Tanz CRND TorontoT. MurakamiA. MyashitaY. WakutaniG. Schmitt-UlmsM. ZhenF. ChenP. FraserP. St George-Hyslop
ColumbiaN. Shnieder
CambridgeS. QamarR. DoddA. CostaC. HoltQ. LinM. VendruscoloG. KaminskiC. KaminskiE. ReesY. LiG. Tartaglia
The Wellcome TrustNational Institutes of Health Research
Canadian Institutes of Health ResearchMedical Research Council
Alzheimer Society of Ontario
Functional Genomics: A new type of protein folding disorder causing neurodegeneration
In 2011, we decided to revaluate the role of protein aggregates using a transgenic worm (C. elegans) model
ALS/FTD mutant FUS, but not WT FUS causes intracytoplasmicaccumulation of FUS assemblies that biochemically and morphologically
resemble those in human ALS/FTD neurons
Conventional amyloids not soluble in 2%SDS and 8M Urea
WT-FUS FUS501
C. Elegans model replicates key features of human FUSopathy.. Toxicity is driven by LC domain
WT FUS model: FUS located in nucleus (physiological); No 8M urea soluble assemblies; No neurotoxicity.
Mutant FUS model: Nuclear and cytoplasmic assemblies; 8M Urea soluble assemblies correlate with toxicity. Assemblies closely resemble those in human FUS_ALS/FTD
tissues.
LC domain is necessary & sufficient for toxicity
What is it about the Low Complexity (LC) domains of FUS that makes them
neurotoxic"?
FUS-LC domain drives assembly of FUS into cytoplasmic liquid droplets in cells
FUS-LC domain drives assembly of FUS into liquid droplets in recombinant protein preps.
0.5 M FUS 100mM NaCL
.. and form visible gels that cycle (4C Jelly - 23C Liquid)
Warmed to 23C
Cooled to 4C
1mM FUS 300mM NaCL
What impact do FUS mutations have on liquid droplet and reversible hydrogel formation?
We devised two assays to look at the formation and stability of liquid droplets and of reversible gels;
What we found is that:
ALS/FTD mutations cause phase transition from liquid droplet / reversible hydrogels into irreversible fibrous hydrogels
Full-length FUS FUS-LC domains
FUS(WT)
FUS(R522G)
FUS(P525L)
FUS501
FUS(R495X)
FUS(R524S)
Time = 0 min
FUS-LC(WT)
Time = 0 min
FUS-LC(G156E)
Time = 50 min Time = 50 min
FUS-LC(S96del)
Time = 50 min
ALS/FTD mutations cause phase transition from liquid droplet into stable hydrogels
Wild type mutant mutant
020406080
100120140
Prop
ortio
n of
dro
plet
sre
mai
ning
at t
= 5
0 m
in
** # # #
To explore the effects of ALS/FTD mutations on these phase transitions, we exploited
the 4 Jelly - 23 liquid cycling assay
23C
4C
WT and benign polymorphic FUS-LC cycle well..ALS/FTD mutants cycle poorly and form stable irreversible hydrogels
Wild type FUS
Benign polymorphism
ALS/FTD Mutant FUS
Droplet reversible gel irreversible gel
num
ber o
f cyc
les
befo
re ir
reve
rsib
ility
0
1
2
3
4
5
6*
N.S.
23C 4C 23C
60
50
R Urea R UreaM.W.(kDa)
Western blot
FUS
Human spinal cordi ii
iii iv
v vi
vii viii
Recombinant FUS protein gels
Recombinant irreversible FUS gels have same solubility and structure as mutant FUS assemblies from human ALS/FTD CNS tissue
FUS liquid droplet and reversible gels have low viscosities (< 3 kPa.s) like P-granules.
Mutant irreversible assemblies high viscosities (> 10 kPa.S)
Wild type FUS
ALS/FTD Mutant FUS
N.S.
**
Liquid Reversible Irreversiblegel gel
0
5
10
15
20
25
30 ***
visc
osity
(kPa
s)
FUS(
LC)-W
TFU
S(LC
)-S96
del
FUS(
LC)-G
156E
FUS(
LC)-W
TFU
S(LC
)-S96
del
FUS(
LC)-G
156E
FUS(
LC)-W
TFU
S(LC
)-S96
del
FUS(
LC)-G
156E
20nm bead tracking
Liquid Gel
Does the liquid droplet / reversible gel transition have a physiologic function? Does it allow regulated capture, transport and
release of RNP granule cargo (other ribonucleoproteins (RNPs), RNAs etc?
Does irreversible gel affect this function?
0
200
400
600
800
GFP
-SM
N re
leas
e **
Gel/Liquid G L G L G L G G G G
GFP-SMN
N.S.
GFP-STAU1
0
200
400
600
800
GFP
-STA
U1
rele
ase
N.S.
*****
Gel/Liquid G L G L G L G G G G
Wild type FUS gels capture ribonucleoproteins (SMN, STAU), but release the cargo when the FUS gel reverts to liquid.
