Simultaneous multiplexed in situ fluorescence imaging of neuronal proteins and messenger RNAs

神经元蛋白和信使 RNA 的同步多重原位荧光成像

• 批准号: 9289191
• 负责人: Mark Bathe
• 金额: $ 41.46万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9289191
• 关键词: Alzheimer' s Disease; Animal Model; Antibodies; Autistic Disorder; Binding; Biological Assay; CRISPR/Cas technology; Cell model; Cells; Collaborations; Complex; Cytoskeletal Proteins; Dendrites; Deoxyribonucleases; Development; Disease; Disease model; Ensure; FMR1; Fluorescent Probes; Fluorescent in Situ Hybridization; Gene Deletion; Genes; Genetic Transcription; Genetic Variation; Health; Homeostasis; Human; Hybrids; Image; Imagery; Imaging Techniques; In Situ; Individual; Institutes; Label; Mediating; Mental disorders; Messenger RNA; Methods; Microscopy; Molecular; Morbidity - disease rate; Mus; Nature; Neurodegenerative Disorders; Neuronal Plasticity; Neurons; Nucleic Acid Hybridization; Nucleic Acid Probes; Nucleic Acids; Patients; Phenotype; Proteins; Proteomics; RNA-Binding Proteins; Reaction; Regulation; Regulatory Pathway; Resolution; Sampling; Schizophrenia; Small Interfering RNA; Spatial Distribution; Specimen; Staining method; Stains; Synapses; Techniques; Technology; Toes; Transcript; Variant; Vertebral column; Work; antibody conjugate; base; design; fluorescence imaging; genetic manipulation; genome wide association study; genome-wide; high resolution imaging; imaging approach; imaging platform; induced pluripotent stem cell; innovation; interest; knock-down; neuronal circuitry; novel; novel therapeutics; nucleic acid structure; patient population; protein distribution; sample fixation; screening; single molecule; small molecule; transcription factor; transcriptome sequencing

PROJECT SUMMARY The development, functional activity, and plasticity of neuronal circuits rely critically on the spatially controlled expression and regulation of synaptic proteins and their messenger RNAs (mRNAs). Genome-wide association studies have revealed extensive polygenic variation in synaptic proteins in association with diseases including autism, schizophrenia, and Alzheimer's. A molecular understanding of how these complex genetic variations impact neuronal synapse development, plasticity, and homeostasis is crucial for the development of new therapies to treat these diseases. Fluorescence imaging offers the potential to characterize neuronal synapse protein and mRNA levels and localizations in situ; however, current imaging approaches can simultaneously interrogate no more than four of the several dozen molecules of interest in any given neuronal sample. To overcome this obstacle, we propose to develop a transformative fluorescence imaging assay that enables simultaneous, highly multiplexed, high-throughput molecular characterization of protein and mRNA expression levels and localizations in intact neurons. To this end, we will develop an innovative labeling strategy that exploits transiently binding fluorescent nucleic acids to enable multiple rounds of imaging of intact specimens, using both standard and super-resolution microscopy. In conjunction, we will develop ultra-bright fluorescent probes based on hybridization chain reaction and structured nucleic acids for visualization of single mRNA molecules. We will apply both standard confocal and super-resolution imaging to characterize spatial distributions and molecular interactions of synaptic proteins and regulatory mRNA-binding proteins, including Fragile-X Mental Retardation Protein (FMRP) in both mouse and human induced pluripotent stem cell models. Using this approach, we will characterize the impact of gene deletions associated with autism on the levels and localizations of more than 10 synaptic and cytoskeletal proteins, as well as examine the interactions of FMRP with dozens of mRNAs in intact dendritic arbors, spines, and synapses. We intend our technique to become broadly useful as a platform technology for the study of the molecular impacts of genetic variations in psychiatric diseases, including autism and schizophrenia. Consequently, we will develop our imaging platform in close collaboration with the Stanley Center at the Broad Institute of MIT and Harvard. The high-throughput nature of our imaging approach ensures that it will be useful for development of novel methods of treating psychiatric diseases using small-molecule and gene-editing approaches.

项目总结 神经元回路的发育、功能活动和可塑性在很大程度上依赖于空间控制的 突触蛋白及其信使RNA的表达和调控。全基因组关联 研究表明，突触蛋白的广泛多基因变异与疾病有关，包括 自闭症、精神分裂症和阿尔茨海默氏症。了解这些复杂的遗传变异是如何 冲击性神经元突触的发育、可塑性和动态平衡对新的 治疗这些疾病的疗法。荧光成像提供了表征神经元突触的可能性 蛋白质和信使核糖核酸水平和原位定位；然而，目前的成像方法可以同时 在任何给定的神经元样本中，询问几十个感兴趣的分子中不超过四个。至 克服这一障碍，我们建议开发一种变革性的荧光成像分析方法，使 同时、高度多路、高通量的蛋白质和信使核糖核酸表达的分子表征 完整神经元的水平和定位。为此，我们将开发一种创新的标签策略， 利用瞬时结合的荧光核酸对完整的标本进行多轮成像， 同时使用标准和超分辨率显微镜。我们将联合开发超亮荧光材料 基于杂交链式反应和结构核酸的单链mRNA显像剂 分子。我们将同时应用标准共焦成像和超分辨率成像来表征空间 突触蛋白和调节mRNA结合蛋白的分布和分子相互作用，包括 脆性X智力低下蛋白(FMRP)在小鼠和人类诱导的多能干细胞模型中的表达。 使用这种方法，我们将表征与自闭症相关的基因缺失对水平和 定位10多个突触和细胞骨架蛋白，以及检查FMRP的相互作用 在完整的树枝、棘突和突触中有数十个mRNA。我们打算把我们的技术变成 广泛用作研究人类遗传变异的分子影响的平台技术 精神疾病，包括自闭症和精神分裂症。因此，我们将开发我们的成像平台 与麻省理工学院斯坦利中心和哈佛大学密切合作。高吞吐量 我们的成像方法的性质确保了它将有助于开发新的治疗方法 使用小分子和基因编辑方法的精神疾病。