Peroxiredoxin

in

human

brain

molecular

forms

cellular

distribution

and

association

with

Alzheimer

's

disease

pathology

Peroxiredoxin

is

an

antioxidant

enzyme

and

is

the

member

of

the

peroxiredoxin

family

Using

two-dimensional

electrophoresis

and

Western

blotting

we

have

shown

for

the

first

time

that

in

human

control

and

brain

tissue

of

patient

's

with

Alzheimer

's

disease

-LRB-

AD

-RRB-

this

enzyme

exists

as

three

major

and

five

minor

forms

with

pIs

from

to

Using

specific

cellular

markers

we

have

shown

that

peroxiredoxin

is

present

in

astrocytes

with

very

low

levels

in

neurons

but

not

detectable

in

microglia

or

oligodendrocytes

In

control

brains

there

was

a

very

low

level

of

peroxiredoxin

staining

in

astrocytes

that

was

confined

to

a

halo

around

the

nucleus

In

AD

there

were

marked

increases

in

the

number

and

staining

intensity

of

peroxiredoxin

positive

astrocytes

in

both

gray

and

white

matter

in

the

midfrontal

cortex

cingulate

hippocampus

and

amygdala

Confocal

microscopy

using

antibodies

to

Aβ

peptide

tau

and

peroxiredoxin

showed

that

peroxiredoxin

positive

astrocytes

are

closely

involved

with

diffuse

plaques

and

to

a

lesser

extent

with

neuritic

plaques

suggesting

that

plaques

are

producing

reactive

oxygen

species

There

appeared

to

be

little

astrocytic

response

to

tau

containing

neurons

Although

peroxiredoxin

positive

astrocytes

were

seen

to

make

multiple

contacts

with

tau

positive

neurons

there

was

no

intraneuronal

colocalization

In

brain

tissue

of

patients

with

AD

many

blood

vessels

exhibited

peroxiredoxin

staining

that

appeared

to

be

due

to

the

astrocytic

foot

processes

These

results

suggest

that

oxidative

stress

conditions

exist

in

AD

and

that

peroxiredoxin

is

an

important

antioxidant

enzyme

in

human

brain

defenses

Introduction

Alzheimer

's

disease

-LRB-

AD

-RRB-

is

a

neurodegenerative

disorder

characterized

by

the

progressive

decline

in

memory

language

cognition

and

motor

functions

The

neuropathological

hallmarks

of

AD

are

the

accumulation

of

extracellular

amyloid

plaques

containing

the

amyloid

beta

peptide

-LRB-

Aβ

-RRB-

and

intraneuronal

neurofibrillary

tangles

containing

hyperphosphorylated

microtubule-associated

protein

tau

Although

the

initiating

molecular

event

-LRB-

s

-RRB-

in

AD

are

not

known

oxidative

stress

produced

by

the

generation

of

reactive

oxygen

species

-LRB-

ROS

-RRB-

appears

to

be

a

fundamental

process

contributing

to

the

neuropathophysiology

-LSB-

-RSB-

Whether

oxidative

stress

has

a

role

in

the

cause

or

affect

in

AD

has

been

debated

for

many

years

but

recent

research

has

suggested

that

it

occurs

prior

to

the

onset

of

symptoms

-LSB-

-RSB-

The

histopathological

evidence

for

oxidative

stress

in

AD

includes

lipid

protein

and

DNA

oxidation

There

is

evidence

to

suggest

that

oxidative

stress

is

generated

peripherally

-LSB-

-RSB-

with

the

brain

being

particularly

susceptible

but

there

is

also

a

body

of

evidence

to

suggest

that

amyloid

β

peptide

-LRB-

-RRB-

may

be

linked

to

lipid

peroxidation

-LSB-

-RSB-

which

may

then

be

linked

to

the

phosphorylation

of

tau

-LSB-

-RSB-

Two

reactive

products

of

lipid

peroxidation

are

the

production

of

four

hydroxynonenal

and

acrolein

-LSB-

-RSB-

which

have

been

shown

to

disrupt

membrane

asymmetry

-LSB-

-RSB-

and

may

be

involved

in

the

conformational

changes

to

tau

promoting

the

formation

of

neurofibrillary

tangles

-LSB-

-RSB-

Oxidative

damage

occurs

as

part

of

the

aging

process

but

it

occurs

at

a

much

greater

rate

in

most

neurodegenerative

diseases

-LSB-

-RSB-

The

hallmark

of

protein

oxidation

is

the

generation

of

protein

carbonyls

which

are

markedly

elevated

in

AD

-LSB-

-RSB-

There

is

overwhelming

evidence

to

implicate

oxidative

stress

in

the

pathogenesis

of

AD

and

this

