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