Pablo R. Moya

How the Serotonin Story is Being Rewritten By New Gene-Based Discoveries Principally Related to SLC6A4, the Serotonin Transporter Gene, Which Functions To Influence All Cellular Serotonin Systems

Discovered and crystallized over sixty years ago, serotonins important functions in the brain and body were identified over the ensuing years by neurochemical, physiological and pharmacological investigations. This 2008 M. Rapport Memorial Serotonin Review focuses on some of the most recent discoveries involving serotonin that are based on genetic methodologies. These include examples of the consequences that result from direct serotonergic gene manipulation (gene deletion or overexpression) in mice and other species; an evaluation of some phenotypes related to functional human serotonergic gene variants, particularly in SLC6A4, the serotonin transporter gene; and finally, a consideration of the pharmacogenomics of serotonergic drugs with respect to both their therapeutic actions and side effects. The serotonin transporter (SERT) has been the most comprehensively studied of the serotonin system molecular components, and will be the primary focus of this review. We provide in-depth examples of gene-based discoveries primarily related to SLC6A4 that have clarified serotonins many important homeostatic functions in humans, non-human primates, mice and other species.

A Haplotype Containing Quantitative Trait Loci for SLC1A1 Gene Expression and Its Association With Obsessive-Compulsive Disorder

CONTEXT Recent evidence from linkage analyses and follow-up candidate gene studies supports the involvement of SLC1A1, which encodes the neuronal glutamate transporter, in the development of obsessive-compulsive disorder (OCD). OBJECTIVES To determine the role of genetic variation of SLC1A1 in OCD in a large case-control study and to better understand how SLC1A1 variation affects functionality. DESIGN A case-control study. SETTING Publicly accessible SLC1A1 expression and genotype data. PATIENTS Three hundred twenty-five OCD probands and 662 ethnically and sex-matched controls. INTERVENTIONS Probands were assessed with the Structured Clinical Interview for DSM-IV, the Yale-Brown Obsessive Compulsive Scale, and the Saving Inventory-Revised. Six single-nucleotide polymorphisms (SNPs) were genotyped. Multiple testing corrections for single-marker and haplotype analyses were performed by permutation. RESULTS Gene expression of SLC1A1 is heritable in lymphoblastoid cell lines. We identified 3 SNPs in or near SLC1A1 that correlated with gene expression levels, 1 of which had previously been associated with OCD. Two of these SNPs also predicted expression levels in human brain tissue, and 1 SNP was further functional in reporter gene studies. Two haplotypes at 3 SNPs, rs3087879, rs301430, and rs7858819, were significantly associated with OCD after multiple-testing correction and contained 2 SNPs associated with expression levels. In addition, another SNP correlating with SLC1A1 gene expression, rs3933331, was associated with an OCD-hoarding subphenotype as assessed by 2 independent, validated scales. CONCLUSIONS Our case-control data corroborate previous smaller family-based studies that indicated that SLC1A1 is a susceptibility locus for OCD. The expression and genotype database-mining approach we used provides a potentially useful complementary approach to strengthen future candidate gene studies in neuropsychiatric and other disorders.

Anxiety and affective disorder comorbidity related to serotonin and other neurotransmitter systems: obsessive–compulsive disorder as an example of overlapping clinical and genetic heterogeneity

Individuals with obsessive–compulsive disorder (OCD) have also been shown to have comorbid lifetime diagnoses of major depressive disorder (MDD; rates greater than 70%), bipolar disorder (rates greater than 10%) and other anxiety disorders (e.g. panic disorder, post-traumatic stress disorder (PTSD)). In addition, overlap exists in some common genetic variants (e.g. the serotonin transporter gene (SLC6A4), the brain-derived neurotrophic factor (BDNF) gene), and rare variants in genes/chromosomal abnormalities (e.g. the 22q11 microdeletion syndrome) found across the affective/anxiety disorder spectrums. OCD has been proposed as a possible independent entity for DSM-5, but by others thought best retained as an anxiety disorder subtype (its current designation in DSM-IV), and yet by others considered best in the affective disorder spectrum. This review focuses on OCD, a well-studied but still puzzling heterogeneous disorder, regarding alterations in serotonergic, dopaminergic and glutamatergic neurotransmission in addition to other systems involved, and how related genes may be involved in the comorbidity of anxiety and affective disorders. OCD resembles disorders such as depression, in which gene × gene interactions, gene × environment interactions and stress elements coalesce to yield OC symptoms and, in some individuals, full-blown OCD with multiple comorbid disorders.

