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.