How oxidative, metabolic and fibril stress effects Alzheimer's Disease.

1. George Perry
The 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

2. Collaborators
Case Western
Reserve University
Mark A. Smith
Lawrence 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 Petersen
Gemma Casadesus
Centro Biologia Molecular, Madrid
Jesús Avila
University of Maryland, Baltimore
Rudy Castellani
Universitat de Valencia
Jose Viña
University of Chile
Ricardo Maccioni
UTSA
Paula I. Moreira
University of Coimbra
University of Wisconsin - Madison
Craig Atwood
Justin Shenk
Catarina Oliveira
Masao Nakamura
University of Yamanashi
Akihiko Nunomura
Massimo Tabaton
Xinglong Wang
Hyoung-gon Lee
Maria Santos
Sonia Correia
Renato Santos
Cinvestav
Raúl Mena
Francisco Garcίa-Sierra
Siddhartha Mondragón-Rodrίguez
Clyde Phelix
Orkid Coskuner
Edwin Barea-Rodriguez
Texas A&M University
Ian Murray

3. 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.

4. Oxidative stress is an early event in
AD.
.
..
..
.
t
Glycation
Normal
Neuron
? Pre-NFT I-NFT E-NFT
FREE CARBONYLS
80HG
HNE
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

5. • ATP production
• key metabolites
• Ca2+ buffering
• free radicals/
ROS generation
• regulation of
PCD
Mitochondria are Critical
to Neuronal Function

6. 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
Wild type probe
Chimera probe
4977 bp deletion
Deleted mtDNANormal mtDNA
8482 10897
8454 10941
13475
13475
8454
8482/13460
13460
ACACAAACTACCACCTACCTCCCTCACCA
TTGGCAGCCTA GCATT
CAACAACCTATTTAGCTGTTCCCCAACCTT
TTCCTCCGACCCCCT
16,569 bp 11,592 bp
OHOH
4977 bp deletion contains ATPase subunit 8, subunit 6, cytochrome-c oxidase subunit III, and
NADH-coenzyme Q oxidoreductase subunit 3, 4 and 5.

7. Examination of
neurons in biopsy
specimens shows
normal mitochondria
(A), mitochondria with
broken cristae (B), and
lipofuscin with
vacuoles (C).
0
0.2
0.4
0.6
0.8
1
Numberperm
2
AD
Control
0
0.2
0.4
0.6
0.8
1
Sizeinm
2
0
2
4
6
8
10
Percentageofarea
Total
mitochondria
Normal
mitochondria
Brokencristae
mitochondria
Vacuolar
lipofuscin
Electrondense
lipofuscin
*
Mitochondria
0
0.2
0.4
0.6
0.8
1
Numberperm
2
AD
Control
0.6
0.8
1
m
2
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.

8. Mitochondria components are in autophagosomes

9. No Fusion Wild Type No Fission
Mitochondrial dynamics

10. Microtubules are reduced specifically in
pyramidal neurons.
Numbers of microtubules
decrease with normal aging
0
1
2
3
4
0-1
1-2
2-3
3-4
4-5
5-6
6-7
7-8
8-9
9-10
10-11
11-12
12-13
13-14
Number of microtubules/m²
Numberofneurons
0
1
2
3
4
0-1
1-2
2-3
3-4
4-5
5-6
6-7
7-8
8-9
9-10
10-11
11-12
12-13
13-14
Number of microtubules/m²
Control
AD -PHF
Control
AD -PHF
Number of microtubules /m2
#ofnon-pyramidalneurons#ofpyramidalneurons
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

11. Could the mitochondrial problem be related to reduced
axonal transport?
O -
2 2 O2
Fe
HH
mitochondria
lysosome
Alzheimer Disease
Normal
FeII
2

12. Catalytic redox activity is cytoplasmic and
increased in AD…
Alzheimer Control
0
5
10
15
20
25
30
35
control(n=5) AD (n=6)
Meandensity
Control (n=5) AD (n=6)
RelativeDensity
p=0.002
Pyramidal neurons AD
cases have significantly
greater levels of
cytoplasmic staining than
age-matched controls,
p=0.002.

13. Nucleic acid damage, as well as redox active
metals are cytosolic
8-hydroxyguanosine Redox active metals

14. 0
50
100
150
200
250
300
non-ox ox
RealtiveDenstiyof8OHGformation
tRNA
rRNA
Ribosomal RNA is 13 times more susceptible to
oxidation than transfer RNA

15. Conformation plays a major role in this binding
ability. After denaturation with formamide, the
iron-binding capacity is dramatically reduced.
0
20
40
60
80
100
120
native denatured
Meandensity
tRNA
rRNA

16. 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.

17. Rabbit reticulocyte ribosome in vitro translation assay
The Fenton reaction causes
RNA cleavage …
…with consequent decrease in
protein synthesis.

18. RNA is a dominant binding site for the redox active
metals released into the cytoplasm
O - -
-
2 2 O2
Fe
HH
mitochondria
lysosome
2
Fe(II)RNA2
RNA
Fe(III)OxRNA
O
2H O
OxRNA
Alzheimer Disease

19. Aβ reduces
copper-induced
oxidation
Normal
B
Ratio of Cu2+ to Ab1-42 (Cu2+/Ab1-42)
0
2
4
6
8
10
12
14
0 1 2 3 4
ascorbate-oxidaseactivity
(M/min)
0
5
10
15
20
25
30
0 1 2 3 4
HNE(M)
Ab1-42 + Cu2+
Cu2+
A
B
Ratio of Cu2+ to Ab1-42 (Cu2+/Ab1-42)
0
2
4
6
8
10
12
14
0 1 2 3 4
ascorbate-oxidaseactivity
(M/min)
B
Ratio of Cu2+ to Ab1-42 (Cu2+/Ab1-42)
0
2
4
6
8
10
12
14
0 1 2 3 4
ascorbate-oxidaseactivity
(M/min)
0
5
10
15
20
25
30
0 1 2 3 4
HNE(M)
Ab1-42 + Cu2+
Cu2+
A
0
5
10
15
20
25
30
0 1 2 3 4
HNE(M)
Ab1-42 + Cu2+
Cu2+
0
5
10
15
20
25
30
0 1 2 3 4
HNE(M)
Ab1-42 + Cu2+
Cu2+
A

20. 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 binding
Cu binding

21. Oxidative damage (8OHG, blue) decreases with increased
amyloid - b (brown)
17 yr.
61 yr.
31 yr.
0
5
10
15
20
0 10 20 30 40 50 60 70
Age
Neuronal8
0
5
10
15
20
0-9 y 10-19 y 20-29 y 30 y & over
Age Groups
Neuronal8OHG
0
1
2
3
%AmyloidBurden
0
5
10
15
20
25
0 1 2 3
% Amyloid Burden
Neuronal8OHG
*
**
†
(n=5) (n=6) (n=5) (n=6)
a
b
c
In vivo

22. 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 ApoE4
AD lacking ApoE 4
Control

23. 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

25. Are Amyloid-b and t
Protective Responses
Against a Cauldron
of Oxidative
Stressors in
Alzheimer Disease?

32. Figure 1. Biotinylated τ binding to Alzheimer’s brain sections.

34. Figure 3. Solid-phase τ binding assay.

35. Figure 4. Antisera to βPP714-734 Immunostatin senile plaques.

36. 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.