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Neuronal DEATH in Alzheimer’s Disease
Molecular Events Linking Disease Pathologies During Neuronal Demise
Jack ReifertFNS 10/30/09
100 billion
10 billion (10%)
200 million (0.2%)
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Alzheimer’s Disease Quick Facts:Over 100 years since the first diagnosis
Mark P. Mattson et al., Nature Reviews Neuroscience V-7, P-286, 2006.
Most common form of dementia - 4.5 to 4.7 million Americans
Average duration from detectable symptoms to death = 8 years
Chronic, age dependent disease (10% over 65, >40% over 85)
Diagnosis only complete with detection of post-mortem pathologies
Clues from inherited forms?
Vulnerability of aged neurons?
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2 Pathologies = 2 Protein Aggregates
No Aggregates Tangles Plaques & Tangles
AD FTDP PD HD ALS CJD
Nature Medicine 10, 1055 - 1063 (2004)
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Amyloid Beta Plaques
βγ
Amyloid-βInsoluble Fibers /Plaques
Soluble Oligomers
AICD Extracellular Domain
Amyloid Precursor Protein (APP)
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W. E. KLUNK AND C. A. MATHIS, UNIVERSITY OF PITTSBURGH
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MAPT / Tau Tangles
Cairns et al., Journal of Pathology V-204 P-438 2004
N C4411 45 74 103
Microtubule Binding Domains
Alzheimer’s disease brain
Assembled in-vitro from recombinant tau with ∆K280 FTDP-17 mutation
Eckhard Mandelkow et al., Brain Pathology 17 (1) , 83–90 , January 2007
Calpain / Caspase Cleavage Sites
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Amyloid Cascade Hypothesis
APP Dysfunction
FAD
?
Plaques
TauDysfunction
Tangles
FTDP-17
CellDeath
LOF - MTs
GOF - Aggregation
Human Genetics:
Biochemistry:
Pathology: Other?
Experimental Evidence: - Aβ treatment on wt mouse neurons in culture = cell death while Aβ treatment on tau -/- neurons = no cell death
Rapoport et al. (2002). PNAS.
- Tau -/- mice crossed with APP Alzheimer’s mouse model alleviates age . related memory deficits observed in the mouse models with tau present.
Robberson et al. (2007). Science.
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Killing Neurons with Amyloid BetaNeuronal Cell Death Induced by a Dose Titration of Amyloid Beta over Indicated
Times. Normalized ATP Content Measurement.
4 Hours 8 Hours 24 Hours 48 Hours 72 Hours0
10
20
30
40
50
60
70
80
90
100
110
0.6µΜ
1.2µΜ
2.5µΜ
5µΜ
10µΜ
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Tau Tubulin
T=0
4 Hr
8 Hr
24 Hr
LDH Release Data. Timecourse of Amyloid Beta Treatments @ 10µΜ.
20 Μιν 40 Μιν 1 Ηουρ 2 Ηουρσ 4 Ηουρσ 8 Ηουρσ 24 Ηουρσ 48 Ηουρσ0
1
2
3
4
5
6
Αµ ψλοιδ Βετα ≅ 10υΜΜοχκ Τρεατεδ
Τιµ ε Αφτερ Αβετα Τρεατµ ε ντ
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Rapid Destruction of Tau
PanTau
GAPDH
10 min 20 min 40 min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours- + - + - + - + - + - +- + - + - +- +3 min
0
0.2
0.4
0.6
0.8
1
1.2
Untreated 3 Min 10 Min 20 Min 40 Min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours
Upper Band
Lower Band
Total Tau
Fold Difference in Band Intensity
Upper Tau BandLower Tau BandTotal Tau
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Tau Disappearance - A Closer Look
25
10 - + - + - + - + - + - + - + - + - +10Õ 20Õ 40Õ 1Hr 2Hr 4Hr 8Hr 24Hr 48Hr
52
3min 48HrAmyloid Beta
3min 48HrAmyloid Beta
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5 Min 10 Min 15 Min 30 Min 1 Hour 2 Hour
150% 475% 325% 250% 225% 140%
Time:% Calpain Activity:
% Caspase Activity: 160% 150% 135% 120% 45% 40%
N C4411 45 74 103
4 Hour
75%
20%
25 341 421Caspase Cleavage Sites
25711 44 230 254 267 310 340 394Calpain Cleavage Sites
Protease Cleavage of Tau - Low MW
52 kD
17 kD
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Tau Phosphorylation
APP Dysfunction
FAD
?
