ALS and RNA binding proteinsas therapeutic target

M2 Majima Sohei
September 26th, 2019

Table of contents- Introduction- TDP-43 & aggregation- Stress Granule as ALS treatment target- SummaryTable of contents- Introduction- TDP-43 & aggregation- Stress Granule as ALS treatment target- SummaryALS (Amyotrophic Lateral Sclerosis)- Most frequent Motor Neuron Disease (MND)- Degeneration in motor neuron in the cerebral
cortex and spinal cord -> muscle weakness- ~10000 patients in Japan (2013)- 90% of ALS is sporadic, 10% is familial.

Lou Gehrig

ice bucket challenge
Hum, Mutat. 2013, 34, 812

Treatment of ALS- Riluzole- Neuroprotective reagent, inhibits glutamate release- Edaravone- Radical scavenger- Both only slow progression by several weeks-> Other target is necessary.

riluzoleedaravone

http://www.als.gr.jp/public/als_info.html

https://www.neurology-jp.org/guidelinem/pdf/als2013_04.pdf

Characteristics of ALS- RNA-binding proteins, such as FUS & TDP-43
are involved- Excess Stress Granules are observed

TDP-43 (TAR DNA binding Protein 43)- Found in ubiquitin-positive aggregates- Dominant inheritance in familial ALS10- Shuttles nucleus and cytoplasm- Localized to SGs under stressed condition- Both WT and mutants aggregations are observed

Brain Research. 2012, 1462, 16

FUS (Fused in Sarcoma)- RNA binding protein- Responsible gene of ALS6
- Shuttles nucleus and cytoplasm- Localized to SGs under stressed condition- Overexpression of WT FUS cause cell death- FUS mutants aggregates

Brain Research. 2016, 1647, 65

Stress Granule (=SG)-One of the membrane-less organelles in cells
Liquid-Liquid phase separation (=LLPS)Interrupt translation of proteins under stress condition
RNA-binding domain (RBD) and Prion-like domain (PLD)
is important for localization-mRNARibosome
RNA binding proteins
HDAC6
RNA helicase
Cell Reports. 2018, 22, 1401

Table of contents- Introduction- TDP-43 & aggregation- Stress Granule as ALS treatment target- Summary

Questions for TDP-43 and ALS

StressBinding to RNA impairs TDP-43 aggregation- Strong affinity to GU repeats- Addition of RNA impairs
aggregation

J. Biol. Chem. 2019, 294, 6696

TDP-43 is acetylated by CBP- K145 & K192 in RRM mainly acetylated.

Nature Communications. 2015, 6, 5845

TDP-43 acetylation results in aggregation- KQ mutant … acetylation mimic- KQ mutant more likely to aggregate
Nature Communications. 2015, 6, 5845

TDP-43 acetylation impairs RNA binding
Nagative control-RNA binding was quantified by CLIP(Crosslinkingimmunoprecipitation) assay.
- KQ mutant showed low affinity to RNA.

TDP-43 can be aggregated in droplet=LCD- TDP-43 LCD(Low Complexity Domain, = GRR) forms
droplet under physiological condition.
J. Biol. Chem. 2019, 294, 6306

TDP-43 can be aggregated in droplet- Atomic force microscopy
image- Fibril was observed.
- High concentration of TDP43 gets aggregated

J. Biol. Chem. 2019, 294, 6306

TDP-43 aggregation summary

StressTable of contents- Introduction- TDP-43 & aggregation- Stress Granule as ALS treatment target- SummaryFUS-GFP as SG marker- Dimercaprol & emetine … reduce SGs- FUS-GFP localizes in SGs under arsenate stress.
-> Use of FUS-GFP as SG marker

BioRxiv. doi.org/10.1101/721001

In cell screening of compounds- 47 compounds hit

In vitro screening of compounds

Hit compounds

Several SG decreased with compounds

Lipoamide prevent fibril formation- G156E FUS mutant … forms fibril- Lipoamide prevents fibril formation, too- Lipoamide keeps droplet soft

iPS-derived neuron as ALS model- FUS P525L mutant … ALS-associated NLS mutation- iPS cells from ALS patient- Material accumulation is observed in FUS P525L neuron

Lipoamide reduces aggregation in cell

- Lipoamides prevented fibrilLipoamide recovers motor defects- D. melanogaster- Expression of human
FUS P525L/R521C- Food supplementation
with lipoamide- Climbing ability
recoveredTable of contents- Introduction- TDP-43 & aggregation- Stress Granule as ALS treatment target- SummarySummary- New target is necessary for ALS treatment- TDP-43 / FUS aggregation is one of thereasons of ALS- TDP-43 aggregation mechanism is still notuncleared- Inhibition of SGs / protein aggregation may
prevent ALS

TDP-43 mutants- Mutation in TDP-43 Glysine-rich domain in ALS10patients.
- C-terminal and RRM is important for SG localization.

- TDP-43 aggregation is also found in the sporadic ALS
patients

https://www.jstage.jst.go.jp/article/jsnt/34/2/34_72/_pdf/-char/ja
Frontiers in Molocular Neuroscience. 2019, 12, 25

Acetylation and stress granules- A-485 … HAT inhibitor- Tubacin … HDAC6 inhibitor- SG volume change by inhibitors

SG formation process

Stress-> Incorporation of HDAC6 to SGs-> Deacetylation of IDR Lysine-> SG maturation
Nature Chemical Biology. 2019, 15, 51