Towards

a

pathway

definition

of

Parkinson

's

disease

a

complex

disorder

with

links

to

cancer

diabetes

and

inflammation

We

have

previously

established

a

first

whole

genome

transcriptomic

profile

of

sporadic

Parkinson

's

disease

-LRB-

PD

-RRB-

After

extensive

brain

tissue-based

validation

combined

with

cycles

of

iterative

data

analysis

and

by

focusing

on

the

most

comparable

cases

of

the

cohort

we

have

refined

our

analysis

and

established

a

list

of

highly

dysregulated

priority

genes

that

are

considered

to

form

the

core

of

the

diseased

Parkinsonian

metabolic

network

The

substantia

nigra

pathways

now

under

scrutiny

contain

more

than

genes

whose

association

with

PD

is

known

from

the

literature

Of

those

more

than

genes

belong

to

the

highly

significantly

dysregulated

group

identified

in

our

dataset

Apart

from

the

complete

list

of

priority

genes

we

present

pathways

revealing

PD

hub

as

well

as

peripheral

network

genes

The

latter

include

Lewy

body

components

or

interact

with

known

PD

genes

Biological

associations

of

PD

with

cancer

diabetes

and

inflammation

are

discussed

and

interactions

of

the

priority

genes

with

several

drugs

are

provided

Our

study

illustrates

the

value

of

rigorous

clinico-pathological

correlation

when

analysing

high-throughput

data

to

make

optimal

use

of

the

histopathological

phenome

or

morphonome

which

currently

serves

as

the

key

diagnostic

reference

for

most

human

diseases

The

need

for

systematic

human

tissue

banking

following

the

highest

possible

professional

and

ethical

standard

to

enable

sustainability

becomes

evident

Introduction

Our

understanding

of

Parkinson

's

disease

-LRB-

PD

-RRB-

is

largely

incomplete

However

the

pace

of

discovery

in

this

field

is

rapidly

accelerating

It

took

more

than

years

for

the

key

region

of

neuronal

damage

the

substantia

nigra

to

be

identified

-LSB-

-RSB-

and

it

took

almost

years

for

the

first

disease-causing

mutation

to

be

discovered

-LSB-

-RSB-

Not

even

years

later

the

first

whole

genome

transcriptome

analysis

had

been

performed

-LSB-

-RSB-

and

a

number

of

other

microarray

studies

focusing

on

known

sequences

were

carried

out

-LRB-

e.g.

-LSB-

-RSB-

-RRB-

We

now

provide

the

complete

list

of

highly

dysregulated

PD

nigral

genes

derived

from

a

brain

tissue-validated

whole

genome

expression

microarray

data

set

In

addition

predicted

interactions

of

a

number

of

these

genes

are

reported

as

potential

drug

targets

We

would

like

to

emphasise

that

the

neurohistological

validation

that

is

so

crucial

for

our

work

and

which

has

already

led

to

the

identification

of

two

novel

Lewy

body

components

predicted

on

the

basis

of

this

dataset

-LSB-

-RSB-

could

not

have

been

performed

without

generous

brain

donations

In

addition

the

iterative

analysis

performed

combining

histological

phenome

-LRB-

morphonome

-RRB-

data

and

clinical

criteria

within

silico

data

mining

would

not

have

been

possible

without

significant

advances

in

computing

notably

virtual

machine

technology

It

is

readily

apparent

that

a

publication

of

this

format

requires

the

Internet

as

it

would

not

have

been

possible

to

publish

its

almost

hyperlinked

files

on

paper

which

are

provided

as

electronic

supplemental

material

PD

is

a

severely

disabling

neurodegenerative

disorder

second

in

frequency

only

to

Alzheimer

's

disease

and

has

a

significant

socio-economic

impact

Unlike

in

Alzheimer

's

disease

however

the

brain

region

taking

the

brunt

of

the

disease

process

is

rather

well

circumscribed

In

addition

there

is

widespread

consensus

on

diagnostic

criteria

both

clinically

and

neuropathologically

-LRB-

-RRB-

This

is

at

least

in

part

due

to

the

fact

that

the

leading

motor

symptoms

are

less

complex

and

easier

to

recognize

and

define

than

the

clinical

signs

in

disorders

mainly

affecting

higher

brain

functions

such

as

cognition

Furthermore

there

is

symptomatic

treatment

for

PD

pointing

to

key

pathways

involved

All

these

are

important

prerequisites

when

working

with

high

throughput

technologies

such

as

microarrays

which

require

precise

tissue

sampling

because

the

procedures

employed

are

both

laborious

and

expensive

Major

known

pathways

involved

in

PD

include

the

ubiquitin-proteasome

system

dysfunction

of

which

may

lead

to

abnormal

protein

deposition

mitochondrial

failure

and

decreased

expression

of

synaptic

proteins

-LSB-

-RSB-

Oxidative

stress

has

been

traditionally

implicated

in

the

aetiology

of

the

disease

but

the

changes

observed

could

be

secondary

The

concept

of

neuroinflammation

has

become

very

popular

recently

-LSB-

-RSB-

but

our

own

work

in

this

field

does

not

currently

support

a

role

for

microgliosis

as

a

driver

of

the

disease

process

-LSB-

-RSB-

The

effective

failure

of

recent

studies

employing

non-steroidal

anti-inflammatory

drugs

supports

this

notion

-LSB-

-RSB-

Thus

there

are

leads

and

popular

ideas

but

the

big

picture

of

PD

pathogenesis

is

still

missing

A

true

understanding

of

PD

and

its

subtypes

will

require

integrated

knowledge

from

several

system

biological

levels

ranging

from

genomics

to

proteomics

and

metabonomics

as

well

as

clinical

data

and

neuroimaging

Through

this

study

we

aim

to

contribute

a

validated

transcriptomic

data

layer

Materials

and

methods

Data

set

used

The

CEL

files

used

for

this

study

have

been

deposited

at

the

National

Center

for

Biotechnology

Information

Gene

Expression

Omnibus

-LRB-

-RRB-

with

GEO

Series

accession

number

-LRB-

scheduled

release

date

January

-RRB-

The

patient

samples

employed

have

been

described

previously

-LSB-

-RSB-

This

data

set

is

based

on

Affymetrix

and

gene

chips

set

and

has

been

extensively

validated

over

a

period

of

years

using

qRT-PCR

immunocytochemistry

and

in

situ

hybridisation

to

cellularly

back-map

sequences

of

interest

-LSB-

-RSB-

Data

analysis

procedures

employed

In

silico

analyses

were

performed

with

the

help

of

programme

packages

from

different

suppliers

Our

initial

microarray

data

analysis

was

performed

using

ArrayAssist

-LRB-

Stratagene

-RRB-

but

there

were

intermittent

problems

with

some

versions

of

the

software

-LRB-

-RRB-

We

have

repeated

our

analysis

using

newer

-LRB-

Linux

and/or

Windows

-RRB-

and

the

latest

version

of

this

program

-LRB-

ArrayAssist

for

Windows

Stratagene

-RRB-

In

addition

we

have

reproduced

our

results

using

an

independent

software

package

for

microarray

analysis

PathwayStudio

The

-LRB-

GC

-RRB-

RMA

algorithm

which

has

become

a

gold

standard

for

Affymetrix

microarray

normalisation

was

applied

in

all

cases

In

addition

PathwayAssist

-LRB-

Ariadne

-RRB-

and

Pathway

Architect

-LRB-

Stratagene

-RRB-

were

employed

during

phases

of

our

work

For

ease

of

use

most

software

installations

were

performed

in

virtual

machines

-LRB-

VMware

Workstation

and

Fusion

respectively

-RRB-

and

two

virtual

machines

were

frequently

run

in

parallel

on

Windows

Linux

or

Macintosh

platforms

The

ability

to

create

virtual

machine

snap

shots

of

critical

stages

of

the

analysis

proved

invaluable

for

the

backtracking

of

results

and

to

allow

comparability

over

time

It

proved

essential

in

the

case

of

the

most

complex

network

analyses

where

software

stability

proved

to

be

a

factor

We

have

used

the

microarray

data

in

a

hypothesis

generating

rather

than

hypothesis

testing

way

-LRB-

cf.

