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Santangelo AM, de Souza FJS, Franchini LF,
Bumaschny VF, Low MJ, Rubinstein M (2007) Ancient
Exaptation of a CORE-SINE Retroposon into a Highly Conserved Mammalian
Neuronal Enhancer of the Proopiomelanocortin Gene. Plos
Genetics (en prensa)
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Bumaschny VF, de Souza FJ, López Leal
R, Santagenlo A, Levi DH, Baetscher M, Low MJ, Rubinstein
M (2007) Transcriptional Regulation of Pituitary POMC
Is Conserved at the Vertebrate Extremes in Spite of Great Promoter
Sequence Divergence. Mol Endocrinol (en prensa)
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Noaín D, Avale ME, Wedemeyer C, Peper
M, Rubinstein M (2006) Identification of Brain
Neurons Expressing the Dopamine D4 Receptor Gene using BAC Transgenic
Mice. Eur J Neurosci 24:2429-2438.
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de Souza FS, Bumaschny VF, Low MJ, Rubinstein
M. (2005) Subfunctionalization of Expression and Peptide
Domains Following the Ancient Duplication of the Proopiomelanocortin
Gene in Teleost Fishes. Mol Biol Evol. 22:2417-2427.
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de Souza FJS,. Santangelo AM, Bumaschny VF,
Avale ME, Smart JL, Low MJ, Rubinstein M (2005)
Identification of Neuronal Enhancers of the Proopiomelanocortin
Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting.
Mol Cell Biol 25:3076-86.
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Avale ME, Nemirovsky SI, Raisman-Vozari R,
Rubinstein M (2004) Elevated Serotonin is Involved
in Hyperactivity but not in the Paradoxical Effect of Amphetamine
in Mice Neonatally Lesioned with 6-Hydroxydopamine. J
Neurosci Res 78:289-96.
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Overstreet L, Hentges S, Bumaschny V, Souza
FJ, Smart J, Santangelo A, Low M, Westbrook G, Rubinstein
M (2004) A Transgenic Marker for Newly Born Granule Cells
in Dentate Gyrus. J Neurosci 23:3251-3259
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Avale ME, Falzone TL, Gelman DM, Low MJ, Grandy
DK, Rubinstein M (2004)The dopamine D4 receptor
is essential for hyperactivity and impaired behavioral inhibition
in a mouse model of attention deficit/hyperactivity disorder.
Mol. Psychiatry 9(7):718-26.
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Gelman DM, Noaín DC, Avale ME, Otero
V,. Low MJ, Rubinstein M (2003) Transgenic Mice
Engineered to Target Cre/LoxP-Mediated DNA Recombination into
Catecholaminergic Neurons. Genesis 36:196-202.
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Heisler LK, Cowley MA, Tecott LH, Fan W, Low
MJ, Smart JL, Rubinstein M, Tatro J, Holstege
H, Lee C, Cone R, Elmquist J (2002) Activation of Central Melanocortin
Pathways by Fenfluramine. Science 297:609-611
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Díaz-Torga G, Feierstein C, Libertun
C, Gelman D, Kelly MA, Low MJ, Rubinstein M,
Becú-Villalobos D (2002) Disruption of the D2 dopamine
receptor alters GH and IGF-I secretion and causes dwarfism in
male mice. Endocrinology 143:1270-1279.
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Falzone TL, Gelman DG, Young JI, Grandy DK,
Low MJ, Rubinstein M (2002) Absence of dopamine
D4 receptors results in enhanced reactivity to unconditioned,
but not conditioned, fear. Eur J Neurosci 15:158-64
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Low MJ, Otero-Corchon V, Parlow AF, Ramirez
JL, Kumar U, Patel YC Rubinstein M (2001) Somatostatin
is required for masculinization of growth hormone–regulated
hepatic gene expression but not of somatic growth. J Clin
Invest 107:1571-1580.
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Rubinstein M, Cepeda C, Hurst
RS, Altemus KL, Ariano MA, Falzone TL, Kozell LB, Meshul CK, Bunzow
JR, LowMJ, Levine MS, Grandy DK (2001) Dopamine D4 Receptor-Deficient
Mice Display Cortical Hyperexcitability. J Neurosci 21:3756-3763.
