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

LIPS：一种用于树突棘蛋白质合成时空控制的新技术

• 批准号: 9037179
• 负责人: WENBIAO GAN
• 金额: $ 52.59万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-23 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9037179
• 关键词: 5' Untranslated Regions; Animal Behavior; Animals; Arabidopsis; Behavior; Binding; Brain; Chimeric Proteins; Dendrites; Dendritic Spines; Disease; Dominant-Negative Mutation; Ensure; Exhibits; Exposure to; FMRP; Generations; Goals; Image; Individual; Kinetics; Knock-out; Learning; Life; Light; Link; Mammalian Cell; Mediating; Memory; Mental Health; Messenger RNA; Methods; Microscopy; Monitor; Morphology; Mus; Mutation; Neurologic; Neurons; Optics; Pathogenesis; Phytochrome; Process; Protein Biosynthesis; Proteins; Protocols documentation; RNA; RNA Recognition Motif; RNA-Protein Interaction; Recruitment Activity; Research Personnel; Resolution; Ribosomes; Role; Site; Structure; Synapses; Synaptic plasticity; Techniques; Technology; Testing; Translating; Translations; Vertebral column; aptamer; awake; base; design; insight; interest; irradiation; learned behavior; mRNA tagging; neural circuit; new technology; novel; optogenetics; prevent; protein expression; public health relevance; research study; response; restoration; spatiotemporal; synaptic function; temporal measurement; time interval; two-photon

 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.

 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因子，最终控制着行为背后的神经回路。我们对突触连接如何与动物行为相关的理解的重大进展来自于经颅双光子。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，无法直接操纵特定的蛋白质含量。突触和棘，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，我们描述了一种使用双光子照射以棘特异性方式控制蛋白质合成的新方法。这种方法称为 LIPS：光诱导蛋白质合成，利用编码感兴趣蛋白质的“光响应”mRNA。树突或脊柱的双光子照射，光激活蛋白被招募到 mRNA 的 5'UTR，从而导致局部翻译，因此，LIPS 将使大脑中的蛋白质合成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，具体目标是 该应用的目的是（1）生成和优化招募两种拟南芥光敏色素的光激活形式的RNA适体：Cry2和PhyB。这些实验将产生第一个光调节的RNA-蛋白质相互作用；为了制造光调节 mRNA，我们将把这些适体整合到特定的 mRNA 中，然后优化用于控制树突和蛋白质合成的双光子照射方案。 (3) 使用 LIPS 剖析 FMRP 和 FMRP 结构域在脊柱重塑中的作用。该实验旨在研究 FMRP 在个体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 直接研究脊柱中 FMRP 水平的变化与脊柱周转的关系，并确定 LIPS 介导的 FMRP 恢复是否在脊柱中进行。 Fmr1 KO 树突导致脊柱稳定性恢复。 总之，LIPS 提供了神经元内蛋白质表达的前所未有的时空控制，这将通过启用两个光子研究来改变。研究人员控制树突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。