Ryan M. Smith

Beta-adrenergic Modulation of Cognitive Flexibility during Stress

Stress-induced activation of the locus ceruleus-norepinephrine (LC-NE) system produces significant cognitive and behavioral effects, including enhanced arousal and attention. Improvements in discrimination task performance and memory have been attributed to this stress response. In contrast, for other cognitive functions that require cognitive flexibility, increased activity of the LC-NE system may produce deleterious effects. The aim of the present study was to determine the effect of pharmacological modulation of the LC-NE system on stress-induced impairments in cognitive flexibility performance in healthy individuals. Cognitive performance, plus psychological and physiological parameters for 16 adults without any history of anxiety disorders, was assessed during four test sessions: stress and no-stress, with each condition tested after administration of propranolol and placebo. The Trier Social Stress Test, a public-speaking and mental arithmetic stressor, was presented to participants for the stress sessions, whereas a similar, but nonstressful, control task (reading, counting) was utilized for the no-stress sessions. Tests of cognitive flexibility included lexical-semantic and associative problem-solving tasks (anagrams, Compound Remote Associates Test). Visuo-spatial memory and motor processing speed tests served as control tasks. Results indicate that (1) stress impaired performance on cognitive flexibility tasks, but not control tasks; (2) compared to placebo, cognitive flexibility improved during stress with propranolol. Therefore, psychological stress, such as public speaking, negatively impacts performance on tasks requiring cognitive flexibility in normal individuals, and this effect is reversed by beta-adrenergic antagonism. This may provide support for the hypothesis that stress-related impairments in cognitive flexibility are related to the noradrenergic system.

Intronic Polymorphisms Affecting Alternative Splicing of Human Dopamine D2 Receptor Are Associated with Cocaine Abuse

The dopamine receptor D2 (encoded by DRD2) is implicated in susceptibility to mental disorders and cocaine abuse, but mechanisms responsible for this relationship remain uncertain. DRD2 mRNA exists in two main splice isoforms with distinct functions: D2 long (D2L) and D2 short (D2S, lacking exon 6), expressed mainly postsynaptically and presynaptically, respectively. Two intronic single-nucleotide polymorphisms (SNPs rs2283265 (intron 5) and rs1076560 (intron 6)) in high linkage disequilibrium (LD) with each other have been reported to alter D2S/D2L splicing and several behavioral traits in human subjects, such as memory processing. To assess the role of DRD2 variants in cocaine abuse, we measured levels of D2S and D2L mRNA in human brain autopsy tissues (prefrontal cortex and putamen) obtained from cocaine abusers and controls, and genotyped a panel of DRD2 SNPs (119 abusers and 95 controls). Robust effects of rs2283265 and rs1076560 on reducing formation of D2S relative to D2L were confirmed. The minor alleles of rs2283265/rs1076560 were considerably more frequent in Caucasians (18%) compared with African Americans (7%). Also, in Caucasians, rs2283265/rs1076560 minor alleles were significantly overrepresented in cocaine abusers compared with controls (rs2283265: 25 to 9%, respectively; p=0.001; OR=3.4 (1.7–7.1)). Several SNPs previously implicated in diverse clinical association studies are in high LD with rs2283265/rs1076560 and could have served as surrogate markers. Our results confirm the role of rs2283265/rs1076560 in D2 alternative splicing and support a strong role in susceptibility to cocaine abuse.

Pharmacogenomics of the RNA World: Structural RNA Polymorphisms in Drug Therapy

The use of pharmacogenomic biomarkers can enhance treatment outcomes. Regulatory polymorphisms are promising biomarkers that have proven difficult to uncover. They come in two flavors: those that affect transcription (regulatory single‐nucleotide polymorphisms (rSNPs)) and those that affect RNA functions such as splicing, turnover, and translation (termed structural RNA SNPs (srSNPs)). This review focuses on the role of srSNPs in drug metabolism, transport, and response. An understanding of the nature and diversity of srSNPs and rSNPs enables clinical scientists to evaluate genetic biomarkers.

Nicotinic α5 receptor subunit mRNA expression is associated with distant 5′ upstream polymorphisms

CHRNA5, encoding the nicotinic α5 subunit, is implicated in multiple disorders, including nicotine addiction and lung cancer. Previous studies demonstrate significant associations between promoter polymorphisms and CHRNA5 mRNA expression, but the responsible sequence variants remain uncertain. To search for cis-regulatory variants, we measured allele-specific mRNA expression of CHRNA5 in human prefrontal cortex autopsy tissues and scanned the CHRNA5 locus for regulatory variants. A cluster of six frequent single-nucleotide polymorphisms (rs1979905, rs1979906, rs1979907, rs880395, rs905740, and rs7164030), in complete linkage disequilibrium (LD), fully account for a >2.5-fold allelic expression difference and a fourfold increase in overall CHRNA5 mRNA expression. This proposed enhancer region resides more than 13 kilobases upstream of the CHRNA5 transcription start site. The same upstream variants failed to affect CHRNA5 mRNA expression in peripheral blood lymphocytes, indicating tissue-specific gene regulation. Other promoter polymorphisms were also correlated with overall CHRNA5 mRNA expression in the brain, but were inconsistent with allelic mRNA expression ratios, a robust and proximate measure of cis-regulatory variants. The enhancer region and the nonsynonymous polymorphism rs16969968 generate three main haplotypes that alter the risk of developing nicotine dependence. Ethnic differences in LD across the CHRNA5 locus require consideration of upstream enhancer variants when testing clinical associations.