Lentivirally mediated GSK-3β silencing in the hippocampal dentate gyrus induces antidepressant-like effects in stressed mice.

Inhibition of glycogen synthase kinase-3 (GSK-3) by pharmacological tools can produce antidepressant-like effects in rodents. However, the GSK-3 isoform(s) and brain region(s) involved in regulating these behavioural effects remain elusive. We studied the effects of bilateral intra-hippocampal injections of lentivirus-expressing short-hairpin (sh)RNA targeting GSK-3β on behavioural performance in mice subjected to chronic stress. Pre-injection of lentivirus-expressing GSK-3β shRNA into the hippocampal dentate gyrus significantly decreased immobility time in both forced swim and tail suspension tests, while the locomotor activity of these mice was unchanged. These results suggest that lentiviral GSK-3β shRNA injection induces antidepressant-like effects in chronically stressed mice. Under these conditions, the expression levels of GSK-3β were persistently and markedly reduced in the hippocampus following GSK-3β shRNA injection. To our knowledge, this is the first demonstration that a single injection of lentivirus-expressing GSK-3β shRNA in the hippocampal dentate gyrus of chronically stressed mice has antidepressant-like effects elicited by gene silencing.

Altered 5-HT2C receptor agonist-induced responses and 5-HT2C receptor RNA editing in the amygdala of serotonin transporter knockout mice

BackgroundThe serotonin 5-HT2C receptor (5-HT2CR) is expressed in amygdala, a region involved in anxiety and fear responses and implicated in the pathogenesis of several psychiatric disorders such as acute anxiety and post traumatic stress disorder. In humans and in rodent models, there is evidence of both anxiogenic and anxiolytic actions of 5-HT2C ligands. In this study, we determined the responsiveness of 5-HT2CR in serotonin transporter (SERT) knockout (-/-) mice, a model characterized by increased anxiety-like and stress-responsive behaviors.ResultsIn the three-chamber social interaction test, the 5-HT2B/2C agonist mCPP decreased sociability and sniffing in SERT wildtype (+/+) mice, both indicative of the well-documented anxiogenic effect of mCPP. This 5-HT2C-mediated response was absent in SERT -/- mice. Likewise, in the open field test, the selective 5-HT2C agonist RO 60-0175 induced an anxiogenic response in SERT +/+ mice, but not in SERT -/- mice. Since 5-HT2CR pre-mRNA is adenosine-to-inosine (A-to-I) edited, we also evaluated the 5-HT2CR RNA editing profiles of SERT +/+ and SERT -/- mice in amygdala. Compared to SERT +/+ mice, SERT-/- mice showed a decrease in less edited, highly functional 5-HT2C isoforms, and an increase in more edited isoforms with reduced signaling efficiency.ConclusionsThese results indicate that the 5-HT2CR in the amygdala of SERT -/- mice has increased RNA editing, which could explain, at least in part, the decreased behavioral responses to 5-HT2C agonists in SERT -/- mice. These alterations in 5-HT2CR in amygdala may be relevant to humans with SERT polymorphisms that alter SERT expression, function, and emotional behaviors.

