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.

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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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Mitochondria0

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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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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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rons

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Control

AD -PHF

Control

AD -PHF

Number of microtubules /m2# of

non

-pyr

amid

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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Rea

ltive

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OH

G fo

rmat

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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

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idas

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tivity

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E (

M)

Ab1-42 + Cu2+

Cu2+

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B

Ratio of Cu2+ to Ab1-42 (Cu2+/Ab1-42)

02468101214

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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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asco

rbat

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idas

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tivity

(M

/min

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Ab1-42 + Cu2+

Cu2+

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Cu2+

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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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A g e G r o u p s

Neuronal 8OHG

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Neuronal 8O

HG

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†

( 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.