Role of miR-8 and Ena in Synaptic Structure and Function

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DESCRIPTION (provided by applicant): Given that disruption of synapse development and function has been show to play a role in a broad range of neurological disorders from mental retardation to neurodegeneration, further understanding of the molecular components involved in the establishment of appropriate synaptic contacts is important for our comprehension of these disease states, and potentially for the discovery of drug targets and therapeutics. Recent studies have demonstrated that post-transcriptional regulation of protein synthesis and the modulation of actin-cytoskeleton dynamics play a critical role in the formation and physiology of neuronal connections. MicroRNA are exquisite regulators of post-transcriptional gene expression and have emerged as excellent candidates to regulate and integrate activity-dependent protein synthesis and actin dynamics at the synapse. My long-term objective is to define the structural, functional, and cellular basis by which microRNA control cytoskeletal dynamics during synaptic development. As an initial step, we will perform a comprehensive study of the role of the conserved microRNA, miR-8, and its functional target Ena, at the synapse. Using microRNA silencing P elements (miR-SP), a novel and versatile transgenic-based technology we have engineered to eliminate microRNA activity with spatial and temporal specificity, we have discovered that miR-8 promotes neuromuscular junction (NMJ) growth by muscle-specific repression of Ena, a well-established regulator of actin dynamics. We will use a broad array of genetic, physiological, imaging and molecular approaches to study the role of miR-8 depletion and Ena overexpression at the synapse. Specifically I will 1) assess whether the neurotransmitter (NT) release apparatus or sensitivity of postsynaptic NT receptors is affected by ubiquitous and/or muscle-specific depletion of miR-8 using electrophysiological analysis; 2) examine the effect of miR-8 depletion and Ena overexpression to the postsynaptic and presynaptic ultrastructural apparatus at the electron microscopy level; and 3) test a series of Ena mutant transgenes to determine which conserved sequence elements are critical for the localization and function of Ena at the NMJ. This will allow us to determine the possible effectors and cellular mechanisms involved in Ena and miR-8-dependent regulation of NMJ morphogenesis. Abnormal synapse morphology and function has been shown to underlie neuropathologies such as Fragije X Mental Retardation, Spinal Muscular Atrophy, and autism. Understanding the function of genes critical for appropriate synapse formation has the potential to yield advances in therapeutics to ameliorate or cure these neuronal disorders, and hence, our proposed research has great significance to biomedicine.