LIPS: A novel technology for spatial and temporal control of protein synthesis in dendritic spines

 描述(申请人提供)：突触中的蛋白质是突触可塑性的基本调节器，最终控制着构成行为的神经回路。在我们理解突触连接如何与动物行为联系方面的一个重大进展来自于对活着的动物树突棘的经颅双光子成像。然而，尽管双光子显微镜取得了进步，但大多数实验都是观察性的。研究人员缺乏直接操纵特定突触和脊椎上的蛋白质含量的能力，这阻碍了他们努力破译突触蛋白质在学习、记忆、行为和疾病中的作用。在这项应用中，我们描述了一种新的方法，使用双光子照射来控制清醒动物中脊椎特异的蛋白质合成。这种方法被称为LIPS：光诱导蛋白质合成。嘴唇利用一种“光响应型”信使核糖核酸编码一种感兴趣的蛋白质。该信使核糖核酸在正常情况下不会被翻译。然而，当双光子照射树突或脊椎时，光激活蛋白被招募到mRNA的5‘UTR区，导致局部翻译。因此，嘴唇将允许蛋白质合成在活动物的大脑中以前所未有的空间和时间分辨率实现。为了开发一种简单而强大的光基因技术，使研究人员能够研究基本上任何蛋白质对突触功能的作用， 这一应用是(1)产生和优化RNA适配子，以招募两种拟南芥光敏色素的光激活形式：Cry2和PhyB。这些实验将导致第一个光调控的RNA-蛋白质相互作用的产生；(2)优化大脑皮层神经元中的嘴唇。为了制造光调控的mRNAs，我们将把这些适配子掺入特定的mRNAs。然后，我们将优化双光子照射方案，以控制活体动物树突和脊柱中的蛋白质合成；(3)使用LIPs来剖析FMRP和FMRP结构域在脊柱重塑中的作用。本实验旨在研究FMRP在活体小鼠大脑皮质单个树突棘水平上调节树突棘动力学的作用。在这里，我们将使用LIPS来直接询问脊柱中FMRP水平的变化与脊柱翻转的相关性，并确定LIPS介导的FMRP在Fmr1 KO树突中的修复是否能够恢复脊柱的稳定性。总之，这些实验将为FMRP如何控制活着的小鼠的脊柱重塑提供深入的见解。总而言之，LIPs为神经元内的蛋白质表达提供了前所未有的时空控制。LIPS将改变双光子研究，使研究人员能够控制棘突和树突的蛋白质组成，并监测特定蛋白质对树突形态和突触可塑性等过程的影响。

 DESCRIPTION (provided by applicant): The proteins in synapses are the fundamental regulators of synaptic plasticity, which ultimately controls the neural circuits that underlie behavior. A major advance in our understanding of how synaptic connectivity is linked to animal behavior comes from transcranial two-photon imaging of dendritic spines in living animals. However, despite the advances made by two-photon microscopy, most experiments have been observational. Researchers lack the ability to directly manipulate the protein content at specific synapses and spines, hindering their efforts to decipher the roles of synaptic proteins in learning, memory, behavior, and disease. In this application, we describe a novel method to use two-photon irradiation to control protein synthesis in a spine- specific manner in awake animals. This method is called LIPS: light-induced protein synthesis. LIPS utilizes a "light-responsive" mRNA encoding a protein of interest. This mRNA is designed so that it is not translated under normal conditions. However, upon two-photon irradiation of a dendrite or spine, a light-activated protein is recruited to the 5'UTR of the mRNA, resulting in localized translation. Thus, LIPS will allow protein synthesis to be achieved with unprecedented spatial and temporal resolution in the brains of live animals. In order to develop a simple and robust optogenetic technology to allow researchers to study the role of essentially any protein on synaptic function, the specific aims of this application are (1) To generate and optimize RNA aptamers that recruit light-activated forms of two Arabidopsis phytochromes: Cry2 and PhyB. These experiments will result in the generation of the first light-regulated RNA-protein interactions; (2) To optimize LIPS in cortical neurons. To make light-regulated mRNAs, we will incorporate these aptamers into specific mRNAs. We will then optimize a two-photon irradiation protocol for controlling protein synthesis in dendrites and spines in live animals; (3) To use LIPS to dissect the role of FMRP and FMRP domains in spine remodeling. The experiments in this aim are designed to investigate the role of FMRP in regulating dendritic spine dynamics at the level of individual dendritic spines in the cortex of live mice. Here we will use LIPS to directly interrogate how varying the level of FMRP in spines correlates with spine turnover and we will determine if LIPS-mediated restoration of FMRP in Fmr1 KO dendrites results in restoration of spine stability. Together, these experiments will provide insight into how FMRP controls spine remodeling in living mice. In summary, LIPS provides unprecedented spatiotemporal control of protein expression within a neuron. LIPS will transform two photon studies by enabling researchers to control the protein composition of spines and dendrites and monitor the effects of specific proteins on processes like dendritic spine morphology and synaptic plasticity.