is

most

likely

an

early

event

in

the

disease

process

Less

is

known

however

of

how

the

brain

responds

to

oxidative

insults

There

are

a

number

of

cellular

antioxidant

defenses

whose

primary

role

is

to

convert

the

ROS

into

unreactive

compounds

These

defense

mechanisms

include

superoxide

dismutase

which

works

together

with

selenium-dependent

glutathione

peroxidase

catalase

and

more

recently

the

peroxiredoxin

family

These

enzymes

transform

the

superoxide

radical

to

hydrogen

peroxide

which

in

turn

is

converted

to

water

The

distribution

and

changes

in

expression

of

the

antioxidant

enzymes

have

not

been

well

studied

as

the

other

aspects

of

the

disease

process

Cellular

selenium-dependant

glutathione

peroxidase

-LRB-

-RRB-

has

long

been

regarded

as

the

major

cellular

antioxidant

enzyme

but

there

are

conflicting

reports

regarding

the

changes

in

activities

of

this

enzyme

in

AD

Early

reports

suggest

that

there

is

no

change

in

activities

in

AD

brains

-LSB-

-RSB-

while

other

researchers

suggest

that

there

is

a

decrease

in

activity

-LSB-

-RSB-

There

are

no

reports

indicating

compensatory

increase

in

activity

however

cell

models

overexpressing

are

more

resistant

to

Aβ-mediated

toxicity

suggesting

that

increased

levels

of

is

protective

-LSB-

-RSB-

There

is

considerable

evidence

to

implicate

Aβ

toxicity

and

the

generation

of

ROS

and

in

particular

hydrogen

peroxide

as

key

steps

in

contributing

to

the

neurotoxicity

in

AD

We

have

previously

reported

that

peroxiredoxin

also

termed

Cys-peroxiredoxin

or

nonselenium

glutathione

peroxidase

is

upregulated

in

Parkinson

's

disease

-LRB-

PD

-RRB-

-LSB-

-RSB-

This

enzyme

has

no

amino

acid

homology

to

any

known

sequence

of

the

glutathione

peroxidase

enzymes

and

has

both

antioxidant

and

phospholipase

activity

and

can

reduce

hydroxyperoxides

-LSB-

-RSB-

Given

that

the

brain

is

particularly

vulnerable

to

oxidative

damage

due

to

its

high

energy

and

oxygen

requirements

high

levels

of

transition

metals

and

peroxidizable

fatty

acids

suggests

that

peroxiredoxin

could

play

a

key

antioxidant

role

in

neuroprotection

There

are

six

members

of

the

peroxiredoxin

family

at

this

stage

with

peroxiredoxins

being

the

members

while

peroxiredoxin

is

the

sole

member

-LSB-

-RSB-

This

enzyme

is

bifunctional

with

of

its

function

as

a

peroxidase

and

the

remainder

as

a

phospholipase

Peroxiredoxin

has

not

specifically

been

examined

in

brain

tissue

of

AD

patients

and

the

aims

of

this

study

are

to

examine

the

molecular

forms

cellular

distribution

and

association

with

AD

pathology

This

study

focused

on

the

cingulate

amygdala

hippocampus

and

cortical

regions

in

six

AD

and

six

control

brains

Materials

and

methods

Antibodies

Primary

antibodies

Peroxiredoxin

-LRB-

rabbit

-RRB-

was

obtained

from

Antibody

Technology

Australia

Pty

Ltd

Adelaide

Australia

Tau

-LRB-

mouse

-RRB-

was

obtained

from

Novacastra

Laboratories

Ltd

Newcastle

UK

Tau

-LRB-

goat

-RRB-

was

obtained

from

Santa

Cruz

Biotechnology

Inc.

California

USA

Amyloid

beta

-LRB-

mouse

-RRB-

was

obtained

from

Novacastra

Laboratories

Ltd

Newcastle

UK

Glial

fibrillary

acidic

protein

-LRB-

GFAP

-RRB-

-LRB-

mouse

-RRB-

was

obtained

from

Novacastra

Laboratories

Ltd

Newcastle

UK

Human

neuronal

protein

HuC/HuD

-LRB-

mouse

-RRB-

was

obtained

from

Molecular

Probes

Inc.

Eugene

USA

Human

HLA

-LRB-

-RRB-

-LRB-

mouse

-RRB-

was

obtained

from

Dakocytomation

Denmark

A/S

Myelin

basic

protein

-LRB-

MBP

-RRB-

-LRB-

mouse

-RRB-

was

obtained

from

Novacastra

Laboratories

Ltd

Newcastle

UK

Secondary

antibodies

Donkey

anti

sheep

was

obtained

from

Jackson

Immunoresearch

West

Grove

USA

Donkey

antirabbit

and

were

obtained

from

Jackson

Immunoresearch

West

Grove

USA

Goat

antimouse

Alexa

was

obtained

from

Molecular

Probes

Inc.

