Characterization of a Functional Comt1 Haplotype in Inbred Strains of Mice

We have pursued a genome-wide approach to examining strain-specific variations in gene expression in the brain of 29 inbred strains of mice. The highest association was found within the locus of the Catechol-O-Methyltransferase (Comt1) gene, coding for an ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catecholamines. In human and mouse, COMT has been associated with multiple behavioral phenotypes, including pain sensitivity and stress response. Multiple brain regions in 29 inbred strains of mice were analyzed for Comt1 expression levels using a genome wide array. Differential expression levels, validated with qPCR, were observed for Comt1. A B2 Short Interspersed Nucleotide Element (SINE) was identified as an insertion in the 3'UTR of Comt1 in 14 strains of a shared haplotype. Experiments using mammalian expression vectors of full length cDNA clones with and without the SINE element present demonstrate the SINE haplotype (+SINE) to have greater Comt1 enzymatic activity. Within strains examined to date, +SINE mice have increased enzymatic function, decreased sensitivity for thermal and chemical- induced pain assays and behavioral differences in several anxiety assays. These results suggest that a haplotype, defined by a 3'UTR B2 SINE element, regulates Comt1 expression and mouse behavior

Social approach and repetitive behavior in eleven inbred mouse strains

Core symptoms of autism include deficits in social interaction, impaired communication, and restricted, repetitive behaviors. The repetitive behavior domain encompasses abnormal motoric stereotypy, an inflexible insistence on sameness, and resistance to change. In recent years, many genetic mouse models of autism and related disorders have been developed, based on candidate genes for disease susceptibility. The present studies are part of an ongoing initiative to develop appropriate behavioral tasks for the evaluation of mouse models relevant to autism. We have previously reported profiles for sociability, preference for social novelty, and resistance to changes in a learned pattern of behavior, as well as other functional domains, for 10 inbred mouse strains of divergent genetic backgrounds. The present studies extend this multi-component behavioral characterization to several additional strains: C58/J, NOD/LtJ, NZB/B1NJ, PL/J, SJL/J, SWR/J, and the wild-derived PERA/EiJ. C58/J, NOD/LtJ, NZB/B1NJ, SJL/J, and PERA/EiJ demonstrated low sociability, measured by time spent in proximity to an unfamiliar conspecific, with 30% to 60% of mice from these strains showing social avoidance. In the Morris water maze, NZB/B1NJ had a persistent bias for the quadrant where the hidden platform was located during acquisition, even after nine days of reversal training. A particularly interesting profile was found for C58/J, which had low social preference, poor performance in the T-maze, and overt motoric stereotypy. Overall, this set of tasks and observational methods provides a strategy for evaluating novel mouse models in behavioral domains relevant to the autism phenotype

Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

Abstract The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (K i  = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions

Linkage scan of alcohol dependence in the UCSF Family Alcoholism Study

Ample data suggest alcohol dependence represents a heritable condition, and several research groups have performed linkage analysis to identify genomic regions influencing this disorder. In the present study, a genome-wide linkage scan for alcohol dependence was conducted in a community sample of 565 probands and 1080 first-degree relatives recruited through the UCSF Family Alcoholism Study. The Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) was used to derive DSM-IV alcohol dependence diagnoses. Although no loci achieved genome-wide significance (i.e., LOD score > 3.0), several linkage peaks of interest (i.e., LOD score > 1.0) were identified. When the strict DSM-IV alcohol dependence diagnosis requiring the temporal clustering of symptoms served as the phenotype, linkage peaks were identified on chromosomes 1p36.31–p36.22, 2q37.3, 8q24.3, and 18p11.21–p11.2. When the temporal clustering of symptoms was not required, linkage peaks were again identified on chromosomes 1p36.31–p36.22 and 8q24.3 as well as novel loci on chromosomes 1p22.3, 2p24.3–p24.1, 9p24.1–p23, and 22q12.3–q13.1. Follow-up analyses were conducted by performing linkage analysis for the 12 alcohol dependence symptoms assessed by the SSAGA across the support intervals for the observed linkage peaks. These analyses demonstrated that different collections of symptoms often assessing distinct aspects of alcohol dependence (e.g., uncontrollable drinking and withdrawal vs. tolerance and drinking despite health problems) contributed to each linkage peak and often yielded LOD scores exceeding that reported for the alcohol dependence diagnosis. Such findings provide insight into how specific genomic regions may influence distinct aspects of alcohol dependence

Molecular genetic mechanisms of allelic specific regulation of murine Comt expression

A functional allele of the mouse catechol-O-methyltransferase (Comt) gene is defined by the insertion of a B2 short interspersed repeat element in its 3′-untranslated region (UTR). This allele has been associated with a number of phenotypes, such as pain and anxiety. In comparison with mice carrying the ancestral allele (Comt+), ComtB2i mice show higher Comt mRNA and enzymatic activity levels. Here, we investigated the molecular genetic mechanisms underlying this allelic specific regulation of Comt expression. Insertion of the B2 element introduces an early polyadenylation signal generating a shorter Comt transcript, in addition to the longer ancestral mRNA. Comparative analysis and in silico prediction of Comt mRNA potential targets within the transcript 3′ to the B2 element was performed and allowed choosing microRNA (miRNA) candidates for experimental screening: mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667. Cell transfection with each miRNA downregulated the expression of the ancestral transcript and COMT enzymatic activity. Our in vivo experiments showed that mmu-miR-667-3p is strongly correlated with decreasing amounts of Comt mRNA in the brain, and lentiviral injections of mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667 increase hypersensitivity in the mouse formalin model, consistent with reduced COMT activity. In summary, our data demonstrate that the Comt+ transcript contains regulatory miRNA signals in its 3′-untranslated region leading to mRNA degradation; these signals, however, are absent in the shorter transcript, resulting in higher mRNA expression and activity levels

Pain modality- and sex-specific effects of COMT genetic functional variants

The enzyme catechol-O-methyltransferase (COMT) metabolizes catecholamine neurotransmitters involved in a number of physiological functions including pain perception. Both human and mouse COMT genes possess functional polymorphisms contributing to inter-individual variability in pain phenotypes such as sensitivity to noxious stimuli, severity of clinical pain and response to pain treatment. In this study, we found that the effects of Comt functional variation in mice are modality-specific. Spontaneous inflammatory nociception and thermal nociception behaviors were correlated the most with the presence of the B2 SINE transposon insertion residing in the 3’UTR mRNA region. Similarly, in humans, COMT functional haplotypes were associated with thermal pain perception and with capsaicin-induced pain. Furthermore, COMT genetic variations contributed to pain behaviors in mice and pain ratings in humans in a sex-specific manner. The ancestral Comt variant, without a B2 SINE insertion, was more strongly associated with sensitivity to capsaicin in female versus male mice. In humans, the haplotype coding for low COMT activity increased capsaicin-induced pain perception in women, but not men. These findings reemphasize the fundamental contribution of COMT to pain processes, and provide a fine-grained resolution of this contribution at the genetic level that can be used to guide future studies in the area of pain genetics

COMT gene locus: new functional variants

Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3′ untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes

Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

Abstract The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (Ki = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.</p