# Elucidating the molecular mechanism underlying ALS/FTD-linked FUS mutations with single-molecule resolution

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2021 · $46,036

## Abstract

PROJECT SUMMARY
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD) are severe neurodegenerative
diseases that affect more than 30,000 people in the United States. ALS/FTLD, which exist on a pathological
spectrum, is hallmarked by the progressive loss of neurons. This leads to defects in motor neuron function and
eventually death, and there is no known cure for ALS/FTLD. A key molecular phenotype of ALS/FTLD is the
aggregation of proteins such as Fused in sarcoma (FUS), an RNA-binding protein with over 70 mutations linked
to ALS/FTLD. Importantly, FUS undergoes liquid-liquid phase-separation (LLPS) to form condensates that likely
contribute to its functions in RNA metabolism and the DNA damage response. However, this normally-reversible
LLPS is altered by Fus mutations, which instead favor a sol-gel transition that forms aggregates that are toxic to
neurons. This aggregation drives ALS/FTLD in patients. FUS mutations are heterozygous, indicating that this
pathological phenotype occurs despite the presence of wild-type FUS. How mutant FUS interacts with wild-type
FUS – if at all – is unknown. Wild-type FUS may decelerate progression of FUS-driven ALS/FTLD by breaking
apart mutant FUS aggregates. As mutant FUS ages, it might escape recovery by wild-type FUS (gain-of-toxicity).
Alternatively, wild-type FUS might get trapped in pathogenic aggregates (loss-of-function). Investigating the
interactions between wild-type and mutant FUS is critically important for understanding the molecular
mechanism underlying ALS/FTLD, and for developing effective treatments. Therefore, I propose to interrogate
the wild-type/mutant FUS interaction in vitro and in vivo with single-molecule techniques. I plan to address three
fundamental questions: (1) Do wild-type and mutant FUS interact? (2) What is the RNA binding phenotype of
mixed wild-type and mutant FUS? (3) How does mutant FUS aging alter wild-type/mutant interactions? I will use
a combination of single-molecule assays (single-molecule FRET, single-molecule FUS nucleation, and single-
molecule tracking) and ensemble assays (in vitro droplet formation, RNA splicing, and droplet fusion by optical
tweezers). This proposal is strengthened by contributions from four collaborators (see support letters), all of
whom are experts on ALS or LLPS. These collaborations and other professional development opportunities will
drive my preparation for a postdoctoral position. I will also focus on improving my scientific communication, and
I plan to become an expert on single-molecule techniques to uniquely position myself for my future academic
career. Altogether, this proposal will develop and use single-molecule techniques to better understand the early
steps of ALS pathogenesis.

## Key facts

- **NIH application ID:** 10148559
- **Project number:** 5F31NS113439-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Kevin Rhine
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 5
- **Project period:** 2020-05-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10148559

## Citation

> US National Institutes of Health, RePORTER application 10148559, Elucidating the molecular mechanism underlying ALS/FTD-linked FUS mutations with single-molecule resolution (5F31NS113439-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10148559. Licensed CC0.

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