-LSB-

-RSB-

-RRB-

Since

our

original

predictions

based

on

the

ArrayAssist

dataset

turned

out

to

be

very

reliable

with

respect

to

subsequent

in

situ

tissue

validation

results

we

have

performed

our

refined

analysis

by

means

of

this

programme

following

extensive

comparison

with

readings

generated

by

the

latest

versions

of

ArrayAssist

and

PathwayStudio

Anyone

intending

to

reproduce

our

results

is

referred

to

the

original

CEL

files

-LRB-

GEO

ID

-RRB-

The

following

original

cases

were

excluded

from

our

refined

analysis

to

obtain

more

homogeneous

cohorts

taking

newly

obtained

histological

and

expression

data

into

account

and

as

well

as

the

sample

of

medial

substantia

nigra

from

Thus

nine

control

nigra

and

PD

nigra

samples

remained

in

the

study

To

find

differentially

expressed

genes

the

p

value

cut

off

was

kept

at

-LRB-

differential

expression

-RRB-

Multiple

testing

corrections

-LRB-

FDR

Bonferroni

-RRB-

were

carried

out

for

comparative

purposes

The

top

genes

identified

on

the

basis

of

probes

are

referred

to

as

the

priority

genes

Hierarchical

clustering

was

executed

on

both

rows

and

columns

using

ArrayAssist

-LRB-

Pearson

centred

distance

metric

centroid

linkage

rule

-RRB-

Similarity

images

were

produced

to

visualise

the

quality

of

clustering

results

-LRB-

-RRB-

In

addition

self-organising

map

clustering

was

performed

on

both

rows

and

columns

using

a

Euclidean

distance

metric

in

ArrayAssist

-LRB-

maximum

number

of

iterations

number

of

grid

rows

number

of

grid

columns

initial

learning

rate

initial

neighbourhood

radius

grid

topology

hexagonal

neighbourhood

type

bubble

-RRB-

For

finding

cell

processes

regulated

by

the

differentially

expressed

genes

PathwayStudio

software

-LRB-

version

-RRB-

was

used

The

top

up-regulated

priority

genes

showing

a

differential

expression

were

selected

and

the

find

common

targets

algorithm

was

employed

in

the

build

pathway

tool

setting

the

cell

process

filter

option

The

procedure

was

repeated

for

diseases

entity

type

Cell

processes

and

disease

conditions

showing

the

highest

number

of

biological

associations

i.e.

the

strongest

probabilistic

relationship

based

on

literature

evidence

to

the

group

of

top

up-regulated

priority

genes

were

selected

Furthermore

known

interactions

between

all

dysregulated

genes

were

identified

using

the

ResNet

database

of

molecular

interactions

which

has

been

derived

from

the

published

literature

by

means

of

a

natural

language

processing

technology

called

Medscan

-LSB-

-RSB-

A

filter

for

the

parameters

promoter

binding

expression

and

regulation

was

applied

in

the

latter

case

For

additional

validation

the

commercial

PathArt

database

-LRB-

PathwayAssist

plug-in

Jubilant

Biosys

-RRB-

containing

a

large

number

of

manually

curated

pathways

was

queried

A

number

of

GEO

datasets

-LRB-

e.g.

astrocytes

and

microglia

in

culture

-RRB-

were

downloaded

to

evaluate

cell-type

specificity

of

expression

of

individual

priority

genes

A

search

for

novel

secreted

biomarkers

was

also

performed

of

which

-LSB-

-RSB-

may

be

one

To

retrieve

known

drug

interactions

of

the

priority

genes

more

than

database

objects

were

checked

in

ResNet

-LRB-

PathwayStudio

-RRB-

Subsequently

all

known

interactions

of

individual

relevant

drugs

contained

in

ResNet

were

retrieved

and

the

results

overlaid

with

the

set

of

priority

genes

Drugs

scoring

high

in

terms

of

the

number

of

their

known

interactions

with

the

priority

genes

were

identified

and

the

relationship

to

PD

analysed

employing

the

PubMed

database

-LRB-

-RRB-

Results

The

extended

set

of

PD

priority

genes

Screening

of

histological

and

clinical

parameters

combined

with

iterative

cycles

of

data

mining

enabled

us

to

identify

the

most

comparable

cases

in

the

cohorts

which

are

detailed

in

The

number

of

highly

significantly

dysregulated

genes

-LRB-

p

-RRB-

increased

from

-LRB-

probes

-RRB-

to

-LRB-

probes

-RRB-

while

of

the

originally

identified

genes

-LSB-

-RSB-

were

confirmed

The

complete

list

of

all

PD

priority

genes

is

provided

in

It

should

be

mentioned

that

there

are

still

some

sequences

that

show

highly

significant

dysregulation

in

PD

on

which

there

is

no

information

available

from

ResNet

the

IDconverter

database

-LSB-

-RSB-

or

NetAffx

However

all

probes

-LRB-

c

-RRB-

were

used

for

the

clustering

-LRB-

Fig.

and

-RRB-

Information

on

the

Affymetrix

probes

available

from

the

public

IDconverter

database

is

provided

in

shows

the

probe

information

not

found

in

IDConverter

or

the

ResNet

database

respectively

that

was

obtained

via

NetAffx

-LRB-

-RRB-

Thirty-one

priority

genes

have

been

previously

designated

PD

candidate

genes

-LRB-

-RRB-

-LRB-

-RRB-

and

it

is

worth

noting

that

are

considered

AD

candidate

genes

as

well

-LRB-

-RRB-

with

SNCA

SNCG

TF

and

being

present

in

both

lists

A

PubMed

search

revealed

that

another

priority

genes

have

known

functional

links

to

PD

-LRB-

-RRB-

Finally

whole

genome/whole

human

body

clustering

using

an

independent

data

set

derived

from

different

organ

samples

which

is

based

on

Affymetrix

Human

Genome

Plus

arrays

allowed

categorical

separation

of

all

nervous

tissue

samples

including

three

different

ganglia

-LRB-

-RRB-

Fig.

showing

the

results

of

unsupervised

hierarchical

clustering

of

the

probes

encompassing

the

priority

genes

-LRB-

corresponding

Affymetrix

probe

IDs

and

expression

data

are

provided

online

in

-RRB-

The

algorithm

separates

the

control

-LRB-

left

-RRB-

from

the

Parkinson

's

disease

group

-LRB-

right

-RRB-

-LRB-

rows

red

expression

above

average

black

average

expression

green

below

average

-RRB-

An

explanation

of

sample

designations

patient

data

and

a

quantitative

colour

code

are

also

provided

in

Gene

ontology

and

pathway

analysis

of

the

differentially

expressed

genes

The

statistically

most

significant

overlap

-LRB-

Fisher

Exact

test

-RRB-

between

the

priority

genes

and

GO

groups

was

found

for

GO

ID

-LRB-

membrane

-RRB-

and

GO

IDs

-LRB-

protein

binding

or

protein

degradation

tagging

activity

respectively

-RRB-

Further

details

are

provided

in

the

supplemental

material

-LRB-

-RRB-

The

main

cell

processes

predicted

to

be

influenced

by

the

top

up-regulated

priority

genes

-LRB-

genes

showing

differential

expression

-RRB-

are

illustrated

in

Fig.

