Tools for manipulating local protein synthesis in the brain

 描述(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。据推测，与自闭症谱系障碍(ASD)相关的很大一部分遗传缺陷可能通过一个共同的机制-突触蛋白质合成失调而导致疾病。真核细胞起始因子4E(EIF4E)与eIF4G的相互作用是帽依赖蛋白合成的限速步骤。在eIF4E过度表达或竞争性抑制物4EBP2被敲除的小鼠模型中，表现出自闭症样行为。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，ASD中改变的神经回路显示出非常高的空间和时间复杂性，因此ASD的治疗干预可能需要在特定的时间窗口针对相关的神经回路，而不是广泛地针对大脑的所有区域。这很难用像4EGI-1这样的小分子或任何目前可用的分子工具来实现。我们建议开发基因编码的光控(‘光遗传’)工具，允许控制4E-4G相互作用。具体地说，我们将开发：(I)OPTO-4EBP2，这是一种允许蓝光控制阻止4E-4G相互作用的工具。从概念上讲，这是一种遗传编码的、基于蛋白质的可逆版本的小分子抑制剂4EGI，它被发现可以逆转小鼠模型中的自闭症样行为。(Ii)OPTO-4E。该工具将允许蓝灯触发对本地翻译的上调。它可以用来测试离散大脑区域蛋白质合成的时间上调是否会导致类似ASD的行为。我们的方法是分两步走的。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。第二阶段是优化，这是有效的体内功能的关键。我们将开发一种基于细胞的4E-4G相互作用实验，该方法基于使用二聚化依赖的荧光蛋白的荧光筛选。这种方法将使我们能够快速筛选数以千计的OPTO-4EBP2和OPTO-4E设计。我们创造的光遗传工具将使对突触可塑性机制的基础研究成为可能。此外，这些工具将使我们有可能确定以4E-4G相互作用为目标并进行适当的时空控制是否是ASD治疗干预的有效方法。

 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.