Synaptic plasticity is important for normal development and for injury-induced repair. The normal aging process and neurodegenerative disorders are associated with impaired plasticity. Motor neuron diseases involve a period of plastic compensatory changes that are eventually overwhelmed by the disease process. Synaptic plasticity is also an important characteristic of Drosophila neuromuscular junctions (NMJ). I have identified novel regulators of Drosophila NMJ arborization, including the mir-2 microRNAs and the protein Commissureless2 (Comm2). Like the better-known Commissurelessl (Comm), Comm2 may regulate the Robo-Slit pathway but whereas Comm affects this pathway during axon guidance, Comm2 may control synaptic branching. I intend to establish how these molecules control NMJ morphology and identify the pathways in which they function through acheiving the three specific aims listed below. 1. Establish if Mir-2 Regulates Comm2 Function. My preliminary data indicate that mir-2 microRNAs affect NMJ branching as does Comm2, one of their predicted targets. The working hypothesis of this aim is that mir-2 functions by regulating Comm2. The ability of mir-2 to down-regulate translation of comml mRNA through its 3'UTR will be determined by in vitro luciferase-assays. In vivo regulation of Comm2 by mir-2 will be established by determining the genetic interactions between mir-2 and Comm2. 2. Determine the role of Comm2 in NMJ Growth. The working hypothesis for this aim, based on my preliminary data, is that Comm2 negatively regulates synaptic arborization. Normal Comm2 expression will be determined by in situ hybridizations. 'Rescue'experiments of the Comm2 loss-of-function mutant will be performed with UAS-Comm2 and GAL4 drivers to prove if Comm2 is essential for normal NMJ branching. 3. Establish the Signaling Pathways Regulated by Comm2. The working hypothesis is that Comm2 functions by controlling the Robo-Slit pathway. Comm2 shares identity with a known regulator of this pathway and my preliminary data indicate that pathway members are present at the larval NMJ. The ability of Comm2 to inhibit Robo receptors will be determined by Comm2 ectopic over-expression experiments. Whether Comm2 acts through Robo receptors at the NMJ will be determined by immunohistochemistry and genetic interactions that will establish if these molecules act in the same pathway. Motor Neuron diseases are devastating disorders that cannot yet be successfully treated. Through understanding the mechanisms that synaptic plasticity, especially synaptic sprouting and branching, in a model organism such as Drosophila we may one day be able to induce these changes in human patients.
