Tools for manipulating local protein synthesis in the brain

 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。据推测，与自闭症谱系障碍（ASD）相关的遗传缺陷的显著部分可能通过共同的机制-突触处蛋白质合成的失调引起疾病。真核起始因子4 E（eIF 4 E）与eIF 4G的相互作用是帽依赖性蛋白质合成的限速步骤。eIF 4 E过表达或竞争性抑制剂4 EBP 2被敲除的小鼠模型显示出自闭症样行为。4 E-4G相互作用的小分子抑制剂4 EGI-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中改变的神经回路表现出非常高的空间和时间复杂性，使得ASD中的治疗干预可能需要在特定时间窗口期间针对相关神经回路，而不是广泛地针对大脑的所有区域。这很难用像4 EGI-1这样的小分子或实际上用任何目前可用的分子工具实现。我们建议开发允许控制4 E-4G相互作用的遗传编码的光控（“光遗传学”）工具。具体来说，我们将开发：（i）opto-4 EBP 2，一种允许蓝光控制阻断4 E-4G相互作用的工具。从概念上讲，这是一种遗传编码的，基于蛋白质的和可逆的小分子抑制剂4 EGI，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。它可以用来测试离散脑区蛋白质合成的时间上调是否会导致ASD样行为。我们的方法分两个阶段。首先，我们将进行第一代opto-4 EBP 2和opto-4 E工具的结构设计。第二阶段是优化，这对于有效的体内功能至关重要。我们将开发一种基于细胞的4 E-4G相互作用测定，其基于使用二聚化依赖性荧光蛋白的荧光筛选。这种方法将使我们能够快速筛选成千上万的opto-4 EBP 2和opto-4 E设计。我们创造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，这些工具将使人们有可能确定以适当的时空控制为目标的4 E-4G相互作用是否是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.