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）的缺失导致发育性脑障碍，其特征是突触连接受损和脑发育晚期活动依赖性调节中断。FMRP的缺失与一系列神经发育障碍（ndd）有关，其症状包括智力残疾、自闭症和儿童癫痫。我们的实验室建立了强大的果蝇疾病模型，并显示人类FMRP在该模型中显示出完全的功能守恒。我们已经多次证明，这个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。在这个修改后的竞争性更新提案中，我请求您的迫切需要的支持，使我们能够继续利用这个美妙的遗传系统，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导致的大脑发育缺陷。实验方法将针对定义明确的蘑菇体（MB）回路，这是一个接收来自多种感觉模式输入的大脑中心，介导联想学习和记忆巩固。在第一个目标中，我们将测试FMRP调节MB回路中适当的兴奋性(E)与抑制性(I)突触平衡的发展的假设。我们提出了遗传和药理学的方法来纠正E和I缺陷独立，以测定恢复的结构，功能和行为输出缺陷在零突变状态。我们假设这将是一个富有成效的治疗干预的新途径。在第二个目标中，我们研究突触活动和FMRP功能在形成MB回路成熟中的相互依赖性。我们假设突触活动通过fmrp依赖机制调节E与I突触消除（“修剪”）相对于稳定。我们将在分阶段的发育试验中使用转基因活性阻滞剂（例如破伤风毒素）和光电流（例如光门控离子通道）在目标E与I神经元中的组合，检查对照与FMRP丢失和功能获得突变体的结果。同时，我们将使用转基因[Ca2+]报告器来绘制MB电路发育过程中活性依赖性E与I的变化。在第三个目标中，我们解决了FMRP作为翻译调节剂的作用，控制MB电路成熟过程中发育阶段适当的蛋白质合成。我们建议在发育时间过程中描述脑蛋白质组，我们已经建立了FMRP功能。这种迫切需要的发育分析以前从未在任何疾病模型中做过。总之，这些目标旨在最大限度地利用强大且经过验证的果蝇疾病模型。我的实验室是唯一一个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人们理解和设计治疗这种最常见的认知功能障碍和自闭症谱系障碍的遗传原因。

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.