Metabolic and Biochemical Alterations in Alzheimer Disease: Facts ...

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
We are deeply saddened that Mark A. Smith, PhD,
Professor of Pathology at Case Western Reserve University
died unexpectedly in December 2010
In Memory: Prof. Mark A. Smith
This talk by Professor Smith on 'Metabolic and Biochemical Alterations in Alzheimer's Disease'
has now been published with the help and guidance of his family and colleagues
and we thank them for ensuring this talk could be made available
Amongst his many contributions to the advancement of human health,
Professor Smith served as Editor-in-Chief of the Journal of Alzheimer's Disease
The journal has a tribute page dedicated to his memory located at
www.j-alz.com/marksmith.html
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Metabolic and Biochemical Alterations
in Alzheimer Disease:
Facts and Fictions
Prof. Mark A. Smith
Case Western Reserve University
Disclosures: Medivation, Neuropharm, Neurotez, Voyager Pharmaceuticals
Funding: Alzheimer Association, John Douglas French Foundation, NIH
2
The brain and Alzheimer disease
Alzheimer disease attacks nerve cells
in several regions of the brain
A. Cerebral Cortex: involved
in conscious thought and language
B. Basal forebrain: has large numbers
of neurons containing i acetylcholine,
li
a chemical important in memory
and learning
C. Hippocampus: essential
to memory storage
The earliest signs of Alzheimer's
are found in the nearby entorhinal
cortex (not shown)
3
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1

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
What causes selective neuronal loss in AD?
• Almost every physiological/pathological mechanism
that a cell can elicit has been implicated!
• And most are probably involved…
• At some point…
4
The usual suspects
5
Proposed chronology of changes in AD
Tauist and BAPtist
Tau phosphorylation
[Neurofibrillary tangles]
Amyloid-β deposition
[Senile plaque]
Amyloid-β deposition
[Senile paque]
Neuro nal death:
dem mentia
Tau phosphorylation
[Neurofibrillary tangles]
6
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2

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Our hypothesis
Tau phosphorylation
[Neurofibrillary tangles]
Fundamental
disease
mechanisms
Neurona al death:
deme entia
Amyloid-β deposition
[Senile plaque]
7
Clues to disease mechanism
Pathological lesions are very
insoluble, possibly crosslinked?
60
Since the prevalence of AD
is strictly age-dependent,
cause must also be
valence
Percent prev
50
40
30
20
10
0
65-69 70-74 75-79 80-84 85-89 90-94 95+
Age
Oxidative stress/modifications?
8
Oxidative modifications
affect all cellular macromolecules
AD Ctl
AD Ctl
Lipid peroxidation/protein adduction
(4-HNE)
Protein oxidation
(free carbonyl groups)
AD Ctl AD Ctl
Nucleic acids
(8-OH-guanosine)
Glycoxidation
(carboxymethyllysine)
9
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3

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
What is relationship?
Pathology versus oxidative stress
Causes
Tau phosphorylation
[neurofibrillary tangles]
Amyloid-β deposition
[senile plaque]
Consequences
Oxidative stress
Oxidative stress
10
ALZ-50
Normal
neuron
?
. . .
.
? Pre-NFT I-NFT E-NFT
HO-1
Nitrotyrosine
Glycation/age
HNE
Amyloid deposition occurs after oxidative stress!
Tau phosphorylation/NFT occur after oxidative stress!
11
Any theory on AD
has to account for NFT
and amyloid-β
In other words,
if they are not the cause,
they may be the consequence
12
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4

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Oxidative stress
induces Tau phosphorylation
ab PHF1/ab 7.51 ratio
(%control)
C A2 A5
Gomez-Ramos et al., 2003 J Neurosci Res 71: 863-870
ab PHF1/ab 7.51 ratio
(%control)
C A2 A5
13
Stress-activated protein kinases
and phospho-τ: a complete overlap
p-p38
P-τ
p-JNK
P-τ
14
Oxidative stress
mediates amyloid-β production
Both βAPP and amyloid-β increased
3-4 fold after oxidative insult
15
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5

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Amyloid-targeted approaches
have been unsuccessful (so far…ever?)
Reasons: Too late! Not strong enough! Wrong target!
The American Journal of Geriatric Pharmacotherapy (2009) M.N. Sabbagh
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Is amyloid an antioxidant?
Neuronal oxidative stress decreases
following amyloid-β deposition
R=-0.97, p

