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 生物分子凝聚物（BMC）作为一种普遍存在的区室化特征而出现 细胞内。这些结构可以通过特定蛋白质的液-液相分离形成， RNA来自细胞质或核质的其余部分，并且可以执行重要的功能（例如， 酶促反应、信号调节）。RNA结合蛋白TDP-43的BMC是 假设成核病理聚集体，并导致肌萎缩性神经元死亡 侧索硬化症（ALS）。然而，BMC很难用传统技术表征 因为它们缺乏膜，并且在稀释时可以溶解，因此具有挑战性（或 不可能净化。此外，询问疾病相关BMC的毒性作用， 由于难以控制类液体或类固体的诱导， 在神经元内凝聚。在这里，我们建议开发一个新的技术平台， Phase-seq，结合了来自不同领域的两种方法-细胞内生物物理 蛋白质缩合的重建和RNA组织的基因组图谱-以解决 目前研究神经元中BMC的方法学局限性。这个工具结合了光遗传学 用尖端基因组学方法（SPRITE）在神经元（COCOCONT）中诱导BMC， 询问BMC的RNA组成和功能。首先，我们将奠定基础， 通过在原代神经元中建立SPRITE来创建RNA的全基因组图谱， 这些高度特化的细胞中的RNA相互作用，并证实我们可以在这些细胞中识别RNA。 通过诱导应激颗粒来诱导单个BMC。然后我们将开发早期和晚期阶段的模型 ALS病理通过诱导TDP-43 BMC在神经元中与Coplets，一种新的技术， 用于以严格的时间和空间分辨率对浓缩物进行受控的光遗传诱导。 最后，我们将联合收割机SPRITE和神经元中的Coplets结合起来，创建一个新的平台Phase-seq 用于同时诱导BMC并通过定位RNA表征其功能 选民。这些实验将使我们能够获得有价值的洞察RNA组织中， 神经元BMC，确定TDP-43冷凝物如何导致神经元死亡，并确定 ALS的新治疗途径。我们预计，Phase-seq平台将广泛用于 适用于询问神经元中内源性BMCs的功能及其后果 异常的骨髓细胞。