Eugene

USA

Goat

antirabbit

alkaline

phosphatase

was

obtained

from

Sigma

Chemical

Company

St

Louis

USA

Brain

tissue

The

brain

tissue

was

obtained

from

the

National

Health

and

Medical

Research

Council

South

Australian

Brain

Bank

Brains

were

removed

at

autopsy

from

clinically

diagnosed

AD

cases

and

confirmed

pathologically

postmortem

The

control

cases

were

obtained

from

people

who

died

of

unrelated

causes

without

diagnosed

neurological

pathology

that

was

confirmed

postmortem

The

brains

were

bisected

and

one

half

snap-frozen

at

°C

and

the

other

immersion

fixed

in

buffered

formaldehyde

Tissue

blocks

were

processed

and

embedded

in

paraffin

and

μm

sections

were

cut

and

mounted

onto

gelatin-coated

slides

Table

provides

a

list

of

the

brain

regions

examined

and

the

case

details

Table

of

cases

used

in

cell

countingCase

numberSexAge

-LRB-

years

-RRB-

DiagnosisRegionPMI

-LRB-

h

-RRB-

Braak

stageNIA

A

H

MC

A

H

MC

A

H

MC

A

H

A

H

A

H

A

H

A

H

A

H

A

H

A

H

A

H

postmortem

index

AD

Alzheimer

's

disease

NIA

Reagan

high

or

low

likely-hood

of

AD

M

male

F

female

C

cingulate

A

amygdala

H

hippocampus

MC

midfrontal

cortex

C

control

ADL

adeno

lung

carcinoma

Unk

unknown

CF

cardiac

failure

SS

septic

shock

DM

disseminated

malignancy

HVD

hypertensive

vascular

disease

Brain

homogenate

Frozen

tissue

from

midfrontal

cortex

of

three

AD

brains

and

three

control

cases

were

homogenized

in

mM

Tris

mM

EDTA

sodium

azide

μl/ml

pepstatin

and

leupeptin

and

mM

of

phenylmethylsulfonyl

fluoride

Five

milliliters

of

homogenization

buffer

was

used

per

gram

of

brain

tissue

and

homogenized

using

six

passes

of

a

motorized

Wheaton

Teflon

pestle

tissue

grinder

Homogenates

were

centrifuged

-LRB-

g

-RRB-

for

min

to

remove

particulate

matter

and

snap-frozen

at

°C

Table

provides

the

details

of

these

cases

Table

of

cases

used

for

PAGE

and

Western

blotsCase

numberSexAge

-LRB-

years

-RRB-

DiagnosisRegionPMI

-LRB-

h

-RRB-

Braak

stageNIA

postmortem

index

AD

Alzheimer

's

disease

F

female

M

male

MC

midfrontal

cortex

C

control

HVD

hypertensive

vascular

disease

ABD

anoxic

brain

damage

SS

septic

shock

Sample

preparation

Aliquots

-LRB-

μl

-RRB-

of

Alzheimer

's

and

control

brain

homogenates

underwent

a

clean

up

as

per

the

Amersham

Biosciences

protocol

The

samples

were

then

resolubilized

in

μl

of

TUC

pH

buffer

containing

M

urea

M

thiourea

CHAPS

dithiothreitol

IPG

buffer

pH

Samples

were

then

assayed

for

total

protein

content

Prior

to

rehydration

of

the

immobilized

pH

gradient

strips

trace

amounts

of

bromophenol

blue

was

added

to

samples

Table

provides

a

list

of

cases

used

in

the

electrophoresis

polyacrylamide

gel

electrophoresis

-LRB-

PAGE

-RRB-

Isoelectric

focusing

Isoelectric

focusing

was

performed

on

cm

immobilized

pH

gradient

strips

-LRB-

pH

Amersham

Pharmacia

-RRB-

using

an

IPGphor

-LRB-

Amersham

Pharmacia

-RRB-

isoelectric

focusing

unit

Samples

were

applied

in

gel

rehydration

-LRB-

V

overnight

-RRB-

using

M

urea

M

thiourea

CHAPS

dithiothreitol

pharmalyte

pH

with

a

trace

of

bromophenol

blue

as

the

re-swell

buffer

with

a

total

loading

volume

of

μl

including

sample

Stained

gels

received

a

μg

protein

load

and

Western

blotting

gels

received

a

μg

load

IEF

was

performed

for

kV

h

-LRB-

V

min

V

min

V

min

V

h

V

hold

to

end

-RRB-

SDS

PAGE

SDS

PAGE

was

performed

using

acrylamide

gels

Immobilized

pH

gradient

strips

were

incubated

in

SDS

equilibration

buffer

containing

w/v

dithiothreitol

for

min

followed

by

SDS

equilibration

buffer

containing

w/v

iodoacetamide

for

min

Strips

were

then

placed

above

the

second

dimension

gels

and

overlaid

with

low

melting

point

agarose

in

Tris

glycine

gel

running

buffer

Electrophoresis

was

performed

at

V

for

h

at

°C

Protein

gels

were

visualized

using

SYPRO

ruby

fluorescent

stain

-LRB-

incubated

for

h

-RRB-

and

scanned

using

a

Typhoon

Variable

Mode

Imager

-LRB-

Amersham

Biosciences

-RRB-

Western

blotting

Separated

proteins

were

transferred

onto

low

fluorescence

PVDF

membrane

by

semi-dry

electroblotting

at

for

h

After

blocking

the

membranes

were

incubated

in

affinity-purified

rabbit

antiperoxiredoxin

antibodies

-LRB-

-RRB-

overnight

at

°C

followed

by

goat

antirabbit

alkaline

phosphatase

antibodies

-LRB-

-RRB-

-LRB-

Sigma

-RRB-

for

h

and

visualized

using

ECF

substrate

and

a

Typhoon

Variable

Mode

Imager

Immunohistochemistry

Localization

of