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
19
“All mutations significantly increased Aβ42/Aβ40 in vitro by significantly decreasing Aβ40
with accumulation of APP C-terminal fragments, a sign of decreased PSEN activity”
20
But what is the mechanism?
APP/PS* [Aβ42/Aβ40] AD
Or
APP/PS* Aβ ?
AD
21
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7

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Genetic factors of AD are associated
with increased oxidative stress
or elevated vulnerability to oxidative stress
Cell lines Transgenic/knock in mice Postmortem brain
A βPP gene Eckert et al., 2001 Smith et al., 1998 Bogdanovic et al., 2001
mutation Marques et al., 2003 Takahashi et al., 2000 Nunomura et al., 2004
PS1 gene Guo et al., 1997 Guo et al., 1997 Nunomura et al., 2004
mutation Leutner et al., 2000
LaFontaine et al., 2002
PS2 gene Hashimoto et al., 2002
mutation
ApoEε4 gene Miyata and Smith, 1996 Montine et al., 1996
polymorphism Ramassamy et al., 1999
Tamaoka et al., 2000
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Therapeutics
People always misunderstand me!
23
Potential mechanism of reactive oxygen
species generation in Alzheimer disease
Redox active metals
Hormonal changes
leptin/LH
Mitochondria/metabolic alterations
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8

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Oxidative modifications
affect all cellular macromolecules
AD Ctl
AD Ctl
Lipid peroxidation/protein p adduction
(4-HNE)
Causes?
Protein oxidation
(free carbonyl groups)
AD Ctl AD Ctl
Nucleic acids
(8-OH-guanosine)
Glycoxidation
(carboxymethyllysine)
25
Mitochondrial abnormalities:
5Kb-deleletion
AD Ctl AD Ctl
Normal mtDNA
16,569 bp
OH
Deleted mtDNA
11,592 bp
OH
13475
13460
4977 bp
deletion
8454 10941
8482 10897
8482/13460
13475
8454
Wild type probe
Chimera probe
CAACAACCTATTTAGCTGTTCCCCAACCTT ACACAAACTACCACCTACCTCCCTCACCA
TTCCTCCGACCCCCT
TTGGCAGCCTA GCATT
4977 bp deletion contains ATPase subunit 8, subunit 6, cytochrome-c oxidase
subunit III, and NADH-coenzyme Q oxidoreductase subunit 3, 4 and 5
26
Mitochondria/redox metal abnormalities
predict sites of oxidative damage
mtDNAr5Kb
8-OHG
Nitrotyrosine
27
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9

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Oxidative damage directly correlates
with mtDNA
mtDNA
(optical
density
X100)
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0 2 4
R 2 = 0.8374
6 8 10 12 14 16
8OHG (optical density X100)
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A
Abnormal mitochondrial distribution
in AD brains
B
C
E
J Neurosci., 29, 9090-103 (2009)
29
PET SCAN-profound metabolic
decrease as an early indicator of AD
Control
Alzheimer
30
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10

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
However, oxidative damage
is apparently restricted to cytoplasm
?
Oxidative modifications
31
H 2 O 2 ? mitochondria 32
Hydrogen peroxide
generates oxidative radicals
Alzheimer disease
(+ H 2 O 2 )
Alzheimer disease
(- H 2 O 2 )
Aged-matched
control
Proc Natl Acad Sci U S A. 94, 9866-8 (1997)
33
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11

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
In situ oxidation of 3,3’ diaminobenzidine
(chelation)
Alzheimer disease 10mM DFX 10mM DTPA
Proc Natl Acad Sci U S A., 94, 9866-8 (1997)
34
Iron histochemistry
AD
Ctl
DFX
Proc Natl Acad Sci U S A., 94, 9866-8 (1997)
DFX/Fe
35
Redox active metals are also in cytoplasm
36
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12

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Control cases lacking endogenous redox
active metals…
AD
Control
… have metal (Fe 2+ /Fe 3+ ) binding sites that produce ROS
if supplied with excess H 2 O 2
Control case untreated and after addition of Fe 2+ /Fe 3+
J Neurochem., 74, 270-9 (2000)
37
Summary: oxidative stress
Proliferation
H 2 O 2
Degradation
Heme oxygenase-1
Redox-metals
ROS
What causes these changes?
38
Predictions
• Therapeutics/preventatives (antioxidants)
• Models (cell/animal)
39
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13

