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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