peroxiredoxin

Immunohistochemical

staining

of

AD

and

control

tissue

was

carried

out

as

previously

described

-LSB-

-RSB-

The

cellular

distribution

of

peroxiredoxin

was

compared

with

the

staining

obtained

with

antibodies

to

specific

cellular

markers

Antibodies

to

GFAP

-LRB-

astrocytes

-RRB-

HLA

-LRB-

-RRB-

-LRB-

microglia

-RRB-

MBP

-LRB-

oligodendrocytes

-RRB-

and

HuC/HuD

-LRB-

neurons

-RRB-

were

used

to

map

the

distribution

of

peroxiredoxin

Colocalization

of

peroxiredoxin

Aβ

and

Tau

Brain

sections

were

incubated

with

rabbit

antiperoxiredoxin

mouse

anti

Aβ

peptide

and

goat

anti

tau

antibodies

overnight

and

detected

with

secondary

antibodies

conjugated

to

fluorescent

fluorophores

Alexa

and

respectively

Sections

were

examined

using

a

Bio-Rad

Confocal

laser

scanning

microscope

and

Bio-Rad

software

package

as

previously

described

-LSB-

-RSB-

Cell

counting

Cell

counting

was

carried

out

blind

on

masked

sections

and

peroxiredoxin

positive

astrocytes

were

counted

at

magnification

with

a

graticule

eye

piece

-LRB-

-RRB-

using

an

Olympus

microscope

on

brain

tissue

from

six

AD

and

six

control

cases

In

each

case

five

regions

were

randomly

selected

to

give

a

uniform

representation

of

the

white

and

gray

matter

in

the

cingulate

hippocampus

amygdala

and

midfrontal

cortex

The

number

of

positive

cells

from

each

of

the

five

areas

was

averaged

and

divided

by

the

area

of

the

graticule

eyepiece

and

expressed

as

The

data

was

then

analyzed

using

a

one-way

ANOVA

that

examined

the

interactions

listed

in

Table

The

significance

was

determined

at

level

of

probability

Table

examined

in

cell

counting

dataInteractions

examinedF

valueCritical

F

-LRB-

P

-RRB-

Significance

-LRB-

-RRB-

Are

there

differences

in

AD

-LRB-

C

A

H

MC

-RRB-

gray

matter

cell

counts

NSAre

there

differences

in

AD

-LRB-

C

A

H

MC

-RRB-

white

matter

cell

counts

NSAre

there

differences

in

control

-LRB-

C

A

H

MC

-RRB-

gray

matter

cell

counts

NSAre

there

differences

in

control

-LRB-

C

A

H

MC

-RRB-

white

matter

cell

counts

NSAre

there

differences

between

gray

and

white

matter

cell

counts

in

AD

tissue

SAre

there

differences

between

gray

and

white

matter

cell

counts

in

control

tissue

SAre

there

differences

in

gray

matter

cell

counts

between

control

and

AD

tissue

SAre

there

differences

in

white

matter

cell

counts

between

control

and

AD

tissue

SAD

Alzheimer

's

disease

NS

not

significant

S

significant

C

cingulate

A

amygdala

H

hippocampus

MC

midfrontal

cortex

Results

Molecular

forms

of

peroxiredoxin

in

human

brain

We

have

previously

shown

that

this

antibody

is

specific

for

a

kDa

monomer

in

rat

lung

and

human

brain

and

the

N-terminal

sequence

is

identical

to

peroxiredoxin

-LSB-

-RSB-

Two-dimensional

PAGE

and

Western

blotting

of

AD

and

control

brain

homogenates

indicated

that

this

protein

exists

as

three

major

and

five

minor

forms

as

shown

in

Fig.

d.

Although

the

loadings

were

consistent

the

five

minor

forms

could

not

be

detected

on

all

gels

For

comparison

the

spots

were

labeled

-LRB-

Fig.

-RRB-

Fig.

b

A

two-dimensional

PAGE

gel

and

blot

of

AD

brain

tissue

-LRB-

Case

-RRB-

and

a

corresponding

control

brain

gel

and

blot

panels

-LRB-

c

d

-RRB-

-LRB-

Case

-RRB-

pI

is

indicated

between

the

gel

and

blots

and

the

molecular

weight

of

the

standards

-LRB-

Std

-RRB-

in

kilo

Daltons

is

shown

on

the

Y-axis

The

gels

were

stained

with

SYPRO

ruby

fluorescent

stain

The

box

indicates

the

location

of

the

molecular

forms

of

peroxiredoxin

Some

spots

are

shown

on

the

blots

to

indicate

the

range

of

peroxiredoxin

pIsFig

composite

figure

at

higher

magnification

showing

the

range

of

molecular

forms

in

three

cases

of

AD

tissue

-LRB-

a

and

-RRB-

and

three

cases

of

control

tissue

-LRB-

b

and

-RRB-

The

range

of

molecular

forms

is

shown

but

not

all

forms

are

present

in

all

cases

Although

there

are

slight

variations

between

blots

the

respective

pIs

are

as

follows

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

The

respective

pIs

of

the

eight

forms

are

and

The

three

major

forms

were

and

and

the

average

pIs

of

these

were

and

for

AD

tissue

and

and

for

control

tissue

These

major

forms

were

consistent

in

all

homogenates

The

intensity

of

the

three

major

forms

was

analyzed

using

the

Typhoon

imager

and

the

intensity

of

spots

and

was

reversed

in

the

AD

and

control

homogenates

-LRB-

Fig.

