Mol Cell: a novel mechanism of amyotrophic lateral sclerosis and frontotemporal dementia

It is well known that protein aggregation is a common pathological mechanism
in many neurodegenerative diseases.
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) have pathological inclusions formed by a variety of RNA-binding proteins, including FUS, TDP-43, TIA1, TAF15, EWSR1, and hnRNPA1
.
They have intrinsic disordered regions (IDRs) that promote aggregation and are often rich in repeats of arginine and glycine
.
In addition, they bind RNA, promoting the formation
of phase-separated condensate.
Once formed, condensate can be transformed into fibrous solids, similar to the pathological inclusion bodies observed during autopsy of ALS/FTLD neurons
.
FUS is one such highly disordered protein and has been shown to perform liquid-liquid phase separation (LLPS).

Some biological macromolecules, such as proteins and nucleic acids, can be condensed into liquid-like membrane-free condensate through LLPS, forming membrane-free compartments within cells and participating in various biological activities
.
FUS can form LLPS
by interacting with either homomorphic FUS or RNA.

On December 17, 2020, SuaMyong's team at Johns Hopkins University published an article entitled "ALS/FTLD-LinkedMutations in FUS Glycine Residues Cause Accelerated Gelation and ReducedInteractions with Wild-Type FUS" on Molecular Cell.
This study suggests that accelerated gelatination of FUS due to glycine mutations may be an important mechanism for FUS-mediated ALS/FTLD pathogenesis, while the lack of physical connection with wild-type (WT) FUS proteins may lead to more serious pathology
.

The study mainly focuses on the effect of WT FUS on the binding of mutant FUS to RNA, oligomerization, and condensate formation and fusion, focusing on answering four basic questions: (1) Can WT and mutant FUS interact to form mixed condensate with RNA backbone? (2) At what stage do WT and mutation FUS bind and separate from each other? (3) What is the RNA binding phenotype of WT and mutant FUS? (4) How does this interaction change as the mutant FUS matures?

Studies have shown that glycine (G) mutations begin to lose association with WT FUS at the earliest stages of FUS-RNA nucleation and condensate formation.
The arginine (R) mutant physically interacts with WT at all stages, promoting the recovery
of R mutation defects.
This recovery effect is reduced if WT FUS is added to the aging mutant FUS condensate

Key Results:

Figure 1.
Glycine mutations in FUS result in condensate separation

(1) G mutation and WTFUS form independent condensate

To test whether WT and mutant FUS clump together to form phase-separated droplets, Cy3- and Cy5-FUS of equal molar ratios are merged together
under droplet formation conditions.
The results showed that the formation of mixed droplets could be observed by mixing RNA with Cy3-WT and Cy5-WT FUS, and that Cy5-labeled R244C mutations could also be efficiently mixed
with Cy3-WTFUS.
However, the Cy5-G156E mutation does not mix easily with Cy3-WT and mostly forms independent droplets
.
(Figure 1)

(2) WT FUS and G mutations cannot bind to the same RNA

The research team developed a single-molecule two-color nucleation assay in which the binding
of color-coded WT and mutant FUS on the RNA backbone was observed in real time by total internal reflection (TIRF) microscopy.
Unlabeled fractional double-stranded U50 RNA is bound to the surface of a single molecule by biotin-NeutrAvidin ligation and flows into TEV-lysed Cy3 and/or Cy5-labeled FUS mixture
while recording single molecule video.
As individual molecules of color-coded FUS bind to RNA, the fluorescence intensity increases
in a stepwise manner.
The results showed that the mixture of G mutation and WT FUS exhibited higher fluorescence intensity than other mixtures, suggesting that G mutation FUS and WT FUS could not interact
.

Figure 2.
The functional defect of the G156E - FUS cannot be recovered by WT FUS

(3) WT FUS cannot compensate for G mutation FUS functional defects

In RNA-FUS interactions, R-mutated FUS binds to WT FUS, compensating for some of its functional defects
due to mutations.
However, the G-mutated FUS cannot interact with WT FUS, so the functional defects caused by the mutation cannot be recovered
.
(Figure 2)

Figure 3.
Mutant FUS captures new WT FUS entering the mutant condensate

(4) Aging mutation FUS affects WT FUS function

To assess the interaction of mutant FUS with WTFUS after aging, the research team mixed Cy5-mutant FUS with Cy3-WT FUS with pre-aging for 3 hours
.
The control group Cy5-WT and Cy3-WT combine rapidly to form mixed droplets
within 10-20 min.
The aging Cy5-G156E and Cy3-WT have not yet bind
.
Cy3-WT is added to aged Cy5-R244C for 1 hour and the two are gradually mixed, which is slightly longer than WT-WT mixing
.
(Figure 3)

Summary:

How do ALS/FTLD-associated FUS variants cause disease onset by altering interactions? Although some pull-down studies have not found interactions between WT FUS and mutant FUS, RNA metabolism such as splicing can be affected
by FUS mutations.
Two common pathological features of neurons in ALS/FTLD patients have been identified so far are aggregation and mislocalization
of FUS with other RNA-binding proteins.
By studying the point mutation of 3 glycine residues of FUS, the team provided a new idea for the pathogenesis of ALS/FTLD, that is, mutant FUS can change the properties of proteins and can reject
WT FUS.
But how does ALS/FTLD progress in cells? This may require further investigation into how the binding of mutant FUS to RNA regulates the liquid-liquid phase separation of FUS in cells, which will also help to identify targets that intervene in the gelatinization of mutant FUS
.