Experimentally Induced Diabetes in Transgenic AD Mice Accelerates

Brain Pathology Giulio Maria Pasinetti, M.D., Ph.D.Mount Sinai School of Medicine

New York, NY

Pasinetti © 2006

Pasinetti © 2006

• Rotterdam Study- Type II diabetes (non insulin dependant diabetes; NDDM) doubles RR (1.9) of AD incidence even when cases with cerebrovascular disorders were excluded (Ott et al., 1999).

• Insulin resistance, a major feature of Type II diabetes, is a significant risk factor pure AD (>2 fold RR). Association of diabetes and AD is strong among carriers of the ApoE4(Peila 2002)

• Therapeutic evidence that certain insulin sensitizing drugs may beneficially influence AD:- biguanide (e.g. metformin)- glitazones (insulin sensitizing & PPAR-activating

actions)

Type II Diabetes - Insulin Resistance A Risk Factor for Alzheimer’s disease?

The Potential Role of Diabetesin Alzheimer’s Disease

GeneticPredisposition

Aging

AD Dementia• Amyloid ?• NFT plaques ?• Others?

Lifestyle factors(high caloric intake)

InsulinResistance

Type II Diabetes• Dislipidemia• ↑Glycerides• Hyperinsulinemia

VAD (Cardiovascular risk factors)• Hypertension• Atherosclerosis

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Diet Induced Diabetes in mouse models of Alzheimer’s Disease type Neuropathology

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• Does insulin resistance promote AD type neuropathology through mechanisms that involve generation (e.g. γ-secretase) or impaired clearance (e.g. insulin degrading enzyme) of Aβ?

• The mechanism associated with insulin resistance mediated amyloidosis may involve abnormal regulation of insulin receptor (IR) functions in the brain.

• If insulin resistance promotes Aβ generation, are insulin sensitizing- anti-diabetic drugs beneficial to AD type amyloidneuropathology?

Pasinetti © 2006

Potential Roles of Diet Induced Diabetes in Alzheimer’s Disease Neuropathology

Scheme of Treatment

Age(months)

• Aβ generation (secretase acticities)• Aβ clearance - IDE• AD-type neuropathology

3

Start High Calorie Diabetogenic Diet

on Tg 2576

2416

Ongoing• Therapeutic efficacy of insulin sens.• Brain pathology by non invasive MRI•Relationship with cognition

86

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Potential Roles of Diet Induced Diabetes

in Alzheimer’s Disease Neuropathology

Tg2576 mice

Standard diet HF diet

Dietary composition

20%20%Protein60%20%Carbohydrate20%60%Fat

CTLHigh FatEffects of diabetogenic high fat diet• obesity • fat pat deposition• triglyceride• insulin• hyperglycemia• = serum cholesterol content • insulin resistance – Gluc Toler test

Pasinetti © 2006

Control GroupInsulin Resistant Group

0

500

1000

1500

Hippocampalformation

CerebralCortex

Inso

lubl

e A

β1-

40(n

g/g)

**0

25

50

75

100

Inso

lubl

e A

β1-

42(n

g/g)

6 Mo 6 Mo

Hippocampalformation

CerebralCortex

*

Aβ1-40 Aβ1-42

Aβ(5M Guanidine extractable)

Diet Induced Diabetes Promotes Aβ Peptide Content in the Brain

6 Mo 6 Mo

*

8 Mo

*

8 Mo 8 Mo

*

8 Mo

*

-------------------------------------------------------------------

• Means + SEM, n= 3-4 per group; *P <0.01 vs control group

• Diet induced insulin resistance lasted for 3-5 months respectively starting at 3 month of age

Diet Induced Diabetes Promotes AD-type β-amyloid Plaque Neuropathology in the

Tg2576 Mouse BrainStereological assessments of amyloid burden

Tg Insulin

resistantTg

control

* ***

Am

yloi

d bu

rden

( %

of c

ereb

ral

cort

ex v

olum

e x

10-4

)

75

50

20

0

15

10

5

0

Am

yloid plaque volume

(x 103

µm3)

Tg ControlTg Diet Induced Diabetes

-------------------------------------N=4-6, P=0.01 ANOVA

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Pasinetti © 2006

Fibrillization

APP

Aβ

ApoEε4

Non-amyloidogenicpathway

Uptake/Clearance

?

