Genetic and Developmental Analyses of Fragile X Mental Retardation Protein

脆性 X 智力低下蛋白的遗传和发育分析

• 批准号: 10744864
• 负责人: Kendal Broadie
• 金额: $ 51.62万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10744864
• 关键词: Afferent Neurons; Animals; Automobile Driving; Behavior; Behavioral; Binding; Biological Assay; Brain; Brain Mapping; Cell surface; Cells; Chlorides; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive; Complex; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Disease; Disease model; Dissection; Dynamin; Eating; Electron Microscopy; Endosomes; Environment; Exhibits; Exposure to; FMR1; Fragile X Syndrome; Funding; Genetic; Genetic Diseases; Genetic Models; Goals; Heritability; Impairment; Insulin Receptor; Interneurons; Interstitial Collagenase; Knock-out; Label; Learning; Life; Ligands; Light; Link; MAP Kinase Gene; Maps; Matrix Metalloproteinases; Mediating; Membrane; Memory; Messenger RNA; Modeling; Molecular; Monitor; Multiple Lentigines; Neuroglia; Neurons; Neuropil; Noonan Syndrome; Odorant Receptors; Odors; Output; PTPN11 gene; Pathway interactions; Phagocytes; Phagocytosis; Phase; Phenotype; Phosphatidylserines; Phosphoric Monoester Hydrolases; Phosphotransferases; Polysaccharides; Process; Proteins; Publishing; Reporter; Role; Sensory; Signal Pathway; Signal Transduction; Sorting; Specific qualifier value; Structure; Surface; Synapses; Testing; Tetanus Toxin; Transgenic Organisms; Translational Regulation; Transmission Electron Microscopy; Visualization; WNT Signaling Pathway; Work; autism spectrum disorder; brain circuitry; critical period; experience; extracellular; gain of function; gene environment interaction; gene interaction; gene product; genetic manipulation; in vivo; insulin secretion; insulin-like peptide; loss of function; mutant; neural circuit; neurotransmission; olfactory sensory neurons; optogenetics; patch clamp; programs; receptor; response; sensor; tool; transgene expression

Early life sensory experience remodels brain circuitry to optimize behavioral output. This competitive renewal continues our genetic dissection of the molecular mechanisms through which synaptic connections are refined based on sensory experience. We use different odorant cues to activate different classes of odorant receptor neurons, downstream projection neurons, and brain learning/memory center Kenyon cells to test the directional remodeling of connectivity and function. In three parallel circuit channels, we use in vivo CRISPR knockout, conditional gene manipulations and transgenic trans-synaptic connectivity mapping to test both neuron and glia class-specific roles in activity-dependent remodeling. These mechanisms go awry in numerous disease states of cognitive and autism spectrum disorders. We directly test here genetic models of Fragile X syndrome (FXS), Noonan syndrome (NS), Noonan syndrome with Multiple Lentigines (NSML) and Neurobeachin (NBEA) autism spectrum disorder (ASD). In Aim 1, we test neuron-specific Fragile X Mental Retardation Protein (FMRP) roles in odorant sensory experience-dependent critical period brain circuit remodeling in 3 distinct circuit channels. We target tetanus toxin light chain (TeTXLC) and excitatory (CsChrimson)/inhibitory (GtAcR) optogenetic tools to odorant sensory neurons, projection neurons, and Kenyon cells to dissect activity-dependent mechanisms. Based on a preliminary screen, we identify channel-selective chloride transporters and Wnt signaling ligands to test their critical period roles. In Aim 2, we test glia-specific FMRP roles in experience-dependent critical period synaptic remodeling by blocking phagocytic pruning at three different levels. We test neuron-to-glia signaling mechanisms in the FXS disease model, including both synapse-secreted insulin like peptide (ILP) “find me” signals and surface phosphatidylserine (PS) “eat me” signals. We test the roles of direct FMRP mRNA targets 1) ESCRT-III Shrub and 2) glial-secreted matrix metalloproteinase 1 (MMP1) in glial synapse phagocytosis and critical period synaptic remodeling. We use single neuron labeling (miniSOG) to visualize glial phagocytosis via transmission electron microscopy (TEM). In Aim 3, we test FMRP translation regulation of PKA/ERK signaling pathways in sensory experience-dependent circuit remodeling. We use new transgenic cAMP reporter (cAMPr) and separation of phases-based activity reporter of kinase (SPARK) to visualize signaling in vivo in response to experience during the critical period; compartmentalized within odorant sensory neurons, projection neurons and Kenyon cells in our 3 parallel circuit channels. We test roles of direct FMRP mRNA target Rugose/NBEA acting as a PKA anchor, with downstream PKA pathway kinase Meng-Po. Using an ERK-SPARK reporter and whole-cell patch-clamp recording to monitor functional remodeling, we test roles of direct FMRP mRNA target Corkscrew/SHP2 acting as an ERK pathway phosphatase. Our ongoing program orders molecular mechanism intersections between Fragile X syndrome (FXS), Noonan syndrome (NS), Noonan syndrome with Multiple Lentigines (NSML), and NBEA-associated autism spectrum disorder in critical period brain circuit remodeling.