A

hyperlinked

version

of

this

figure

providing

details

on

all

genes

and

their

interactions

is

available

online

-LRB-

-RRB-

A

search

of

canonical

pathways

and

signalling

pathways

in

PathwayStudio

yielded

the

signalling

pathway

which

shares

six

priority

genes

as

the

highest

ranking

result

-LRB-

-RRB-

Fig.

Cell

processes

predicted

to

be

influenced

by

the

top

up-regulated

priority

genes

-LRB-

genes

showing

a

differential

expression

-RRB-

based

on

in

silico

analysis

employing

the

ResNet

database

-LRB-

PathwayStudio

Ariadne

-RRB-

P

and

expression

values

of

all

probes

can

be

found

in

An

online

version

of

this

figure

with

hyperlinks

is

provided

as

b

The

three

disease

conditions

showing

the

strongest

biological

association

with

the

group

of

top

up-regulated

priority

genes

which

may

serve

as

drivers

of

the

disease

process

underlying

PD

In

silico

analysis

was

performed

employing

the

ResNet

database

of

molecular

interactions

-LRB-

Pathway-Studio

Ariadne

-RRB-

An

online

version

of

this

figure

with

hyperlinks

is

provided

as

Predicted

interactions

of

a

subset

of

the

priority

genes

are

shown

in

Fig.

A

total

of

known

connections

were

retrieved

from

ResNet

This

figure

is

provided

for

orientation

purposes

Hyperlinked

permutations

of

this

figure

showing

details

of

all

genes

and

their

interactions

are

available

online

-LRB-

-RRB-

Fig.

super

pathway

not

stratified

for

cell

type

illustrating

known

direct

interactions

between

the

PD

priority

genes

-LRB-

regulation

expression

and

promoter

binding

only

-RRB-

A

total

of

interactions

-LRB-

relations

-RRB-

are

shown

and

any

unlinked

entities

were

removed

Display

style

by

effect

cellular

layout

colour

codes

promoter

binding

violet

green

positive

regulation

red

negative

regulation

grey

and/or

broken

lines

unknown

-LRB-

Resnet

database

unedited

-RRB-

Blue

shading

around

selected

genes

indicates

their

involvement

in

the

cellular

process

and

disease

conditions

depicted

in

Fig.

and

b

respectively

Permutations

of

this

figure

with

hyperlinks

are

provided

online

as

Known

components

of

Lewy

bodies

and

how

they

relate

to

the

group

of

priority

genes

are

presented

in

Fig.

The

probes

for

most

of

these

components

were

found

to

cluster

together

in

the

self-organising

map

shown

in

A

hyperlinked

version

of

Fig.

with

expression

values

overlaid

is

available

online

-LRB-

-RRB-

Interactions

of

known

PD

genes

with

the

priority

genes

of

this

study

are

demonstrated

in

Fig.

-LRB-

-RRB-

Fig.

pathway

containing

proteins

found

in

Lewy

bodies

-LSB-

-RSB-

Priority

genes

of

this

study

are

marked

by

the

blue

shading

An

online

version

of

this

figure

with

hyperlinks

and

an

overlay

of

expression

values

is

provided

as

Symmetrical

layout

display

by

effect

-LRB-

PathwayStudio

-RRB-

Fig.

of

known

PD

genes

-LRB-

marked

yellow

-RRB-

with

the

priority

genes

of

this

study

Expression

value

overlay

red

indicates

up

regulation

and

blue

indicates

down-regulation

in

PD

nigra

An

online

version

of

this

figure

with

hyperlinks

and

an

overlay

of

significance

values

is

provided

as

Cellular

layout

display

by

effect

-LRB-

ResNet

-RRB-

We

did

not

observe

an

effect

for

gender

-LRB-

cf.

-LSB-

-RSB-

-RRB-

Hierarchical

clustering

of

the

male

and

female

PD

patients

on

the

basis

of

the

expression

values

of

the

priority

genes

did

not

separate

the

groups

nor

did

a

whole

genome

clustering

omitting

sex

chromosomal

sequences

Relationship

to

diseases

and

drug

interactions

A

search

of

the

ResNet

database

identified

three

disease

conditions

that

showed

the

strongest

probabilistic

relationship

to

the

group

of

top

up-regulated

priority

genes

cancer

diabetes

and

inflammation

as

illustrated

in

Fig.

A

hyperlinked

version

of

this

figure

providing

details

on

all

genes

and

their

interactions

is

available

online

-LRB-

-RRB-

Known

drug

interactions

of

some

of

the

priority

genes

were

retrieved

from

the

ResNet

database

through

checking

of

more

than

database

objects

It

is

noteworthy

that

drugs

such

as

clozapine

cocaine

and

haloperidol

which

are

used

in

the

treatment

of

PD

or

which

cause

Parkinsonian

side

effects

appear

to

interact

with

a

large

number

of

PD

priority

genes

-LRB-

-RRB-

A

search

of

the

ResNet

database

also

yielded

information

on

the

interactions

of

two

cytostatic

drugs

paclitaxel

and

vincristine

with

the

priority

genes

identified

in

this

study

-LRB-

-RRB-

Both

paclitaxel

and

vincristine

have

been

reported

to

induce

parkinsonian

side

effects

-LSB-

-RSB-

Hub

vs

peripheral

genes

A

number

of

genes

known

to

have

numerous

interactions

with

other

genes

were

found

amongst

the

priority

genes

These

represent

so-called

network

hubs

and

include

NFKBIA

ACHE

and

TH

as

well

as

about

others

-LRB-

to

connectivities

-RRB-

Figure

and

b

contain

a

number

of

hubs

However

almost

of

the

priority

genes

have

no

known

interactions

according

to

the

ResNet

database

although

the

cellular

localisation

is

known

in

some

instances

Such

genes

may

be

called

peripheral

genes

of

the

human

interactome

-LSB-

-RSB-

Apparently

peripheral

priority

genes

with

still

unknown

pathway

connections

and

cellular

localisation

are

ASMTL

FARSLB

IPW

MANEAL

NCDN

OGDHL

PRKY

UBPH

Up-regulated

peripheral

genes

are

shown

in

Table

Following

the

above

criteria

a

number

of

Lewy

body

components

and

genes

known

to

interact

with

the

established

PD

genes

seem

peripheral

to

main

brain

metabolic

pathways

but

their

exact

status

remains

to

be

determined

Table

peripheral

priority

genes

Discussion

Our

study

reveals

a

significant

up-regulation

of

substantia

nigra

genes

in

PD

which

have

known

biological

associations

with

cancer

diabetes

and

inflammation

This

includes

major

hub

genes

-LSB-

-RSB-

such

as

somatic

mutations

of

which

can

cause

cancer

This

is

of

note

as

forms

part

of

a

molecular

network

that

integrates

tumour

suppression

and

ageing

-LSB-

-RSB-

is

another

cancer

and

Parkinson

's

disease-associated

protein

-LSB-

-RSB-

and

it

is

of

special

interest

in

the

present

context

that

the

ubiquitin-proteasomal

pathway

has

an

established

role

in

neoplastic

processes

-LSB-

-RSB-

Furthermore

both

parkin

and

might

be

tumour

suppressor

genes

and

it

has

been

suggested

that

although

cancer

is

rare

in

PD

unraveling

the

link

between

PD

and

cancer

-LSB-

-RSB-

may

open

a

therapeutic

window

for

both

diseases

-LSB-

-RSB-

The

finding

of

a

molecular

biological

association

between

diabetes

and

PD

is

not

truly

surprising

either

-LSB-

-RSB-

Thirdly

the

link

of

PD

with

inflammation

which

emerges

from

our

unsupervised

analysis

seems

almost

expected

considering

the

very

lively

debate

of

this

topic

in

the

literature

The

whole

genome

transcriptome

data

presented

here

certainly

justify

additional

scrutiny

of

the

underlying

mechanisms

in

relation

to

PD

pathogenesis

The

problem

of

defining

what

causes

PD

at

a

system

level

has

become

more

complex

with

the

recent

finding

that

disease-relevant

genes

may

reside

at

the

periphery

of

disease

networks

It

is

interesting

to

note

that

the

neighbour

of

a

disease

node

appears

more

likely

to

be

another

disease

protein

which

also

preferentially

interacts

with

other

disease

nodes

-LSB-

-RSB-

Proteins

that

are

associated

with

the

same

disease

show

a

increased

tendency

to

interact

with

each

other

than

those

not

associated

with

the

same

disease

-LSB-

-RSB-

This

should

direct

our

attention

also

to

genes

that

do

not

form

major

network

hubs

but

which

are

either

likely

to

be

involved

in

PD

on

cell

biological

grounds

-LRB-

e.g.

