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Genes in flies: in search of biological pathways and models of RLS Subhabrata Sanyal Emory University Gene/Homolog Mutations Disease/ Therapy Phenotypes • An implicated gene • Manipulate strongly conserved homolog • Ability to assay expected phenotypes • Link back to Human disease/therapy Why flies? Drosophila as a model organism Behavior: systems Organs: circuits Cellular: neurons Molecular: signaling GFP Techniques to manipulate genes: GAL4-UAS GAL4 Gene of Interest UAS elements Dopaminergic neurons in the fly TH-GAL4 UAS-GFP UAS-RNAi(BTBD9) • An implicated gene … Bric-a-brac Tramtrack Broad Complex Stefansson, H., Rye DB. et. al. N Engl J Med 2007 Winkelmann J., Schormair, B. et. al. Nat Genetics 2007 The Fly homolog of BTBD9 is CG1826 Homology Between Drosophila and Human BTBD9 48% identity 63% similarity BTB domain conserved The Fly gene region: Human Drosophila CG1826 is expressed in the nervous system •Manipulate strongly conserved homolog Transposon Excision to generate Deletions P-element 5’-UTR Coding region of GOI Precise 3’-UTR + Transposase Imprecise A B C D Deletion mutants in CG1826 All deletion mutants are alive • Ability to assay expected phenotypes • Focus on sleep Features of sleep • • • • • • • • • Humans Drosophila Period of immobility Increased arousal threshold EEG correlates Molecular correlates Drug-dependent effects Gender and individual differences Circadian regulation Homeostatic regulation Fragmentation with aging • Ability to assay expected phenotypes • An implicated gene • Manipulate strongly conserved homolog • Ability to assay expected phenotypes Results of BTBD9 knockdown in flies? Knockout of BTBD9/CG1826 disrupts sleep Total sleep time Average number of sleep epochs Average length of sleep epoch Wild-type (n=112) CG1826 ex13 (n=16) BTBD9/CG1826 knockdown exclusively in TH neurons is sufficient for sleep disruption Average number of sleep epochs Constant darkness 110 100 ‡ 90 ♦ 80 70 60 50 130 % of wild-type Total sleep time % of wild-type 12h:12h light:dark Wild-type (n=112) CG1826 RNAi (n=100) TH control (n=32) THxCG1826 RNAi (n=32) 120 110 100 90 80 Average length of sleep epoch % of wild-type 120 110 100 90 80 70 60 50 * § *‡ p<0.05 p<0.01 § p<0.005 ♦ p<0.000001 General locomotion is normal in BTBD9/CG1826 mutants % of total flies 100 90 80 Wild-type CG1826 RNAi 70 60 50 40 30 20 10 0 0/5 1/5 2/5 3/5 4/5 Number of successful climbs 5/5 • Link back to Human disease/therapy a. Link to dietary iron (iron chelators/more iron) b. Better link to dopamine (agonists/antagonists) c. Genetic interactors (per, hth, Ataxin etc.) d. Pharmacology (Mirapex, Requip) e. Model for drug testing, pathway analysis. Novel directions • Gene-gene and gene-environment interactions • BTBD9 function in neural development and plasticity • Consequences of sleep loss on cognitive functions Acknowledgements • Amanda Freeman (FIRST Fellowship) • David Rye and lab. members, Emory Emory URC and NARSAD