-RRB-

Minor

forms

and

were

present

in

AD

homogenates

but

absent

in

the

control

tissue

while

minor

form

was

present

in

control

tissue

but

absent

in

AD

tissue

-LRB-

Fig.

-RRB-

Cellular

distribution

of

peroxiredoxin

in

control

and

AD

brain

tissue

Using

immunohistochemical

markers

for

specific

human

brain

cell

types

we

have

shown

that

peroxiredoxin

is

abundant

in

reactive

astrocytes

-LRB-

Fig.

c

-RRB-

in

AD

tissue

and

present

in

very

low

levels

in

neurons

-LRB-

Fig.

l

-RRB-

We

were

not

able

to

detect

peroxiredoxin

in

either

microglia

-LRB-

Fig.

f

-RRB-

or

oligodendrocytes

-LRB-

Fig.

i

-RRB-

Fig.

localization

of

peroxiredoxin

-LRB-

-RRB-

with

GFAP

-LRB-

astrocyte

marker

-RRB-

-LRB-

a

b

GFAP

c

merged

image

bar

μM

-RRB-

-LRB-

microglia

marker

-RRB-

-LRB-

d

e

f

merged

image

bar

μM

-RRB-

MBP

-LRB-

oligodendrocyte

marker

-RRB-

-LRB-

g

h

MBP

i

merged

image

bar

μM

-RRB-

Hu

-LRB-

neuronal

marker

-RRB-

-LRB-

j

k

Hu

l

merged

image

bar

μM

-RRB-

Cell

localization

was

performed

using

AD

tissue

There

was

a

marked

contrast

between

the

staining

of

control

and

reactive

astrocytes

in

AD

Reactive

astrocytes

in

AD

were

hypertrophied

with

abundant

staining

of

peroxiredoxin

while

in

control

astrocytes

the

staining

was

confined

to

a

halo

around

the

nucleus

-LRB-

Fig.

-RRB-

The

increased

staining

of

peroxiredoxin

was

seen

in

all

regions

examined

and

in

both

gray

and

white

matter

but

the

staining

was

not

evenly

distributed

and

some

regions

on

the

same

section

were

heavily

labeled

but

adjacent

regions

showed

little

staining

-LRB-

Fig.

-RRB-

Fig.

immersion

image

-LRB-

-RRB-

of

typical

astrocyte

seen

in

AD

cortex

-LRB-

a

-RRB-

and

in

control

cortex

-LRB-

b

-RRB-

Fig.

immunohistochemistry

-LRB-

DAB

substrate

-RRB-

of

peroxiredoxin

staining

of

astrocytes

in

AD

gray

matter

-LRB-

a

-RRB-

AD

white

matter

-LRB-

b

-RRB-

control

tissue

gray

matter

-LRB-

c

-RRB-

and

control

tissue

white

matter

-LRB-

d

-RRB-

All

images

at

magnification

Cell

counting

In

AD

and

control

tissue

peroxiredoxin

was

colocalized

with

GFAP

indicating

that

the

upregulation

is

occurring

in

astrocytes

which

is

similar

to

what

was

found

in

PD

and

dementia

with

Lewy

bodies

-LSB-

-RSB-

Cell

counting

was

conducted

on

midfrontal

cortex

cingulate

amygdala

and

hippocampus

brain

regions

and

in

gray

and

white

matter

to

determine

if

specific

areas

or

matter

type

were

specifically

affected

These

are

summarized

in

Fig.

A

one-way

ANOVA

was

performed

comparing

the

cell

counts

between

AD

and

control

tissue

in

different

regions

by

tissue

and

matter

type

The

results

of

the

interactions

examined

are

shown

in

Table

Fig.

bar

chart

showing

the

range

of

cell

counts

in

different

brain

regions

in

gray

and

white

matter

in

control

tissue

and

in

AD

tissue

WM

white

matter

GM

gray

matter

Cing

cingulate

Amy

amygdala

MFC

midfrontal

cortex

Hip

hippocampus

Counts

are

shown

as

mean

SE

N

in

all

cases

Colocalization

of

peroxiredoxin

and

AD

pathology

The

major

defining

pathology

in

AD

is

the

presence

of

extracellular

plaques

and

intracellular

hyperphosphorylated

tau

in

the

form

of

tangles

Plaques

are

present

in

brain

tissue

at

different

stages

of

development

ranging

from

diffuse

plaques

to

fibrillar

and

neuritic

plaques

It

is

generally

considered

that

diffuse

plaques

do

not

contain

tau

but

neuritic

plaques

contain

both

Aβ

and

tau

Colocalization

of

peroxiredoxin

and

the

Aβ

peptide

a

major

component

of

plaques

and

tau

was

carried

out

to

determine

if

peroxiredoxin

positive

astrocytes

were

associated

with

AD

pathology

Tau

pathology

Tau

deposits

in

the

form

of

neurofibrillary

tangles

were

observed

in

many

neurons

and

neurites

and

were

often

surrounded

by

peroxiredoxin

astrocytes

which

made

multiple

contacts

-LRB-

Fig.

