Protein Misfolding in NeurodegenerativeDisease

Rick MorimotoDepartment of Biochemistry, Molecular

Biology and Cell BiologyNorthwestern University

Evanston, IL, USA

2008 Philip Hauge Abelson Advancing Science SeminarScience, Stress, and Human Health

October 24, 2008

Biology Encyclopedia Forum

The word "stress" defines in biology the changes suffered by anorganism due to a value too high or too low of an environmental factorthat induces tension and exhaustion. Softpedia

The stress response is the human body's reaction to anything thatthrows off the balance inside it—injury, infection, fear, exercise, or pain.The body reacts with an alarm phase, then a resistance phase, duringwhich it tries to fix the imbalance, and then, if that fails, an exhaustionphase.

Different views of STRESS RESPONSES

What is stress to the celland how does this affectthe organism?

Different views of STRESS RESPONSES

RadiationRadiation

Temperature (Heat)Temperature (Heat)

OxygenOxygen

Heavy metalsHeavy metals

Chemically reactivemoleculesChemically reactivemolecules

OsmoticOsmotic

Pathology and DiseasePathology and Disease

Infectious diseaseInflammationMetabolic diseaseCancerNeurodegenerative

diseases

Infectious diseaseInflammationMetabolic diseaseCancerNeurodegenerative

diseases

Cell Stress Response: Detection, Adaptation & Survival

FEVER & INFLAMMATIONISCHEMIA & OXIDANT INJURY

TISSUE INJURY & REPAIR

AGING

VIRAL & BACTERIAL INFECTION

PATHOPHYSIOLOGICAL STATEPATHOPHYSIOLOGICAL STATE

CANCER

HEAVY METALS

INHIBITORS OF ENERGY METABOLISM

HEAT SHOCKAMINO ACID ANALOGUES

OXYGEN-FREERADICALS

ENVIRONMENTAL STRESSENVIRONMENTAL STRESS

HEAT SHOCKPROTEINS

HSF

HSE

MISFOLDED PROTEINS

HSF

HSF

ONCOGENES &PROTO-ONCOGENES

DEVELOPMENT & DIFFERENTIATION

GROWTH FACTORS

CELL CYCLE

CELL GROWTH & DEVELOPMENTCELL GROWTH & DEVELOPMENT

PARKINSONS DISEASEPRION DISEASES

ALZHEIMERS DISEASE

HUNTINGTONS DISEASE

PROTEIN MISFOLDING DISEASES PROTEIN MISFOLDING DISEASES

ALS

Back to the basics: Biochemistry 101

• proteins are essential constituents of the cells

• proteins are the key subunits of molecular machines of life

• proteins, however are highly sensitive to their environmentand readily go awry

• consequently, proteins are very much at risk during ageing

• What happens and how does the cell protect itself from thestress of misfolded and damaged proteins?

Protein folding is an essential function of all cells

Dobson, Nature (2003)

Each image 1 µm x 1 µm

Slide courtesy of Jeff Kelly based on dataof Teplow, Glabe, Krafft, and Lansbury

Aβ amyloidogenesis involves the transition from solubleprotein to toxic oligomers and fibrils in AD

Cell death

Disease

protofibrils

Aβ

The Stress of Misfolded and Damaged Proteins

normal neuron neurons expressing toxicHuntington’s protein

Protein Conformational Diseases

Metabolic Transport

Insulin TransthyretinProlactin Apolipoprotein A1Atrial natriuretic factor LactoferrinIslet amyloid polypeptideProcalcitoninα1-antitrypsin

Nervous systemImmunity

Aβ peptideImmunoglobulin light chain α-synucleinImmunoglobulin heavy chain TauSerum amyloid A SOD1β2-microglobulin HuntingtinCystatin C AtaxinsLysozyme PrionFibrinogen α

Neurodegenerativediseases

Lou GehrigALS

Jacob Javits - ALS

Woody GuthrieHuntington’s disease

Mo UdallParkinson’s disease

Ronald Reagan - Alzheimer’s disease

Muhammed Ali & Michael J.Fox - Parkinson’s disease

Huntington’s disease

Movie courtesy of the Venezula HD Project

Neurodegenerative Diseases

~ 6.5 million AmericansSelective Neuronal lossAging associatedDementia

Thought processesMemoryLanguagePersonality

Loss of Motor Control Fatal

Many human diseases are associated with the chronicexpression of misfolded & damaged proteins

Soto (2003)

Plaques and tangles Lewy bodies

Amyloid plaques

Aggregates

Intranuclear inclusions

Alzheimer’s Parkinson’s

PolyQ diseases Prions

Amyotrophic lateralsclerosis

Neurodegenerative diseases arediseases of protein misfolding.