Lewy

body

components

-RRB-

or

which

interact

with

PD

causing

genes

A

significant

fraction

of

the

genes

identified

in

this

study

still

represent

functionally

ill-characterised

entities

The

on-line

material

of

this

manuscript

illustrates

some

of

the

pathways

and

biological

association

networks

that

emerge

from

our

analysis

Networks

are

now

recognized

to

pervade

all

aspects

of

human

biology

and

the

question

where

function

lies

within

a

cell

is

shifting

from

a

simple

focus

on

genes

to

the

understanding

that

behind

each

cellular

function

there

is

a

discernible

network

module

consisting

of

genes

transcription

factors

RNAs

enzymes

and

metabolites

-LSB-

-RSB-

However

network

medicine

is

still

in

its

infancy

and

the

present

study

may

be

the

first

where

an

iterative

multidimensional

tissue

analysis

approach

has

been

applied

to

a

human

neurological

disorder

The

ultimate

goal

of

such

analyses

is

the

precise

cellular

localisation

of

all

expressed

human

disease

genes

in

their

affected

tissues

The

present

PD

dataset

has

so

far

yielded

two

novel

components

of

Lewy

bodies

-LSB-

-RSB-

but

much

more

back-mapping

work

will

need

to

be

performed

For

instance

the

exact

mechanism

of

cell

death

in

PD

is

still

unknown

-LSB-

-RSB-

Recent

evidence

has

suggested

that

one

mechanism

linked

to

the

death

of

terminally

differentiated

neurons

is

aberrant

re-entry

into

the

cell

cycle

and

possible

connections

between

oxidative

stress

and

unscheduled

cell

cycle

re-entry

in

PD

have

been

proposed

-LSB-

-RSB-

However

as

neuroscientists

we

may

have

to

move

beyond

the

description

of

the

cell

cycle

that

has

been

propagated

by

those

in

the

cancer

field

because

the

regulation

of

the

cell

cycle

in

the

neuron

is

much

more

nuanced

-LRB-

K

Herrup

-RRB-

This

raises

the

possibility

that

some

of

the

data

supporting

cell

processes

such

as

mutagenesis

in

this

study

may

have

to

be

re-read

and

interpreted

in

a

modified

way

It

is

worth

noting

in

this

context

that

absence

of

RET

signalling

in

mice

causes

progressive

and

late

degeneration

of

the

nigrostriatal

system

-LSB-

-RSB-

We

would

also

like

to

point

out

that

the

present

study

provides

additional

evidence

for

the

importance

of

changes

in

the

neuronal

cytoskeleton

in

PD

-LSB-

-RSB-

because

neurofilament

subunit

as

well

as

microtubuli-associated

protein

genes

were

found

to

be

highly

dysregulated

Dysregulation

of

signal

transduction

heat

shock

and

synaptic

proteins

also

featured

very

prominently

The

view

that

the

nigral

genes

presented

here

are

relevant

for

sporadic

PD

is

supported

by

the

finding

that

their

pattern

of

expression

is

characteristic

of

nervous

tissue

-LRB-

-RRB-

It

is

further

clear

from

our

data

that

the

human

substantia

nigra

in

PD

does

not

represent

dead

tissue

but

that

there

is

an

active

ongoing

disease

process

understanding

of

which

may

hold

the

key

to

halting

PD

The

dysregulated

priority

genes

may

reside

at

the

core

of

the

disease

process

and

could

serve

as

novel

targets

for

therapeutic

intervention

This

idea

is

supported

by

the

observation

that

a

number

of

priority

genes

interact

with

drugs

whose

actions

are

associated

with

a

Parkinsonian

clinical

phenotype

The

uncertainty

whether

inflammatory

processes

truly

represent

a

causative

factor

in

the

aetiology

of

PD

-LSB-

-RSB-

requires

an

answer

Our

own

work

and

that

of

others

suggests

a

direct

role

of

primary

glial

degeneration

in

the

pathogenetic

process

underlying

PD

-LSB-

-RSB-

This

means

that

PD

extends

beyond

the

neuron

The

disease

is

also

not

confined

to

the

substantia

nigra

anatomically

Detailed

cellular

back-mapping

of

all

priority

genes

to

brain

tissue

will

help

to

settle

these

questions

New

algorithms

are

required

to

explain

the

links

of

PD

as

defined

in

the

living

and

in

the

microscope

with

the

underlying

high-throughput

datasets

Collection

of

links

to

Electronic

Supplemental

Material

-LRB-

ESM

-RRB-

List

of

all

highly

dysregulated

genes

-LRB-

priority

genes

-RRB-

contained

in

the

ResNet

database

-LRB-

Ariadne

-RRB-

List

of

the

Affymetrix

probes

identifying

highly

dysregulated

sequences

including

all

priority

genes

P

and

differential

expression

values

for

the

Affymetrix

probes

Publicly

available

information

on

the

Affymetrix

probes

from

IDconverter

-LRB-

-RRB-

Information

on

gene

sequences

not

found

in

the

ResNet

database

obtained

via

NetAffx

-LRB-

-RRB-

List

of

priority

genes

that

are

published

PD

candidate

genes

-LRB-

-RRB-

List

of

priority

genes

that

are

published

AD

candidate

genes

List

of

priority

genes

with

known

functional

links

to

PD

-LRB-

PubMed

-RRB-

The

literature

references

for

this

table

can

be

found

in

Gene

Ontology

analysis

of

the

genes

Signalling

pathway

analysis

of

the

genes

-LRB-

PathwayStudio

-RRB-

Hierarchical

clustering

of

the

probes

-LRB-

dendrogram

description

clustering

on

rows

and

columns

Pearson

centred

distance

metric

centroid

linkage

rule

-RRB-

the

clustering

separates

PD

cases

-LRB-

green

right

-RRB-

from

controls

-LRB-

red

left

-RRB-

designation

of

samples

as

indicated

in

the

dataset

submitted

to

GEO

-LRB-

see

Materials

and

methods

section

-RRB-

Abbreviations

used

F

female

M

male

LN

lateral

substantia

nigra

MN

medial

substantia

nigra

CON

PDC

controls

PD

Parkinson

's

disease

followed

by

the

number

indicating

the

age

of

each

subject

-LRB-

cf.

Moran

et

al.