α-secretase

1. β-secretase

2. Neprilysin

Degradation

APPMutations

Synthetic Pathway 2. γ-secretase

1. IDE

amyloidosis

Potential Mechanisms Through which Diet Induced Diabetes May Influence AD β-amyloidois in the Brain

γ

Plasma membrane

CytoplasmFull length APPCOOH

P6 (CTF-γ)

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C8

In Vitro γ-Secretase Assaybased on detection of the putative C-terminal fragment of Aβ-PP (P6)

(Sastre & Haass, 2001)

Isolate membrane fraction(100,000 × g)

Incubate at 37oC for 2 h Un-reacted control (4 oC)

Control

Electrophoretic detection of C8 immunoreactive γ−CTF

αβ

Quantification of C-Terminal Fragment (CTF)-γ of APP as Index of γ-Secretase Activity

This in vitro γ-secretase assay allows APP/CTF-γ to be cleaved while membrane bound

APP

Aβ

1. β-secretase

degradation

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Diet Induced Diabetes Coincides with Increased γ-Secretase Activity in the Brain

Factors affecting Aβ production

2. γ-secretase

amyloidosis

6 KDa

105 KDaHolo-APP

α,β- CTFγ -CTF

Diet: Control Diabetes Control Diabetes

No Incubation Control

0

100

200

γ-C

TF/ h

olo-

APP

( as

% o

f LF)

0

25

50

75

Hol

oA

PP(O

pita

lDen

sity

)

γ-CTF / holo-APP holo-APP

Diets: Control Ins. Res. Control Ins. Res.

*

13 kDa10 kDa

•Diet induced insulin resistance lasted for 5 months starting at 3 month of age

•Means + SEM, n= 3 per group; *P <0.01 vs control group

Generation of CTF-γ of APP as Index of γ-Secretase activity

Major Cleavage Sites for Metallopeptidasesin the holo-APP Protein May Predict Degradation

Pasinetti © 2006

SignalPeptide

Full length APPIntracellular

DomainExtracellular

Domain

COOHNH2

A = angiotensin-converting enzymeE = ECE-1M = MNP-9N = NEPI = IDE

KM / DAE / FRHD / SG / YEVH / H / QK / L / V / F / F / AEDVGSNK / GA / IIG / L / MVG / GVV / IA / TVI

N A N I I E E N,E, I N N M

M I I I M M

γ-secretase(producing Aβ 1-40

or Aβ 1-42) β-secretase

Role of Insulin Degrading Enzyme in AD

Pasinetti © 2006

• Aβ peptide levels in brain are inversely correlated with IDE and IDE influence γ−CTF degradation (Miller et al., 2003).

• IDE regulates the elevation of insulin, and its hypofunction (IDE KO) promotes Aβ generation in vivo ( Farris et al., 2003).

• Reduced hippocampal IDE in late onset AD associated most strongly with APOE4 allelic content (Cook et al., 2003).

APP

Aβ

2. Neprilysin

Pasinetti © 2006

degradation

Diet induced diabetes in Tg2576 Mice Coincides with Decreased IDE Expression and Activity

in the Brain Factors affecting

Aβ production

1. IDE

amyloidosis

0 20 40 600

20

40

60

Time (min)D

egra

ded

Insu

lin(%

CPM

)

0 20 40 6040

60

80

100

Time (min)

Und

egra

ded

Insu

lin(%

CPM

)

0

50

100

150

IDE

Con

tent

(% o

f Con

trol

die

t)

*

Control GroupInsulin Resistant Group

IDE 110KDa

Insu

lin R

esis

tant

Gro

up

Con

trol

Gro

up

APP 105KDa

A B

Control Group

Insulin ResistantGroup

Control Group

Insulin ResistantGroup

…while resulting in decreased degraded

insulin (Aβ)

*

*

*

*

Insulin resistance results in increased levels of

undegraded insulin (Aβ)

• Means + SEM, n= 3 per group; *P <0.01 vs control group

• Diet induced insulin resistance lasted for 5 months starting at 3 month of age

Pasinetti © 2006

Escape latencies for APP mice improved during the the learning phase of water maze testing(consistent at this age) while Insulin Resistant APP mice maintained longer latencies

(suggesting that insulin resistance coincided with memory impairment).

0 1 2 3 4 5 6 7 80

10

20

30

40

50

Insulin ResistantAPPAPP

Day(4 trials / day)

time

(s)

Diet Induced Diabetes Coincides with Spatial Memory Impairment in a Water Maze Behavior Test

(8 months old Tg 2576 mice following 5 month diet leading to insulin resistance)

ANOVA p=0.0016 (effect of treatment on learning)

1 2 3 4

Insulin ResistantAPP

Target

Quadrant

APP mice showed a preference for the formerplatform location during the spatial probe test

While Insulin Resistant APP mice swam randomly across the 4 quadrants, suggesting impaired spatial learning

0

20

40

% D

ista

nce

Targ

et /

Dis

tanc

e Q

uadr

ant

1 2 3 40

20

40

APP

Target* *

Quadrant

% D

ista

nce

Targ

et /

Dis

tanc

e Q

uadr

ant

* P<0.05 vs target* P<0.05 vs target

The Role of Diet Induced Diabetes in Alzheimer’s Disease

What is the mechanism through which diabetes may

promotes Aβ processing?