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Antioxidant diet is protective
Nutrients per 1000
kilocalories
Vitamin A (RE)
α carotene (mcg)
β Carotene (mcg)
Pro-A carotene (mcg)
Lutein (mcg)
Lycopene (mcg)
Vitamin C (mg)
Vitamin E (a TE)
Alzheimer disease cases
n=104
855
294
1921
2231
972
666
74.6
5.6
Controls
n=223
983
389
2370
2809
1214
927
86.7
5.9
p Value
0.001
0.001
0.003
0.001
0.015015
0.001
0.007
NS
Servings per day
Yellow, green vegetables
Vitamin C fruits, vegetables
2.0
2.4
2.3
2.6
0.022
NS
40
Vitamin E and AD
Therapeutic
Preventative
ormance
Cognitive Perfo
Vitamin E
Control
Time
Sana et al., 1997 Petersen et al., 2005
41
Rather than capturing radicals,
a better therapeutic strategy
would be to reduce their production
42
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Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Only one non-amyloid drug in phase III: 2010
• IVIg or Intravenous immunoglobulin (Baxter)
• Monoclonal Anti-AB Antibody (Elan-Wyeth; Lilly & Co.)
• Semagacestat or LY-450139 (Lilly & Co.)
• Dimebon (Medivation)
– Neuroprotective agent, thought to work by protecting mitochondria in brain cells
Lin T. Nat Medicine. 2006; Doody et al., The Lancet, 2008
43
Oxidative stress: cellular/animal models
• Cell death!!
• Selective cell death!! e.g., CA1 neurons
• Oxidative stress increases amyloid-β
–(e.g., Beal SOD/APP)
• Antioxidants decrease amyloid-β
– e.g., Papolla w/ melatonin
– Cole w/ curcumin
– But at best partial models….certainly not AD
44
Perhaps
• Oxidative stress is necessary, but not sufficient
• So what else is necessary?
45
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15

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
What causes selective neuronal loss in AD?
• Almost every physiological/pathological mechanism
that a cell can elicit has been implicated!
• And most are probably involved….
• At some point…
46
Fundamental mechanisms
• Luteinizing hormone
– Higher in AD/DS
– High LH = poor cognition (receptor-dependent)
– Agonist rescues OVX-mediated declines
– Menopause is major risk factor for AD
• Leptin
– Low in AD
– In vitro leptin reduces phospho-tau through GSKβ
– In vitro/vivo (CNDR8) leptin
reduces amyloid/improves cognition
• Cell cycle alterations
47
Cell cycle related proteins in AD
• Cell cycle markers
• Telomeres/telomerase
• Bi-nucleation
• Chromosome instability
Causes or consequences?
Neurobiology of Aging, 21, 783-796, 2000
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16

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Establishment of a new animal model
CaMKII-MYC mice
MYC is specifically induced in cerebral cortical and hippocampal neurons by depletion of doxycyline
CaMKII
promotor
tTA
Dox
Activation
Inactivation
tetO
p
MYC
C-MYC (now referred to as MYC) is a member of a family of proto-oncogenes comprising
C-MYC, N-MYC, and L-MYC; MYC encodes a transcription factor that, as part of a heterodimeric
complex with MAX, regulates the expression of a multitude of genes involved in regulating cellular
proliferation and growth; Overexpression of MYC is commonly associated with
tumorigenesis; Where MYC exerts its neoplastic function by inducing autonomous cellular
proliferation and cellular growth, blocking differentiation, and inducing genomic destabilization
Am J Pathol., 174, 891-7 (2009)
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Cell cycle re-entry in CaMKII-MYC mice
MYC-ON
MYC-OFF
PCNA
NeuN
NeuN
Ki-67
Am J Pathol., 174, 891-7 (2009)
50
Cell cycle re-entry in CaMKII-MYC mice (2)
Am J Pathol., 174, 891-7 (2009)
51
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17

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Selective neurodegeneration
in CaMKII-MYC mice
MYC-ON
MYC-OFF
Am J Pathol., 174, 891-7 (2009)
52
Selective neurodegeneration
in CaMKII-MYC mice (2)
TUNEL assay
GFAP
MYC-ON
Am J Pathol., 174, 891-7 (2009)
MYC-OFF
53
Cognitive deficits in CaMKII-MYC mice
The Morris water maze (WMM)
T-maze
Escape latency
(distance moved)
850
810
770
730
690
650
On Off
1 2 3
Traning (day)
40
35
On
30
25
20
15
10
% time in target quadrant
Am J Pathol., 174, 891-7 (2009)
Off
54
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18

