Presentation made by Dr. Peter St. George-Hyslop on October 30, 2015 at the Alzforum-hosted live webinar titled "Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?" More information and the recording of the session available at http://www.alzforum.org/webinars/fluid-business-could-liquid-protein-herald-neurodegeneration

1. Tanz CRND Toronto
T. Murakami
A. Myashita
Y. Wakutani
G. Schmitt-Ulms
M. Zhen
F. Chen
P. Fraser
P. St George-Hyslop
Columbia
N. Shnieder
Cambridge
S. Qamar
R. Dodd
A. Costa
C. Holt
Q. Lin
M. Vendruscolo
G. Kaminski
C. Kaminski
E. Rees
Y. Li
G. Tartaglia
The Wellcome Trust
National Institutes of Health Research
Canadian Institutes of Health Research
Medical Research Council
Alzheimer Society of Ontario
Functional Genomics: A new type of protein
folding disorder causing neurodegeneration

2. In 2011, we decided to revaluate the
role of protein aggregates using a
transgenic worm (C. elegans) model…

3. ALS/FTD mutant FUS, but not WT FUS causes intracytoplasmic
accumulation 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

4. 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

5. What is it about the Low Complexity (LC)
domains of FUS that makes them
“neurotoxic"?

6. FUS-LC domain drives assembly of FUS into
cytoplasmic liquid droplets in cells…

7. FUS-LC domain drives assembly of FUS into
liquid droplets in recombinant protein preps
.
0.5 μM FUS 100mM NaCL

8. .. and form visible gels that cycle
(4ºC Jelly - 23ºC Liquid)
Warmed to 23ºC
Cooled to 4ºC
1mM FUS 300mM NaCL

9. 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

10. 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
0
20
40
60
80
100
120
140
Proportionofdroplets
remainingatt=50min
**
# # #

11. To explore the effects of ALS/FTD mutations
on these phase transitions, we exploited
the 4º Jelly - 23º liquid cycling assay
23ºC
4ºC

12. 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
numberofcycles
beforeirreversibility
0
1
2
3
4
5
6
*
N.S.
23°C 4°C 23°C

13. 60
50
R Urea R Urea
M.W.
(kDa)
Western blot
FUS
Human spinal cord
i 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

14. 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 Irreversible
gel gel
0
5
10
15
20
25
30
***
viscosity(kPas)
FUS(LC)-WT
FUS(LC)-S96del
FUS(LC)-G156E
FUS(LC)-WT
FUS(LC)-S96del
FUS(LC)-G156E
FUS(LC)-WT
FUS(LC)-S96del
FUS(LC)-G156E
20nm bead tracking
Liquid Gel

15. • 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?

16. 0
200
400
600
800
GFP-SMNrelease
**
Gel/Liquid G L G L G L G G’ G G’
GFP-SMN
N.S.
GFP-STAU1
0
200
400
600
800
GFP-STAU1release 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)
Release
4ºC 23ºC 4ºC 23ºC
FUS-LC (WT) FUS-LC (Mutant)
No release

17. FUS(WT)
FUS(G156E)
FUS(S96del)
GELLED
REVERTED TO LIQUID (WT) ;
IRREVERSIBLE GEL (MUTANTS)LIQUID
0
1.3 µm
4
SMN Stau1 SMN Stau1 SMN Stau1
Diffusion
coefficient
µm2/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
Reversible
FUS(WT)
Irreversible
FUS(G156E)

18. • 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?

19. Non-injected
FUS(WT)
FUS(P525L)
10 μm
FUS501
anti-
puromycin
antibody
Phase
Contrast
0.8
1.0
Newproteinsynthesis
1.2
0.6
0.4
0.2
0
N.S.
1.4
1.6
***
**
***
Non-injected
Wild type FUS
LC domain
ALS/FTD Mutant FUS
ALS/FTD mutant FUS and FUS-LC domain reduce new
protein synthesis in cultured Xenopus neurons

20. 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?

21. TAKE HOME POINTS
1. 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 convential
amyloids.
5. These irreversible hydrogels cause neurotoxicity by disturbing
RNP granule function, and inhibiting RNA metabolism and protein
translation.

22. Tanz CRND Toronto
T. Murakami
A. Myashita
Y. Wakutani
G. Schmitt-Ulms
M. Zhen
F. Chen
P. Fraser
P. St George-Hyslop
Columbia
N. Shnieder
Cambridge
S. Qamar
R. Dodd
A. Costa
C. Holt
Q. Lin
M. Vendruscolo
G. Kaminski
C. Kaminski
E. Rees
Y. Li
G. Tartaglia
Acknowledgements
The Wellcome Trust
National Institutes of Health Research
Canadian Institutes of Health Research
Medical Research Council
Alzheimer Society of Ontario

24. 0.8
1.0
NewProteinsynthesis
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

25. 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
8
numberofpab-1positivegranules(/neuron)
4
6
N.S.
N.S.
0
2
basal condition heat shock
10