Studying circadian (~24hr) rhythms offers an excellent opportunity to understand a key brain function at molecular, cellular and circuit levels as well as possibly identifying novel targets for therapies for sleep disorders and jetlag. Studies in Drosophila identified the first circadian clock gene, which is conserved in humans and linked to an inherited human sleep disorder. Clock genes function in central brain pacemaker neurons to control whole animal behavioral rhythms. The endogenous molecular and neural rhythms of pacemaker neurons provide a unique model to study how gene expression controls daily changes in neuronal signaling which, at least in Drosophila, includes rhythms in structural plasticity. To identify novel regulators of circadian rhythms, we generated whole genome expression profiles from a group of purified master pacemaker neurons, the Drosophila LNvs. We identified a set of 10 genes that are expressed with a daily rhythm in a clock-dependent manner and which are much more highly expressed in LNvs than in other differentiated neurons. Of these 10 genes, four encode previously identified core clock genes such as period. Here, we propose to study CG33275, one of the other 6 genes. CG33275 is an unstudied gene which likely encodes a Guanine nucleotide Exchange Factor (GEF) that activates a Rho family GTPase. CG33725 is the Drosophila ortholog of human Puratrophin, which has been linked with a hereditary form of spinocerebellar ataxia, but is unstudied at the molecular level. We refer to CG33275 as dPuratrophin (dPura) and believe that basic studies of this gene in Drosophila could help explain the disease-association of human Puratrophin. Rhythmic dPura expression could impose circadian rhythms on the activity of a Rho family GTPase, which have also not yet been implicated in circadian rhythms. Here, we first aim to determine if dPura is indeed a GEF using genetics and biochemistry. Using genetics, we will test which of the 6 Drosophila Rho GTPase family members genetically interact with dPura in clock neurons to regulate circadian behavior. We will complement these in vivo experiments with in vitro biochemical experiments that directly measure dPura GEF activity.
Our second aim i s to identify the role of dPura in LNvs by characterizing dPura mutants we have identified that strongly alter circadian behavioral rhythms. Specifically, we will ask if dPura mutants show altered circadian gene expression, structural plasticity and/or intracellular trafficking in LNvs that could underlie the behavioral defects. Since GEFs are often activated by extracellular signals, dPura could help pacemaker neurons integrate internal clock time (via rhythmic expression) with external signals. Given that mouse Puratrophin also shows circadian expression in the brain, our studies should give insight into both fly and mammalian circadian rhythms as well as helping understand Puratrophin function in general.
Circadian pacemaker neurons are an excellent model to study the genetic contribution to changes in neuronal activity and plasticity. We have identified a novel regulator in pacemaker neurons - dPura, the fly ortholog of a gene associated with spinocerebellar ataxia in humans. We propose to identify dPura's molecular target in pacemaker neurons (likely conserved in humans) and how dPura regulates pacemaker neuron function.
|Petsakou, Afroditi; Sapsis, Themistoklis P; Blau, Justin (2015) Circadian Rhythms in Rho1 Activity Regulate Neuronal Plasticity and Network Hierarchy. Cell 162:823-35|
|Collins, Ben; Blau, Justin (2013) A plastic clock. Neuron 78:580-2|