Genetic and Developmental Analyses of Fragile X Mental Retardation Protein

描述（由申请人提供）：脆性X智力迟钝蛋白（FMRP）缺失导致发育性脑障碍，其特征为发育晚期脑中突触连接受损和活动依赖性调节中断。FMRP缺失与一系列神经发育障碍（NDD）相关，其症状包括智力残疾、自闭症和儿童癫痫。本实验室建立了功能强大的果蝇疾病模型，并在该模型中显示人FMRP具有完全的功能保守性。我们已经多次证明，该模型提供了对人类疾病状态的分子和细胞基础的直接见解。在这个修订后的竞争性更新提案中，我请求您迫切需要的支持，使我们能够继续利用这个奇妙的遗传系统，以及果蝇大脑中相对简单的学习/记忆神经回路及其完全表征的行为输出，来测试关于FMRP丢失的核心假设，并提出干预措施来纠正由此产生的发育性大脑缺陷。实验方法将针对定义明确的蘑菇体（MB）回路，这是一个接受来自多种感觉方式的输入以介导联想学习和记忆巩固的大脑中心。在第一个目标，我们将测试的假设，FMRP调节适当的兴奋性（E）与抑制性（I）的MB电路内的突触平衡的发展。我们提出了遗传和药理学的手段，以纠正E与I的缺陷独立，测定恢复的架构，功能和行为输出缺陷的无效突变状态。我们假设这将是一个富有成效的新途径的治疗干预。在第二个目标中，我们研究了突触活动和FMRP功能在塑造MB回路成熟中的相互依赖性。我们假设突触活动通过FMRP依赖性机制相对于稳定性调节E对I突触消除（“修剪”）。我们将在阶段性发育试验中使用转基因活性阻断剂（例如破伤风毒素）和光电流（例如光门控离子通道）在靶向E vs. I神经元中的组合，检查对照组与FMRP丧失和功能获得突变体相比的结果。同时，我们将使用转基因[Ca 2 +]报告绘制活动依赖性E与I的变化在MB电路的发展。在第三个目标中，我们解决的作用，FMRP作为一个翻译调节控制发育阶段适当的蛋白质合成过程中MB电路成熟。我们建议在我们已经建立的FMRP功能的发育时间过程中表征脑蛋白质组。这种迫切需要的发育分析以前从未在任何疾病模型中进行过。总之，这些目标旨在最大限度地利用强大和经过验证的果蝇疾病模型。我的实验室是唯一准备从事这项工作的实验室，我真的相信我们可以极大地帮助理解和设计治疗认知功能障碍和自闭症谱系障碍的最常见遗传原因。

DESCRIPTION (provided by applicant): Loss of Fragile X Mental Retardation Protein (FMRP) causes a developmental brain disorder characterized by impaired synaptic connectivity and disrupted activity-dependent modulation in the late developing brain. Loss of FMRP is associated with a range of neurodevelopmental disorders (NDDs) with symptoms including intellectual disability, autism and childhood epilepsy. Our laboratory established the powerful Drosophila disease model and showed human FMRP displays total functional conservation in this model. We have repeatedly proven this model provides direct insights into the molecular and cellular bases of the human disease state. In this revised competitive renewal proposal, I ask for your urgently needed support to allow us to continue to take advantage of this wonderful genetic system, and relative simplicity of the learning/memory neural circuit in the Drosophila brain and its thoroughly-characterized behavioral output, to test core hypotheses regarding FMRP loss and proposed interventions to correct the resultant developmental brain defects. Experimental approaches will target the well-defined Mushroom Body (MB) circuit, a brain center receiving input from multiple sensory modalities to mediate associative learning and memory consolidation. In the first aim, we will test the hypothesis that FMRP regulates the development of the appropriate excitatory (E) vs. inhibitory (I) synaptic balance within the MB circuit. We propose genetic and pharmacological means to correct E vs. I defects independently, to assay restoration of architectural, functional and behavioral output defects in the null mutant state. We hypothesize this will be a fruitful new avenue for therapeutic intervention. In the second aim, we examine the inter-dependence of synaptic activity and FMRP function in shaping MB circuit maturation. We hypothesize that synaptic activity regulates E vs. I synapse elimination ("pruning") relative to stabilization via a FMRP-dependent mechanism. We will use a combination of transgenic activity blockers (e.g. tetanus toxin) and photocurrents (e.g. light-gated ion channels) in targeted E vs. I neurons in staged developmental trials, examining outcomes in controls compared to FMRP loss and gain-of-function mutants. In parallel, we will use transgenic [Ca2+] reporters to chart activity-dependent E vs. I changes during MB circuit development. In the third aim, we tackle the role of FMRP as a translational regulator controlling development stage appropriate protein synthesis during MB circuit maturation. We propose to characterize the brain proteome over the developmental time course we have established for FMRP function. Such desperately needed developmental profiling has never before been done in any disease model. Together, these aims are designed to make maximal use of the powerful and proven Drosophila disease model. My lab is the only lab poised to pursue this work, and I truly believe we can aid enormously in providing understanding and devising treatments for this most common heritable cause of cognitive dysfunction and autism spectrum disorder.