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

 描述（由申请人提供）：突触中的蛋白质是突触可塑性的基本调节剂，其最终控制作为行为基础的神经回路。我们对突触连接如何与动物行为联系的理解的一个重大进展来自活体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棘的蛋白质含量的能力，阻碍了他们破译突触蛋白在学习，记忆，行为和疾病中的作用的努力。在本申请中，我们描述了一种新的方法，使用双光子照射，以控制蛋白质合成的脊椎特异性的方式在清醒的动物。这种方法被称为LIPS：光诱导蛋白质合成。LIPS利用编码感兴趣的蛋白质的“光响应”mRNA。这种mRNA被设计成在正常条件下不被翻译。然而，在树突或棘的双光子照射后，光激活蛋白被募集到mRNA的5 'UTR，导致局部翻译。因此，LIPS将允许在活体动物的大脑中以前所未有的空间和时间分辨率实现蛋白质合成。为了开发一种简单而强大的光遗传学技术，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， 本申请是（1）产生和优化RNA适体，其募集两种拟南芥光敏色素的光激活形式：Cry 2和PhyB。这些实验将导致第一个光调节RNA-蛋白质相互作用的产生;（2）优化皮层神经元中的LIPS。为了制造光调控的mRNA，我们将这些适体掺入特定的mRNA中。然后我们将优化一种双光子照射方案，用于控制活体动物树突和棘中的蛋白质合成;（3）利用LIPS来剖析FMRP和FMRP结构域在棘重塑中的作用。本实验旨在研究FMRP在小鼠大脑皮层单个树突棘水平上调控树突棘动力学的作用。在这里，我们将使用LIPS直接询问脊柱中FMRP水平的变化如何与脊柱转换相关，我们将确定LIPS介导的Fmr 1 KO树突中FMRP的恢复是否会导致脊柱稳定性的恢复。总之，这些实验将为FMRP如何控制活体小鼠的脊柱重塑提供深入了解。总之，LIPS提供了前所未有的时空控制神经元内的蛋白质表达。LIPS将改变双光子研究，使研究人员能够控制棘和树突的蛋白质组成，并监测特定蛋白质对树突棘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.