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（由适用提供）：突触中的蛋白质是突触可塑性的基本调节剂，最终控制了行为行为的神经回路。我们对合成连通性如何与动物行为联系起来的主要进步来自活动物中树突状棘的thranial两光子成像。然而，尽管两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突上蛋白质含量的能力，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。在此应用中，我们描述了一种使用两光子辐照以在清醒动物中特异性方式控制蛋白质合成的新方法。该方法称为嘴唇：光诱导的蛋白质合成。 Lips利用了编码感兴趣蛋白质的“轻响应性” mRNA。该mRNA的设计使得它不会在正常条件下翻译。然而，在对树突或脊柱的两光子照射后，将光激活的蛋白募集到mRNA的5'UTR中，从而导致局部翻译。这就是嘴唇将通过空前的空间和临时分辨率在活动物的大脑中实现蛋白质合成。为了开发一种简单，强大的光遗传技术，使研究人员可以研究任何蛋白质在突触功能上的作用， 该应用程序是（1）生成和优化RNA适体，这些适体募集了两种拟南芥植物色素的光激活形式：Cry2和Phyb。这些实验将导致产生第一个光调节的RNA - 蛋白质相互作用。 （2）优化皮质神经元中的嘴唇。为了制作轻度调节的mRNA，我们将将这些适体纳入特定的mRNA中。然后，我们将优化一种两光子辐照方案，用于控制活动物中树突和刺中的蛋白质合成； （3）使用LIPS剖析FMRP和FMRP结构域在脊柱重塑中的作用。此目标中的实验旨在研究FMRP在活小鼠皮质中单个树突状棘水平上调节树突状棘动力学中的作用。在这里，我们将使用LIPS直接询问棘突中FMRP水平与脊柱周转率相关的如何变化，我们将确定LIPS介导的FMR1 fMRP是否恢复了FMR1 KO Dendrites中的FMRP是否会导致脊柱稳定性的恢复。这些实验将共同洞悉FMRP如何控制活小鼠中的脊柱重塑。总而言之，LIPS对神经元内蛋白质表达的史无前例的时空控制。 LIPS通过使研究人员能够控制棘突和树突的蛋白质组成，并监测特定蛋白对树突状脊柱形态和突触可塑性等过程的影响，从而改变两项光子研究。