Increased gene expression of diacylglycerol kinase eta in bipolar disorder

Bipolar disorder (BD) is a highly heritable neuropsychiatric illness characterized by recurrent episodes of depression and mania or hypomania. Recent genome-wide association studies of BD have proposed novel candidates, including the gene encoding the η isoform of diacylglycerol kinase (DGKH; Baum et al. 2008a). The association of this gene with BD was replicated at the haplotype level in a Sardinian sample (Squassina et al. 2009), but not for individual SNPs or as a predictor of lithium response (Manchia et al. 2009). Diacylglycerol kinase (DGK) regulates the intracellular concentration of diacylglycerol, which is a main component of inositol signalling systems targeted by the major anti-BD agent, lithium (reviewed in van Blitterswijk et al. 2000; see also Harwood, 2005, for discussion of the inositol-depletion hypothesis and lithium and BD). DGKH is highly expressed in brain and is also glucocorticoid-inducible and stress-responsive (Klauck et al. 1996; Murakami et al. 2003). Here, we present evidence that gene expression of DGKH is increased in BD. We quantified DGKH mRNA by real-time quantitative polymerase chain reaction in a collection of 100 post-mortem brain-tissue samples donated by The Stanley Medical Research Institute. All samples were derived from the dorsolateral prefrontal cortex and originated from three diagnostic groups: BD (n = 31), schizophrenia (n = 35) and unaffected controls (n = 34). We used intron-spanning primers TGA CAG CAC AGA AAC AGA TGA AT and GGA GAC CGA GGT GCA GTT T as well as a fluorescent probe (Universal Probe Library no. 69, Roche Applied Science, USA) under amplification conditions described previously (Wendland et al. 2009). Experiments were done after the specimen code was broken and they were thus unblinded. Using G*Power version 3.1.2 (Faul et al. 2007), we determined our sample had some power (56%) to detect medium effect sizes (f = 0.25) and excellent power (94%) for large effect sizes (f = 0.4) in a fixed-effects omnibus analysis of variance test. The entire dataset has been uploaded to The Stanley Medical Research Institute databank (http://www.stanleyresearch.org/brain). We did not apply multiple testing correction for all comparable expression genetics studies uploaded to this databank. All experiments were conducted under protocols approved by the Institutional Review Board of the National Institute of Mental Health Division of Intramural Research Programs in Bethesda, MD. One-way analysis of variance showed that the three diagnostic groups differed significantly in their DGKH mRNA expression levels (F2,97 = 4.44, p = 0.01). As shown in Fig. 1, BD samples displayed significantly increased DGKH levels (Tukey’s post-hoc analysis, p <0.05), with the mean expression level being about 25% higher in BD than in controls. We also quantified two known DGKH transcript variants (Murakami et al. 2003) with transcript-specific probes but did not observe significant differences; moreover, we did not observe significant sex or age effects (data not shown). Fig. 1 Expression levels of DGKH mRNA in post-mortem brain tissue samples of three diagnostic groups. A total of 100 samples [bipolar disorder (BD, n = 31), schizophrenia (SZ, n = 35) and unaffected controls (n = 34)] were analysed by quantitative polymerase ... We genotyped all samples for two SNPs within the genomic DGKH locus, rs1170191 and rs1012053, that remained associated with BD in a meta-analysis (Baum et al. 2008b). Allelic and genotypic frequencies did not significantly differ between the three diagnostic groups for both SNPs. There was no association of either SNP with DGKH mRNA levels, even after inclusion of diagnosis as covariate (data not shown). Our data suggest that increased gene expression of DGKH is involved in the pathogenesis of BD. Interestingly, the BD disorder group did not differ significantly from the schizophrenia group (although the latter was also not significantly different from normal controls, Fig. 1), raising the possibility that the observed increase is not specific for BD. The average expression level was 20%higher in schizophrenia donors compared to controls, but this did not reach statistical significance in the post-hoc test. Larger post-mortem and other functional studies with greater power might clarify the role of DGKH in other psychiatric disorders. The DGKH over-expression hypothesis described above can be tested in future genetics studies once expression quantitative trait loci have been identified for DGKH. Further, model organisms over-expressing Dgkh may allow further insight into pathogenetic mechanisms of BD. The lack of correlation of SNPs that first implicated DGKH in BD with expression levels suggests that other variants conferring allele-specific expression may result in stronger association signals with the disorder. It thus seems warranted to identify variants correlating with high levels of DGKH mRNA or gain-of-function coding polymorphisms as carriers of these alleles can be postulated to be at increased risk for BD. It is our hope that this study might contribute to the elucidation of the pathogenesis of this debilitating disorder.

miR-15a and miR-16 regulate serotonin transporter expression in human placental and rat brain raphe cells

The serotonin transporter (SERT) is a key regulatory molecule in serotonergic transmission implicated in numerous biological processes relevant to human disorders. Recently, it was shown that SERT expression is controlled by miR-16 in mouse brain. Here, we show that SERT expression is regulated additionally by miR-15a as well as miR-16 in human and rat tissues. This post-transcriptional regulation was observed and characterized in reporter assays and likewise when endogenous SERT expression was evaluated in human placental choriocarcinoma JAR cells and rat brain raphe RN46A cells - two cell lines that endogenously express SERT. Similar effects for miR-16 to those of miR-15a were found in both human and rat cell lines. The effects of miR-15a and miR-16 were comparable in extent to those originally reported for miR-16 in mice. These findings represent a novel layer of complexity for SERT expression regulation exerted by the mir-15a/16 cluster, whose genes are adjacently located at human chromosome 13q14.3.