c

-RRB-

Although

astrocytes

made

many

contacts

with

neurons

most

neurons

had

very

low

levels

of

peroxiredoxin

and

it

did

not

appear

to

be

upregulated

or

to

be

colocalized

with

tau

in

AD

Fig.

c

Confocal

localization

of

tau

within

neurons

-LRB-

a

-RRB-

and

peroxiredoxin

positive

astrocytes

-LRB-

b

-RRB-

and

-LRB-

c

-RRB-

the

merged

image

in

AD

brain

tissue

-LRB-

bar

μM

-RRB-

d

f

Confocal

localization

of

Aβ

peptide

-LRB-

d

Alexa

green

-RRB-

and

peroxiredoxin

positive

astrocytes

-LRB-

e

-RRB-

and

-LRB-

f

-RRB-

the

merged

image

in

a

diffuse

plaque

in

AD

brain

tissue

-LRB-

bar

μM

-RRB-

g

j

Confocal

localization

of

Aβ

peptide

-LRB-

Alexa

green

-RRB-

and

peroxiredoxin

positive

astrocytes

-LRB-

blue

-RRB-

in

four

different

plaques

in

AD

brain

tissue

-LRB-

bar

μM

-RRB-

k

Immunohistochemical

staining

-LRB-

DAB

substrate

-RRB-

showing

peroxiredoxin

staining

of

astrocytes

and

astrocyte

foot

processes

in

the

walls

of

blood

vessels

in

the

midfrontal

cortex

in

Alzheimer

's

disease

-LRB-

-RRB-

Arrows

indicate

peroxiredoxin

positive

astrocytic

processes

Plaques

Diffuse

plaques

contained

strongly

stained

aggregations

of

Aβ

peptide

Peroxiredoxin

staining

was

confined

to

astrocytes

and

their

projections

that

were

in

close

proximity

to

plaques

-LRB-

Fig.

h

-RRB-

Some

astrocytes

were

observed

within

plaques

but

generally

the

astrocyte

cell

bodies

were

on

the

periphery

with

their

processes

projecting

into

the

plaques

Some

plaques

contained

the

beginnings

of

a

dense

core

of

amyloid

and

took

on

a

wagon

wheel

appearance

-LRB-

Fig.

j

-RRB-

Diffuse

plaques

and

those

with

Aβ

beginning

to

aggregate

appeared

to

elicit

a

stronger

peroxiredoxin

astrocytic

response

than

those

with

a

dense

core

of

Aβ

Peroxiredoxin

staining

of

blood

vessels

In

many

regions

of

the

AD

brain

there

appeared

to

be

strong

peroxiredoxin

staining

in

the

walls

of

many

blood

vessels

which

could

be

seen

to

have

multiple

contacts

with

strongly

staining

activated

astrocytes

-LRB-

Fig.