But ALS/FTD mutant FUS gels retain SMN and STAU1 cargo
Reversible gel(wild type)
Liquid (wild type)
Irreversible gel(ALS mutant)
Release4C 23C 4C 23C
FUS-LC (WT) FUS-LC (Mutant)
No release
FUS(WT)
FUS(G156E)
FUS(S96del)
GELLED REVERTED TO LIQUID (WT) ;
IRREVERSIBLE GEL (MUTANTS)LIQUID
0
1.3 m
4
SMN Stau1 SMN Stau1 SMN Stau1
Diffusion coefficient
m2/s
Imaging of single RNP cargo molecules (SMN, STAU) show free motion of cargo when wild type gels revert to liquid,
but retention of cargo by irreversible mutant FUS gels
ReversibleFUS(WT)
IrreversibleFUS(G156E)
What effect would sequestration of RNP cargo have on neuronal function?
RNP granules are known to be important for local control of RNA metabolism and new protein synthesis
Could irreversible fibrillar hydrogel formation sequester RNP granule cargo and block new protein synthesis in axon terminals?
Non-injected
FUS(WT)
FUS(P525L)
10 m
FUS501
anti-puromycinantibody
PhaseContrast
0.8
1.0
New
pro
tein
syn
thes
is
1.2
0.6
0.4
0.2
0
N.S.1.4
1.6
***
*****
Non-injectedWild type FUSLC domainALS/FTD Mutant FUS
ALS/FTD mutant FUS and FUS-LC domain reduce new protein synthesis in cultured Xenopus neurons
Inhibition of local protein synthesis
dispersed monomer
liquid droplet
FUS mutation
local protein synthesis
regulation of local RNA
metabolism and translation
reversible hydrogel
Irreversible fibrous hydrogel
Similar effects on nuclear RNA transcription, splicing?
TAKE HOME POINTS1. Assembly of mutant RNA binding proteins into pathological
intraneuronal deposits is functionally important in ALS/FTD caused by mutations in FUS (and other related RNA-binding proteins: TDP43);
2. Assembly is driven by low complexity (LC) domain, which physiologically drive formation of liquid protein droplets and reversible hydrogels
3. Mutant hydrogels become irreversible if gelled too often or too long..
4. These irreversible hydrogels represent a novel type of neurotoxic misfolded protein that are different from conventialamyloids.
5. These irreversible hydrogels cause neurotoxicity by disturbing RNP granule function, and inhibiting RNA metabolism and protein translation.
Tanz CRND TorontoT. MurakamiA. MyashitaY. WakutaniG. Schmitt-UlmsM. ZhenF. ChenP. FraserP. St George-Hyslop
ColumbiaN. Shnieder
CambridgeS. QamarR. DoddA. CostaC. HoltQ. LinM. VendruscoloG. KaminskiC. KaminskiE. ReesY. LiG. Tartaglia
Acknowledgements
The Wellcome TrustNational Institutes of Health ResearchCanadian Institutes of Health Research
Medical Research CouncilAlzheimer Society of Ontario
0.8
1.0
New
Pro
tein
syn
thes
is
1.2
0.4
0.6
N.S.
***
**
0
0.2
1.4
1.6
Thapsigargin - - + + - - - -PERKi - + - + - + - +
N.S. N.S.
The reduced protein synthesis is not to due activation of unfolded protein response (UPR) because PERK inhibition (neurons) or
PERK RNAi knock down (C elegans) does not rescue mutant-FUS inhibition of protein synthesis
Control
Thapsigargin
PERKi
The reduced protein synthesis is not to due activation of unfolded protein response (UPR) because stress granules are not increased
in vivo in mutant FUS animals compared to wild type animals
8nu
mbe
r of p
ab-1
pos
itive
gra
nule
s (/n
euro
n)
4
6
N.S.
N.S.
0
2
basal condition heat shock
10
Functional Genomics: A new type of protein folding disorder causing neurodegenerationIn 2011, we decided to revaluate the role of protein aggregates using a transgenic worm (C. elegans) modelALS/FTD mutant FUS, but not WT FUS causes intracytoplasmic accumulation of FUS assemblies that biochemically and morphologically resemble those in human ALS/FTD neuronsC. Elegans model replicates key features of human FUSopathy.. Toxicity is driven by LC domainWhat is it about the Low Complexity (LC) domains of FUS that makes them neurotoxic"?Slide Number 6Slide Number 7.. and form visible gels that cycle (4C Jelly - 23C Liquid)What impact do FUS mutations have on liquid droplet and reversible hydrogel formation?ALS/FTD mutations cause phase transition from liquid droplet into stable hydrogelsTo explore the effects of ALS/FTD mutations on these phase transitions, we exploited the 4 Jelly - 23 liquid cycling assayWT and benign polymorphic FUS-LC cycle well..ALS/FTD mutants cycle poorly and form stable irreversible hydrogelsSlide Number 13FUS liquid droplet and reversible gels have low viscosities (< 3 kPa.s) like P-granules. Mutant irreversible assemblies high viscosities (> 10 kPa.S)Slide Number 15Wild type FUS gels capture ribonucleoproteins (SMN, STAU), but release the cargo when the FUS gel reverts to liquid. But ALS/FTD mutant FUS gels retain SMN and STAU1 cargo Slide Number 17Slide Number 18ALS/FTD mutant FUS and FUS-LC domain reduce new protein synthesis in cultured Xenopus neuronsSlide Number 20TAKE HOME POINTSAcknowledgementsSlide Number 23The reduced protein synthesis is not to due activation of unfolded protein response (UPR) because PERK inhibition (neurons) or PERK RNAi knock down (C elegans) does not rescue mutant-FUS inhibition of protein synthesis The reduced protein synthesis is not to due activation of unfolded protein response (UPR) because stress granules are not increased in vivo in mutant FUS animals compared to wild type animals