Dopamine depletion shifts behavior from activity based reinforcers to more sedentary ones and adenosine receptor antagonism reverses that shift: Relation to ventral striatum DARPP32 phosphorylation patterns.

The mesolimbic dopamine (DA) system plays a critical role in behavioral activation and effort-based decision-making. DA depletion produces anergia (shifts to low effort options) in animals tested on effort-based decision-making tasks. Caffeine, the most consumed stimulant in the world, acts as an adenosine A1/A2A receptor antagonist, and in striatal areas DA D1 and D2 receptors are co-localized with adenosine A1 and A2A receptors respectively. In the present work, we evaluated the effect of caffeine on anergia induced by the VMAT-2 inhibitor tetrabenazine (TBZ), which depletes DA. Anergia was evaluated in a three-chamber T-maze task in which animals can chose between running on a wheel (RW) vs. sedentary activities such as consuming sucrose or sniffing a neutral odor. TBZ-caffeine interactions in ventral striatum were evaluated using DARPP-32 phosphorylation patterns as an intracellular marker of DA-adenosine receptor interaction. In the T-maze, control mice spent more time running and much less consuming sucrose or sniffing. TBZ (4.0 mg/kg) reduced ventral striatal DA tissue levels as measured by HPLC, and also shifted preferences in the T-maze, reducing selection of the reinforcer that involved vigorous activity (RW), but increasing consumption of a reinforcer that required little effort (sucrose), at doses that had no effect on independent measures of appetite or locomotion in a RW. Caffeine at doses that had no effect on their own reversed the effects of TBZ on T-maze performance, and also suppressed TBZ-induced pDARPP-32(Thr34) expression as measured by western blot, suggesting a role for D2-A2A interactions. These results support the idea that DA depletion produces anergia, but does not affect the primary motivational effects of sucrose. Caffeine, possibly by acting on A2A receptors in ventral striatum, reversed the DA depletion effects. It is possible that caffeine, like selective adenosine A2A antagonists, could have some therapeutic benefit for treating effort-related symptoms.

Intracellular calcium chelation with BAPTA-AM modulates ethanol-induced behavioral effects in mice.

Calcium (Ca(2+)) has been characterized as one of the most ubiquitous, universal and versatile intracellular signaling molecules responsible for controlling numerous cellular processes. Ethanol-induced effects on Ca(2+) distribution and flux have been widely studied in vitro, showing that acute ethanol administration can modulate intracellular Ca(2+) concentrations in a dose dependent manner. In vivo, the relationship between Ca(2+) manipulation and the corresponding ethanol-induced behavioral effects have focused on Ca(2+) flux through voltage-gated Ca(2+) channels. The present study investigated the role of inward Ca(2+) currents in ethanol-induced psychomotor effects (stimulation and sedation) and ethanol intake. We studied the effects of the fast Ca(2+) chelator, BAPTA-AM, on ethanol-induced locomotor activity and the sedative effects of ethanol. Swiss (RjOrl) mice were pretreated with BAPTA-AM (0-10 mg/kg) 30 min before an ethanol (0-4 g/kg) challenge. Our results revealed that pretreatment with BAPTA-AM prevented locomotor stimulation produced by ethanol without altering basal locomotion. In contrast, BAPTA-AM reversed ethanol-induced hypnotic effects. In a second set of experiments, we investigated the effects of intracellular Ca(2+) chelation on ethanol intake. Following a drinking-in-the-dark methodology, male C57BL/6J mice were offered 20% v/v ethanol, tap water, or 0.1% sweetened water. The results of these experiments revealed that BAPTA-AM pretreatment (0-5 mg/kg) reduced ethanol consumption in a dose-dependent manner while leaving water and sweetened water intake unaffected. Our findings support the role of inward Ca(2+) currents in mediating different behavioral responses induced by ethanol. Our results are discussed together with data indicating that ethanol appears to be more sensitive to intracellular Ca(2+) manipulations than other psychoactive drugs.

A GFP-tagged muscleblind C protein isoform reporter construct.

Drosophila muscleblind (mbl), the ortholog of human Muscleblind-like 1 (MBNL1) gene involved in Myotonic Dystrophy (DM), gives raise to protein isoforms MblA to G. The specific functions and subcellular distribution of isoforms are still largely unknown. To overcome the lack of isoform-specific antibodies we generated transgenic flies that express a GFP:MblC fusion protein under the control of the Gal4/UAS system. The reporter fusion protein was able to functionally complement mbl loss of function mutations, demonstrating activity, and accumulated predominantly in adult muscle nuclei. The fluorescent nature of the reporter makes it appropriate for live imaging detection of MblC protein isoform.

