk for depressive episodes. Long term, practitioners may individualize treatment of patients with IBS using genotype as one of the factors.

Norepinephrine transporter mRNA is elevated in the locus coeruleus following short- and long-term desipramine treatment.

In situ hybridization for the norepinephrine transporter (NET) mRNA was performed in animals receiving short-term (2 days) and long-term (4 weeks) treatment with desipramine (DMI; 10 mg/kg, intraperitoneal). Following short-term and long-term DMI treatment, a significant (P < 0.05) increase in hybridization of 35S-labeled oligonucleotides to NET mRNA in the locus coeruleus was observed compared to that observed in vehicle-treated animals. The mechanism of this increase in transporter mRNA or its involvement in the therapeutic effects of anti-depressants remains to be determined.

Transcriptome profiling of human hippocampus dentate gyrus granule cells in mental illness

This study is, to the best of our knowledge, the first application of whole transcriptome sequencing (RNA-seq) to cells isolated from postmortem human brain by laser capture microdissection. We investigated the transcriptome of dentate gyrus (DG) granule cells in postmortem human hippocampus in 79 subjects with mental illness (schizophrenia, bipolar disorder, major depression) and nonpsychiatric controls. We show that the choice of normalization approach for analysis of RNA-seq data had a strong effect on results; under our experimental conditions a nonstandard normalization method gave superior results. We found evidence of disrupted signaling by miR-182 in mental illness. This was confirmed using a novel method of leveraging microRNA genetic variant information to indicate active targeting. In healthy subjects and those with bipolar disorder, carriers of a high- vs those with a low-expressing genotype of miR-182 had different levels of miR-182 target gene expression, indicating an active role of miR-182 in shaping the DG transcriptome for those subject groups. By contrast, comparing the transcriptome between carriers of different genotypes among subjects with major depression and schizophrenia suggested a loss of DG miR-182 signaling in these conditions.

Response to Psychosocial Treatment in Poststroke Depression Is Associated With Serotonin Transporter Polymorphisms

Background and Purpose— The Living Well With Stroke study has demonstrated effectiveness of a brief psychosocial treatment in reducing depressive symptoms after stroke. The purpose of this analysis was to determine whether key variables associated with prevalence of poststroke depression also predicted treatment response. Methods— Response to a brief psychosocial/behavioral intervention for poststroke depression was measured with the Hamilton Rating Scale for Depression. Analysis of covariance models tested for interaction of potential predictor variables with treatment group on percent change in Hamilton Rating Scale for Depression from pre- to post-treatment as an outcome. Results— Initial depression severity, hemispheric location, level of social support, age, gender, and antidepressant adherence did not interact with the treatment with respect to percent change in Hamilton Rating Scale for Depression when considered 1 at a time. Participants who carried 1 or 2 s-alleles at the 5-HTTLPR serotonin transporter polymorphism or 1 or 2 9- or 12-repeats of the STin2 VNTR polymorphism had significantly better response to psychosocial treatment than those with no s-alleles or no 9- or 12-repeats. Conclusions— Opposite to the effects of antidepressant drug treatment with selective serotonin reuptake inhibitors, the Living Well With Stroke psychotherapy intervention was most effective in 5-HTTLPR s-allele carriers and STin2 VNTR 9- or 12-repeat carriers. Clinical Trial Registration— URL: www.clinicaltrials.gov/ct/show/NCT00194454?order_1. Unique identifier: NCT00194454.

Living Well with Stroke: Design and Methods for a Randomized Controlled Trial of a Psychosocial Behavioral Intervention for Poststroke Depression

BACKGROUND Depression is a sufficiently common sequela of a completed stroke to warrant intervention to improve mood, social, and functional outcome. Pharmacologic trials suggest short-term mood improvement from antidepressant treatment but no studies to date have determined whether these short-term gains can be enhanced and extended by a brief psychosocial/behavioral intervention delivered by advanced practice nurses. In addition, drug trials have not reported on functional outcomes such as limitations in ability, limitations in participation, and overall quality of survival. This randomized controlled trial was designed to evaluate the short- and long-term efficacy of a new brief psychosocial/behavioral intervention adjunctive to antidepressant treatment in reducing poststroke depression and improving functional outcomes. METHODS A total of 101 survivors of ischemic stroke with poststroke depression were randomly assigned to receive a brief psychosocial/behavioral intervention plus antidepressant or usual care, including antidepressants. RESULTS The primary outcome was reduction in depressive symptom severity (Hamilton Depression Rating Scale) at 12 months after stroke. Secondary outcomes were reductions in limitations in activity (Barthel Index), reduction in limitation in participation, and overall stroke impact (Stroke Impact Scale) at 6, 12, and 24 months poststroke. Factors influencing best response to psychosocial intervention were also explored. CONCLUSION This article provides detail on the design and treatment methods of this randomized trial in progress. Findings from this study provide important information regarding the long-term efficacy of such a behavioral intervention in reducing poststroke depression and subsequent impaired aspects of psychosocial and physical recovery.