Genetic and Developmental Analyses of Fragile X Syndrome

描述(申请人提供)：脆性X综合征(FRAX)是一种发育性大脑疾病，具有发育中的大脑神经元结构发育和功能可塑性异常，导致智力低下、学习障碍和自闭症。我的实验室建立了果蝇FRAX模型，并证明它提供了对人类疾病状态的分子和细胞基础的直接洞察。在这个修订的提案中，我请求您的支持，以利用这个模型和果蝇大脑的相对简单，来测试FRAX的核心假设和纠正大脑缺陷的干预措施。首先，我将测试在明确的脑神经回路形成中对脆性X智力低下蛋白(FMRP)的时间要求，以及这些回路正常行为的维持表现。我的目标是两个定义非常明确的大脑电路：1)调节规律运动周期的生物钟电路，2)调节嗅觉学习/记忆巩固的蘑菇体(MB)电路。这一目标将表征这些回路的发育及其在FMRP存在和不存在的情况下的突触连接。将使用转基因条件FMRP表达系统来检验这一假设，即在神经回路发育的短暂时期特定需要FMRP来建立正常行为的维持。在离散发育窗口表达FMRP的转基因动物将被测试解剖神经回路的形成、突触结构和功能，以及昼夜活动和嗅觉学习和记忆巩固的行为输出。其次，我将测试FMRP在感觉通道、电和突触神经传递活动依赖的脑神经回路发育变化中的假设作用。FMRP功能和大脑电路活动之间的相互作用将在双突变组合中进行分析，并使用一系列经过验证的转基因工具，这些工具可以增加或减少目标大脑区域的电活动。代谢性神经递质受体信号的具体作用将通过基因突变和药理学研究相结合的方法在胆碱能和谷氨酸能脑区进行分析。这些通路的时间作用将通过在大脑发育的特定阶段定时应用药物来评估，以确定治疗干预的时间窗口。FMRP在神经传递活性诱导的磷酸化下游的局部翻译控制中的作用将通过使转基因成分磷酸化或去磷酸化的模拟蛋白来测试。必须认识到，这些假设的功能从未在活体或动物模型中进行过测试。第三，我将使用新的蛋白质组学技术来筛选在大脑发育的特定时期发生的大脑蛋白质变化，无论是否存在FMRP。同时，将进行系统的正向遗传筛查，以直接确定dfmr1基因相互作用因素。总而言之，这些目标旨在最大限度地利用经过验证的果蝇FRAX遗传模型。我是唯一一个准备继续这项工作的人，并且真的相信我可以在理解和治疗脆性X综合征方面提供巨大的帮助。公共卫生相关性：脆性X综合征是一种发育性大脑疾病，是智力低下和自闭症谱系障碍最常见的遗传形式。这项建议研究这种疾病在发育中的大脑的分子和细胞基础，并直接测试旨在纠正大脑发育异常的治疗干预措施。

DESCRIPTION (provided by applicant): Fragile X Syndrome (FraX) is a developmental brain disorder with abnormal neuron architecture development and functional plasticity of the developing brain, causing mental retardation, learning disabilities and autism. My lab established the Drosophila FraX model and proved it provides direct insights into molecular and cellular bases of the human disease state. In this revised proposal, I ask your support to take advantage of this model, and the relative simplicity of the Drosophila brain, to test core hypotheses of FraX and interventions to correct brain defects. First, I will test the temporal requirements for the Fragile X Mental Retardation Protein (FMRP) in defined brain neural circuit formation and the maintained manifestation of normal behaviors from these circuits. I target two extremely well-defined brain circuits; 1) the circadian clock circuit that regulates regular motor activity cycles, and 2) the mushroom body (MB) circuit that mediates olfactory learning/memory consolidation. This aim will characterize the development of these circuits and their synaptic connectivity in the presence vs. absence of FMRP. A transgenic conditional FMRP expression system will be used to test the hypothesis that FMRP is required specifically during a transient period of neural circuit development to establish maintenance of normal behaviors. Transgenic animals expressing FMRP during discrete developmental windows will be tested for anatomical neural circuit formation, synaptic structure and function, and the behavior outputs of circadian activity and olfactory learning and memory consolidation. Second, I will test the hypothesized role of FMRP in sensory modality, electrical and synaptic neurotransmission activity-dependent changes in brain neural circuit development. The interaction between FMRP function and brain circuit activity will be assayed in double mutant combinations and with an array of proven transgenic tools that either increase or decrease electrical activity in targeted brain regions. The specific role of metabotropic neurotransmitter receptor signaling will be assayed in both cholinergic and glutamatergic brain regions using a combination of genetic mutants and pharmacological studies. The temporal roles of these pathways will be assessed with timed application of drugs during defined stages of brain development to define temporal windows for therapeutic intervention. The role of FMRP in local translation control downstream of a neurotransmission activity-induced phosphorylation will be tested by making transgenic constitutively phosphorylated or desphosphorylated mimic proteins. It is imperative to appreciate that these hypothesized functions have never been tested in vivo, in an animal model. Third, I will use new proteomic technologies to screen for brain protein changes occurring during specific periods of brain development, in the presence and absence of FMRP. In parallel, systematic forward genetic screens will be pursued to directly identify dfmr1 genetic interactors. Together, these aims are designed to make maximal use of the proven Drosophila FraX genetic model. I am the only one poised to pursue this work and truly believe that I can aid enormously in the understanding and treatment of Fragile X Syndrome. PUBLIC HEALTH RELEVANCE: Fragile X Syndrome is a developmental brain disorder that is the most commonly inherited form of both mental retardation and autism spectrum disorders. This proposal studies the molecular and cellular basis of this disease in the developing brain and directly tests therapeutic interventions aimed at correcting the brain developmental abnormalities.