Pasinetti © 2006

Pasinetti © 2006

pY—

pY——pY

—pY

Insulin

Ras IRS

Changes in Signaling Pathway

β-Amyloid Generation

IR-p

Y11

6 2/1

163 /I

R(I P

)/IR

/ Ac t

i n(%

of C

ontr

ol)

Control Insulin Resistant 0

50

100

150

*

0

50

100

150

IR /

Act

in(%

of C

ontr

ol)

Control Insulin Resistant

8 month old Tg 2576 AD mice, 5 months of diet

IR-β

Actin 42KDa

95KDaControl

Insulin Resistant

95KDapY1162/1163 IR-βIR-β

Control

95KDa

Insulin Resistant

Insulin Resistance

Diet Induced Diabetes Influences Insulin Receptor PY 1162/1163-

IR in Absence of Detectable Change in Insulin Receptor Expression in the Cerebral Cortex

Implication in IR mediated signal transduction

Pasinetti © 2006

pY—

pY——pY

—pY

Insulin

Insulin Resistance

Ras

Raf

MAPK

IRS

Akt

GSK-3αGSK-3β

0

50

100

150

P-M

APK

/Tot

al M

APK

(% o

f Con

trol

)

*

Control InsulinResistant

MAPK

0

100

200 *G

SK-3

α,β

/ Act

in(%

of C

ontr

ol) *

GSK-3α GSK-3β

GSK-3α (pY217)GSK-3β (pY217)

Actin

Con

trol

Insu

linR

esis

tant

42kDa

Con

trol

Insu

linR

esis

tant

Total MAPK 42/44

P-MAPK 42/44

Control InsulinResistant Control Insulin

Resistant

Altered Insulin Receptor PY 1162/1163-IR in Diabetic Tg 2576 Mice Coincides with Decreased MAP Kinase Phosphorylation and

and Increase GSK-α and GSK-β Phosphorylation in the Cerebral Cortex

8 month old Tg 2576 AD mice, 3 months of diet

Pasinetti © 2006

Activation of GSK-3 Correlates With Induction of γ-secretase activity in the brain of

“diabetic” Tg2576 mice

“Active” GSK-3α

100

200

γ-C

TF/ h

olo-

APP

(as

% o

f con

trol

)

0

25

50

75

Holo A

PP / Actin

(Optical D

ensity)0

*

105 KDa

6 KDa

Holo-APP

CTF-α, βCTF- γ

13 kDa10 kDa

+ + - -Incubation:

TgControl

TgInsulin

Resistant

TgControl

TgInsulin

Resistant

25 50 75 100 125 1500

100

200γ-

secr

etas

e ac

tivity

(% o

f con

trol

)

Phospho [S21]GSK-3α

25 50 75 100 125 1500

100

200

γ-se

cret

ase

activ

ity(%

of c

ontr

ol)

Phospho [S9]GSK-3β

Tg controlTg insulin resistant

Correlation of γ -secretase activity with GSK-3 activities

γ-secretaseactivity

“Active” GSK-3β

A B

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Pasinetti © 2006

IRβ silencing decreases AKT and promotes GSK (decreased S9 and S21 phosphorylation)

consistent with the evidence in “diabetic”Tg2576 mice

A

Neg. ControlSi-IRβ

0

25

50

75

100

125

pS47

3 -A

kt /

Act

in(%

of c

ontr

ol)

** **

0

25

50

75

100

125

Akt

/ Act

in(%

of c

ontr

ol)

pS473-Akt

Akt

Actin

60 kDa

60 kDa

42 kDa

Neg.Cont. Si-IRβ B

0

25

50

75

100

125

*

0

25

50

75

100

125

GSK

-3α

/ Act

in(%

of c

ontr

ol)

pS21

-GSK

-3α

/ Act

in(%

of c

ontr

ol)

pS21-GSK-3α

GSK-3α

Actin

51 kDa

51 kDa

42 kDa

Neg.Cont. Si-IRβ C

0

25

50

75

100

125

0

25

50

75

100

125

GSK

-3β

/ Act

in(%

of c

ontr

ol)

pS9 -

GSK

-3β

/ Act

in(%

of c

ontr

ol)

*

pS9-GSK-3β

GSK-3β

Actin

46 kDa

46 kDa

42 kDa

Neg.Cont. Si-IRβ

Means + SEM, n= 3 per groupin 2 independent studies*P <0.01 vs control group

Pasinetti © 2006

Neuronal IR - KO in “NIRKO” mice recapitulates altered signalling in the brain

observed in “diabetic” Tg2576 mice

A NIRKO(+/-)WT

IR total

actin

IRβ

Expr

essi

on(%

of W

T C

otnr

ol)

0

25

50

75

100

125

*

BWTNIRKO(+/-)

pS473-Akt

**

0

25

50

75

100

125

**

Akt

/ Act

in(%

of c

ontr

ol)

Akt

C

pS9-GSK-3β0

25

50

75

100

125

**GSK

-3β

/ Act

in(%

of c

ontr

ol)

**

pS21-GSK-3βGSK-3β

- Hemizygous NIRKO, collaboration with Dr. Accili- Did not detect for GSK3α

Pasinetti © 2006

Insulin Resistance Diabetes

The Role of Diabetes in AD Lifestyle factors (e.g. diet)

γ-secretaseAccelerating factor in AD amyloid

neuropathology Neurological

Controls/ MCI AD Dementia

Aβ-generationIDE

Pasinetti © 2006