-LSB-

-RSB-

-RRB-

LN

M

LN

M

M

LN

M

LN

F

LN

M

LN

M

LN

M

LN

F

LN

F

LN

F

M

M

LN

M

LN

M

M

F

M

LN

M

An

explanation

for

the

colour

coding

is

provided

in

The

labels

used

for

the

clustering

and

the

corresponding

Affymetrix

probe

set

IDs

are

explained

in

The

four

probes

at

the

very

bottom

of

the

figure

-LRB-

XIST

X

-LRB-

inactive

-RRB-

specific

transcript

-RRB-

in

and

identify

the

female

patients

in

our

refined

cohort

They

served

as

an

internal

control

Column

similarity

image

for

Self-organising

map

corresponding

to

-LRB-

dendrogram

description

clustering

on

rows

and

columns

Euclidean

distance

metric

maximum

number

of

iterations

number

of

grid

rows

number

of

grid

columns

initial

learning

rate

initial

neighbourhood

radius

grid

topology

hexagonal

neighbourhood

type

bubble

-RRB-

Hierarchical

clustering

of

the

probes

using

whole

genome

array

datasets

-LRB-

Affymetrix

Human

Genome

Plus

arrays

-RRB-

representing

individual

organ

samples

including

brain

regions

and

three

ganglia

-LSB-

-RSB-

GSM

numbers

refer

to

the

respective

file

names

in

the

complete

dataset

which

comprises

whole

genome

arrays

-LRB-

GEO

database

GSE

ID

-RRB-

There

is

a

complete

separation

of

nervous

tissue

-LRB-

left

-RRB-

from

other

organs

on

the

basis

of

the

probes

The

designations

of

all

samples

and

their

code

numbers

are

provided

in

Online

version

-LRB-

with

links

-RRB-

of

Fig.

Online

version

-LRB-

with

links

-RRB-

of

Fig.

-LRB-

XIST

was

not

included

in

this

analysis

-RRB-

Online

permutations

of

Fig.

Layout

by

cellular

localisation

with

links

Symmetrical

layout

with

links

Hierarchical

layout

with

links

Known

components

of

Lewy

bodies

are

indicated

by

the

blue

highlighting

-LRB-

this

figure

is

identical

to

except

that

it

has

a

lower

resolution

-RRB-

Online

version

of

Fig.

with

expression

values

overlaid

-LRB-

red

indicates

up

and

blue

indicates

down-regulation

in

PD

nigra

respectively

-RRB-

The

blue

shading

of

the

priority

genes

has

been

replaced

with

yellow

Hyperlinked

online

version

of

Fig.

