Oxidative, Metabolic, and Fibril Stress in Alzheimer Disease
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Transcript of Oxidative, Metabolic, and Fibril Stress in Alzheimer Disease
George PerryThe University of Texas at San Antonio
“From Neurodegeneration to Brain Health”
Case Western Reserve University
Oxidative, Metabolic, and Fibril Stress in Alzheimer Disease
Cleveland, Ohio 25 October 2013
Collaborators
Case Western Reserve University
Mark A. SmithLawrence M. Sayre
Takeda Chemical Company
Keisuke Hirai
Asahikawa Medical College
Xiongwei Zhu
Tohoku University School of Medicine
Atsushi Takeda
University of Texas-Brownsville
Luis Colom
University of Genova
Robert PetersenGemma Casadesus
Centro Biologia Molecular, Madrid
Jesús Avila
University of Maryland, Baltimore
Rudy Castellani
Universitat de Valencia
Jose Viña
University of ChileRicardo Maccioni
UTSA
Paula I. Moreira
University of Coimbra
University of Wisconsin - MadisonCraig Atwood
Justin Shenk
Catarina Oliveira
Masao Nakamura
University of YamanashiAkihiko Nunomura
Massimo Tabaton
Xinglong Wang
Hyoung-gon Lee
Maria Santos
Sonia CorreiaRenato Santos
Cinvestav
Raúl MenaFrancisco Garcίa-Sierra
Siddhartha Mondragón-Rodrίguez Clyde Phelix
Orkid Coskuner
Edwin Barea-Rodriguez
Texas A&M University
Ian Murray
Definition of Oxidative Stress
1. Classic definition: The production of reactive oxygen in excess of antioxidant mechanisms
2. Evolving definition: Altered homeostatic balance resulting from oxidant insult.
Oxidative stress is an early event in AD.
......
t
GlycationNormalNeuron
? Pre-NFT I-NFT E-NFT
FREE CARBONYLS
80HGHNE
Potential Mechanisms of Reactive Oxygen Species
Generation in apparently normal neurons in Alzheimer Disease
Active microglia
Redox active metals
Amyloid-b
Advanced glycation endproducts
Mitochondria??
Sources of reactive oxygen
• ATP production• key metabolites• Ca2+ buffering• free radicals/ ROS generation• regulation of
PCD
Mitochondria are Critical to Neuronal Function
Alzheimer Control
In situ hybridization of mtDNA
Immunolocalization of Cytochrome oxidase 1 is increased in AD
Alzheimer disease Control
Mitochondrial components are increased in Alzheimer disease
Wi l d type probe
Chi mera probe
4977 bp del et i on
Deleted mtDNANormal mtDNA
8482 108978454 10941
1347513475
8454
8482/ 1346013460
ACACAAACTACCACCTACCTCCCTCACCATTGGCAGCCTA GCATT
CAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCT
16, 569 bp 11, 592 bpOHOH
4977 bp del et i on cont ai ns ATPase subuni t 8, subuni t 6, cyt ochrome-c oxi dase subuni t I I I , and NADH-coenzyme Q oxi dor educt ase subuni t 3, 4 and 5.
Examination of neurons in biopsy specimens shows normal mitochondria (A), mitochondria with broken cristae (B), and lipofuscin with vacuoles (C).
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F i g u r e 7
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Mitochondria0
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F i g u r e 7
Percentage area of intact mitochondria is significantly decreased in cases of AD (n=8) as compared to age-matched controls (n=5). p=0.012
While mtDNA is increased, mitochondria are not.
Mitochondria components are in autophagosomes
No Fusion Wild Type No Fission
Mitochondrial dynamics
Microtubules are reduced specifically in pyramidal neurons.
R2 = 0.9841p=0.016
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4
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8
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12
60 65 70 75 80
Age (yr)
Num
ber
or m
icro
tubu
les/
m²
Numbers of microtubules decrease with normal aging
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Num
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f neu
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Control
AD -PHF
Control
AD -PHF
Number of microtubules /m2# of
non
-pyr
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al n
euro
ns
#
of p
yram
idal
neu
rons
Control
AD -PHF
AD +PHF
Control
AD -PHF
AD +PHF
Control
AD -PHF
AD +PHF
Pyramidal neurons
Non-pyramidal neurons
p=0.000004
p=0.90
Microtubules (arrowheads) remain intact even in close proximity to paired helical
filaments (*)PHF
MT
Could the mitochondrial problem be related to reduced axonal transport?