Genetic and chemical modifiers of a CUG toxicity model in Drosophila.

Non-coding CUG repeat expansions interfere with the activity of human Muscleblind-like (MBNL) proteins contributing to myotonic dystrophy 1 (DM1). To understand this toxic RNA gain-of-function mechanism we developed a Drosophila model expressing 60 pure and 480 interrupted CUG repeats in the context of a non-translatable RNA. These flies reproduced aspects of the DM1 pathology, most notably nuclear accumulation of CUG transcripts, muscle degeneration, splicing misregulation, and diminished Muscleblind function in vivo. Reduced Muscleblind activity was evident from the sensitivity of CUG-induced phenotypes to a decrease in muscleblind genetic dosage and rescue by MBNL1 expression, and further supported by the co-localization of Muscleblind and CUG repeat RNA in ribonuclear foci. Targeted expression of CUG repeats to the developing eye and brain mushroom bodies was toxic leading to rough eyes and semilethality, respectively. These phenotypes were utilized to identify genetic and chemical modifiers of the CUG-induced toxicity. 15 genetic modifiers of the rough eye phenotype were isolated. These genes identify putative cellular processes unknown to be altered by CUG repeat RNA, and they include mRNA export factor Aly, apoptosis inhibitor Thread, chromatin remodelling factor Nurf-38, and extracellular matrix structural component Viking. Ten chemical compounds suppressed the semilethal phenotype. These compounds significantly improved viability of CUG expressing flies and included non-steroidal anti-inflammatory agents (ketoprofen), muscarinic, cholinergic and histamine receptor inhibitors (orphenadrine), and drugs that can affect sodium and calcium metabolism such as clenbuterol and spironolactone. These findings provide new insights into the DM1 phenotype, and suggest novel candidates for DM1 treatments.

Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis.

BACKGROUND: Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene. METHODOLOGY/PRINCIPAL FINDINGS: We used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression. CONCLUSIONS/SIGNIFICANCE: Taken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.

Muscleblind isoforms are functionally distinct and regulate alpha-actinin splicing.

Drosophila Muscleblind (Mbl) proteins control terminal muscle and neural differentiation, but their molecular function has not been experimentally addressed. Such an analysis is relevant as the human Muscleblind-like homologs (MBNL1-3) are implicated in the pathogenesis of the inherited muscular developmental and degenerative disease myotonic dystrophy. The Drosophila muscleblind gene expresses four protein coding splice forms (mblA to mblD) that are differentially expressed during the Drosophila life cycle, and which vary markedly in their ability to rescue the embryonic lethal phenotype of muscleblind mutant flies. Analysis of muscleblind mutant embryos reveals misregulated alternative splicing of the transcripts encoding Z-band component alpha-Actinin, which can be replicated in human cells expressing a Drosophilaalpha-actinin minigene and epitope-tagged Muscleblind isoforms. MblC appreciably altered alpha-actinin splicing in this assay, whereas other isoforms had only a marginal or no effect, demonstrating functional specialization among Muscleblind proteins. To further analyze the molecular basis of these differences, we studied the subcellular localization of Muscleblind isoforms. Consistent with the splicing assay results, MblB and MblC were enriched in the nucleus while MblA was predominantly cytoplasmic. In myotonic dystrophy, transcripts bearing expanded non-coding CUG or CCUG repeats interfere with the function of human MBNL proteins. Co-expression of CUG repeat RNA with the alpha-actinin minigene altered splicing compared with that seen in muscleblind mutant embryos, indicating that CUG repeat expansion RNA also interferes with Drosophila muscleblind function. Moreover MblA, B, and C co-localize with CUG repeat RNA in nuclear foci in cell culture. Our observations indicate that Muscleblind isoforms perform different functions in vivo, that MblC controls muscleblind-dependent alternative splicing events, and establish the functional conservation between Muscleblind and MBNL proteins both over a physiological target (alpha-actinin) and a pathogenic one (CUG repeats).

The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing.

