Nina Tang Sherwood - US grants

The overall goal of this research proposal is to understand the short- term modulation of synaptic transmission by neurotrophins. There is increasing evidence that neurotrophin expression and neuronal excitability are intimately related. Neurotrophins are thus emerging, beyond traditional "growth and survival factors", as potential mediators of synaptic plasticity. Given the fundamental importance of rapid synaptic changes in nervous system functioning, a role for neurotrophins in the rapid mediation of synaptic plasticity is compelling. Furthermore, the likely involvement of these growth factors in such diseases as Alzheimer's and Parkinson's underscores the importance of understanding neurotrophin effects in the nervous system. Three questions will be investigated to understand the specific, short-term effects of neurotrophins on synaptic transmission: 1) How is synaptic transmission in neurons modulated by each member of the neurotrophin family? 2) At what locus, presynaptic or postsynaptic, do neurotrophins act to modulate synaptic transmission? 3) What are the specific molecular mechanisms by which neurotrophins modulate transmission? The proposed experiments will be addressed in CA1 hippocampal pyramidal neurons, which express the neurotrophins and their receptors in abundance, and are well-studied as a model of neuronal plasticity. Whole-cell patch clamp methods will be used to characterize synaptic currents in dissociated, "autaptic" neurons -- isolated neurons which have formed synapses on themselves. The use of such a simplified, yet functionally relevant model of a synapse will enable the understanding of specific neurotrophin effects on synaptic transmission, and help to elucidate the general role of neurotrophins in neuronal plasticity.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the regulation of the cytoskeleton in neuronal development and function. In particular we focus on the microtubule cytoskeleton, which is essential to many aspects of neuronal function, including neurite outgrowth, synapse formation and remodeling, and transport of cellular components between the cell body and distal compartments. Although dynamic instability, the process by which microtubules grow and shrink at their ends, remains the predominant mechanism by which microtubule polymer length is thought to be regulated, the importance of an additional mechanism known as microtubule severing is becoming increasingly apparent. Microtubule severing proteins are ATPases that bind to and cut the microtubule in the central, more energetically stable region of the polymer. They are found in phyla ranging from plants to humans, and have been shown to play critical roles in growth, cell division and nervous system function. In humans, mutations in the microtubule severing protein Spastin are the major cause of Autosomal Dominant Hereditary Spastic Paraplegia, a debilitating neurodegenerative disease in which the longest axon tracts of the central nervous system degenerate. Our work in Drosophila has shown that loss of Spastin leads to a paucity of microtubules in distal synaptic boutons, as well as reduced synaptic transmission. These animals exhibit severely compromised motor behavior, reminiscent of the human disease. The goal of this proposal is to understand the cell biological events in which microtubule severing is utilized in neurons, and the regulation of these processes. Three severing proteins are expressed in the developing fly nervous system: spastin, katanin-60 (kat60), and kat-like. Each may function in distinct cell types, developmental stages, or cell compartments; alternatively, they may be functionally redundant. Based on their embryonic expression patterns, mutant phenotypes, and specificity of regulatory proteins we have identified through genetic screens, we hypothesize that each protein has an independent role(s) in neuronal function, but may be partially redundant. To test this hypothesis we will: 1) characterize neuronal morphology and microtubule distribution in single and combinatorial loss-of- function mutants in these genes, 2) elucidate the roles of the spastin genetic regulators DPak3, Cdk5, and Pctaire, and 3) execute in vivo screens for genetic modifiers of kat60 and kat-like. We will exploit the strengths of Drosophila to achieve these goals, focusing on a combination of genetic and imaging approaches that are well-established in the lab. PUBLIC HEALTH RELEVANCE: The goals of this project are to elucidate the roles and regulation of the three proteins predicted to sever neuronal microtubules in the fly: spastin, kat60, and kat-like. Misregulation of microtubules is implicated in a host of human diseases, including Hereditary Spastic Paraplegia (caused by mutations in spastin), Alzheimer's Disease, and many forms of cancer. An understanding of these proteins will therefore be broadly beneficial to human health, revealing pathways controlling microtubule growth and/or loss, and thereby providing additional targets for therapeutic intervention. [unreadable] [unreadable]