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.

项目摘要 神经元电路的发育，功能活性和可塑性严格依赖于空间控制的 突触蛋白及其信使RNA（mRNA）的表达和调节。全基因组关联 研究揭示了突触蛋白的广泛多基因变异与包括在内的疾病 自闭症，精神分裂症和阿尔茨海默氏症。对这些复杂遗传变异的分子理解 影响神经元突触的发展，可塑性和稳态对于新的发展至关重要 治疗这些疾病的疗法。荧光成像具有表征神经元突触的潜力 蛋白质和mRNA水平以及原位定位；但是，当前的成像方法可以同时 在任何给定的神经元样品中，询问几十个感兴趣的分子中的四个。到 克服这一障碍，我们建议开发一种变革性的荧光成像测定法，以实现 蛋白质和mRNA表达的同时，高度多重的高通量分子表征 完整神经元的水平和局部化。为此，我们将制定一种创新的标签策略， 利用瞬时结合的荧光核酸，以实现多个完整标本的成像， 使用标准和超分辨率显微镜。结合起来，我们将发展超光线荧光 基于杂交链反应和结构化核酸的探针，用于可视化单个mRNA 分子。我们将同时应用标准共聚焦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 小鼠和人类诱导的多能干细胞模型中的脆弱X智力低下蛋白（FMRP）。 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 超过10种突触和细胞骨架蛋白的局部定位，并检查FMRP的相互作用 完整的树突状乔木，刺和突触中有数十个mRNA。我们打算我们的技术成为 作为一种平台技术，可用于研究遗传变异的分子影响 精神病，包括自闭症和精神分裂症。因此，我们将开发成像平台 与麻省理工学院和哈佛大学的史丹利中心密切合作。高通量 我们的成像方法的性质确保它对于开发新颖的治疗方法将是有用的 使用小分子和基因编辑方法的精神病疾病。