Neuronal

pentraxin

II

is

highly

upregulated

in

Parkinson

's

disease

and

a

novel

component

of

Lewy

bodies

Neuronal

pentraxin

II

-LRB-

-RRB-

is

the

most

highly

upregulated

gene

in

the

Parkinsonian

substantia

nigra

based

on

our

whole

genome

expression

profiling

results

We

show

here

that

it

is

a

novel

component

of

Lewy

bodies

and

Lewy

neurites

in

sporadic

Parkinson

's

disease

-LRB-

PD

-RRB-

is

also

known

as

the

neuronal

activity-regulated

protein

-LRB-

Narp

-RRB-

which

is

secreted

and

involved

in

long-term

neuronal

plasticity

Narp

further

regulates

AMPA

receptors

which

have

been

found

to

mediate

highly

selective

non-apoptotic

cell

death

of

dopaminergic

neurons

is

found

in

close

association

with

alpha-synuclein

aggregates

in

both

substantia

nigra

and

cerebral

cortex

in

PD

but

unlike

alpha-synuclein

gene

expression

which

is

down-regulated

in

the

Parkinsonian

nigra

could

represent

a

driver

of

the

disease

process

In

view

of

its

profound

-LRB-

-RRB-

upregulation

and

its

established

role

in

synaptic

plasticity

as

well

as

dopaminergic

nerve

cell

death

is

a

very

interesting

novel

player

which

is

likely

to

be

involved

in

the

pathway

dysregulation

which

underlies

PD

Introduction

Neuronal

pentraxins

constitute

a

family

of

proteins

that

are

homologous

to

C-reactive

and

acute-phase

proteins

in

the

immune

system

and

are

thought

to

be

involved

in

activity-dependent

synaptic

plasticity

-LSB-

-RSB-

The

neuronal

pentraxins

-LRB-

-RRB-

and

the

neuronal

pentraxin

receptor

were

identified

as

synaptic

proteins

that

bind

to

affinity

columns

of

the

snake

venom

toxin

taipoxin

which

presynaptically

blocks

neurotransmission

and

the

luminal

calcium-binding

protein

-LSB-

-RSB-

-LSB-

-RSB-

is

also

known

as

neuronal

activity-regulated

pentraxin

-LRB-

Narp

-RRB-

which

was

first

identified

as

an

immediate

early

gene

responding

to

the

induction

of

seizures

in

rat

hippocampus

-LSB-

-RSB-

The

expression

of

this

protein

is

regulated

by

synaptic

activity

and

it

induces

the

formation

of

new

excitatory

synapses

and

the

regulation

of

alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid

-LRB-

AMPA

-RRB-

type

glutamate

receptor

clustering

at

established

synapses

-LSB-

-RSB-

Importantly

dopaminergic

neurons

are

selectively

susceptible

to

excitotoxicity

mediated

via

AMPA

receptor

activation

with

the

resulting

cell

death

being

non-apoptotic

-LSB-

-RSB-

A

second

line

of

research

that

makes

a

highly

interesting

gene

in

the

context

of

Parkinson

's

disease

-LRB-

PD

-RRB-

research

concerns

its

expression

in

hypothalamic

nerve

cells

that

co-express

hypocretin/orexin

-LSB-

-RSB-

and

which

are

prodigiously

affected

by

the

neurodegeneration

in

PD

These

nerve

cells

have

been

implicated

in

the

sleep

disturbances

that

are

often

found

in

PD

patients

-LSB-

-RSB-

Using

