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

额颞叶痴呆(FTD)和肌萎缩侧索硬化症(ALS)是两种致命的神经变性 没有治愈或有效治疗的疾病。FTD/ALS的一个共同病理特征是存在 具有固有无序区的各种RNA结合蛋白(RBPs)形成的细胞质内含物 (IDR)。这些限制性商业惯例包括FUS，以及TDP-43、TAF15和EWSR1，它们都是应激的组成部分 颗粒剂(SG)。Sgs是一种液体状的瞬时细胞质无膜细胞器，当细胞处于 通过一种名为液-液相分离(LLP)的过程来压缩限制性商业惯例和核糖核酸。因为 SGS富含FTD/ALS疾病限制性商业惯例，如FUS，如果应激，本质上容易聚集 颗粒未及时清除，FUS可通过反常相变形成固体纤维状聚集体。 可通过隔离其他蛋白质和RNA并损害压力而导致毒性和神经退化 颗粒动力学。我们假设，药物能够逆转反常的FUS相变和纤化 可恢复应激颗粒的动力学和功能，从而减轻FUS毒性。这样做的长期目标是 该项目包括开发具有逆转病理异常阶段的治疗潜力的此类药物 FU的转变和聚集，并阐明了这些因素的作用机制。在我的博士后生涯中 经过训练，我们发现核进口受体Kapβ2可以作为一种蛋白伴侣和一种 解聚酶通过与C-核定位信号结合来防止和逆转FUS聚集 FUS的终点。因此，我们假设其他与fus结合的生物分子也可以预防和逆转其 相变和晶化。在这项建议中，我们寻求将FUS结合的短RNA发展为 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的大肠杆菌中，与FUS蛋白结合的RNA阻止了FUS聚集。我们将首先 定义具有可减轻FU的相分离和聚集的特定序列的RNA寡核苷酸， 以及与FTD/ALS有关的其他限制性商业惯例。因为核糖核酸酶能迅速消化RNA聚合物 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 具有更高的细胞稳定性。这些寡核苷酸的活性将通过体外相分离进行测试。 由纯化的限制性商业惯例重组的系统。然后我们将定义RNA的作用机制 减少FUS组装的寡核苷酸。由于相分离的动态性和非均质性 FUS系统，我们将结合整体生物物理测量和单分子荧光分析来 实现这一目标。在最终目的中，我们将测试rna解聚是否可以恢复应激颗粒的动力学。 并解救细胞内的FUS毒性。在我们的研究中发现的这些短RNA寡核苷酸具有巨大的潜力 可作为FTD/ALS患者的RNA治疗药物。

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.