早期生活的感觉经验重塑大脑回路，以优化行为输出。这次竞争性的续约 继续我们对突触连接得以完善的分子机制的遗传解剖 基于感官体验。我们使用不同的气味线索来激活不同类别的气味受体 神经元、下游投射神经元和大脑学习/记忆中心Kenyon细胞来测试定向神经元的功能。 重塑连接和功能。在三个平行的电路通道中，我们使用体内CRISPR敲除， 条件基因操作和转基因跨突触连接映射来测试神经元和神经胶质细胞 在活动依赖性重塑中的类特异性作用。这些机制在许多疾病状态中出错 认知障碍和自闭症谱系障碍。我们在这里直接测试脆性X综合征（FXS）的遗传模型， 努南综合征（NS）、努南综合征伴多发性长痣（NSML）和神经海滩（Nebeachin）自闭症 谱系障碍（ASD）。在目标1中，我们测试了神经元特异性脆性X智力迟钝蛋白（FMRP）的作用， 在气味感觉经验依赖的关键期，3个不同回路通道的脑回路重塑。 我们靶向破伤风毒素轻链（TeTXLC）和兴奋性（CsChrimson）/抑制性（GtAcR）光遗传学工具 气味感觉神经元，投射神经元，和凯尼恩细胞解剖活动依赖机制。 基于初步筛选，我们鉴定了通道选择性氯离子转运蛋白和Wnt信号配体， 测试他们的关键时期角色。在目标2中，我们测试了胶质细胞特异性FMRP在经验依赖性关键期中的作用， 在三个不同的水平上阻断吞噬细胞修剪来抑制突触重塑。我们测试神经元到神经胶质的信号传递 FXS疾病模型中的机制，包括突触分泌的胰岛素样肽（ILP）“找到我” 信号和表面磷脂酰丝氨酸（PS）“吃我”信号。我们测试了直接FMRP mRNA靶点的作用， 1)ESCRT-III Shrub和2）胶质突触吞噬作用中胶质分泌的基质金属蛋白酶1（MMP 1）， 关键期突触重塑。我们使用单神经元标记（miniSOG）来观察胶质细胞的吞噬作用， 透射电子显微镜（TEM）。在目标3中，我们测试PKA/ERK信号转导的FMRP翻译调节， 感觉经验依赖性电路重塑的途径。我们使用新的转基因cAMP报告基因（cAMPr） 和分离基于相的激酶活性报告子（SPARK），以可视化体内应答中的信号传导 在关键时期的经验;划分在气味感觉神经元，投射神经元， 和凯尼恩细胞在我们的3个平行电路通道中。我们测试了FMRP mRNA直接靶向Rugose/Numbers的作用， 作为PKA锚，与下游PKA途径激酶Meng-Po。使用ERK-SPARK报告基因， 全细胞膜片钳记录监测功能重塑，我们测试直接FMRP mRNA靶点的作用， Corkscrew/SHP 2作为ERK途径磷酸酶。我们正在进行的项目 脆性X综合征（FXS）、努南综合征（NS）、努南综合征与多发性骨髓瘤之间的交叉 Lentigines（NSML）和NBEA相关的自闭症谱系障碍在关键期脑回路重塑。