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Jack Reifert, DeeAnn Hartung, Stuart Feinstein
Neurons in Distress – Linking Amyloid Beta Induced Stress on Hippocampal Neurons to Tau Biochemistry
Annual NRI Retreat - 2009
Disease Related Changes to MAPT (Tau)
N C4411
Microtubule Binding Domains
Nature Medicine 10, 1055 - 1063 (2004)
Eckhard Mandelkow et al., Brain Pathology 17 (1) , 83–90 , January 2007
Accumulation of NFT’s:
- Fragmentation
- Hyperphosphorylation
- Aggregation
25711 44 230 254 267 310 340 394Potential Calpain Cleavage Sites
45 74 103
Amyloid Beta Pathology in Alzheimer’s Disease
Nature Medicine 10, 1055 - 1063 (2004)
Amyloid-Insoluble Fibers /Plaques
Soluble Oligomers
AICD Extracellular Domain
Amyloid Precursor Protein (APP)
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
Upper Tau BandLower Tau BandTotal Tau
Amyloid Beta Induces Rapid Degradation of Full Length Tau
25
10 - + - + - + - + - + - + - + - + - +10’ 20’ 40’ 1Hr 2Hr 4Hr 8Hr 24Hr 48Hr
52
Protease Mediated Tau Degradation?
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:
Tau
GAPDH
% Caspase Activity: 160% 150% 135% 120% 45% 40%
4 Hour
75%
20%
CytoTox-ONE Raw Data on 15 DIV Hippo Neurons Treated withAmyloid Beta
Time Zero 20 Min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours0
100000
200000
300000
400000
500000
600000
700000
Untreated
Amyloid Beta @ 10uM
Media Alone Line
Time's
Control Cell Line - Alive
50 - 75%
Corresponding Neuronal Viability
Protease Activity
Tau Degradation
Activation of Tau Targeting Kinases Whole GSK
pGSK3 ( 9)Ser
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
3GSK B
3pGSK B
Whole GSKα/3pGSK (9) - Inactive
GSK3
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
CDK-5
Tau Phosphorylation Changes?
pTau 181 Light Decreased
pTau 199 Robust Decreased
pTau 202 None NO
pTau 212 Robust Decreased
pTau 217 Robust Decreased
pTau 231 Robust Decreased
pTau 235 Robust Decreased
pTau 262 Light Decreased
pTau 396 None NO
pTau 404 Robust Decreased
pTau 396/404 (PHF-1) Robust DecreasedpTau 413 Light Decreased
Phospho-Tau Antibody
Detected in Untreated Sample?
Increased Signal with A
?Treatment
- + - + - + - +- +20 min 40 min 1 Hour 2 Hours 8 Hours4 Hours
- +
PHF1
Summary:
-Tau degradation precedes cell death.
- While full length tau disappears, low MW tau fragments are retained.
- Survival signaling cascades are perturbed by amyoid beta treatment resulting in GSK3 kinase activation.
- Protease activation by amyloid beta may be responsible for tau degradation and CDK-5 activation.
- Phospho-specific tau epitopes are detected robustly in untreated samples, and no increase is seen with amyloid beta treatment.
Cairns et al., Journal of Pathology V-204 P-438 2004
xxxxxx
Extra Slide -Tau Related Diseases
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
A 10@ μM
+ CaspaseInhibitor
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 + / - C a l p a i n I n h i b i t o r .
( 0 % = B a c k g r o u n d 1 0 0 % = U n t r e a t e d C e l l s ) ( n = 2 x T r i p l i c a t e w e l l s )
1 min 5 min 10 min 15 min 30 min 1 hour 2 hours 4 hours
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
A @ 1 0 u M
w / C a l p a i n I
T i m e A f t e r A T r e a t m e n t
Aβ @ 10μM
+ Calpain Inhibitor
C a s p a s e 9 A c t i v i t y 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
A b e t a a t 1 0 u M .
1 min 5 min 15 min 30 min 1 Hour 2 Hours 4 Hours
0
2 5 0 0
5 0 0 0
7 5 0 0
1 0 0 0 0
1 2 5 0 0
1 5 0 0 0
1 7 5 0 0
2 0 0 0 0
A b e t a @ 1 0 u M
w / C a s p - 9 I n h i b i t o r
D M S O - C e l l D e a t h C o n t r o l
L i v e C e l l C o n t r o l s
C a s p a s e 9 I n h i b i t o r A l o n e
S t a u r o s p o r i n e C o n t r o l
T i m e A f t e r A b e t a T r e a t m e n t
Extra Slide - Protease Assays
0.1 1 100
25
50
75
100
4 Hours8 Hours
24 Hours
48 Hours
72 Hours
Concentration of Amyloid Beta (μM)
Per
cent
Cel
l Dea
th
Time of AmyloidBeta Treatment
ATP Luminescent Cell Viability Assay(Cell-Titer Glow® - Promega # G7570)
Extra Slide - ATP Cell Viability
Extra Slide - ERK / AKT Signaling
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
GAPDH
- + - + - + - + - + - + - + - + - +- +10 min 20 min 40 min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours 48 Hours3 min
pERK 1/2
Whole ERK
Active
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
Whole ERK 1/2pERK1/2 - Active