O -2 2 O2
Fe
HH
mitochondria
lysosome
Alzheimer Disease
Normal
FeII2
Catalytic redox activity is cytoplasmic and increased in AD…
Alzheimer Control
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control (n=5) AD (n=6)
Mea
n de
nsity
Control (n=5) AD (n=6)
Rel
ativ
e D
ensi
ty p=0.002
Pyramidal neurons AD cases have significantly greater levels of cytoplasmic staining than age-matched controls, p=0.002.
Nucleic acid damage, as well as redox active metals are cytosolic
8-hydroxyguanosine Redox active metals
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non-ox ox
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ltive
Den
stiy
of 8
OH
G fo
rmat
ion tRNA
rRNA
Ribosomal RNA is 13 times more susceptible to oxidation than transfer RNA
Conformation plays a major role in this binding ability. After denaturation with formamide, the iron-binding capacity is dramatically reduced.
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Mea
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tRNA
rRNA
Oxidized RNA, detected by 8OHG, is only present in ribosomes purified from Alzheimer disease
Ribosomal RNA was immunoprecipitated with a monoclonal antibody against 8OHG and followed by RT-PCR using specific primers for human 28s and 18s rRNA fragments.
Rabbit reticulocyte ribosome in vitro translation assayThe Fenton reaction causes RNA cleavage …
…with consequent decrease in protein synthesis.
RNA is a dominant binding site for the redox active metals released into the cytoplasm
O - -
-
2 2 O2
Fe
HH
mitochondria
lysosome
2Fe(II)RNA2 RNA
Fe(III)OxRNA
O
2H O
OxRNA
Alzheimer Disease
Aβ reduces copper-induced oxidation
Normal
B
Ratio of Cu2+ to Ab1-42 (Cu2+/Ab1-42)
02468101214
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asco
rbat
e-ox
idas
e ac
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)
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HN
E (
M)
Ab1-42 + Cu2+
Cu2+
A
B
Ratio of Cu2+ to Ab1-42 (Cu2+/Ab1-42)
02468101214
0 1 2 3 4
asco
rbat
e-ox
idas
e ac
tivity
(M
/min
)
B
Ratio of Cu2+ to Ab1-42 (Cu2+/Ab1-42)
02468101214
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tivity
(M
/min
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HN
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Ab1-42 + Cu2+
Cu2+
A
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E (
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Ab1-42 + Cu2+
Cu2+
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Ab1-42 + Cu2+
Cu2+
A
Amyloid plaque cores purified from brain demonstrate characteristic Congo red birefringence (A), are strongly reactive with antisera to Ab 1-42 (B), but not with irrelevant antisera (C).
A
B
C
Spectra of plaque cores (B) are virtually identical to synthetic peptide (A). Intensity at 1604 is indicative of Zn binding and at 1278 for Cu binding, both increased in purified cores.
B sheet
Zn bindingCu binding
Oxidative damage (8OHG, blue) decreases with increased amyloid - b (brown)
17 yr.
61 yr.
31 yr.
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A g e
Neuronal 8OHG
C o n t r o lD S
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1 0
1 5
2 0
0 - 9 y 1 0 - 1 9 y 2 0 - 2 9 y 3 0 y & o v e r
A g e G r o u p s
Neuronal 8OHG
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% Amyloid Burden
0
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1 0
1 5
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0 1 2 3% A m y lo i d B u r d e n
Neuronal 8O
HG
*
* *
†
( n = 5 ) ( n = 6 ) ( n = 5 ) ( n = 6 )
a
b
c
In vivo
Levels of neuronal 8OHG in AD decrease with:
A. increasing levels of amyloid (p=0.002 in cases with ApoE 4 and p=0.05 in cases lacking ApoE4) and
B. the duration of the disease (p<0.03).
AD with ApoE4AD lacking ApoE 4Control
Levels of 8OHG immunoreactivity are significantly increased in familial AD cases (FAD ) cases (n=13, avg age 59 yr) compared with controls (n=15,avg age 66 yr). One presymptomatic case with PS-1 mutation shows a similar level to average FAD cases (A).
In (FAD) there is a significant inverse correlation of Ab42 burden (B) but not with Ab 40 (C).
Familial AD Cases
Ab 42Ab 42
Ab 40
Are Amyloid-b and t Protective Responses Against a Cauldron of Oxidative Stressors in Alzheimer Disease?
Figure 1. Biotinylated τ binding to Alzheimer’s brain sections.
Figure 3. Solid-phase τ binding assay.
Figure 4. Antisera to βPP714-734 Immunostatin senile plaques.
Summary• Oxidative damage is an early feature of
Alzheimer disease.
• Mitochondria abnormalities are critical to oxidative abnormalities through Fe/Cu oxidation of RNA.
• Aβ may play a protective antioxidant role.
• Nucleated assembly through AβPP-tau interactions may be critical to development of Alzheimer pathology.