a

whole

genome

expression

dataset

of

the

human

Parkinsonian

nigra

-LSB-

-RSB-

we

have

identified

as

the

most

highly

upregulated

gene

in

a

cohort

of

sporadic

PD

cases

Here

we

demonstrate

that

labels

Lewy

bodies

in

paraffin

sections

and

that

there

is

dysregulation

of

also

in

PD

frontal

cortex

Materials

and

methods

Tissue

samples

from

human

SN

and

frontal

cortex

Some

of

the

sporadic

PD

cases

used

in

this

study

and

several

controls

have

been

described

previously

-LSB-

-RSB-

Information

on

the

cases

employed

for

this

project

is

provided

in

Supplemental

Table

Brain

tissue

was

obtained

from

the

UK

Parkinson

's

Disease

Society

Brain

Bank

at

Imperial

College

London

The

diagnostic

work

was

performed

in

the

Department

of

Neuropathology

following

international

neuropathological

consensus

criteria

for

PD

-LRB-

-RRB-

which

require

use

of

alpha-synuclein

as

a

molecular

diagnostic

marker

Ethics

committee

approval

was

available

for

all

human

tissue

work

of

this

study

For

the

dissection

of

the

substantia

nigra

tissue

-LRB-

medial

and

lateral

nigra

were

prepared

separately

-RRB-

the

portion

of

the

midbrain

containing

the

substantia

nigra

was

separated

from

the

brainstem

distal

to

the

mamillary

bodies

-LRB-

angle

cut

-RRB-

at

the

level

of

the

third

cranial

nerve

and

the

superior

colliculi

A

slice

approximately

mm

in

depth

was

prepared

containing

both

SN

and

the

cross-sectioned

red

nucleus

at

the

level

of

its

greatest

diameter

Then

the

SN

was

dissected

from

the

surrounding

nuclei

and

the

pars

reticulata

Once

the

SN

was

isolated

it

was

divided

into

two

portions

the

medial

SN

and

the

lateral

SN

In

all

cases

where

frontal

lobe

was

used

the

superior

frontal

gyrus

was

sampled

at

the

same

level

Quantitative

real-time

polymerase

chain

reaction

Total

RNA

was

extracted

from

PD

and

control

cases

and

transcribed

using

the

RETROscript

kit

-LRB-

Applied

Biosystems

Warrington

UK

-RRB-

following

the

manufacturer

's

protocol

Primers

for

the

internal

control

genes

were

as

before

-LSB-

-RSB-

Predesigned

Taqman

primers

for

-LRB-

-RRB-

were

purchased

-LRB-

Applied

Biosystems

-RRB-

and

Taqman

mastermix

-LRB-

Applied

Biosystems

-RRB-

was

used

Each

reaction

was

run

in

triplicate

using

an

ABI

PRISM

Sequence

Detector

-LRB-

Applied

Biosystems

-RRB-

For

every

gene

tested

a

negative

control

was

run

without

cDNA

template

Data

analysis

for

real-time

PCR

-LRB-

DART-PCR

Version

-LSB-

-RSB-

-RRB-

was

used

to

determine

relative

differences

in

mRNA

expression

between

PD

cases

and

controls

-LRB-

also

see

Supplemental

Figure

-RRB-

In

situ

hybridization

-LRB-

ISH

-RRB-

ISH

was

performed

using

radioactive

oligonucleotide

probes

as

described

previously

-LSB-

-RSB-

Oligonucleotides

were

designed

using

NetPrimer

software

-LRB-

Premier

BioSoft

