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.

项目概要 生物分子凝聚体（BMC）正在成为分隔的普遍特征 这些结构可以通过特定蛋白质的液-液相分离而形成。 来自细胞质或核质其余部分的 RNA 可以执行重要功能（例如， RNA 结合蛋白 TDP-43 的酶促反应、信号调节）。 使肌萎缩侧索硬化症中的病理聚集成核并导致神经元死亡 然而，侧索硬化症（ALS）很难用传统技术来表征。 因为它们缺乏膜并且可以在稀释时溶解，因此具有挑战性（或 不可能）此外，研究与疾病相关的 BMC 的毒性作用也已被证实。 由于难以控制类液体或类固体的诱导，因此一直存在问题 在这里，我们建议开发一个新技术平台， Phase-seq，结合了不同领域的两种方法——生物物理细胞内方法 蛋白质浓缩的重建和 RNA 组织的基因组图谱 – 解决 目前研究神经元 BMC 的方法存在局限性，该工具结合了光遗传学。 使用尖端基因组学方法 (SPRITE) 在神经元 (Corelet) 中诱导 BMC 首先，我们将为探究 BMC 的 RNA 组成和功能奠定基础。 Phase-seq 通过在初级神经元中建立 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，并证实我们可以识别其中的 RNA 然后我们将通过诱导应力颗粒来开发单个 BMC 的早期和晚期模型。 ALS 病理学通过 Corelets 诱导神经元中的 TDP-43 BMC，这是一项新技术，可以 用于具有严格时间和空间分辨率的凝聚物的受控光遗传学诱导。 最后，我们将在神经元中结合 SPRITE 和 Corelet 创建 Phase-seq，一个新颖的平台 通过绘制 RNA 同时诱导 BMC 并表征其功能 这些实验将使我们能够深入了解 RNA 的组织。 神经元 BMC，确定 TDP-43 凝聚物如何导致神经元死亡，并确定 我们预计 Phase-seq 平台将得到广泛应用。 适用于探究神经元内源性 BMC 的功能及其后果 疾病中异常的 BMC。