On-demand neuronal condensate interactomes using new optogenomics tools

使用新的光基因组学工具按需神经元凝聚体相互作用

• 批准号: 10685497
• 负责人: Clifford P Brangwynne
• 金额: $ 20.25万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10685497
• 关键词: ALS pathology; ALS patients; Address; Affect; Amyotrophic Lateral Sclerosis; Axon; Biochemical Reaction; Biology; Biophysics; Cell Death; Cell physiology; Cells; Cellular Stress; Communities; Complex; Cytoplasm; Cytoplasmic Granules; Dendrites; Development; Disease; Disease model; Gene Expression; Genomic approach; Genomics; Individual; Liquid substance; Maps; Measures; Membrane; Messenger RNA; Methodology; Methods; Modeling; Molecular; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Nucleoplasm; Oxidative Stress; Pathologic; Phase; Physical condensation; Play; Proteins; Public Health; RNA; RNA Splicing; RNA Transport; RNA-Binding Proteins; Regulation; Resolution; Rest; Rodent; Signal Transduction; Solid; Structure; TDP-43 aggregation; Techniques; Technology; Toxic effect; Translations; Work; biophysical techniques; experimental study; genome-wide; genomic tools; innovation; insight; neuron loss; new technology; novel; novel therapeutics; optogenetics; protein TDP-43; protein reconstitution; recruit; stress granule; technology platform; tool

PROJECT SUMMARY Biomolecular condensates (BMCs) are emerging as a ubiquitous feature of compartmentalization within cells. These structures can form by liquid-liquid phase separation of particular proteins and RNAs from the rest of the cytoplasm or nucleoplasm and can perform important functions (e.g. enzymatic reactions, signaling regulation). BMCs of the RNA-binding protein TDP-43 are hypothesized to nucleate pathological aggregates and lead to neuron death in Amyotrophic Lateral Sclerosis (ALS). However, BMCs are difficult to characterize with traditional techniques because they lack membranes and can dissolve upon dilution and, therefore, are challenging (or impossible) to purify. Additionally, interrogating toxic effects of disease-associated BMCs has been problematic because of the difficulty of controlled induction of liquid-like or solid-like condensates within neurons. Here, we propose the development of a new technology platform, Phase-seq, that combines two approaches from different fields – biophysical in-cellulo reconstitution of protein condensation and genomic mapping of RNA organization – to address current methodological limitations for studying BMCs in neurons. This tool combines optogenetic induction of BMCs in neurons (Corelets) with a cutting-edge genomics approach (SPRITE) to interrogate the RNA compositions and functions of BMCs. First, we will lay the groundwork for Phase-seq by establishing SPRITE in primary neurons to create a genome-wide map of RNA- RNA interactions in these highly specialized cells and confirm that we can identify RNAs within individual BMCs by inducing stress granules. We will then develop models of early and late stage ALS pathology by inducing TDP-43 BMCs in neurons with Corelets, a new technology that allows for controlled optogenetic induction of condensates with tight temporal and spatial resolution. Finally, we will combine SPRITE and Corelets in neurons to create Phase-seq, a novel platform for simultaneous induction of BMCs and characterization of their functions by mapping RNA constituents. These experiments will allow us to gain valuable insight into RNA organization in neuronal BMCs, determine how TDP-43 condensates may lead to neuron death, and identify novel therapeutic avenues for ALS. We anticipate that the Phase-seq platform will be broadly applicable for interrogating the functions of endogenous BMCs in neurons and the consequences of aberrant BMCs in disease.

项目摘要 生物分子冷凝物（BMC）正在成为隔室化的无处不在的特征 在细胞内。这些结构可以通过特定蛋白质和 来自其余细胞质或核质的RNA，可以执行重要功能（例如， 酶促反应，信号调节）。 RNA结合蛋白TDP-43的BMC为 假设是核病理骨料，并导致肌萎缩性神经元死亡 侧硬化症（ALS）。但是，BMC很难用传统技术来表征 因为它们缺乏膜，可以在稀释时溶解，因此是挑战（或 不可能）净化。此外，询问与疾病相关的BMC的毒性作用具有 由于难以控制液体样或固体样的诱导难以控制的问题 神经元内的凝结。在这里，我们建议开发一个新技术平台， 相序列结合了来自不同领域的两种方法 - 生物物理内细胞内 RNA组织的蛋白质凝结和基因组图的重建 - 以解决 当前研究神经元中BMC的方法学局限性。该工具结合了光遗传学 用最先进的基因组学方法（Sprite）诱导BMC在神经元（曲线）中的诱导 询问BMC的RNA组成和功能。首先，我们将为 通过在原发性神经元中建立精灵来创建RNA-基因组图图， 这些高度专业的细胞中的RNA相互作用，并确认我们可以识别RNA 通过诱导的应激颗粒单个BMC。然后，我们将开发早期和晚期的模型 ALS诱导的TDP-43 BMC在带有Corelets的神经元中，这是一种新技术，允许 用于控制临时和空间分辨率紧密的冷凝物的光遗传学诱导。 最后，我们将在神经元中结合精灵和珊瑚，以创建一个新型平台，这是一个新型平台 通过映射RNA，用于简单地诱导BMC并表征其功能 构成。这些实验将使我们能够在RNA组织中获得宝贵的见解 神经元BMC，确定TDP-43冷凝物如何导致神经元死亡，并确定 ALS的新型热途径。我们预计相序平台将广泛 适用于询问内源性BMC在神经元中的功能和后果 疾病中异常的BMC。