International

-RRB-

Two

oligonucleotides

complementary

to

mRNA

at

nts

-LRB-

AGCCACAGGCAGATGGTGAAGGCGTACAGCTCAG

probe

A

-RRB-

and

nts

-LRB-

TCGACGTTATTGTCCACCCACGG-GATGATGTTGC

probe

B

-RRB-

were

synthesized

-LRB-

Sigma

Genosys

Poole

UK

-RRB-

Oligonucleotides

were

radioactively

end-labelled

with

-LRB-

Perkin-Elmer

UK

-RRB-

using

terminal

deoxynucleotidyl

transferase

-LRB-

Promega

UK

-RRB-

Snap

frozen

unfixed

post-mortem

tissue

blocks

of

the

frontal

cortex

and

SN

were

cut

on

a

cryostat

-LRB-

μm

-RRB-

collected

onto

Superfrost

Plus

slides

-LRB-

VWR

-RRB-

and

stored

at

°C

Before

use

tissue

sections

were

fixed

for

min

in

fresh

paraformaldehyde

in

M

phosphate

buffer

-LRB-

pH

-RRB-

Pre-hybridisation

treatment

included

acetylation

in

M

acetic

M

triethanolamine

dehydration

in

graded

alcohol

-LRB-

ethanols

-RRB-

and

delipidation

in

chloroform

Hybridisation

with

the

oligonucleotide

probe

was

performed

overnight

at

°C

Post-hybridisation

sections

were

washed

in

standard

saline

citrate

solutions

with

increasing

stringencies

The

sections

were

dehydrated

rapidly

through

graded

alcohols

-LRB-

ethanol

-RRB-

air-dried

dipped

in

autoradiographic

emulsion

-LRB-

GE

Healthcare

Amersham

UK

-RRB-

and

exposed

for

weeks

The

autoradiographic

emulsion

was

developed

sections

were

counterstained

with

toluidine

blue

-LRB-

VWR

-RRB-

for

Nissl

substance

and

mounted

for

visualisation

using

bright

field

microscopy

for

the

counterstain

and

epi-polarized

illumination

for

the

silver

grains

of

the

autoradiographic

emulsion

Control

slides

run

to

check

for

specificity

of

hybridization

included

competition

with

an

excess

of

oligonucleotide

-LRB-

times

-RRB-

and

the

use

of

two

probes

to

different

portions

of

the

mRNA

of

interest

which

produced

identical

patterns

of

signal

Black

and

white

photographs

of

emulsion-coated

sections

were

taken

using

a

microscope

provided

with

a

Hamamatsu

digital

camera

-LRB-

Herrsching

Germany

-RRB-

and

HiPic

software

-LRB-

Herrsching

-RRB-

to

capture

images

Digital

colour

images

were

captured

with

a

Retiga

monochrome

camera

using

a

color

option

provided

by

a

RGB-HM-S

filter

-LRB-

QImaging

Burnaby

BC

Canada

-RRB-

and

QCapture

software

-LRB-

QImaging

-RRB-

Adobe

Photoshop

was

used

to

assemble

images

Immunohistochemistry

Paraffin

embedded

tissue

sections

-LRB-

μm

-RRB-

were

used

for

all

stainings

No

antigen

retrieval

was

required

for

whereas

for

alpha-synuclein

-LRB-

SNCA

-RRB-

antibody

staining

sections

were

treated

with

formic

acid

for

min

-LSB-

-RSB-

Adjacent

sections

from

SN

were

stained

with

goat

polyclonal

antibody

-LRB-

Santa

Cruz

Biotechnology

Inc.

-RRB-

and

mouse

monoclonal

anti-SNCA

-LRB-

BD

Transduction

Laboratories

catalogue

number

-RRB-

overnight

-LRB-

°C

-RRB-

Secondary

antibodies

used

were

gG-HRP

-LRB-

Santa

Cruz

Biotechnology

Inc.

