Tools for manipulating local protein synthesis in the brain

操纵大脑局部蛋白质合成的工具

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：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。据研究，很大一部分遗传缺陷与自闭症谱系障碍（ASD）相关。）可能通过一种常见机制引起疾病 - 突触蛋白质合成失调。真核起始因子 4E (eIF4E) 与 eIF4G 的相互作用是帽依赖性蛋白质合成的限速步骤。 eIF4E 过度表达或竞争性抑制剂 4EBP2 被敲除的模型表现出类似自闭症的行为，4E-4G 相互作用的小分子抑制剂 4EGI-1 在这些小鼠模型中显示出逆转自闭症症状的令人兴奋的潜力。然而，自闭症谱系障碍中改变的神经回路表现出非常高的空间和时间复杂性，因此自闭症谱系障碍的治疗干预可能需要在特定的时间窗口内针对相关的神经环路，而不是广泛地在大脑的所有区域，这很难通过像 4EGI-1 这样的小分子或任何当前可用的分子工具来实现，我们建议开发允许控制的基因编码光控制（“光遗传学”）工具。具体而言，我们将开发：(i) opto-4EBP2，一种允许蓝光控制阻断 4E-4G 相互作用的工具。从概念上讲，这是一种基于基因编码的蛋白质。以及小分子抑制剂 4EGI 的可逆版本，该抑制剂被发现可以逆转小鼠模型中的自闭症样行为。 (ii) opto-4E 该工具将允许蓝光触发局部翻译的上调。离散大脑区域中蛋白质合成的时间上调会导致类似 ASD 的行为，首先，我们将对第一代 opto-4EBP2 和 opto-4E 工具进行基于结构的设计。阶段是我们将开发一种基于细胞的 4E-4G 相互作用测定法，该测定法基于使用二聚化依赖性荧光蛋白的荧光筛选，该方法将使我们能够快速筛选数千种 opto-4EBP2 和 opto-4E。我们创建的光遗传学工具将允许对突触可塑性机制进行基础研究，此外，这些工具将可以确定以适当的时空控制为目标的 4E-4G 相互作用是否是一种有效的方法。用于 ASD 的治疗干预。