Neuroscience Retreat 2009

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


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 262 Light Decreased

pTau 396 None NO


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


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


ERK

pERK

Whole ERK 1/2pERK1/2 - Active

Documents

Neuroscience Retreat 2009