Our long-term aim is to use a genetic approach in Drosophila to elucidate molecular mechanisms of neural signaling. Here we focus on synaptic development and neurodegeneration. Among our collection of ts-paralytic mutants, we have discovered nwk, which causes extensive synaptic overgrowth and vacu, which causes severe neurodegeneration. These mutants provide novel starting points for dissecting mechanisms regulating synaptic growth and neuronal viability. Our goals are to determine the in vivo functions of these genes using genetic, molecular, morphological, and electrophysiological techniques to characterize the mutants and to understand how they cause the observed phenotypes. Nwk is an SH3-containing adaptor protein belonging to a family that is conserved from yeast to humans. It localizes to periactive zones of synaptic boutons, a region specialized for regulation of synaptic growth. We hypothesize that Nwk is a component of a regulatory pathway that links extracellular cues to synaptic growth and plasticity via regulation of the actin cytoskeleton. We will analyze the structure and function of Nwk by characterizing new point mutations as well as nwk transgenes in which specific motifs are mutated. A possible functional link with RhoGAP will be tested in transgenic flies. We will examine the effect of nwk on activity-dependent synaptic functions and learning behavior to determine if normal physiological and behavioral plasticity requires Nwk. We will dissect the nwk signaling pathway by analyzing epistatic interactions with other synaptic growth mutations, screening for nwk enhancers and suppressors, and identifying interacting proteins in yeast two-hybrid screens, vacu causes reduced lifespan and age-dependent loss of motor behavior associated with appearance of massive spongiform neuropathology throughout the CNS. We will determine the molecular basis of this defect by identifying and characterizing the encoded protein. Electrophysiological studies will be performed to clarify the relationship between altered neural activity and neurodegeneration. The underlying mechanisms of neurodegeneration will be analyzed by ultrastructural studies, tests for apoptosis, examination of epistatic interactions, and screens for genetic suppressors. These studies will advance our understanding of the molecules and mechanisms that mediate synaptic growth and plasticity and neuronal viability. These processes are fundamental to nervous system function in all higher organisms and they are disrupted in many human diseases. Thus, our studies should have broad biological and medical significance.
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