The

gene

colours

range

in

this

figure

indicates

significance

high

white

low

red

The

blue

shading

indicates

that

the

respective

gene

is

a

priority

gene

Known

drug

interactions

of

some

of

the

priority

genes

as

derived

from

the

ResNet

database

-LRB-

more

than

database

objects

were

checked

-RRB-

It

is

noteworthy

that

drugs

such

as

clozapine

cocaine

and

haloperidol

which

are

used

in

the

treatment

of

PD

or

which

cause

Parkinsonian

side

effects

appear

to

interact

with

an

especially

large

number

of

PD

priority

genes

Clozapine

Cocaine

Haloperidol

This

signalling

pathway

was

identified

based

on

a

search

of

canonical

pathways

and

signalling

pathways

in

PathwayStudio

and

ranked

highest

-LRB-

also

see

-RRB-

Six

priority

genes

are

represented

in

this

pathway

and

are

marked

by

the

blue

shading

Interactions

of

the

cytostatic

drug

paclitaxel

with

a

total

of

priority

genes

-LRB-

blue

shading

-RRB-

are

shown

Display

by

effect

Hierarchical

layout

Interactions

of

the

cytostatic

drug

vincristine

with

priority

genes

-LRB-

violet

shading

bottom

of

figure

-RRB-

are

illustrated

Red

shaded

genes

also

showed

dysregulation

in

the

PD

nigra

-LRB-

p

-RRB-

Display

by

references

count

-LRB-

darker

blue

indicates

a

larger

number

of

references

supporting

the

respective

connection

-RRB-

Hierarchical

layout

References

for

Designations

and

code

numbers

for

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

lewy bodies
microarrays
network medicine

aposklesis
expression analysis
neurodegeneration
synaptic dysfunction


β-Amyloid 25-35 Peptide Reduces the Expression of Glutamine Transporter SAT1 in Cultured Cortical Neurons β-Amyloid -LRB- Aβ -RRB- peptides may cause malfunction and death of neurons in Alzheimer 's disease . We investigated the effect of Aβ on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex . The cultures were treated with Aβ -LRB- 25-35 -RRB- at 3 and 10 μM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity . In mixed neuronal -- glial cell cultures -LRB- from P1 rats -RRB- , Aβ reduced the concentration of system A glutamine transporter 1 -LRB- SAT1 -RRB- , by up to 50 % expressed relative to the neuronal marker microtubule-associated protein 2 -LRB- MAP2 -RRB- in the same cell . No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons , or on glial system N glutamine transporter 1 -LRB- SN1 -RRB- . In neuronal cell cultures -LRB- from E18 rats -RRB- , Aβ -LRB- 25-35 -RRB- did not reduce SAT1 immunoreactivity , suggesting that the observed effect depends on the presence of astroglia . The results indicate that Aβ may impair neuronal function and transmitter synthesis , and perhaps reduce excitotoxicity , through a reduction in neuronal glutamine uptake . Introduction Alzheimer 's disease -LRB- AD -RRB- is a neurodegenerative disorder , leading to progressive loss of memory , impaired cognition and debilitation . It affects about 2 % of the population in industrialized countries , the risk of having the disease increasing rapidly with age beyond 70 , hence the prevalence of AD will increase several fold in the coming decades -LSB- 1 , 2 -RSB- . AD is characterized by extracellular deposits of β-amyloid -LRB- Aβ -RRB- , associated with dystrophic neuronal processes , degenerating and apoptotic neurons showing neurofibrillary tangles -LRB- i.e. aggregates of hyperphosphorylated tau protein filaments -RRB- . The neurotoxic effects of Aβ and some of its fragments have been demonstrated in in vitro primary cultures from human and rat cortical neurons , in hippocampal slice cultures and following in vivo injection into various areas of the rat and monkey brain -LSB- 3 -RSB- . Aβ peptides cause neuronal cell death and also increase the vulnerability of neurons to excitotoxicity -LSB- 4 -RSB- . Aβ -LRB- 25-35 -RRB- has been frequently used in investigations of Aβ properties as a less expensive and more easily handled substitute for the native full-length peptide , Aβ -LRB- 1-42 -RRB- , the latter being implicated in the pathogenesis of AD . Implicit in these experiments is the assumption that the mechanism of action of Aβ -LRB- 25-35 -RRB- is similar to that of Aβ -LRB- 1-42 -RRB- . Indeed , Aβ -LRB- 25-35 -RRB- mimics the toxicological and aggregational properties of the full-length peptide , though these characteristics are increased ; i.e. , the shorter peptide is more toxic to cultured neurons , exhibits toxicity earlier , causes more membrane protein oxidation , and aggregates faster than the native Aβ -LRB- 1-42 -RRB- -LSB- 5 -RSB- . The amino acids glutamine and glutamate are essential for brain metabolism and function . Glutamate is the major excitatory transmitter of the brain -LSB- 6 -RSB- , particularly in the cerebral cortex and hippocampus , and therefore has important roles in cognition , learning and memory . After synaptic release , it is taken up mainly into astroglia by glutamate transporters -LSB- 7 -RSB- . Perturbed glutamate uptake may cause excitotoxicity and is implicated in the pathogenesis of AD -LSB- 7 -RSB- . Once accumulated into astrocytes , glutamate is converted to the non-excitatory amino acid glutamine by the glial enzyme glutamine synthetase . Glutamine is then returned to neurons via glutamine transporters of system N -LRB- SNs -RRB- and system A -LRB- SATs -RRB- and used as a source for the synthesis of glutamate through its conversion by phosphate-activated glutaminase -LSB- 8 , 9 -RSB- . Glutamate is packaged for exocytosis by vesicular glutamate transporters -LRB- VGLUTs -RRB- -LSB- 10 , 11 -RSB- , or used as precursor of GABA in GABAergic neurons . The aim of this study was to explore the effects of Aβ -LRB- 25-35 -RRB- treatment on main transporters implicated in sustaining synaptic release of amino acids , i.e. the glutamine transporters SN1 and SAT1 and the vesicular glutamate transporters VGLUT1 and VGLUT2 , in two different in vitro models : neuronal cell cultures and mixed cell cultures of rat cerebral cortex . Experimental procedures Cell cultures Neuronal cultures were prepared from embryos of Wistar rats at day 18 of gestation -LRB- E18 -RRB- using a modification of an earlier described method -LSB- 12 -RSB- . In brief , the neocortices of embryos were dissociated by treatment with 0.25 % trypsin/calcium -- magnesium-free buffer/EDTA for 10 min at 37 ○ C . The suspension was settled for 2 min at 2,000 × g and then the cells were mixed in DMEM , triturated and passed through a cell strainer . The cells were cultured in DMEM supplemented with 2 % B27 and 1 % GlutaMAX I . The cells were plated on poly-l-lysine -LRB- 50 μg/ml -RRB- coated glass coverslips at a density 0.5 × 106 cells/ml and the cultures were maintained in a humidified atmosphere of 5 % CO2/95 % air at 37 ○ C for 5 days in vitro before incubation with peptide . Primary mixed cell cultures were prepared from the cerebral cortices of 1-day-old -LRB- P1 -RRB- neonatal Wistar rats and plated on poly-L-lysine-treated coverslips -LRB- see above -RRB- . The dissociated cortical cells were suspended in Neurobasal-A medium containing 2 % B27 and 1 % GlutaMAX I and maintained as described for E18 cells . Rats were kept according to nationally and internationally approved conditions . Culture media and reagents were from Gibco , Invitrogen Corporation -LRB- Carlsbad , CA , USA -RRB- . Incubation with Aβ peptide Neuronal and mixed cell cultures were treated with Aβ -LRB- 25-35 -RRB- -LRB- Sigma-Aldrich , St.Louis , MO , USA -RRB- at final concentrations of 3 and 10 μM , for 12 or 24 h . In this study the B27-containing medium was removed from the cultures at day 6 . Cells were washed twice with DMEM or Neurobasal-A medium and then incubated in medium for 12 and 24 h in the absence or presence of Aβ -LRB- 25-35 -RRB- . Controls received no peptide . Antibodies Antibody to SAT1 was raised in rabbit to a fusion protein corresponding to the N-terminal amino acids 1-71 of SAT1 -LRB- aka GlnT , -LSB- 13 -RSB- ; aka SA2 , -LSB- 14 -RSB- -RRB- , and affinity purified and characterized -LRB- T.T. Solbu & F.A. Chaudhry , to be published -RRB- as described for SN1 -LSB- 15 -RSB- . A GST-fusion peptide was made by cloning the first 213 base pairs from SAT1 into a pGex3X vector -LRB- Amersham Biosciences , GE Healthcare , Oslo , Norway -RRB- . Using primers 5 ′ - cccgggatccttatgatgcatttcaaa-3 ′ and 5 ′ - gcggaattcggttgttcctggaat-3 ′ -LRB- Eurogentech , Herstal , Belgium -RRB- , the 5 ′ end of SAT1 was amplified by PCR and ligated into pGex3X using BamH1 and EcoR1 -LRB- New England Biolabs , Ipswich , MA , USA -RRB- . GST-SAT1 was purified on Gluthatione Sepharose 4B as prescribed by the manufacturer -LRB- Amersham Biosciences ; `` GST Gene Fusion System Handbook '' -RRB- . Antibodies to other transporters were prepared and characterized as described -LRB- VGLUT1 and VGLUT2 : -LSB- 16 -RSB- ; SN1 : -LSB- 15 -RSB- -RRB- . Antibodies to marker proteins -LSB- glial fibrillary acidic protein -LRB- GFAP -RRB- , anti-microtubule associated protein-2A & B -LRB- MAP2 -RRB- -RSB- , raised in mice , were from Sigma-Aldrich . Secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit IgG from Molecular Probes -LRB- Eugene , Oregon , USA -RRB- . Immunocytochemical staining Following exposure to peptide , cells attached to 14-mm glass coverslips were fixed with