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

描述(申请人提供)：脆性X智力低下蛋白(FMRP)是一种RNA结合的翻译调节因子，与几种发育性大脑疾病有关，包括儿童癫痫、自闭症谱系障碍和脆性X综合征。我们的实验室已经证实，在果蝇疾病模型中，人类的FMRP功能是完全保守的，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的缺失以外周和中枢神经系统突触过度生长为特征，包括蘑菇体(MB)学习和记忆中心11，13，58。与人类一样，缺乏dFMRP1的果蝇表现出明显的学习和记忆缺陷，并且不能诱导依赖活动的突触连接修剪12，54。虽然这些行为和细胞表型已经很好地确定了，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力，我试图确定dFMRP1在确定和调节MB学习记忆中心功能特性方面的需求。我假设1。)FMRP调节MB回路2中的感觉表征和记忆巩固。)在果蝇成体神经发育的早期使用阶段，FMRP通过选择性地抑制靶mRNA转录以响应神经元活动而发挥作用，从而发挥活性依赖的MB回路功能。3.)FMRP与其他mRNA结合的翻译调控因子Pumilio和Staufen结合，控制发育阶段特异的蛋白质表达驱动电路组装。我将结合全细胞膜片钳记录、钙离子成像和光遗传操作来验证这些假说36-41。我将把用于可视化MB Kenyon细胞的IR-DIC光学技术集成到我们现有的电生理记录设备中，并遵循现有的程序记录来自KC及其神经投射神经元(PN)36、55、56的气味诱发活动。我将产生钙成像和光遗传学实验所需的遗传库，以及FMRP和已知的mRNA结合FMRP相互作用元件STAU和PUM47，48，64的反式杂合突变体。总之，这些实验有望为理解脆性X相关学习缺陷和感觉超敏的功能相关性做出重大贡献，并为感觉信号转导、表征和巩固的性质提供基本的见解。 公共卫生相关性：脆性X综合征是遗传性智力残疾的主要形式，由单一基因产物--脆性X智力低下蛋白(FMRP)的缺失引起。FMRP的缺失与学习和记忆受损、昼夜节律紊乱、感觉过敏和突触过度生长有关。本研究方案利用功能强大的果蝇遗传系统，确定蘑菇体学习记忆中心FMRP的功能需求。

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.