Mutations enhance the formationof damaged proteins and theappearance of aggregates.

Misfolded proteins andaggregates cause molecularSTRESS and interfere withcellular function.

Unfolded state Intermediatefolded states

Native state

Molecularchaperones

Molecularchaperones

Degradation

Normal turnover

Toxic folds

AmyloidosesHD, PD, ALS, AD

Misfolded states

Cystic Fibrosis

Impropertrafficking

Emphysema

PROTEOSTASIS

Are neurodegenerative diseases a large family of DISTINCTdiseases or a large family of RELATED diseases?

• the genes (Aβ, huntingtin, α-synuclein, tau, SOD1, PrP) associated withneurodegenerative disease are not related

• the cells affected and symptoms can vary greatly

• yet, all are associated with the expression of mutant proteinsthat damage neurons

• a common feature…That neurodegenerative diseasesresult from the expression of misfolded proteins and thesubsequent damage incurred by the cell

• for many, both familial and non-familial forms occur

polyQ, SOD1, α-synuclein, tau, prions

Model systems for protein conformational diseases

In vitro Tissue culture Yeast C. elegans Drosophila Mice

Aggregation toxicity is enhanced by polyQ length and ageing

Morley et al., PNAS, 2002

Day 7 Day 10 Day 4

Q35

0

20

40

60

80

100

0 2 4 6 8 10age (days)

mot

ility

(% c

ontro

l) Q29

Q33 Q35Q40Q82

Jim Morley

0

50

100

150

200

age (days) 0 2 4 6 8 10 12

aggr

egat

es (#

) Q80Q40

Q35Q33

Q29YFP

Protein aggregationProtein aggregation Cellular toxicityCellular toxicity

Q19 Q82 Q35

Proteostasis is regulated by pathways that controllifespan and the heat shock response

• The toxicity of aggregation-prone proteins is suppressed by the insulin-like (ILS) signaling pathway (age-1/daf-2) and activation of daf-16.

• Hsf1 is essential for the ILS pathway & hsf1 itself enhances lifespan.

Morley et al., PNAS, 2002;Mol. Cell. Biol. 2004;Hsu et al., Science, 2003

Mutant SOD1 is highly aggregation-prone but onlymildly toxic

Tali Gidalevitz

FRAPAnalysis

Motility Assay

Expression of α-synuclein forms age-dependentdiffuse aggregates

Van Ham et al.,PLOS Genetics,2008

Expression of aggregation-proneand metastable proteins

Genetic screens for the proteostasis network

Genome-wide genetic screens in C. elegans:enhancers - genes that result in enhanced aggregation toxicitysuppressors - genes that prevent aggregation toxicitychaperone regulation - genes that regulate chaperones

PROTEOSTASIS

PROTEOSTASIS

Proteome protectivegene networks (HSF1, age-1,chaperones)

Five major classes of genes modulate polyQ aggregation

RNA synthesis &processing (41)

Transcription regulation (8)T27F2.1(Skip)

Transcription (2)ama-1 (RPB1)

Splicing (21)T13H5.4 (SAP61)

Proteinsynthesis (62)

Initiation (6)Y39G10AR.8 (eIF-2-γ)

Elongation (4)eft-3 (eEF1A-2)

Ribosomal proteins (46)rps-26 (S26)

Proteinfolding (9)

Chaperonin (6)cct-5 (TCP-1-ε)

Hsp70 (2)hsp-1 (Hsc70)

DnaJ (1)F18C12.2A

Proteintransport (30)

Endocytosis (1)Y105E8A.9 (γ-adaptin)

Nuclear import (5)C53D5.6 (Importin β-3)

Cytoskeleton (6)K01G5.7 (Tubulin β-2)

Proteindegradation (20)

19S (8)rpt-5 (26S 6A)

20S (10)pas-4 (α type 7)

Ub ligase (2)C47E12.5 (E1)

………others genes including 6 ATP synthases and genes of unknown function

Nollen, Garcia et al., PNAS 2004

Why are misfolded proteins a risk to the cell?

normal muscle development isnecessary during embryogenesis

normal musclestructure

worms healthy

paramyosin(ts)normal thick

filaments assembly

15oC

Develop folding sensors that reporton protein homeostasis.