-RRB-

Discussion

Peroxiredoxin

also

known

as

peroxiredoxin

or

non-selenium

glutathione

peroxidase

is

a

human

brain

antioxidant

enzyme

that

is

abundant

in

activated

astrocytes

and

present

in

low

levels

in

neurons

On

exposure

to

hydrogen

peroxide

the

N-terminal

peroxiredoxin

corresponding

to

Cys

is

readily

oxidized

but

as

yet

its

redox

partner

has

not

been

identified

Glutathione

lipoic

acid

and

cyclophilin

have

all

been

implicated

-LSB-

-RSB-

In

addition

to

its

peroxidase

activity

peroxiredoxin

also

has

phospholipase

activity

that

might

be

significant

in

the

brain

which

has

a

high

lipid

content

-LSB-

-RSB-

Peroxiredoxin

staining

of

astrocytes

is

markedly

elevated

in

many

brain

regions

in

PD

and

dementia

with

Lewy

bodies

-LSB-

-RSB-

We

now

show

that

it

is

upregulated

in

the

astrocytes

in

AD

and

describe

for

the

first

time

the

range

of

molecular

forms

of

peroxiredoxin

in

human

brain

in

both

control

and

AD

tissue

Three

major

variants

and

five

minor

forms

were

identified

with

pIs

ranging

from

to

There

were

three

major

differences

in

the

peroxiredoxin

variants

between

control

and

AD

tissue

The

AD

tissue

had

the

two

more

acidic

forms

and

that

were

not

present

in

control

tissue

the

intensity

of

the

major

forms

and

were

reversed

between

AD

and

control

tissue

and

minor

form

was

absent

in

AD

tissue

We

can

only

speculate

on

the

significance

of

these

findings

at

this

stage

but

different

forms

may

have

different

affinities

for

different

substrates

that

might

be

more

abundant

under

various

pathological

conditions

or

the

more

acidic

forms

are

the

result

of

oxidation

-LSB-

-RSB-

At

this

stage

we

can

not

determine

whether

these

differences

are

true

isoforms

or

post-translational

modifications

of

the

same

protein

but

would

suspect

the

latter

These

findings

are

in

agreement

with

two

previous

proteomic

studies

in

an

AD

mouse

model

and

in

human

brain

AD

tissue

which

reported

elevated

levels

of

peroxiredoxin

-LSB-

-RSB-

A

more

recent

study

using

and

cells

treated

with

Aβ

also

reported

increased

levels

of

peroxiredoxin

-LSB-

-RSB-

Aβ-treated

cells

contained

an

extra

acidic

form

which

was

suggested

to

be

the

result

of

oxidation

of

the

catalytic

cysteine

to

a

sulfenic

sulfinic

or

sulfonic

derivative

We

found

two

minor

acidic

forms

in

AD

tissue

which

is

also

consistent

with

this

report

Tissue

selection

is

also

a

variable

that

needs

to

be

considered

While

the

AD

tissue

had

all

the

hallmarks

of

AD

pathology

and

was

easy

to

define

age-matched

control

tissues

are

somewhat

more

variable

All

the

control

tissue

was

from

people

without

clinically

defined

AD

but

most

brains

in

this

age

group

usually

contain

some

neurodegenerative

pathology

The

age

span

of

the

two

groups

was

similar

years

for

the

AD

group

and

years

for

the

control

group

but

the

mean

age

for

the

control

group

was

years

older

We

feel

confident

that

although

some

control

tissue

may

have

had

some

elements

of

subclinical

AD

pathology

the

reduced

level

of

astrocyte

activation

and

the

clear

differences

in

the

cell

counting

would

indicate

that

all

the

control

tissues

were

not

experiencing

high

levels

of

oxidative

stress

as

compared

to

the

AD

tissue

Previously

we

had

shown

that

peroxiredoxin

was

abundant

in

astrocytes

in

PD

and

dementia

with

Lewy

body

tissue

-LSB-

-RSB-

We

have

now

used

cellular

markers

to

other

brain

cell

types

to

show

that

in

addition

to

astrocytes

there

is

a

low

level

in

the

cytoplasm

of

most

neurons

Unlike

astrocytes

neurons

do

not

appear

to

have

increased

peroxiredoxin

staining

in

AD

tissue

We

could

not

detect

any

peroxiredoxin

staining

in

microglia

or

oligodendrocytes

Cell

counting

of

peroxiredoxin

positive

astrocytes

indicated

that

there

was

a

marked

increase

in

both

the

gray

and

white

matter

in

AD

tissue

compared

with

control

tissue

Gray

matter

had

more

peroxiredoxin

positive

astrocytes

than

white

matter

in

both

AD

and

control

tissue

and

this

difference

was

more

pronounced

in

AD

tissue

with

F

values

of

and

respectively

Considering

that

the

major

pathology

-LRB-

plaques

and

tangles

-RRB-

is

located

in

the

gray

matter

it

would

be

expected

that

oxidative

stress

is

more

pronounced

in

this

area

Cell

counting

indicated

that

the

peroxiredoxin

positive

astrocyte

numbers

were

elevated

in

all

regions

examined

suggesting

that

the

insult

activating

astrocytes

is

wide

spread

and

not

confined

to

any

specific

region

in

either

the

gray

or

white

matter

Indeed

extracellular

plaques

were

observed

in

all

regions

examined

in

AD

tissue

Although

all

regions

examined

showed

astrocyte

activation

this

was

not

uniformly

distributed

through

all

areas

with

some

areas

showing

extensive

astrocyte

activation

while

adjacent

areas

were

similar

to

controls

There

are

two

defining

pathologies

in

AD

These

are

the

presence

of

extracellular

plaques

comprising

abundant

deposits

of

Aβ

peptides

which

later

aggregate

to

form

amyloid

-LSB-

-RSB-

and

intracellular

hyperphosphorylated

tau

aggregations

in

the

form

of

paired

helical

filaments

which

are

termed

tangles

-LSB-

-RSB-

The

two

well-described

forms

of

plaques

have

been

termed

diffuse

plaques

and

neuritic

or

senile

plaques

-LSB-

-RSB-

although

other

intermediate

forms

such

as

fibrillar

plaques

have

been

described

-LSB-

-RSB-

Diffuse

plaques

are

spherical

structures

of

less

dense

Aβ

deposits

and

are

considered

an

early

stage

of

plaque

formation

In

diffuse

plaques

aggregations

of

Aβ

exists

as

nonamyloid

deposits

but

as

the

plaque

progresses

the

Aβ

and

other

protein

aggregations

change

from

the

normal

conformation

into

a

β-sheet

structure

termed

amyloid

and

take

up

specific

dyes

like

Congo

red

and

thioflavin

-LSB-

-RSB-

These

plaques

do

not

generally

contain

tau

however

in

many

confocal

images

diffuse

plaques

contain

focal

points

of

tau

which

are

probably

enlarged

neurites

Neuritic

or

senile

plaques

contain

a

dense

core

of

amyloid

enriched

with

Aβ

peptide

with

a

corona

of

less

dense

amyloid

The

diffuse

plaques

appeared

to

elicit

a

stronger

astrocytic

response

based

on

the

level

of

peroxiredoxin

staining

than

the

neuritic

plaques

Our

images

suggest

that

once

the

Aβ

forms

amyloid

and

condenses

it

is

less

stimulating

toward

astrocytes

and

perhaps

the

formation

of

amyloid

is

a

protective

mechanism

to

inactivate

the

toxicity

of

soluble

Aβ

These

observations

are

consistent

with

the

findings

of

Kawaguchi-Niida

et

al.