-RRB-

and

biotinylated

horse

anti-mouse

-LRB-

Vector

Laboratories

-RRB-

respectively

The

ABC

kit

-LRB-

Elite

Vector

Laboratories

-RRB-

was

used

for

visualisation

using

diaminobenzidine

tetrahydrochloride

-LRB-

Sigma

UK

-RRB-

Sections

were

counter-stained

in

Mayer

's

haemalum

For

fluorescence

microscopy

anti-SNCA

-LRB-

-RRB-

and

-LRB-

-RRB-

were

added

to

the

sections

sequentially

-LRB-

each

was

incubated

overnight

at

°C

-RRB-

For

the

detection

of

the

primary

antibodies

sections

were

rinsed

in

M

Tris

buffered

saline

-LRB-

TBS

-RRB-

and

then

incubated

with

either

the

goat

anti-rabbit

IgG

tagged

with

Alexa

Fluor

-LRB-

red

-RRB-

for

h

at

room

temperature

or

the

biotinylated

anti-mouse

antibody

-LRB-

Vector

Laboratories

-RRB-

for

hour

followed

by

incubation

with

streptavidin

Alex

Fluor

-LRB-

green

-RRB-

for

h

at

room

temperature

Finally

tissue

sections

were

rinsed

in

TBS

and

incubated

in

sudan

black

for

min

before

mounting

in

Tris-buffered

glycerol

Western

blotting

Snap

frozen

midbrain

tissue

samples

containing

SN

from

PD

and

control

cases

were

homogenized

with

RIPA

buffer

-LRB-

pH

-RRB-

heated

to

°C

for

min

and

subsequently

centrifuged

-LRB-

rpm

min

-RRB-

The

protein

supernatant

was

removed

and

the

concentration

of

protein

determined

-LRB-

BIO-RAD

protein

reagent

BIO-RAD

UK

-RRB-

using

a

spectrophotometer

Denaturing

gels

were

run

using

μg

of

protein

from

each

sample

in

μl

RIPA

buffer

μl

β-mercaptoethanol

and

μl

lithium

dodecyl

sulphate

sample

buffer

-LRB-

Invitrogen

UK

-RRB-

NuPAGE

Bis

Tris

polyacrylamide

gels

-LRB-

Invitrogen

-RRB-

were

run

in

NuPage

SDS

running

buffer

-LRB-

Invitrogen

-RRB-

Gels

were

blotted

onto

nitrocellulose

membranes

-LRB-

Invitrogen

-RRB-

immersed

in

NuPage

LDS

transfer

buffer

-LRB-

Invitrogen

-RRB-

in

a

cold

room

-LRB-

°C

-RRB-

overnight

applying

V.

Successful

protein

transfer

was

verified

by

staining

the

membrane

in

Ponceau

S

-LRB-

VWR

UK

-RRB-

for

min

Prior

to

overnight

incubation

in

polyclonal

goat

antibody

to

-LRB-

-RRB-

membranes

were

washed

in

phosphate

buffered

saline

-LRB-

PBS

-RRB-

containing

Tween

and

subsequently

rinsed

in

Tween

containing

skimmed

milk

-LRB-

blocking

step

-RRB-

for

min

Incubation

with

the

secondary

antibody

coupled

to

horseradish

peroxidase

-LRB-

Vector

Labs

UK

-RRB-

was

carried

out

for

h

at

room

temperature

Visualization

of

antibody

binding

using

enhanced

chemilluminescence

-LRB-

ECL

Amersham

Biosciences

UK

-RRB-

was

performed

according

to

the

manufacturer

's

instructions

Semi-quantitative

ratings

The

labelled

SN

sections

were

examined

by

two

independent

observers

blinded

to

the

identity

of

the

respective

cases

The

occurrence

of

immunoreactive

profiles

in

the

SN

was

scored

for

the

overall

burden

of

immunoreactive

deposits

using

a

seven-point

scale

ranging

from

to

-LRB-

absent

to

severe

-RRB-

Correlation

analysis

-LRB-

Spearman

Sigmatstat

-RRB-

was

conducted

within

the

PD

cohort

for

mRNA

expression

-LRB-

log

microarray

data

-RRB-

and

the

corresponding

semi-quantitative

immunocytochemical

results

Results

is

a

novel

component

of

Lewy

bodies

We

demonstrate

here

that

protein

in

the

Parkinsonian

substantia

nigra

localizes

to

Lewy

bodies

Lewy

neurites

and

some

glial

cells

is

the

most

upregulated

gene

in

our

microarray

data

set

-LSB-

-RSB-

It

shows

the

greatest

difference

between

disease

and

control

nigrae

-LRB-

probe

P

E

differential

expression

DElog

which

is

equivalent

to

-RRB-

Many

neurons

show

more

than

one

immunoreactive

LB

-LRB-

Fig.

-RRB-

Western

blotting

confirmed

specificity

of

the

antibody

revealing

a

band

which

corresponds

to

the

monomer

-LRB-

kDa

Fig.

-RRB-

Very

strong

upregulation

of

mRNA

expression

in

PD

was

confirmed

by

means

of

quantitative

real

time-PCR

-LRB-

Fig.

-RRB-

Fig.