Plaques
TauDysfunction
Tangles
FTDP-17
CellDeath
LOF - MTs
GOF - Aggregation
Other?
Neuronal DEATH after amyloid beta treatment ~ 50% @ 24 Hours
Rapid protease activity on tau produces low MW fragments
GSK3βCDK5
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Signaling Pathways Investigated:General Pathway Method of Analysis Implication
AKT Phosphorylation vs whole protein Survival / Growth Factor Signaling
NF-κΒ Εξπρεσσιον λεϖελσ ανδ πηοσπηορψλατιον Ινφλαµµατιον
ΜΑΠΚ Πηοσπηορψλατιον ϖσ ωηολε προτειν Γροωτη Φαχτορ Σιγναλινγ
ΕΡΚ Πηοσπηορψλατιον ϖσ ωηολε προτειν Συρϖιϖαλ / Γροωτη Φαχτορ Σιγναλινγ
Αποπτοσισ Χασπασε (προ ανδ χλεαϖεδ φορµ) Ινιτιατιον ανδ Εξεχυτιονερ
Βχατενιν Εξπρεσσιον λεϖελσ ανδ πηοσπηορψλατιον Γροωτη / Συρϖιϖαλ / ∆ιφφερεντιατιον
Προτεασε Συβστρατεσ Φυλλ−λενγτη ϖσ χλεαϖεδ φορµ Νευροναλ Σιγναλινγ / Χελλ ∆εατη
Προ−Αποπτοτιχ Βχλ−2 ΜεµβερσΕξπρεσσιον λεϖελσ ανδ πηοσπηορψλατιον Χελλ ∆εατηΑντι−Αποπτοτιχ Βχλ−2 ΜεµβερσΕξπρεσσιον λεϖελσ ανδ πηοσπηορψλατιον Συρϖιϖαλ
Kinases Targeting Tau:Kinase: Relation to Cell Signaling:
CDK5 Calpain (P35 --> P25)GSK3B AKTMAPK ERK (Direct)MARK/PAR1 ERK (Indirect)PKA OtherSrc Like Tyrosine Kinases Other
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Fold Difference in Band Intensity
Whole GSK
GAPDH
10 min 20 min 40 min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours- + - + - + - + - + - + - + - + - +
Inactive
3 min- +
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Untreated 3 Min 10 Min 20 Min 40 Min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours
GSK3B
pGSK3B
Whole GSK α/βπΓΣΚ3 β (9) − Ιναχτιϖε
35 kD
25 kD
5 Min 10 Min 15 Min 30 Min 1 Hour 2 Hour
150% 475% 325% 250% 225% 140%
Time:% Calpain Activity:
4 Hour
pGSK(S9)
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0
5 0
1 0 0
1 5 0
2 0 0
A K T
E R K
G S K 3 β
Χ ∆ Κ 5 ( Π 2 5 )
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
3min 10min 20min 40min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours
Time After Ab Treatment
% Relative Activity
Survival Kinases
Tau KinasesGFÕs
PI3-K
GFRÕs
PDK1/2
AKT
ERK 1/2
Ras
Cell Death
Calpain
p35
p25CDK-5 GSK3β
Stress!
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Decreased Phosphorylation?
199/202
217
231
396
396/404
Tau-5
KO UN UN 3’ 10’ 20’ 40’ 1 2 4 8 24
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15 DIV Hippocampal Neurons Treated with 10uM Amyloid Beta for Indicated Times. pTauWestern Blot Analysis. (n=3)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
pTau 181
pTau 199/202pTau 205 pTau 217
pTau 231
pTau 235
pTau 396
pTau 400
pTau 396/404pTau 413
Untreated 3 Min 10 Min 20 Min 40 Min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours
BIII TubulinTubulin (DM1A)
Time After Amyloid Beta Treatment
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De-Phosphorylation or Degradation?
pTau 181pTau 199/202pTau 205pTau 217pTau 231
UN 10’ 20’ 40’ 1 2 4 8 24
Tau-1 = de-phospho selective (181-205)
3’
pTau Epitope: Untreated %: 30 Minutes Abeta:pTau 202 14.3pTau 205 11.1pTau 220 30.8pTau 231 23.1pTau 235 18.2pTau 239 53.8pTau 413 25.0
Coverage Range: 145 - 300, 312 - 385 145 - 300, 350 - 385
None!
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Tau Hyperphosphorylation in AD!