Employ temperature-sensitivemutations as highly sensitivereporters of the folding environment.Functionality of a ts mutation reflectsmetastability; folding and clearancein the cell at different temperatures.

Develop folding sensors that reporton protein homeostasis.

Employ temperature-sensitivemutations as highly sensitivereporters of the folding environment.Functionality of a ts mutation reflectsmetastability; folding and clearancein the cell at different temperatures. abnormal muscle

structure

movement defectsembryonic lethality

paramyosin(ts)lack of normal thick

filaments

25oC

0

20

40

60

80

100

15o 17.5o 20o 22.5o 25o

% p

heno

type

15oCts+

Q40?

ts

worms healthy 15oC

25oCembryonic

lethality

15oC

Q40

Q40 15oC

worms healthy

15oC

paramyosin(ts) +Q40

embryonic lethality

Does an aggregation-prone protein cause a globalimbalance in protein folding homeostasis?

Gidalevitz, Ben-Zvi et al., Science, 2006

polyQ exposes the mild folding defect at the permissive temperature! polyQ exposes the mild folding defect at the permissive temperature!

Q40/+Ras;Q40/+

Protein polymorphisms enhance the risk for proteinconformational disease

Gidalevitz, Ben-Zvi et al., Science, 2006

Q40/Q40

Para;Q40/Q40

0

10

20

30

40

50

60

70

80

90

Q40/+ Q40/Q40 Ras;Q40/+ Para;Q40/Q40

#

of

aggre

gat

es

• polyQ aggregation toxicity isenhanced by the expression ofmetastable proteins in the proteome

• this reveals a positive feedbackloop between metastable andconformationally sensitive proteinsand polyQ

• and suggests that protein foldinghomeostasis is delicate!

• polyQ aggregation toxicity isenhanced by the expression ofmetastable proteins in the proteome

• this reveals a positive feedbackloop between metastable andconformationally sensitive proteinsand polyQ

• and suggests that protein foldinghomeostasis is delicate!

The global consequences of an aggregation-proneprotein on cellular folding homeostasis

Gidalevitz, Ben-Zvi et al. Science, 2006; adapted from Bates, Science, 2006

The global consequences of an aggregation-proneprotein on cellular folding homeostasis

Gidalevitz, Ben-Zvi et al. Science, 2006; adapted from Bates, Science, 2006

Implications of “induced” proteostasis collapse

• are there costs of protein polymorphisms to the PROTEOME?

• protein polymorphisms are not a single conformer state;rather they represent a collection of metastable states that fold(or misfold) because of the folding landscape.

• disease associated aggregation-prone proteins, however,tax the network and cause “induced” proteostasis collapse.

• variation in expressed polymorphisms could explaintissue-specificity and differences observed in phenotypesand time of onset.

Day 1 Day 3 25ºCtswt tswt tswt

α-paramyosin

Phalloidin

Merge

Anat Ben-Zvi

0

20

40

60

80

100

0 2 4 6 8 10 12

Para

lysi

s (%

)

Time (days of adulthood)

perlecan(ts)

wt

0

20

40

60

80

100

0 2 4 6 8 10 12

Slow

mov

emen

t (%

)

Time (days of adulthood)

paramyosin(ts)

wt

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Dist

rupt

ed m

uscl

e ce

lls (%

)

Time (days of adulthood)

paramyosin(ts)

wt

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Dist

rupt

ed m

uscl

e ce

lls (%

)

Time (days of adulthood)

Perlecan(ts)

wt

Proteins harboring mutations that affect foldingcollapse during ageing

Organismal & cellular phenotypes

paramyosin misfolding

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16Time (days of adulthood)

ras(ts); daf-16 O/E

Osm

(%

)

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

Osm

(%

)

Time (days of adulthood)

ras(ts); hsf-1 O/E0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

Osm

(%

)

Time (days of adulthood)

ras(ts)

Anat Ben-Zvi

Age-dependent collapse of ras(ts) in neurons issuppressed by Hsf1 and Daf-16

• Activation of the Proteostasis Networks by Daf-16 and Hsf1 can restorefolding and function to multiple proteins with mild folding defects.

