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Molecular and Behavioral Genetics of Mammalian Brain Genes
Summary: Marcelo Rubinstein's mouse transgenic laboratory is interested in understanding how the dopamine D4 and D2 receptors participate in motor planning, learning, emotional behaviors, and responses to psychostimulant drugs. In addition, his laboratory studies the molecular mechanisms that control transcription of the proopiomelanocortin gene in brain neurons.
Molecular and Behavioral Genetics of the Dopamine D4 Receptor in the Mammalian Brain
The neurotransmitter dopamine (DA) participates in the control of motor activity, identification of incentive-salient stimuli, and spatio-temporal integration of goal-oriented behaviors. These functions are mediated by five different DA receptor subtypes, which belong to the superfamily of G protein–coupled receptors. Among all five DA receptors, the D4 receptor (D4R) stands out because of its higher affinity for atypical antipsychotics and the highly polymorphic nature of the human gene (DRD4) in its coding sequence. Given that particular polymorphic alleles have been found to be more prevalent in ADHD probands, DRD4 has been implicated in attention deficit and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric condition characterized by deficits in filtering irrelevant information, poor behavioral inhibition, and hyperactivity. Twin, adoption, and segregation studies have estimated a heritability of 50 to 90 percent for ADHD; furthermore, there is evidence that genes involved in mesocortical DAergic neurotransmission may be candidates for a genetic predisposition to this disorder. For example, impaired behavioral inhibition, loss of attention, and difficulties in concentration are symptoms that indicate malfunction of prefrontal cortical circuits receiving DA innervation. In addition, the indirect dopamine agonists methylphenidate and amphetamine exert therapeutic benefits in ADHD patients. Despite its potential importance in ADHD, functional studies on D4R have been hindered by the lack of sufficiently selective compounds with proven efficacy in vivo. However, some years ago we generated mutant mice lacking D4R
(Drd4-/-), which provided insight into the physiological roles of this receptor subtype. In novel and familiar environments, Drd4-/- mice were shown to be less active than wild-type controls and displayed supersensitivity to the psychomotor stimulant effects of ethanol, methamphetamine, and cocaine. In addition, the mice showed enhanced vigilance in approach/avoidance paradigms and increased excitability in prefrontal cortical neurons.
Studying the expression pattern of Drd4 mRNA and its protein product has proven difficult owing to several technical difficulties. In an effort to identify brain cells that express Drd4, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the Drd4 locus. Our laboratory recently showed that, in the adult mouse brain, Drd4 expression is restricted to neurons of layers V and VI of the prefrontal cortex (PFCx) and discrete groups of neurons of the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. Its prominent expression in the PFCx supports the importance of the D4R in complex behaviors depending on cortical DA transmission and its possible role in the etiology and/or pathophysiology of ADHD. We localized D4Rs in both excitatory glutamatergic pyramidal neurons and inhibitory GABAergic interneurons of the prefrontal cortex. Therefore, it is conceivable that exaggerated or deficient D4R stimulation may alter the exquisite fine-tuning of prefrontal cortical circuits. Given the high prevalence of ADHD in school-age children (3–6 percent) and the fact that most of these young patients are medicated chronically with psychostimulants, it is of fundamental interest to investigate the genetic contributions and molecular mechanisms underlying the neurodevelopmental alterations that occur during onset and progression of the disorder.
We have generated a mouse model that mimics key hallmarks of the human disease, including hyperactivity, paradoxical response to psychostimulants, and poor behavioral inhibition, and we are using this model to investigate the contribution of individual gene mutations to these impairments. We recently reported that these phenotypes are prevented or altered by genetic ablation of the D4R gene or pharmacological manipulation of this receptor subtype, thus demonstrating a direct interaction between D4R stimulation and significant behaviors of this ADHD-like model.
Current studies in my laboratory attempt to combine evolutionary genomics, gene expression analysis in transgenic mice, conditional gene targeting, and a battery of behavioral tests to investigate the participation of the D4R and human polymorphic variants in complex behaviors including locomotion, attention, impulsivity, time perception, and responses to psychostimulant drugs.
Transcriptional Regulation of the Proopiomelanocortin Gene in the Brain
The proopiomelanocortin (POMC) gene encodes a prohormone expressed at significant levels in pituitary endocrine cells and in brain neurons producing a variety of biologically active peptides that mediate several physiological actions, including the stress response, food intake, and stress-induced analgesia. During recent years, POMC hypothalamic neurons have received a great deal of attention because they express receptors for the adipostatic hormone leptin and play a central role in the control of energy homeostasis and body weight regulation. Fat cells release leptin, which, after traveling through the blood, is able to enter the brain to provide information about the body's energy stores. Stimulation of leptin receptors in POMC neurons leads to the release of melanocortins which, in turn, stimulate central melanocortin receptors to decrease food intake and increase metabolic rate. The importance of the central melanocortin pathway in feeding behavior is clearly observed in mice and humans with homozygous Pomc null mutations, both species displaying hyperphagia and early-onset obesity. Even though total POMC deficiency is very rare in humans, POMC is a strong candidate gene to predispose to familial obesity. Several independent genome-wide scans for quantitative trait loci (QTL) have found a highly significant genetic linkage between a relatively narrow region in chromosome 2 containing the POMC locus and obesity-related traits. However, polymorphisms in the coding sequences of POMC that alter the structure or function of POMC peptides apparently do not account for this correlation, suggesting the alternative possibility that mutations in noncoding regulatory sequences may alter the level of POMC RNA transcripts and consequently the concentration of POMC peptides in brain.
In an effort to understand the molecular mechanisms that govern POMC neuronal transcriptional regulation and the role that POMC might play in feeding centers of the brain and the genetics of obesity, we combined functional expression analysis in transgenic mice with in silico phylogenetic footprinting. Given the lack of POMC-expressing neuronal cell lines, we performed a deletion/truncation analysis in transgenic mice; the animals provided a highly faithful and efficient expression system in which to test the ability of different genomic regions to target reporter gene expression to POMC hypothalamic neurons. In collaboration with Malcolm Low (Oregon Health and Science University, Portland, Oregon) we identified two novel distal enhancers, which we named nPE1 and nPE2, that play an essential role in the activation of POMC gene expression in a selected population of hypothalamic arcuate neurons. Our transgenic mouse and phylogenetic analyses demonstrated that (1) a distal genomic region containing nPE1 and nPE2 is necessary and sufficient to direct authentic neuron-specific expression of reporter genes to POMC arcuate neurons; (2) either nPE1 or nPE2 ensures proper reporter expression in POMC arcuate neurons, whereas simultaneous deletion of these two enhancers completely eliminates expression in POMC neurons; (3) nPE1 and nPE2 nucleotide sequences and genomic organization are both highly conserved among mammals but not between mammals and birds, amphibians, or fish; (4) the enhancer activity of mouse and human genomic fragments containing nPE1 and nPE2 is functionally conserved; and (5) POMC expression in the brain and pituitary is controlled by different and independent sets of enhancers. Current studies in my lab aim to further characterize nPE1 and nPE2 function and their evolutionary origin.
Last updated September 2008
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