Graduate Seminar - Halloween 2009

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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%)



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?



2 Pathologies = 2 Protein Aggregates

No Aggregates Tangles Plaques & Tangles

AD FTDP PD HD ALS CJD

Nature Medicine 10, 1055 - 1063 (2004)



Amyloid Beta Plaques

βγ

Amyloid-βInsoluble Fibers /Plaques

Soluble Oligomers

AICD Extracellular Domain

Amyloid Precursor Protein (APP)

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are needed to see this picture.

W. E. KLUNK AND C. A. MATHIS, UNIVERSITY OF PITTSBURGH



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



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.



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


QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.Killing Neurons with Amyloid Beta

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



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



Tau Disappearance - A Closer Look

25

10 - + - + - + - + - + - + - + - + - +10Õ 20Õ 40Õ 1Hr 2Hr 4Hr 8Hr 24Hr 48Hr

52

3min 48HrAmyloid Beta

3min 48HrAmyloid Beta



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


QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.Kinase Activity -

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


QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.Signaling Summary

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


QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.Tau Targeting Kinases


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


GSK3B

pGSK3B

Whole GSK α/βπΓΣΚ3 β (9) − Ιναχτιϖε

35 kD

25 kD


150% 475% 325% 250% 225% 140%


4 Hour

pGSK(S9)


QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.Suggested Kinase Activity

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!



Decreased Phosphorylation?

199/202

217

231

396

396/404

Tau-5

KO UN UN 3’ 10’ 20’ 40’ 1 2 4 8 24


QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTimeª and aTIFF (Uncompressed) decompressorare needed to see this picture.How About Phospho-Tau?

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



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!



Tau Hyperphosphorylation in AD!



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”



Revisit The Model

APP Dysfunction

FAD

?

Plaques

TauDysfunction

Tangles

FTDP-17

CellDeath

GSK3βCDK5

ERKAKT



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



PanTau pTau 217

pTau 231 pTau 396



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).




150% 475% 325% 250% 225% 140%


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


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 1/2Active

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

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

Health & Medicine

Graduate Seminar - Halloween 2009