pentraxin

II

-LRB-

-RRB-

is

strongly

upregulated

in

substantia

nigra

and

cerebral

cortex

of

PD

patients

a

immunocytochemistry

demonstrating

specific

staining

of

Lewy

bodies

in

a

dopaminergic

neuron

b

Western

blotting

confirms

specificity

of

the

antibody

c

qRT-PCR

shows

highly

increased

levels

of

mRNA

in

substantia

nigra

and

to

a

lesser

extent

in

frontal

cortex

The

arbitrary

units

along

the

ordinate

represent

relative

fold

changes

-LSB-

-RSB-

The

control

value

is

error

bars

indicate

SEM

d

e

In

situ

hybridisation

demonstrates

mRNA

expression

in

nerve

cells

as

well

as

glia

in

both

substantia

nigra

and

frontal

cortex

Labelling

is

also

found

over

non-pigmented

neurons

-LRB-

arrow

in

d

SN

-RRB-

The

arrows

in

e

mark

glial

cells

-LRB-

cortex

-RRB-

In

addition

there

is

autoradiographic

signal

in

the

neuropil

-LRB-

asterisks

-RRB-

which

would

be

in

keeping

with

the

presumed

dendritic

expression

of

mRNA

f

immunocytochemistry

of

cortical

neuropil

reveals

occasional

intercellular

patches

of

arborised

labelling

also

possibly

suggestive

of

a

dendritic

localisation

-LRB-

asterisks

-RRB-

The

arrow

points

to

a

immunoreactive

glial

cell

Counterstaining

in

a

and

f

haemalum

and

toluidine

blue

in

d.

Scale

bars

μm

-LRB-

a

f

-RRB-

μm

-LRB-

d

-RRB-

and

μm

-LRB-

e

-RRB-

Localisation

of

and

SNCA

protein

In

situ

hybridisation

revealed

expression

of

mRNA

in

nerve

cells

as

well

as

glia

in

both

SN

and

frontal

cortex

respectively

-LRB-

Fig.

e

-RRB-

immunoreactive

LBs

were

also

observed

in

the

frontal

cortex

and

there

were

ramified

-LRB-

arborised

-RRB-

positive

profiles

of

cell

processes

in

the

neuropil

-LRB-

Fig.

-RRB-

Analysis

of

adjacent

tissue

sections

demonstrated

co-localisation

of

and

SNCA

in

the

same

nerve

cells

-LRB-

Fig.

b

-RRB-

This

was

confirmed

by

dual

immunofluorescence

-LRB-

Fig.

e

-RRB-

Overall

immunoreactive

neuronal

deposits

mapped

very

closely

to

alpha-synuclein

aggregates

both

cellularly

and

subcellulary

and

was

also

observed

in

incidental

Lewy

bodies

-LRB-

for

a

summary

of

cases

used

see

Supplemental

Table

of

Electronic

Supplementary

Material

-RRB-

In

contrast

no

immunoreactive

intracellular

aggregates

were

observed

in

two

cases

of

tauopathy

i.e.

progressive

supranuclear

palsy

where

alpha-synuclein

deposits

are

also

not

found

However

not

all

SNCA

immunoreactive

profiles

were

positive

i.e.

the

number

of

SNCA

labelled

nerve

cells

outnumbered

that

of

stained

ones

-LRB-

about

-RRB-

In

addition

in

some

nerve

cells

SNCA

immunopositivity

was

observed

as

small

granular

cytoplasmic

deposits

-LRB-

aggresomes

-RRB-

that

lacked

immunoreactivity

-LRB-

Fig.

b

-RRB-

However

immunoepitopes

were

found

to

decorate

the

halo

of

LBs

more

distinctly

than

alpha-synuclein

-LRB-

Fig.

-RRB-

Strongly

immunoreactive

Lewy

neurites

-LRB-

Fig.

-RRB-

were

also

found

Cases

with

high

levels

of

protein

expression

showed

a

larger

alpha-synuclein

burden

We

estimate

that

about

one-third

of

the

alpha-synuclein

immunoreactive

protein

deposits

also

stain

for

Labelling

of

normal

appearing

axons

by

was

widespread

in

both

PD

-LRB-

Fig.

-RRB-

and

control

brains

Glial

cytoplasmic

-LRB-

Papp-Lantos

-RRB-

inclusions

of

multiple

system

atrophy

-LRB-

MSA

-RRB-

were

positive

-LRB-

not

shown

-RRB-

and

in

PD

a

sizeable

number

of

glial

cells

expressed

protein

that

appeared

to

be

SNCA

negative

-LRB-

Fig.