Alternative splicing is widely used to generate protein diversity and to control gene expression in many biological processes, including cell fate determination and apoptosis. In this review, we focus on the Muscleblind family of tissue-specific alternative splicing regulators. Muscleblind proteins bind pre-mRNA through an evolutionarily conserved tandem CCCH zinc finger domain. Human Muscleblind homologs MBNL1, MBNL2 and MBNL3 promote inclusion or exclusion of specific exons on different pre-mRNAs by antagonizing the activity of CUG-BP and ETR-3-like factors (CELF proteins) bound to distinct intronic sites. The relative activities of Muscleblind and CELF proteins control a key developmental switch. Defined transcripts follow an embryonic splice pattern when CELF activity predominates, whereas they follow an adult pattern when Muscleblind activity prevails. Human MBNL proteins show functional specializations. While MBNL1 seems to promote muscle differentiation, MBNL3 appears to function in an opposing manner inhibiting expression of muscle differentiation markers. MBNL2, on the other hand, participates in a new RNA-dependent protein localization mechanism involving recruitment of integrin alpha3 protein to focal adhesions. Both muscleblind mutant Drosophila embryos and Mbnl1 knockout mice show muscle abnormalities and altered splicing of specific transcripts. In addition to regulating terminal muscle differentiation through alternative splicing control, results by several groups suggest that Muscleblind participates in the differentiation of photoreceptors, neurons, adipocytes and blood cell types. Misregulation of MBNL activity can lead to human pathologies. Through mechanisms not completely identified yet, expression of transcripts containing large non-coding CUG or CCUG repeat expansions mimics muscleblind loss-of-function phenotypes. Archetypical within this class of disorders are myotonic dystrophies. Our understanding of the biology of Muscleblind proteins has increased dramatically over the last few years, but several key issues remain unsolved. Defining the mechanism of the activity of Muscleblind proteins, their splicing partners, and the functional relevance of its several protein isoforms are just a few examples.

An interspecific functional complementation test in Drosophila for introductory genetics laboratory courses.

Introductory genetics courses often include evolutionary genetics concepts such as sequence homology and functional conservation. It is usually assumed that two sequences showing homology (i.e., sharing a common ancestral sequence) perform the same molecular function. The correlation, however, does not always hold true, and evidence for functional conservation must come from functional studies. In this study we describe a genetics laboratory class that demonstrates functional conservation between the Drosophila protein Muscleblind (Mbl) and its human ortholog MBNL1. We use the Gal4/UAS system to express MBNL1 in a Drosophila mutant background and measure the in vivo activity of the human protein by rescue of mbl mutant phenotype in embryos. As a control, ubiquitous expression of Drosophila MblC, one of the four protein isoforms encoded by the gene, increased by 71% the viability of mbl mutant embryos and greatly reduced the hypercontracted abdomen of mutant larvae. In a parallel experiment, human MBNL1 provided a robust rescue of the embryonic lethality (78%) and improved abdomen hypercontraction as well. Under both conditions, rescued larvae die as first instars, probably due to overexpression effects, lack of alternative protein isoforms, or incomplete expression in critical tissues such as the nervous system. The use of two constructs in the rescue experiment (UAS-mblC and UAS-MBNL1) and the incomplete rescue prompt several questions for students. The fact that a human protein works in a Drosophila cellular context illustrates the use of an in vivo test to prove functional conservation.

Serpent and a hibris reporter are co-expressed in migrating cells during Drosophila hematopoiesis and Malpighian tubule formation.

Motile mesodermal cells contribute several cell types to developing embryos. In Drosophila, blood cell precursors or prohemocytes, are first detected in the procephalic mesoderm by the expression of the GATA transcription factor Serpent. Once specified, a subset of prohemocytes migrate posteriorly to populate most of the embryo and further differentiate as plasmatocytes. Similarly, Drosophila nephrogenesis involves integration of posterior mesodermal cells into the Malpighian tubule primordia where these cells differentiate as stellate cells. Here we investigated the possibility that the immunoglobulin-domain protein Hibris and the GATA factor Serpent were co-expressed in motile mesodermal cells by using the hibris expression reporter P[w(+)]36.1 and antibody staining. We show that P[w(+)]36.1 reproduces the endogenous expression of hibris in several embryonic tissue types and organs, including mesectoderm, early mesoderm, pharyngeal musculature, hindgut, anal plates, posterior spiracles, and antennomaxillary complex. We find that both migrating prohemocytes and posterior mesodermal cells, before their integration into the Malpighian tubule primordia, simultaneously express the hibris reporter and Serpent. We also show that hibris function is not essential for prohemocyte migration out of the procephalic mesoderm NOR maintenance of Serpent expression in prohemocytes.