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：
9337064
负责人：
Andrew Woolley
金额：
$ 17.18万
依托单位：
UNIVERSITY OF TORONTO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-01 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9337064
关键词：
Address Affect Alpha Cell Area Autistic Disorder Behavior Behavioral Binding Biological Assay Brain Brain region Cell Extracts Cells Collection Communities Complex Darkness 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 Structural Models Structure Symptoms Synapses Synaptic plasticity Testing Therapeutic Intervention Time Translation Initiation Translations Up-Regulation Work addiction autism spectrum disorder base design dimer effective therapy experimental study in vivo inhibitor/antagonist mouse model mutant neural circuit novel optogenetics overexpression public health relevance screening small molecule small molecule inhibitor 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（申请人证明）：了解局部蛋白质合成与突触可塑性如何是一种AA - 同样相关的，即假设的含义，即与自闭症谱相关的遗传缺陷的象征（ASD可能通过常见机制引起疾病 - 蛋白质的失调 - 蛋白质的失调具有EIF4G的IF4E的合成是cap依赖的蛋白质合成的速率限制步骤。 Mousels在ASD中的两种程度都在特定的时间窗口中表现出非常多的度。 “光遗传学”工具可以控制4E-4G相互作用。在小鼠模型中逆转自闭症的行为。 Opto-4E工具将基于荧光二聚体的荧光蛋白，开发基于单元格的4E-4G相互作用。此外，这些工具将使ITHER针对4G的相互作用与Appriatempril控制ASAD ASAD方法进行治疗干预ASD。