Developing RNA Oligonucleotides to Mitigate Abberant FUS Phase Transition in FTD/ALS

开发 RNA 寡核苷酸以减轻 FTD/ALS 中异常的 FUS 相变

• 批准号: 10185657
• 负责人: Lin Guo
• 金额: $ 117万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10185657
• 关键词: ALS patients; Affect; Amyotrophic Lateral Sclerosis; Binding; Biochemical; Biological Assay; Biological Models; Biophysics; Cell Nucleus; Cell model; Cells; Cytoplasmic Granules; Cytoplasmic Inclusion; Data; Disease; EWSR1 gene; Escherichia coli; Fluorescence; Goals; Impairment; In Vitro; Interruption; Length; Link; Liquid substance; Methods; Molecular Chaperones; Mutate; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Import; Nuclear Localization Signal; Oligonucleotides; Organelles; Pathologic; Phase; Phase Transition; Play; Polymers; Postdoctoral Fellow; Process; Property; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA-Binding Protein FUS; RNA-Binding Proteins; Ribonucleases; Role; Solid; Stress; Structure; System; TAF15 gene; Testing; Therapeutic; Time; Toxic effect; Training; analog; base; biophysical properties; combat; design; effective therapy; frontotemporal lobar dementia-amyotrophic lateral sclerosis; improved; mutant; neurotoxicity; post-doctoral training; prevent; protein TDP-43; protein aggregation; receptor; reconstitution; recruit; single molecule; stress granule; therapeutic RNA; therapeutically effective

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two fatal neurodegenerative diseases with no cure or effective treatment. A common pathological hallmark of FTD/ALS is the presence of cytoplasmic inclusions formed by various RNA-binding proteins (RBPs) with intrinsically disordered regions (IDRs). These RBPs include FUS, as well as TDP-43, TAF15, and EWSR1, which are all components of stress granule (SG). SGs are liquid-like transient cytoplasmic membraneless organelles that form when cells are under stress through a process called liquid-liquid phase separation (LLPS) that condensates RBPs and RNA. Because SGs are enriched with FTD/ALS disease RBPs such as FUS that is intrinsically aggregation-prone, if stress granules are not cleared in time, FUS can go through aberrant phase transition to form solid fibrillar aggregates that can induce toxicity and neurodegeneration by sequestering other proteins and RNAs and impairing stress granule dynamics. We hypothesize that agents able to reverse aberrant FUS phase transition and fibrillization would restore stress granule dynamics and function, thus mitigating FUS toxicity. The long-term goal of this project includes developing such agents with therapeutic potentials to reverse pathological aberrant phase transition and aggregation of FUS, and elucidating the mechanism of action of these agents. During my postdoc training, we discovered that nuclear import receptor Kapβ2 can function as a protein chaperone and a disaggregase to prevent and reverse FUS aggregation by binding to the nuclear localization signal in the C- terminus of FUS. Thus, we hypothesize that other FUS-binding biomolecules can also prevent and reverse its phase transition and fibrillization. In this proposal, we seek to develop FUS-binding short RNAs as oligonucleotide disaggregases to mitigate aberrant phase transition and toxicity of FUS. Our preliminary data indicate that RNAs that bind to FUS protein in FUS-expressing E. Coli prevent FUS aggregation. We will first define RNA oligonucleotides with specific sequences that can mitigate phase separation and aggregation of FUS, and other RBPs that are implicated in FTD/ALS. Because RNA polymers are rapidly digested by ribonucleases in the cell, to develop RNA-based oligonucleotides for therapeutic purpose, we will also design RNA analogues with higher cellular stability. The activities of these oligonucleotides will be tested using in vitro phase separation system reconstituted from purified RBPs. We will then define the mechanism of action of the RNA oligonucleotides to mitigate FUS assembly. Due to the dynamic and heterogeneous nature of phase separated FUS system, we will combine ensemble biophysical measurements and single-molecule fluorescence assays to achieve this goal. In the final aim, we will test whether RNA disaggregases can restore stress granule dynamics and rescue FUS toxicity in cell. These short RNA oligonucleotides identified in our study have great potential to be developed as RNA therapeutics for FTD/ALS patients.