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Cell cycle
abnormalities
Proliferation:
cancer
Cell death: Alzheimer disease
55
Models and/or Tx????
• Oxidative stress
• Mitochondrial alterations
• Cell cycle alterations
• Hormonal imbalances (leptin/gonadotropins)
Work well as preventatives, not as treatments
Why, why< why?
56
Back to the drawing board?
ALZ-50
Normal
neuron
?
.
.
.. .
? Pre-NFT I-NFT E-NFT
Oxidative stress
Cell cycle changes
57
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19

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Alzheimer disease
Phospho-ERK
Phospho-JNK
Phospho-p38
Mitogenic signal
Oxidative signals
Mech Ageing Dev., 123, 39-46 (2001)
58
Oxidative stress and cell cycle changes
occur earlier than Tau or amyloid-β
ALZ-50
Normal
neuron
. ? .
.. .
? Pre-NFT I-NFT E-NFT
Oxidative stress
Cell cycle changes
But which one is first ?
59
Control cases (pre-Alzheimer?)
Phospho-ERK Phospho-JNK Phospho-p38
Control group 1
Mitogenic signal
Oxidative signals
Control group 2
Mech Ageing Dev., 123, 39-46 (2001)
60
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20

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Alzheimer disease
Phospho-ERK Phospho-JNK Phospho-p38
Mitogenic signal
Oxidative signals
Mech Ageing Dev., 123, 39-46 (2001)
61
Two hit hypothesis
Lancet Neurology, 3, 219-26 (2004)
Hit A
Hit D
Aged not Demented
or
Declining not Atrophied
Hit A
Hit D
Alzheimer disease or demented & aged
62
Are there more than two hits?
Risk
Age ([Age in years]lbs)
Mutations (50lbs)
Menopause*******
Obesity********
ApoE4 (20lbs/Apoe4 allele)
Head injury (10-20lbs)
Smoking (5lbs)
Atherosclerosis (15lbs)
Protection
Reserve (100lbs)
Male gender (10lbs)
Exercise (10lbs)
Education (10lbs)
Antioxidant (10lbs)
ERT (10lbs)
63
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21

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
Menopause
APP/PS1
Obesity
Aβ/tau
Cholesterol
Oxidative
stress
APOE
Aging
STOP
Cytochrome C/
cell death
Ca 2+
buffering
Oxidative
stress
Redox
metals
Synaptic
dysfunction
Metabolic
alterations
64
“The way to get things done
is not to mind who gets the credit for doing them”
Benjamin Jowett (1817-1893), British theologian and classicist
65
Collaborators
Case Western Reserve Univ. Centro Biologia Molecular,
• George Perry (Pathology) Madrid
• Lawrence M. Sayre (Chemistry) • Jesús Avila
• Xiongwei Zhu (Pathology) Harvard University
• Gemma Casadesus
• Ashley Bush
(Neurosciences)
Johns Hopkins University
• Hyoung-gon-Lee (Pathology) • Donald Price
• Robert P. Friedland
University of Barcelona
(Neurology)
• Rudy Castellani (Pathology)
• Luke Szweda
(Physiology/Biophysics)
• Alfred Rimm (Epidemiology)
• Bruce Lamb (Genetics)
• Grace Petot (Nutrition)
• Paul Carey (Biochemistry)
Asahikawa Medical College
• Akihiko Nunomura
• Shigeru Chiba
• Merce Pallas
• Toni Camins
Voyager Pharmaceuticals
• Richard Bowen
Columbia University
• Shi-Du Yan
• David Stern
Takeda Chemical Company
•Keisuke Hirai
Thomas Jefferson University
• Hilary Koprowski
Tohoku University
School of Medicine
• Atsushi Takeda
University of California
San Diego
• Donald Cleveland
University of South Alabama
• Miguel A. Pappolla
University of Genova
• Massimo Tabaton
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22

Metabolic and Biochemical Alterations
in Alzheimer Disease: Facts and Fictions
Prof. Mark A. Smith
“If you don’t have anything smart to say,
say it with an English accent”
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