Common and rare alleles of the serotonin transporter gene, SLC6A4, associated with Tourette's disorder

To evaluate the hypothesis that functionally over‐expressing alleles of the serotonin transporter (SERT) gene (solute carrier family 6, member 4, SLC6A4) are present in Tourettes disorder (TD), just as we previously observed in obsessive compulsive disorder (OCD), we evaluated TD probands (N = 151) and controls (N = 858). We genotyped the refined SERT‐linked polymorphic region 5‐HTTLPR/rs25531 and the associated rs25532 variant in the SLC6A4 promoter plus the rare coding variant SERT isoleucine‐to‐valine at position 425 (I425V). The higher expressing 5‐HTTLPR/rs25531 LA allele was more prevalent in TD probands than in controls (χ2 = 5.75; P = 0.017; odds ratio [OR], 1.35); and, in a secondary analysis, surprisingly, it was significantly more frequent in probands who had TD alone than in those who had TD plus OCD (Fishers exact test; P = 0.0006; OR, 2.29). Likewise, the higher expressing LAC haplotype (5‐HTTLPR/rs25531/rs25532) was more frequent in TD probands than in controls (P = 0.024; OR, 1.33) and also in the TD alone group versus the TD plus OCD group (P = 0.0013; OR, 2.14). Furthermore, the rare gain‐of‐function SERT I425V variant was observed in 3 male siblings with TD and/or OCD and in their father. Thus, the cumulative count of SERT I425V becomes 1.57% in OCD/TD spectrum conditions versus 0.15% in controls, with a recalculated, family‐adjusted significance of χ2 = 15.03 (P < 0.0001; OR, 9.0; total worldwide genotyped, 2914). This report provides a unique combination of common and rare variants in one gene in TD, all of which are associated with SERT gain of function. Thus, altered SERT activity represents a potential contributor to serotonergic abnormalities in TD. The present results call for replication in a similarly intensively evaluated sample.

Rare missense neuronal cadherin gene ( CDH2 ) variants in specific obsessive-compulsive disorder and Tourette disorder phenotypes

The recent finding that the neuronal cadherin gene CDH2 confers a highly significant risk for canine compulsive disorder led us to investigate whether missense variants within the human ortholog CDH2 are associated with altered susceptibility to obsessive-compulsive disorder (OCD), Tourette disorder (TD) and related disorders. Exon resequencing of CDH2 in 320 individuals identified four non-synonymous single-nucleotide variants, which were subsequently genotyped in OCD probands, Tourette disorder probands and relatives, and healthy controls (total N=1161). None of the four variants was significantly associated with either OCD or TD. One variant, N706S, was found only in the OCD/TD groups, but not in controls. By examining clinical data, we found there were significant TD-related phenotype differences between those OCD probands with and without the N845S variant with regard to the co-occurrence of TD (Fisher’s exact test P=0.014, OR=6.03). Both N706S and N845S variants conferred reduced CDH2 protein expression in transfected cells. Although our data provide no overall support for association of CDH2 rare variants in these disorders considered as single entities, the clinical features and severity of probands carrying the uncommon non-synonymous variants suggest that CDH2, along with other cadherin and cell adhesion genes, is an interesting gene to pursue as a plausible contributor to OCD, TD and related disorders with repetitive behaviors, including autism spectrum disorders.

N.N-dimethyl-thioamphetamine and methyl-thioamphetamine, two non-neurotoxic substrates of 5-HT transporters, have scant in vitro efficacy for the induction of transporter-mediated 5-HT release and currents

We studied two non‐neurotoxic amphetamine derivatives (methyl‐thioamphetamine, MTA and N,N‐dimethylMTA, DMMTA) interacting with serotonin (5‐HT) transporters (SERTs) with affinities comparable to that of p‐Cl‐amphetamine (pCA). The rank order for their maximal effects in inducing both [3H]5‐HT release from rat brain synaptosomes or hSERT‐expressing HEK‐293 cells, and currents in hSERT‐expressing oocytes, was pCA » MTA ≥ DMMTA. A correlation between drug‐induced release and currents is also strengthened by the similar bell shape of the dose–response curves. Release experiments indicated that MTA and DMMTA are SERT substrates although MTA is taken up by HEK‐293 cells with a Vmax 40% lower than pCA. The weak effects of MTA and DMMTA in vitro might therefore be due to their properties as ‘partial substrates’ on the mechanisms, other than translocation, responsible for currents and/or release. After either local or systemic in vivo administration, MTA and DMMTA release 5‐HT in a manner comparable to pCA. These findings confirm that the neurotoxic properties of some amphetamine derivatives are independent of their 5‐HT‐releasing activity in vivo. It is worth noting that only those amphetamine derivatives with high efficiency in inducing 5‐HT release and currents in vitro have neurotoxic properties.