Post on 02-Aug-2020
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
Pasinetti © 2006
Diet Induced Diabetes in mouse models of Alzheimer’s Disease type Neuropathology
Pasinetti © 2006
• 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
Pasinetti © 2006
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
Pasinetti © 2006
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)
Pasinetti © 2006
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
Pasinetti © 2006
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
Pasinetti © 2006
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