who

reported

that

the

highly

reactive

carbonyl

crotonaldehyde

generated

during

lipid

peroxidation

is

localized

to

reactive

astrocytes

microglia

and

diffuse

plaques

but

undetectable

in

amyloid

cores

-LSB-

-RSB-

Although

the

activation

of

astrocytes

and

upregulation

of

peroxiredoxin

would

be

considered

a

protective

response

in

cell

culture

soluble

Aβ

has

been

shown

to

stimulate

nitric

oxide

synthetase

and

the

production

of

nitric

oxide

which

is

very

damaging

to

neurons

-LSB-

-RSB-

This

response

is

further

upregulated

by

other

inflammatory

cytokines

such

as

interleukin

1β

and

tumour

necrosis

factor-α

which

are

reported

to

be

released

from

astrocytes

-LSB-

-RSB-

At

this

stage

we

are

not

sure

of

the

net

benefit

to

neural

tissue

of

activated

astrocytes

with

elevated

levels

of

peroxiredoxin

concomitant

with

the

increased

production

of

nitric

oxide

Neurofibrillary

tangles

comprising

hyperphosphorylated

tau

were

observed

in

many

regions

in

AD

tissue

Two

antibodies

were

used

to

localize

tau

a

monoclonal

antibody

-LRB-

Novacastra

-RRB-

and

one

specific

for

paired

helical

filaments

-LRB-

Santa

Cruz

-RRB-

and

both

produced

a

similar

staining

pattern

Neurons

containing

tau

were

not

surrounded

by

activated

astrocytes

as

were

the

diffuse

plaques

and

to

a

lesser

extent

the

neuritic

plaques

suggesting

that

these

cells

are

not

secreting

toxic

or

activating

products

that

are

stimulating

to

astrocytes

In

PD

Lewy

bodies

contained

a

dense

core

of

peroxiredoxin

staining

suggesting

that

it

was

trying

to

detoxify

oxidative

stress

produced

by

Lewy

bodies

but

we

did

not

observe

any

interaction

between

tau

and

peroxiredoxin

in

AD

tissue

Although

there

is

considerable

evidence

to

implicate

oxidative

stress

in

the

hyperphosphorylation

of

tau

this

does

not

appear

to

upregulate

peroxiredoxin

in

tau

positive

neurons

In

some

regions

the

walls

of

blood

vessels

exhibited

strong

peroxiredoxin

staining

which

also

elicited

a

strong

astrocytic

response

in

surrounding

astrocytes

These

activated

astrocytes

had

many

processes

in

contact

with

the

endothelial

cells

Although

vascular

amyloid

deposits

were

seen

in

some

sections

and

is

a

key

feature

of

AD

-LSB-

-RSB-

the

upregulation

of

peroxiredoxin

appeared

to

occur

in

regions

without

distinct

Aβ

staining

Aβ

has

been

shown

to

induce

vascular

vasoconstriction

in

rat

skin

which

can

be

reversed

by

superoxide

dismutase

and

catalase

indicating

that

Aβ

is

capable

of

generating

ROS

within

blood

vessels

-LSB-

-RSB-

The

blood

brain

barrier

consists

of

the

capillary

endothelial

cells

and

the

foot

processes

of

the

astrocytes

and

the

fused

basal

lamina

of

both

cells

The

peroxiredoxin

staining

within

the

capillaries

appears

to

be

in

the

foot

processes

of

the

astrocytes

Whether

this

increased

staining

is

due

to

ROS

generated

from

within

neural

tissue

or

reflects

a

more

systemic

pathology

derived

from

the

circulation

is

unclear

In

conclusion

we

have

shown

for

the

first

time

the

range

of

peroxiredoxin

variants

which

are

either

isoforms

or

post-translational

modifications

in

human

brain

tissue

We

have

also

shown

that

peroxiredoxin

is

primarily

an

astrocytic

enzyme

with

very

low

levels

in

neurons

and

is

not

detectable

in

microglia

or

oligodendrocytes

This

enzyme

is

markedly

elevated

in

astrocytes

in

both

white

and

gray

matter

in

AD

Strongly

staining

peroxiredoxin

astrocytes

appear

to

be

involved

in

the

detoxification

of

diffuse

plaques

and

to

a

lesser

extent

in

neuritic

plaques

but

is

not

associated

with

tau

aggregations

within

neurons

From

this

work

and

previous

work

on

PD

and

dementia

with

Lewy

bodies

we

suggest

that

peroxiredoxin

is

a

major

antioxidant

enzyme

in

human

neural

tissue

B I O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O

peroxiredoxin 6
molecular forms
oxidative stress
astrocytes