Activity-dependent modulation of the Drosophila mushroom body function by FMRP

描述（由申请人提供）：脆性X智力迟钝蛋白（FMRP）是一种rna结合的翻译调节因子，涉及多种发育性脑疾病，包括儿童癫痫、自闭症谱系障碍和脆性X综合征。我们的实验室已经确定，人类FMRP功能在果蝇疾病模型中是完全保守的，并一再证明与哺乳动物生物学直接相关。果蝇FMRP同源基因dFMRP1的缺失，其特征是外周和中枢神经系统的突触过度生长，包括蘑菇体（MB）学习和记忆中心11,13,58。与人类一样，缺乏dFMRP1的果蝇在学习和记忆方面表现出明显的缺陷，并且无法诱导突触连接的活动依赖性修剪12,54。虽然这些行为和细胞表型已经很好地建立起来，但这些表型背后的神经元活动却知之甚少。利用果蝇模型系统的遗传能力，我试图确定dFMRP1在决定和调节MB学习和记忆中心的功能特性方面的要求。我假设。)FMRP调节MB回路中的感觉表征和记忆巩固。在果蝇成体神经发育早期，FMRP通过选择性抑制目标mRNA转录物来响应神经元活动，从而参与活性依赖性MB回路功能。3)。FMRP与其他mrna结合的翻译调控因子Pumilio和Staufen组合相互作用，控制发育阶段特异性蛋白表达驱动电路组装。我将使用全细胞膜片钳记录、Ca2+成像和光遗传学操作的组合来测试这些假设36-41。我将把使用可视化MB Kenyon细胞的IR-DIC光学技术整合到我们现有的电生理记录设备中，并按照现有的程序记录KCs及其支配投射神经元（PN）36,55,56的气味诱发活动。我将生成钙成像和光遗传学实验所需的遗传库，以及FMRP和已知mRNA结合FMRP相互作用物状态和泵的反杂合突变体47,48,64。总之，这些实验为理解脆性X相关的学习缺陷和感觉超敏反应的功能相关性做出了重大贡献，并为感觉信号转导、表征和巩固的本质提供了基本的见解。

DESCRIPTION (provided by applicant): Fragile X Mental Retardation Protein (FMRP) is an RNA-binding translational regulator implicated in several developmental brain disorders including childhood epilepsy, autism spectrum disorder and Fragile X syndrome. Our lab has established that human FMRP function is completely conserved in the Drosophila disease model, and has repeatedly proven direct relevance to mammalian biology60. The loss of the drosophila FMRP homolog, dFMRP1, is characterized by synaptic overgrowth in both the peripheral and central nervous systems, including the mushroom body (MB) learning and memory center11,13,58. As in humans, Drosophila lacking dFMRP1 exhibit pronounced deficits in learning and memory and are unable to induce activity-dependent pruning of synaptic connectivity12,54. Though these behavioral and cellular phenotypes are well established, the neuronal activity underlying these phenotypes is poorly understood. Using the genetic power of the Drosophila model system I seek to determine the requirements of dFMRP1 in determining and modulating the functional properties of the MB learning and memory center. I hypothesize 1.) FMRP regulates sensory representation and memory consolidation in the MB circuit 2.) FMRP functions in activity- dependent MB circuit function by selectively repressing target mRNA transcripts in response to neuronal activity during the early-use period of Drosophila adult neurodevelopment. 3.) FMRP interacts combinatorially with other mRNA-binding translational regulators, Pumilio and Staufen, to control development stage-specific protein expression driving circuit assembly. I will test these hypotheses using a combination of whole-cell patch clamp recording, Ca2+ imaging, and optogenetic manipulations36-41. I will integrate the IR-DIC optic technology used visualize MB Kenyon cells into our existing electrophysiological recording equipment and follow existing procedures for recording odor evoked activity from both KCs and their innervating projection neurons (PN)36,55,56. I will generate the genetic stocks necessary for calcium imaging and optogenetics experiments as well as the trans-heterozygous mutants for both FMRP and known mRNA binding FMRP interactors stau and pum47,48,64. Together these experiments promise significant contributions to understanding the functional correlates of Fragile X associated learning deficits and sensory hypersensitivity, and offer fundamental insights into the nature of sensory signal transduction, representation, and consolidation. PUBLIC HEALTH RELEVANCE: Fragile X Syndrome is the leading form of inherited intellectual disability and is caused by the loss of a single gene product, the Fragile X Mental Retardation Protein (FMRP). Loss of FMRP is associated with impaired learning and memory, circadian dis-rhythmicity, sensory hypersensitivity, and synaptic overgrowth. This research proposal utilizes the powerful Drosophila genetic system to define the functional requirements of FMRP in the mushroom body learning and memory center.