formaldehyde -LRB- 4 % paraformaldehyde , freshly depolymerized , in 0.1 M sodium phosphate buffer pH 7.4 -RRB- for 20 min at room temperature followed by three washes in phosphate buffered saline -LRB- 10 mM phosphate buffer in 150 mM NaCl -RRB- and then blocked with blocking buffer -LSB- 10 % -LRB- v/v -RRB- newborn calf serum -LRB- NCS -RRB- , 3 % -LRB- w/v -RRB- bovine serum albumin -LRB- BSA -RRB- , 0.5 % -LRB- w/v -RRB- Triton X-100 in Tris buffered saline -LRB- TBS -RRB- pH 7.4 -RSB- for 1 h at room temperature . After blocking , sections were incubated with the primary antibodies -LRB- anti-VGLUT1 1:3,000 ; anti-VGLUT2 1:3,000 ; anti-SAT1 2 mg/ml ; anti-SN1 0.3 mg/ml ; anti-MAP2 1:200 ; GFAP 1:500 ; dilutions optimized in pilot experiments -RRB- in primary antibody solution -LSB- 3 % -LRB- v/v -RRB- NCS , 1 % BSA , 0.5 % Triton X-100 , 0.05 % NaN3 in TBS pH 7.4 -RSB- overnight at 4 °C , and then the cells were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 for 1 h at room temperature . The coverslips were then washed three times in fresh antibody solution . Cells on coverslips were sealed to microscopic slides with Fluoromout G water based -LRB- Southern Biotechnology Associates , Birmingham , AL , USA -RRB- . Quantitative analysis of immunoreactivities Dual immunofluorescence for VGLUT1 , VGLUT2 , SAT1 , SN1 , GFAP and MAP2 was examined and quantified with a Zeiss Axioplan 2 microscope equipped with an LSM 5 PASCAL confocal unit , LSM software and a 40x/1 .3 oil PlanApo objective -LRB- Carl Zeiss , Heidelberg , Germany -RRB- . We used standard FICT-rhodamine filter settings . Other settings were optimized initially , and held constant throughout the study so that all sections were digitized under the same conditions of illumination . Care was taken to use similar times for orientation -LRB- with conventional fluorescence -RRB- and adjustments in all preparations , in order to avoid differences in photobleaching . The cells were selected on the criterion that they showed representative morphology -LRB- Fig. 1 -RRB- and strong immunoreactivity for MAP2 -LRB- neurons -RRB- or for SN1 -LRB- in the case of astroglia -RRB- . The cell body and the most proximal parts of the processes were outlined for digitalization of area and of fluorescence intensity for two immunoreactivities -LRB- MAP2 and a transporter -RRB- . The analysis was performed without notice of the treatment group ; the lack of difference in neuronal area between treatment groups suggests that sampling bias did not interfere with the results obtained . Fluorescence intensity is expressed in arbitrary units , without subtraction of background . For each glass coverslip , five fields were randomly sampled and two cells quantified per field , i.e. 10 cells , to obtain an average value for each immunoreactivity and experiment . Fig. 1Classification of cell types . -LRB- a , c , e -RRB- MAP2 positive -LRB- green -RRB- neuronal cells -LRB- arrowheads -RRB- . -LRB- b -RRB- GFAP positive -LRB- red -RRB- astroglial cell -LRB- arrow , same field as a -RRB- . -LRB- d , e -RRB- SN1 positive -LRB- red -RRB- astroglial cell -LRB- arrow , same field as c -RRB- ; SN1 -LRB- in contrast to GFAP -RRB- reveals the entire extent of the astrocyte . -LRB- e -RRB- MAP2 and SN1 immunoreacivities superimposed . -LRB- f -RRB- DIC image -LRB- same field as c -- e -RRB- . Arrowheads and arrows indicate the positions of the nuclei of sample neurons and astrocytes , respectively . Note that all of the 10 cells situated on or next to the astrocyte in -LRB- f -RRB- are identified as MAP2 positive neurons in -LRB- c -RRB- . Double immunofluorescence observed by confocal microscopy in mixed cell cultures from rat cortex at P1 Data analysis Data are expressed as mean ± standard error of the mean -LRB- SEM -RRB- of four different experiments -LRB- four different sets of cultures prepared at four different times -RRB- for P1 and E18 cultures . Advanced statistical analysis was performed according to a linear mixed model regression analysis , using the function lme of the nlme package -LSB- 17 -RSB- in the statistical software R -LRB- http://www.r-project.org/ -RRB- . The fundamental assumptions of the model are that the observations are normally distributed with a variance varying only with protein type , and that they are uncorrelated when they come from different experiments . Log -LRB- for P1 data -RRB- or square root -LRB- for E18 data -RRB- transformations were performed as quantile -- quantile plots showed these transformations to give the best fits to the Gaussian distribution . Results Cell types Cell types were characterized in control cultures incubated without Aβ . In mixed neuronal -- glial cultures -LRB- from cortex of P1 rat pups -RRB- , neurons and astroglia were clearly identified by immunoreactivity for the markers MAP2 and GFAP , respectively -LRB- Fig. 1a , b -RRB- . Double labelling for MAP2 and system N glutamine transporter SN1 , localized in the membranes of astroglia -LSB- 15 -RSB- , again showed the two immunoreactivities in separate cells -LRB- Fig. 1c -- e -RRB- , SN1 displaying the full extension of protoplasmatic-like astrocytes -LRB- i.e. beyond the parts pervaded by GFAP filaments , Fig. 1a , b -RRB- . The MAP2 expressing cells show the morphological characteristics of neurons and usually grow in close apposition to astroglia -LRB- Fig. 1a -- f -RRB- . In neuronal cultures -LRB- from E18 rats -RRB- , MAP2 positive neurons had similar morphology , but no GFAP positive astroglial cells were found -LRB- not shown -RRB- . Double labelling for MAP2 and different transporter proteins showed the vesicular glutamate transporters VGLUT1 or VGLUT2 to be in neurons . A slight signal for SN1 in neurons was close to background levels . The system A glutamine transporter SAT1 was mainly localized in neurons but slight immunoreactivity was seen in some astroglial cells -LRB- Fig. 2a , b -RRB- . In the neurons , SAT1 staining was strong in central areas of the perikaryon and pervaded the processes . Fig. 2SAT1 -LRB- red -RRB- colocalized -LRB- yellow-orange -RRB- with MAP2 -LRB- green -RRB- in neurons in P1 cortical mixed cell cultures . Images show sample cells , insets display framed areas separately for SAT1 immunoreactivity . Cultures were exposed to control medium for 12 h -LRB- a -RRB- or 24 h -LRB- b -RRB- , or to 10 μM of Aβ -LRB- 25-35 -RRB- for 12 h -LRB- c -RRB- or 24 h -LRB- d -RRB- . SAT1 is expressed in the soma and processes of neurons , as revealed by double labelling with antibodies to the somatodendritic neuronal marker MAP2 shown superimposed on SAT1 . Foci of strong perikaryal immunoreactivity suggest intracellular stores of SAT1 . -LRB- Slight SAT1 immunoreactivity occurs also in glia-like perikarya -LRB- b , centre right , arrow -RRB- , immunonegative for MAP2 . -RRB- The immunoreactive cells were morphologically similar in the different conditions -LRB- a -- d -RRB- . Scale bar = 20 μm Survey of preparations double stained for MAP2 and either VGLUT1 or VGLUT2 indicated that a large proportion of the neurons were VGLUT immunoreactive . Similarly , double staining for MAP2 and SAT1 suggested that most of the neurons in the cultures contained SAT1 . Effects of Aβ in mixed cell cultures Cortical mixed cell cultures -LRB- P1 -RRB- were exposed to Aβ . After day 5 in vitro cell cultures were incubated with 3 or 10 μM of Aβ -LRB- 25-35 -RRB- for 12 or 24 h and then processed for immunohistochemistry and quantitative analyses . Neurons with typical morphology and strong MAP2 staining were seen also after exposure to Aβ -LRB- Fig. 2c , d -RRB- . The cells appeared morphologically similar to those in control cultures -LRB- Figs. 1 , 2a , b -RRB- . Transporter immunoreactivities were quantitatively analysed -LRB- Fig. 3a , b -RRB- in neurons with typical morphology and clear MAP2 immunoreactivity . In addition , SN1 was quantified in cells with typical astroglial morphology according to SN1 immunofluorescence . All immunoreactivities showed a tendency for decline on exposure to Aβ -LRB- 25-35 -RRB- , at 12 h as well as at 24 h , but this attained statistical significance only for SAT1 . -LRB- The low SN1 level in neurons is close to background and may not be biologically significant . -RRB- The size of the central areas of the cells , over which the fluorescence intensities were recorded , was independent of peptide exposure in the neurons and only slightly reduced -LRB- by about 16 % -RRB- in the glia -LRB- Fig. 3c , d -RRB- . This indicates that , in the neurons recorded , Aβ treatment did not cause general impairment resulting in a reduction of cell size . Fig. 3Aβ induced a reduction of SAT1 immunoreactivity in neurons in P1 mixed cortical cell cultures . Transporter immunoreactivities -LRB- arbitrary units of fluorescence intensity , FL -RRB- and areas -LRB- μm2 -RRB- were recorded in individual cells in cultures incubated with 0 , 3 or 10 μM Aβ -LRB- 25-35 -RRB- for 12 h -LRB- a , c , e -RRB- or 24 h -LRB- b , d , f -RRB- . Areas -LRB- c , d -RRB- comprising the perikaryon and proximal neurites were outlinded for recording of an average fluorescence intensity in each cell . To cancel out random differences between cells , transporter immunoreactivity was also expressed relative to MAP2 immunoreactivity in the same cell -LRB- e , f -RRB- . All values are for neurons , except for SN1 , which was recorded also in astroglial cells -LRB- area and relative