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）是两种致命的神经退行性疾病

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.