Tools for manipulating local protein synthesis in the brain

操纵大脑局部蛋白质合成的工具

• 批准号: 8806632
• 负责人: Andrew Woolley
• 金额: $ 14.85万
• 依托单位: UNIVERSITY OF TORONTO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-19 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8806632
• 关键词: Address; Affect; Area; Autistic Disorder; Behavior; Behavioral; Binding; Biological Assay; Brain; Brain region; Cell Extracts; Cells; Collection; Communities; Complex; Data; Defect; Dendritic Spines; Deposition; Dimerization; Disease; Elements; Eukaryotic Initiation Factor-4E; Exhibits; Fluorescence; Fluorescence Polarization; Generations; Hela Cells; Hour; In Vitro; Intervention; Kinetics; Knock-out; Length; Libraries; Light; Measures; Memory; Mental Depression; Mental Health; Mental disorders; Methodology; Methods; Molecular; Mutation; Nervous system structure; Neurons; Phase; Point Mutation; Protein Biosynthesis; Proteins; Role; Site; Sorting - Cell Movement; Staging; Structural Models; Structure; Symptoms; Synapses; Synaptic plasticity; Testing; Therapeutic Intervention; Time; Translation Initiation; Translations; Up-Regulation; Work; addiction; autism spectrum disorder; base; design; effective therapy; in vivo; inhibitor/antagonist; mouse model; mutant; neural circuit; novel; optogenetics; overexpression; public health relevance; research study; screening; small molecule; spatiotemporal; tool

 DESCRIPTION (provided by applicant): Understanding how local protein synthesis leads to synaptic plasticity is a fundamental problem, but it is also highly relevant to mental illness. It as been hypothesized that a significant fraction of the genetic defects associated with autism spectrum disorders (ASD) may cause disease through a common mechanism - the dysregulation of protein synthesis at synapses. The interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4G is the rate limiting step for cap-dependent protein synthesis. Mouse models in which eIF4E is overexpressed, or in which the competitive inhibitor 4EBP2 is knocked out, display autistic-like behaviors. A small molecule inhibitor of the 4E-4G interaction, 4EGI-1, shows exciting potential for reversing autistic symptoms in these mouse models. However, the neural circuits that are altered in ASD exhibit very high degrees of both spatial and temporal complexity so that therapeutic interventions in ASD will likely need to be directed at relevant neural circuits during specific time windows, rather than broadly at all areas of the brain. This i hard to achieve with small molecules like 4EGI-1 or indeed with any currently available molecular tool. We propose to develop genetically-encoded light-controlled ('optogenetic') tools that permit control of the 4E-4G interaction. Specifically we will develop: (i) opto-4EBP2, a tool that will permit blue light controlled blocking of the 4E-4G interaction. Conceptually this is a genetically-encoded, protein-based and reversible version of the small molecule inhibitor 4EGI that was found to reverse autism-like behaviors in mouse models. (ii) opto-4E. This tool will permit blue light triggered up-regulation of local translation. It can be used to test whether time up-regulation of protein synthesis in discrete brain regions leads to ASD-like behaviors. Our approach is two-stage. First, we will carry out structure-based design of first-generation opto-4EBP2 and opto-4E tools. The second stage is optimization, which is critical for effective in vivo function. We will develop a cell-based 4E-4G interaction assay based on fluorescence screening using dimerization dependent fluorescent proteins. This methodology will allows us to rapidly screen thousands of opto-4EBP2 and opto-4E designs. The optogenetic tools we create will permit fundamental studies on the mechanisms of synaptic plasticity. In addition, these tools it will make it possible to determine whether targeting the 4E-4G interaction with appropriate spatiotemporal control is a valid approach for therapeutic intervention in ASD.

 描述（由适用提供）：了解局部蛋白质合成如何导致突触可塑性是一个基本问题，但它也与精神疾病高度相关。正如假设的那样，与自闭症谱系障碍（ASD）相关的遗传缺陷的很大一部分可能通过常见机制引起疾病 - 突触时蛋白质合成的失调。真核起始因子4E（EIF4E）与EIF4G的相互作用是依赖帽依赖性蛋白质合成的速率限制步骤。 EIF4E过表达的鼠标模型，或者将竞争性抑制剂4EBP2拆除，显示出准确的行为。 4E-4G相互作用的小分子抑制剂4EGI-1在这些小鼠模型中逆转准确的症状，显示出令人兴奋的潜力。但是，在ASD中改变的神经元电路表现出很高的空间和暂时复杂性，因此在特定时间窗口中，可能需要针对ASD的治疗干预措施，而不是大脑的所有区域都需要针对相关的神经元回路。我很难用小分子（例如4EGI-1）或任何当前可用的分子工具实现这一目标。我们建议开发允许控制4E-4G相互作用的遗传编码的光控制（“光遗传学”）工具。具体而言，我们将开发：（i）OPT-4EBP2，该工具将允许蓝光控制4E-4G相互作用的阻塞。从概念上讲，这是一种一般编码的，基于蛋白质的小分子抑制剂4EGI，发现在小鼠模型中可以逆转类似自闭症的行为。 （ii）opt-4e。该工具将允许蓝光触发本地翻译的上调。它可用于测试离散大脑区域中蛋白质合成的时间上调是否导致类似ASD的行为。我们的方法是两个阶段。首先，我们将对第一代OPT-4EBP2和OPT-4E工具进行基于结构的设计。第二阶段是优化，这对于有效的体内功能至关重要。我们将基于使用二聚化依赖性荧光蛋白的荧光筛选开发基于细胞的4E-4G相互作用测定法。这种方法将使我们能够快速筛选数千个Opt-4EBP2和Opto-4E设计。我们创建的光遗传学工具将允许有关突触可塑性机制的基本研究。此外，这些工具将有可能确定针对适当时空控制的4E-4G相互作用是ASD治疗干预的有效方法。