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N C4411 45 74 103
25 341 421Caspase Cleavage Sites
25711 44 230 254 267 310 340 394Calpain Cleavage Sites
pTau 262pTau 400pTau 396/404pTau 413
PHF “Core”
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Revisit The Model
APP Dysfunction
FAD
?
Plaques
TauDysfunction
Tangles
FTDP-17
CellDeath
GSK3βCDK5
ERKAKT
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Future Directions:Characterize tau fragments by Mass Spec
Determine relative toxicity of fragments alone
Evaluate phosphorylation in relation to protease activity
Culture experimentsIn-vitro aggregation assaysIn-vitro MT polymerization assays
Evaluate importance of observed neuronal culture phenomenon inboth mouse models and human condition
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PanTau pTau 217
pTau 231 pTau 396
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Happy Halloween 2009!Thanks:
1) Feinstein Lab Members / Stu
2) Committee Members : Zach Ma, Joel Rothmann, Les Wilson
3) Friends…..
Funding:NIH (RO1 NS035010)California Dept. of Public Health - Alzheimer’s Research Foundation
((aa) Senile plaques in neocortex ) Senile plaques in neocortex of Alzheimer disease.of Alzheimer disease.
b) NFTs in hippocampus of FTDP-17 (R406W mutation).
© Lewy body in substantia nigra of Parkinson disease.
(d) Intranuclear polyglutamine inclusion in neocortex of Huntington disease.
(e) Ubiquitinylated inclusion in spinal cord motor neuron of ALS.
(f) Protease-resistant PrP in cerebellum of CJD (panel f courtesy of Nigel Cairns).
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5 Min 10 Min 15 Min 30 Min 1 Hour 2 Hour
150% 475% 325% 250% 225% 140%
Time:% Calpain Activity:
Tau
GAPDH
% Caspase Activity: 160% 150% 135% 120% 45% 40%
4 Hour
75%
20%
0
50
100
150
200
AKT
ERK
GSK3βΧ∆ Κ5 (Π25)
200
300
400
500
600
3min 10min 20min 40min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours
Time After Aβ Treatment
% R
elat
ive
Act
ivit
y
Survival Kinases
Tau Kinases
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Untreated 3 Min 10 Min 20 Min 40 Min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours
AKT
pAKT 473
Whole AKTpAKT (473) - Active
- + - + - + - + - + - + - + - + - +- +10 min 20 min 40 min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours3 min
Whole AKT
pAKT(473)Active
GAPDH
Fold Difference in Band Intensity
GAPDH
- + - + - + - + - + - + - + - + - +- +10 min 20 min 40 min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours3 min
pERK 1/2Active
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Untreated 3 Min 10 Min 20 Min 40 Min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours
ERK
pERK
Fold Difference in Band Intensity
Whole ERK 1/2pERK1/2 - Active
C a s p a s e 3 / 7 A c t i v i t y L u m i n e s c e n s e A s s a y . 1 5 D I V H i p p o c a m p a l N e u r o n s
T r e a t e d w i t h 1 0 u M A b e t a . ( n = 2 A v e r a g e o f T r i p l i c a t e W e l l s )
1 min 5 min 15 min 30 min 1 Hour 2 Hours 4 Hours
0
2 5
5 0
7 5
1 0 0
1 2 5
1 5 0
1 7 5
2 0 0
A b e t a @ 1 0 u M
w / C a s p - 3 / 7 I
L i v e C e l l s
S t a u r s p o r i n e T r e a t e d C e l l s
T i m e A f t e r A b e t a T r e a t m e n t
Aβ ≅ 10 µΜ+ Χασπασε Ινηιβιτορ
1 5 D I V H i p p o c a m p a l N e u r o n s t r e a t e d w i t h 1 0 u M A β + / − Χ α λ π α ι ν Ι ν η ι β ι τ ο ρ .
( 0 % = Β α χ κ γ ρ ο υ ν δ 1 0 0 % = Υ ν τ ρ ε α τ ε δ Χ ε λ λ σ ) ( ν = 2 ξ Τ ρ ι π λ ι χ α τ ε ω ε λ λ σ )
1 µιν 5 µιν 10 µιν 15 µιν 30 µιν 1 ηουρ 2 ηουρσ 4 ηουρσ
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
5 0 0
Α β ≅ 1 0 υ Μ
ω / Χ α λ π α ι ν Ι
Τ ι µ ε Α φ τ ε ρ Α β Τ ρ ε α τ µ ε ν τ
Ab @ 10mM
+ Calpain Inhibitor
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