• Ageing at the molecular level results from the collapse of proteostasis.

Daf-16 Hsf1

SYSTEMS BIOLOGY OF PROTEOSTASISSYSTEMS BIOLOGY OF PROTEOSTASIS

PROTEIN HOMEOSTASISPROTEIN HOMEOSTASIS

DAF16DAF16HSF1HSF1

Environment &physiologicalstressors

Environment &physiologicalstressors

OxidativedamageOxidativedamage

MissensemutationsMissensemutations

Post-translationalmodifications

Post-translationalmodifications

Catastrophic collapse of proteostasis during ageingand expression of misfolded proteins

Colla

pse

of P

rote

in H

omeo

stas

is

Time/Ageing

0

100

Loss

of C

ell F

unct

ion 100

0I I I I I I I

Protein HomeostasisNetwork of chaperones &clearance machines

Protein HomeostasisNetwork of chaperones &clearance machines

Catastrophic collapse of proteostasis during ageingand expression of misfolded proteins

Colla

pse

of P

rote

in H

omeo

stas

is

Time/Ageing

0

100

Loss

of C

ell F

unct

ion 100

0I I I I I I I

Expression ofdamagedproteins

Catastrophic collapse of proteostasis during ageingand expression of misfolded proteins

Colla

pse

of P

rote

in H

omeo

stas

is

Time/Ageing

0

100

Loss

of C

ell F

unct

ion 100

0I I I I I I I

Expression ofdamagedproteins

Imbalance in ProteinHomeostasis

Catastrophic collapse of proteostasis during ageingand expression of misfolded proteins

Colla

pse

of P

rote

in H

omeo

stas

is

Time/Ageing

0

100

Loss

of C

ell F

unct

ion 100

0I I I I I I I

Restore Proteostasis

MetastableProtein

EfficientProteasome

AbundantChaperones

InefficientProteasome

DepletedChaperones

Protein Aggregation

HiddenMutationsRevealed

CellularCellularToxicityToxicity

ProteinSequestration

CellularCellular ChallengeChallenge

MisfoldedProtein

Decreased chaperone activity and proteinDecreased chaperone activity and proteinhomeostasis during aging & chronic stresshomeostasis during aging & chronic stress

Toxic SpeciesAccumulation

Therapeutic strategiesTherapeutic strategiesto enhance chaperoneto enhance chaperonelevels and activitylevels and activity

Kai Orton

The proteostasis network protects the proteomeand restores folding and clearance

HSF1 in stress biology, proteostasis & ageing

PolyQ & mut SOD1 causes a global collapse of protein homeostasis

Protein polymorphisms are modifiers of conformational diseases

Contributions of ~350 genes to the Proteostasis Network

Roles of lifespan (DAF-16) and HSF1 stress response networks

Global collapse of protein homeostasis in ageing

Global consequences of misfolded proteins:

Stress and longevity:

Proteostasis Regulators Small molecule regulators of HSF1 and chaperonenetworks

Systems approach to stress biology: Neuronal networks that regulate HSF1

Northwestern UniversityEvanston, Illinois

Tali Gidalevitz

Susana Garcia

Cindy Voisine

NIH (NIGMS, NIA, NINDS)HDSA Coalition for the Cure, ALSADaniel F. and Ada L. Rice Foundation

Veena Prahlad

Julius Anckar, Lea Sistonen (Abo Akademi & BioCity Turku, Finland),Lada Klaic, Rick Silverman (Northwestern) Kevin Morano(UT Houston), Peter Hodder, Franck Maddoux, (Scripps)Jeff Kelly, Bill Balch (Scripps), Andy Dillin (Salk)

Ning Wang

Janine Kirstein

Catarina Silva

Anat Ben-Zvi Patricija vanOosten Hawle

Jesper Pedersen

Kai Orton