Emily Petruccelli and Ralph Hazlewood will Present Their Research on September 12, 2013

Emily’s Abstract:

Drosophila DopEcR, a dual receptor for ecdysteroids and dopamine, modulates ethanol-induced sedation behavior

Steroid hormones exhibit profound effects on behavior through both genomic and non-genomic signaling. Unlike the classic genomic mechanism where steroids act via cognate nuclear hormone receptors, the significance and molecular underpinnings of rapid non-genomic steroid signaling that occurs independently of new mRNA synthesis remains poorly understood. Recently, a Drosophila G-protein coupled receptor named DopEcR was identified as a putative non-genomic steroid receptor. Interestingly, DopEcR activates rapid intracellular signaling cascades in response to both dopamine and the major insect steroid hormone ecdysone when expressed in heterologous cell culture. Our goal is identify the function of DopEcR in vivo to elucidate the role of non-genomic steroid signaling in the nervous system. Since DopEcR is primarily expressed in the fly brain and uses signaling components associated with evolutionarily conserved alcohol-induced behaviors, we examined the responses of DopEcR mutants to ethanol. We found that DopEcR mutants are significantly resistant to ethanol-induced sedation – a phenotype that can predispose animals to alcohol addiction. To further understand DopEcR-dependent regulation of alcohol-induced behavior, we examined the potential genetic interactions of DopEcR with EGFR and cAMP signaling since both pathways have previously been shown to affect ethanol-induced sedation in flies and rodents. Our experiments strongly suggest that DopEcR functions to inhibit EGFR/Erk and activate cAMP signaling following ethanol exposure. Further observation of ethanol-induced behaviors following genetic and pharmacologic manipulations will continue to help elucidate the function and mechanism of DopEcR. Our findings offer important insight into how behavioral response to alcohol is controlled by dopamine and non-canonical steroid signaling.

Ralph’s Abstract:

Heterozygous triplication of regulatory elements is responsible for cavitary optic disc anomalies

Purpose: To identify and characterize the gene that causes autosomal dominant, congenital malformations of the optic nerve known as cavitary optic disc anomaly (CODA) in a multiplex family with 17 affected members. Features of the nerve disease in CODA closely resemble that of glaucoma (excavated optic nerve head appearance) that occurs in the absence of elevated IOP, suggesting that the gene that causes CODA may also contribute to the pathophysiology of glaucoma.

Methods: The gene that causes CODA was previously mapped to a 13.5Mb locus on chromosome 12q14. The proband of the CODA pedigree was tested for copy number variations (CNVs) in the chromosome 12q14 region with custom comparative genomic hybridization using the NimbleGen platform (Madison, WI). CNVs were confirmed with quantitative PCR in the proband and in the remaining 16 affected family members as well as in a panel of 78 controls. Confirmed CNVs were analyzed for their effect on downstream genes using a luciferase reporter gene construct (pGL3, Promega, Madison, WI) in HEK293T cells.

Results: CGH experiments identified a triplication of a 6kb segment of DNA upstream of matrix metalloproteinase 19 (MMP19) in the proband of the CODA pedigree. Quantitative PCR experiments confirmed that the MMP19 promoter CNV is present in the proband as well as in the additional 16 affected family members. No control subjects carried this mutation. Furthermore there were no instances of this variant in the database of genomic variants (projects.tcag.ca/variation). The luciferase reporter gene assay showed that the 6kb sequenced spanned by the CNV in CODA patients increased luciferase activity and functioned as a transcription enhancer. Moreover, a similar analysis of overlapping subdivisions of the DNA sequence in the 6kb CNV showed that a 1kb segment had a strong positive influence (8-fold higher) on downstream gene expression. Immunohistochemical experiments identified robust expression in the optic nerve head and throughout the optic nerve extending to the retrolamniar regions consistent with microarray expression data.

Conclusions: We have identified a copy number variation mutation in the promoter sequence of the MMP19 gene that co-segregates with CODA in our large 17 member pedigree. Moreover we have shown that the CNV spans DNA sequences that powerfully enhance downstream genes (i.e. MMP19). MMP19 is also expressed in the relevant ocular tissues. These functional data strongly suggest that the genetic defects in MMP19 cause CODA and that over-activity of this gene has an important role in the pathogenesis of this optic nerve disease.

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Posted on September 10, 2013, in Student Seminar. Bookmark the permalink. Leave a comment.

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