immunoreactivity for SN1 in glia were 40 times the displayed numbers -RRB- . Background was not subtracted ; the low values for SN1 in neurons are close to non-immunoreactive background . Columns show mean + SEM of measurements from four independent experiments -LRB- n = 4 -RRB- , in each experiment 10 cells were averaged . Asterisks indicate a statistically significant difference compared with control -LRB- * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 -RRB- in a linear mixed model regression analysis -LRB- statistical software R -RRB- . Only SAT1 showed a robust effect of Aβ To adjust for differences in the condition of the individual cell , transporter immunoreactivity was expressed relative to the simultaneously recorded MAP2 immunoreactivity of the same cell -LRB- Fig. 3e , f -RRB- . In this analysis , the effect of Aβ on SAT1 stood out even more clearly , showing the largest percentage changes , whereas there were no statistically significant effects of Aβ on VGLUT1 , VGLUT2 or SN1 in neurons , neither on SN1 in astroglia . After both 12 and 24 h incubation , SAT1 was reduced to about two thirds of the control level by 3 μM of Aβ , and about one half by 10 μM of Aβ . As statistical tests showed similar effects at 12 and 24 h , the data were reanalysed statistically with a simplified linear mixed model assuming the same fixed effect at 12 and 24 h . This analysis gave higher statistical significance for the effect on SAT1 -LRB- P = 0.0009 at 3 μM , P = 0.0000 at 10 μM -RRB- than did the full model reported in Fig. 3 . It confirmed the lack of effect on the other transporters -LRB- P > 0.2 -RRB- , and the lack of effect on perikaryal size -LRB- P > 0.1 -RRB- , except for the size of SN1 positive glia -LRB- P < 0.03 for 3 μM , P < 0.01 for 10 μM -RRB- . Lack of effect of Aβ in neuronal cell cultures Cultured cortical neurons -LRB- E18 -RRB- were exposed to Aβ -LRB- 25-35 -RRB- in the same way as for P1 mixed cell cultures . MAP2 positive neurons had a similar appearance as in P1 cultures , in controls as well as after exposure to Aβ -LRB- Fig. 4a -- d -RRB- . Quantification of the fluorescence intensities -LRB- Fig. 5 -RRB- indicated slight effects of Aβ on SAT1 and SN1 -LRB- Fig. 5a , b -RRB- , but not on the other transporters , or on cell area -LRB- Fig. 5c , d -RRB- . However , when the fluorescence intensity for transporter was normalized to that for MAP2 in the same cell , there were no significant effects of Aβ , except on SN1 -LRB- which in neurons is very low and of questionable biological significance -RRB- . Statistical tests suggested no difference between 12 and 24 h or between 3 and 10 μM . When statistically analysed according to a simplified linear mixed model , assuming the same fixed effect for the different incubation times and concentrations of Aβ , there were no significant effects of Aβ on any of the transporter immunoreactivities -LRB- P ≥ 0.07 -RRB- . Fig. 4SAT1 -LRB- red -RRB- colocalized -LRB- yellow-orange -RRB- with MAP2 -LRB- green -RRB- in neurons in E18 cortical neuronal cell cultures . Sample neurons are shown . The immunoreactive cells were morphologically similar to those in P1 cultures -LRB- Fig. 2 -RRB- . No glia-like cells were present . Cells were incubated in the absence -LRB- a , b -RRB- or presence of 10 μM Aβ -LRB- 25-35 -RRB- -LRB- c , d -RRB- for 12 h -LRB- a , c -RRB- or 24 h -LRB- b , d -RRB- . Labelling was strong in the perinuclear region and processes of neurons . Scale bar = 20 μmFig . 5Lack of effect of Aβ on SAT1 immunoreactivity in E18 cortical neuronal cells -LRB- cultured without glia -RRB- . Data were obtained and presented as in Fig. 3 Finally , to test more directly whether the results differed between the two types of culture used , the MAP2 normalized data for P1 and E18 cultures were combined into one linear mixed model for regression analysis . The fixed effect was now defined as the expected difference in effect of Aβ exposure between P1 and E18 cultures . Assuming the same fixed effect at 12 and 24 h , the effect on SAT1 was significantly different between neurons in P1 and E18 cultures -LRB- P = 0.016 for 3 μM , P = 0.010 for 10 μM of Aβ -RRB- but those on the other transporters were not -LRB- P > 0.3 -RRB- . Assuming similar effect at the two concentrations of Aβ , the significance level was even higher -LRB- SAT1 P = 0.004 ; other transporters P > 0.2 -RRB- . Discussion The main finding of the present study is that Aβ causes a reduction in neuronal SAT1 , a transporter thought to be essential for neuronal uptake of glutamine -LSB- 9 , 13 , 14 -RSB- to support neuronal function including the formation of neurotransmitter in glutamatergic as well as GABAergic neurons . The transmitter of the neurons loosing SAT1 was not directly identified , but as double labelling for MAP2 with VGLUTs and MAP2 with SAT1 both showed major proportions of double labelled cells , they are likely to comprise glutamatergic neurons . The concentrations of Aβ were selected on the basis of literature data to be effective but with low to moderate toxicity levels -LSB- 12 -RSB- . To further minimize confounding influence of indiscriminate toxic damage , the immunoreactivities were measured in individual cells that showed near normal morphology and near normal immunoreactivity for the neuronal marker MAP2 . Therefore , unlike in biochemical studies of whole cultures , cells dying or dead due to toxic effects of Aβ -LSB- 5 -RSB- were excluded from analysis . Further , random variation in the condition of cells was corrected for by expressing the transporter immunoreactivities relative to that of MAP2 , measured in the same cell by double immunofluorescence . Aβ peptides have been found to induce proteolysis of MAP2 in cultured mouse cortical neurons -LSB- 18 -RSB- , but in the cells sampled in the present study such proteolysis must have been minor , as the relation between the values for the different transporters and conditions were similar whether expressed as fluorescence units or relative to MAP2 fluorescence units . The effect of Aβ on neuronal SAT1 was observed in cultures containing neurons in close apposition to astroglial cells , but not in neuronal cultures without glia . This suggests that Aβ does not produce the effect by acting directly on the neurons , but via the glial cells , an idea gaining some support from the observed reduction in astroglial cell size . However , as the astrocytes had only slightly reduced size and normal levels of their marker protein SN1 , the changes in neuronal SAT1 can not be ascribed simply to toxic damage in the glia . The findings are consistent with previous reports that the presence of astrocytes enhances Aβ induced neurotoxicity in hippocampal cell cultures -LSB- 19 -RSB- and that the presence of reactive astrocytes is a characteristic feature of the lesions in AD brain -LSB- 20 -RSB- . Notwithstanding , the effect on neuronal SAT1 can not be ascribed simply to an indiscriminate toxic influence on the neurons from the Aβ exposed glia , because there was no similar effect on VGLUTs , and because the effect was observed in neurons with apparently normal morphology and normal expression of the neuronal marker MAP2 . Could the reduced level of neuronal SAT1 contribute to the impaired resistance of neurons to Aβ when cultured in the presence of glia ? While this does not appear likely , the observed 50 % reduction in glutamine transporter level might possibly be critical in certain conditions . The mechanism by which astroglial cells cause changes in neuronal expression of SAT1 must be determined by future research . As free radicals and oxidative stress play important parts in AD -LSB- 21 -RSB- , it is interesting that crotonaldehyde accumulates in reactive astrocytes in AD brain -LSB- 22 -RSB- . Astrocytes mediate Aβ induced neuronal death through oxidative stress -LSB- 23 -RSB- . One possible mediator of the observed glia-neuron influence is BDNF signalling , which is impaired in cortical neuron cultures exposed to Aβ -LSB- 24 , 25 -RSB- , although Aβ may affect this signalling cascade without the participation of glia -LSB- 24 -RSB- . An attractive possibility is offered by the observation that Aβ -LRB- 25-35 -RRB- causes astrocytes to produce nerve growth factor -LRB- NGF -RRB- , thereby inducing tau hyperphosphorylation and reduced survival in hippocampal neurons -LSB- 26 -RSB- . As the effect is exacerbated by NO , this mechanism may offer a link with the effects of free radicals in AD pathology . It fits with the recent observation that reduction of endogenous tau protects against Aβ toxicity in a mouse model of AD -LSB- 27 -RSB- . The mechanism of vulnerability of neurons in AD appears to involve excitotoxicity -LSB- 4 -RSB- . The increase in glial glutamate transporters induced by Aβ -LSB- 28 -RSB- may represent a measure to limit excitotoxic damage . Similarly , the reduced neuronal levels of SAT1 observed here after exposure to Aβ may limit the production of glutamate , but also of GABA , for synaptic release . It might represent an adaptation to curb excitotoxicity in AD , but might concomitantly impair synaptic function .

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 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 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 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

β-amyloid peptide (25-35)
cell culture
alzheimer’s disease
vesicular glutamate transporter