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Cowley MA, Smart JL, Rubinstein M,
Cerdán MG, Diano S, Horvath TL, Cone RD, Low MJ (2001)
Leptin activates anorexigenic POMC neurons through a neural network
in arcuate nucleus. Nature 411:480-484.
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Phillips TJ, Brown KJ, Burkhart-Kasch S, Wenger
CD, Kelly MA, Rubinstein M, Grandy DK, Low MJ
(1988) Alcohol preference and sensitivity are markedly reduced
in mice lacking dopamine D2 receptors. Nature
Neuroscience 1:610-615.
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Young JI, Otero V, Cerdán MG, Falzone
TL, Chan E-C, Low MJ, Rubinstein M (1998) Authentic
Cell-Specific and Developmentally Regulated Expression of Proopiomelanocortin
Genomic Fragments in Hypothalamic and Hindbrain Neurons of Transgenic
Mice. J Neurosci 18:6631-6640.
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Kelly MA, Rubinstein M, Phillips
TJ, Lessov CN, Burkhart-Kasch S, Zhang G, Bunzow J, Fang Y, Gerhardt
GA, Grandy DK, Low MJ (1998) Locomotor activity in D2 dopamine
receptor deficient mice is determined by gene dosage, genetic
background and developmental adaptations. J Neurosci 18:
3010-3020.
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Cerdán MG, Young JI, Zino E, Falzone
TL, Otero V, Torres HN, Rubinstein M (1998) Accurate
Spatial and Temporal Transgene Expression Driven by a 3.8 Kilobase
Promoter of the Bovine ß-casein Gene in the Lactating Mouse
Mammary Gland. Mol Reprod Dev 49:236-245.
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Rubinstein M, Phillips TJ,
Bunzow JR, Falzone TL, Dziewczapolski G, Pugsley TA, Zhang G,
Fang Y, Larson JL, McDougall JA, ChesterJA, Saez C, Gershanik
O, Low MJ, Grandy DK (1997) Mice Lacking Dopamine D4 Receptors
Are Supersensitive to Ethanol, Cocaine and Methamphetamine. Cell
90:991-1001.
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Kelly MA*, Rubinstein M*,
Asa SL, Zhang G, Saez C, Bunzow JR, Allen RG, Hnasko R, Ben-Jonathan
N, Grandy DK, Low MJ (1997) Pituitary lactotroph hyperplasia and
chronic hyperprolactinemia in dopamine D2 receptor-deficient mice.
Neuron 19:103-113.
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Rubinstein M, Mogil JS, Japón
MA, Chan EC, Allen RG, Low MJ (1996) Absence of Opioid Stress-Induced
Analgesia in Mice Lacking ß-Endorphin by Targeted Mutagenesis.
Proc Natl Acad Sci USA 93:3995-4000.
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Japón MA, Rubinstein M,
Low MJ (1994) In situ hybridizarion analysis of anterior pituitary
hormone gene expression during fetal mouse development.
J Histochem Cytochem 42:1117-1125.
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Rubinstein M, Mortrud M,
Liu B, Low MJ Rat and mouse proopiomelanocortin gene sequences
target tissue-specific expression to the pituitary gland but not
to the hypothalamus of transgenic mice. (1993) Neuroendocrinol.
58:373-380.
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Rubinstein M, Japón
MA, Low MJ Introduction of a point mutation into the genome by
homologous recombination in embryonic stem cells using a replacement
type vector with a selectable marker. (1993) Nucleic Acid
Res. 21:2613-2617.
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Rubinstein M, Liu B, Goodman
RH, Low MJ 1992 Targeted expression of somatostatin in vasopressinergic
magnocellular hypothalamic neurons of transgenic mice. Molec.Cell.
Neurosci. 3:152-161.
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Rubinstein M, Muschietti
JP, Gershanik O, Flawiá MM, Stefano FJE 1990 Adaptative
mechanisms of striatal D1 and D2 dopamine receptors in response
to a prolonged reserpine treatment in mice. J Pharmacol
Exp Therap 252:810-816.
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Rubinstein M, Gershanik O,
Stefano FJE 1988 Different roles of D-1 and D-2 dopamine receptors
involved in locomotor activity of supersensitive mice. Europ
J Pharmacol 148:419-426.
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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.
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