额颞叶痴呆（FTD）和肌萎缩侧索硬化（ALS）是两种致命的神经退行性疾病， 无法治愈或有效治疗的疾病。FTD/ALS的常见病理学标志是存在 由具有固有无序区域的各种RNA结合蛋白（RBP）形成的细胞质内含物 （IDRs）。这些RBP包括FUS，以及TDP-43，TAF 15和EWSR 1，它们都是压力的组成部分 颗粒（SG）。SG是一种液体状的短暂的细胞质无膜细胞器，当细胞处于缺氧状态时形成。 通过一种称为液-液相分离（LLPS）的过程来浓缩RBP和RNA。因为 SG富含FTD/ALS疾病RBP，如FUS，如果应激，其本身具有聚集倾向 颗粒未被及时清除，FUS可通过异常相变形成固体纤维状聚集体 它可以通过隔离其他蛋白质和RNA并削弱压力来诱导毒性和神经退化 颗粒动力学我们假设能够逆转异常FUS相变和凝血的药物 将恢复应激颗粒动力学和功能，从而减轻FUS毒性。长期目标是 项目包括开发具有治疗潜力的药物，以逆转病理异常阶段 FUS的转移和聚集，并阐明这些试剂的作用机制。在我做博士后的时候 训练，我们发现核输入受体Kapβ2可以作为蛋白伴侣和一个蛋白质伴侣。 解聚酶通过结合C-核定位信号来防止和逆转FUS聚集。 FUS的终点。因此，我们假设其他FUS结合生物分子也可以阻止和逆转其表达。 相变和纤维化。在这个提议中，我们寻求开发FUS结合短RNA， 寡核苷酸解聚以减轻FUS的异常相变和毒性。我们的初步数据 表明在表达FUS大肠杆菌中结合FUS蛋白的RNA。大肠杆菌阻止FUS聚集。我们将首先 定义具有特定序列的RNA寡核苷酸，其可以减轻FUS的相分离和聚集， 和其他与FTD/ALS有关的RBP。因为RNA聚合物被核糖核酸酶迅速消化 在细胞中，为了开发用于治疗目的的基于RNA的寡核苷酸，我们还将设计RNA类似物 细胞稳定性更高。这些寡核苷酸的活性将使用体外相分离进行测试 从纯化的RBP重建的系统。然后我们将定义RNA的作用机制 寡核苷酸以减轻FUS组装。由于相分离的动态性和非均质性， FUS系统，我们将结合联合收割机系综生物物理测量和单分子荧光分析， 实现这一目标。在最后的目标中，我们将测试RNA解聚剂是否可以恢复应激颗粒动力学 并拯救细胞内FUS毒性。在我们的研究中发现的这些短RNA寡核苷酸具有很大的潜力， 作为FTD/ALS患者的RNA治疗剂。

项目成果

Regulating Phase Transition in Neurodegenerative Diseases by Nuclear Import Receptors.

• DOI: 10.3390/biology11071009
• 发表时间: 2022-07-04
• 期刊: BIOLOGY-BASEL
• 影响因子: 4.2
• 作者: Girdhar, Amandeep;Guo, Lin
• 通讯作者: Guo, Lin

Liquid-Liquid Phase Separation of TDP-43 and FUS in Physiology and Pathology of Neurodegenerative Diseases.

• DOI: 10.3389/fmolb.2022.826719
• 发表时间: 2022
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5
• 作者: Carey JL;Guo L
• 通讯作者: Guo L

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

定义可逆转具有朊病毒样结构域的 RNA 结合蛋白的有害相变的 RNA 寡核苷酸。

• DOI: 10.1101/2023.09.04.555754
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Guo,Lin;Mann,JacobR;Mauna,JocelynC;Copley,KatieE;Wang,Hejia;Rubien,JackD;Odeh,HanaM;Lin,JiaBei;Lee,BoLim;Ganser,Laura;Robinson,Emma;Kim,KevinM;Murthy,AnastasiaC;Paul,Tapas;Portz,Bede;Gleixner,AmandaM;Diaz,Zamia;C
• 通讯作者: C