Synaptic Signaling and Aberrant RNA Splicing in Autism Spectrum Disorders

Interactions between presynaptic and postsynaptic cellular adhesion molecules (CAMs) drive synapse maturation during development. These trans-synaptic interactions are regulated by alternative splicing of CAM RNAs, which ultimately determines neurotransmitter phenotype. The diverse assortment of RNAs produced by alternative splicing generates countless protein isoforms necessary for guiding specialized cell-to-cell connectivity. Failure to generate the appropriate synaptic adhesion proteins is associated with disrupted glutamatergic and gamma-aminobutyric acid signaling, resulting in loss of activity-dependent neuronal plasticity, and risk for developmental disorders, including autism. While the majority of genetic mutations currently linked to autism are rare variants that change the protein-coding sequence of synaptic candidate genes, regulatory polymorphisms affecting constitutive and alternative splicing have emerged as risk factors in numerous other diseases, accounting for an estimated 40–60% of general disease risk. Here, we review the relationship between aberrant RNA splicing of synapse-related genes and autism spectrum disorders.

Whole transcriptome RNA-Seq allelic expression in human brain

BackgroundMeasuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals.ResultsWe find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals.ConclusionsThese results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.

mRNA Transcript Diversity Creates New Opportunities for Pharmacological Intervention

Most protein coding genes generate multiple RNA transcripts through alternative splicing, variable 3′ and 5′UTRs, and RNA editing. Although drug design typically targets the main transcript, alternative transcripts can have profound physiological effects, encoding proteins with distinct functions or regulatory properties. Formation of these alternative transcripts is tissue-selective and context-dependent, creating opportunities for more effective and targeted therapies with reduced adverse effects. Moreover, genetic variation can tilt the balance of alternative versus constitutive transcripts or generate aberrant transcripts that contribute to disease risk. In addition, environmental factors and drugs modulate RNA splicing, affording new opportunities for the treatment of splicing disorders. For example, therapies targeting specific mRNA transcripts with splice-site–directed oligonucleotides that correct aberrant splicing are already in clinical trials for genetic disorders such as Duchenne muscular dystrophy. High-throughput sequencing technologies facilitate discovery of novel RNA transcripts and protein isoforms, applications ranging from neuromuscular disorders to cancer. Consideration of a genes transcript diversity should become an integral part of drug design, development, and therapy.

Combined effect of maternal serotonin transporter genotype and prenatal stress in modulating offspring social interaction in mice.

Several studies suggest that prenatal stress is a possible risk factor in the development of autism spectrum disorders. However, many children exposed to stress prenatally are born healthy and develop typically, suggesting that other factors must contribute to autism. Genes that contribute to stress reactivity may, therefore, exacerbate prenatal stress‐mediated behavioral changes in the adult offspring. One candidate gene linked to increased stress reactivity encodes the serotonin transporter. Specifically, an insertion/deletion (long/short allele) polymorphism upstream of the serotonin transporter gene correlates with differential expression and function of the serotonin transporter and a heightened response to stressors. Heterozygous serotonin transporter knockout mice show reductions in serotonin transporter expression similar to the human short polymorphism. In this study, the role of prenatal stress and maternal serotonin transporter genotype were assessed in mice to determine whether their combined effect produces reductions in social behavior in the adult offspring. Pregnant serotonin transporter heterozygous knockout and wild‐type dams were placed in either a control condition or subjected to chronic variable stress. The adult offspring were subsequently assessed for social interaction and anxiety using a three‐chamber social approach task, ultrasonic vocalization detection, elevated‐plus maze and an open field task. Results indicated that prenatal stress and reduced serotonin transporter expression of the dam may have the combined effect of producing changes in social interaction and social interest in the offspring consistent with those observed in autism spectrum disorder. This data indicates a possible combined effect of maternal serotonin transporter genotype and prenatal stress contributing to the production of autistic‐like behaviors in offspring.

BrainSeq: Neurogenomics to Drive Novel Target Discovery for Neuropsychiatric Disorders

We outline an ambitious project to characterize the genetic and epigenetic regulation of multiple facets of transcription in distinct brain regions across the human lifespan in samples of major neuropsychiatric disorders and controls. Initially focused on schizophrenia and mood disorders, the goal of this consortium is to elucidate the underlying molecular mechanisms of genetic associations with the goal of identifying novel therapeutic targets. The consortium currently consists of seven pharmaceutical companies and a not-for-profit medical research institution working as a precompetitive team to generate and analyze publicly available archival brain genomic data related to neuropsychiatric illness.

Neuroimaging in psychiatric pharmacogenetics research: the promise and pitfalls.

The integration of research on neuroimaging and pharmacogenetics holds promise for improving treatment for neuropsychiatric conditions. Neuroimaging may provide a more sensitive early measure of treatment response in genetically defined patient groups, and could facilitate development of novel therapies based on an improved understanding of pathogenic mechanisms underlying pharmacogenetic associations. This review summarizes progress in efforts to incorporate neuroimaging into genetics and treatment research on major psychiatric disorders, such as schizophrenia, major depressive disorder, bipolar disorder, attention-deficit/hyperactivity disorder, and addiction. Methodological challenges include: performing genetic analyses in small study populations used in imaging studies; inclusion of patients with psychiatric comorbidities; and the extensive variability across studies in neuroimaging protocols, neurobehavioral task probes, and analytic strategies. Moreover, few studies use pharmacogenetic designs that permit testing of genotype × drug effects. As a result of these limitations, few findings have been fully replicated. Future studies that pre-screen participants for genetic variants selected a priori based on drug metabolism and targets have the greatest potential to advance the science and practice of psychiatric treatment.