-RRB-

Correlation

analysis

for

microarray

expression

data

and

the

corresponding

semi-quantitative

immunocytochemical

case

ratings

showed

a

significant

correlation

-LRB-

r

P

Table

-RRB-

However

there

was

no

association

between

expression

and

duration

of

the

disease

Fig.

of

and

alpha-synuclein

a

b

and

alpha-synuclein

immunolabelling

of

adjacent

tissue

sections

demonstrates

significant

but

not

complete

overlap

between

the

two

proteins

The

large

arrow

in

a

denotes

the

same

Lewy

body-containing

dopaminergic

nigral

neuron

Small

granular

cytoplasmic

deposits

showing

alpha-synuclein

immunoreactivity

in

some

nerve

cells

-LRB-

arrows

in

b

-RRB-

are

negative

These

aggresomes

are

sometimes

difficult

to

distinguish

from

neuromelanin

granules

based

on

staining

alone

but

their

cytoplasmic

distribution

is

also

different

from

the

latter

In

contrast

Lewy

neurites

are

double-labelled

-LRB-

arrow

head

in

b

-RRB-

Neuromelanin

-LRB-

asterisks

in

a

-RRB-

is

unstained

c

d

e

Dual

immunofluorecence

demonstrates

co-localisation

of

neuronal

pentraxin

II

-LRB-

red

c

-RRB-

and

alpha-synuclein

-LRB-

green

d

-RRB-

Double-labelled

structures

-LRB-

arrows

in

c

-RRB-

light

up

in

yellow

in

the

overlay

-LRB-

e

-RRB-

Scale

bar

μm

-LRB-

a

b

-RRB-

and

μm

-LRB-

c

e

-RRB-

respectivelyFig

of

neuronal

pentraxin

II

immunoreactive

deposits

in

the

Parkinsonian

substantia

nigra

a

Lewy

bodies

are

labelled

which

often

show

a

crennelated

surface

-LRB-

also

see

c

-RRB-

Arrows

of

the

inset

point

to

labelled

connections

bridges

between

small

Lewy

bodies

Cross-sectioned

axon

bundles

in

white

matter

are

numerous

-LRB-

longer

arrows

-RRB-

b

Arrows

point

to

Lewy

neurites

in

the

substantia

nigra

c

is

present

in

pre-Lewy

bodies

and

glial

cells

Labelling

can

be

seen

inside

the

cytoplasm

of

nerve

cells

in

the

neighbourhood

and

often

directly

attached

to

mature

Lewy

bodies

-LRB-

long

arrows

-RRB-

Note

the

crenellated

surface

of

compacted

deposits

-LRB-

centre

and

Lewy

body

at

the

bottom

of

the

figure

-RRB-

due

to

hair-like

projections

which

radiate

into

the

cytoplasm

immunoreactivity

is

also

present

in

glial

cells

A

subset

of

astrocytes

as

well

as

possibly

microglia

appear

to

be

labelled

-LRB-

small

arrows

-RRB-

The

short

arrow

in

the

lower

left

points

to

what

appears

to

be

nuclear

immunoreactivity

Scale

bars

-LRB-

a

-RRB-

-LRB-

b

-RRB-

and

-LRB-

c

-RRB-

μm

respectivelyTable

analysis

for

microarray

expression

data

and

the

corresponding

semi-quantitative

immunocytochemical

ratings

for

eight

of

the

PD

casesCase

numberExpression

mRNA

-LRB-

log

-RRB-

Semi-quantitative

ratings

IR

in

.278105587139.800657274368.7396793641.578.445081081.593.4182034440.5127.9801395781148.9652648212156.4144958231

Discussion

The

most

significant

finding

of

this

study

is

the

striking

presence

of

in

classical

Lewy

bodies

and

in

Lewy

neurites

Experimental

studies

have

demonstrated

that

Narp

the

rat

homologue

of

is

important

in

neuronal

development

promoting

neuronal

migration

synapse

formation

-LSB-

-RSB-

and

neurite

outgrowth

-LSB-

-RSB-

Studies

in

neuronal

pentraxin

I

and

neuronal

pentraxin

II

knockout

mice

have

demonstrated

a

role

in

early

synaptic

refinements

in

the

retina

and

dorsal

lateral

geniculate

nucleus

during

development

-LSB-

-RSB-

It

has

also

been

suggested

that

Narp

's

role

in

synaptic

plasticity

may

underlie

the

long-lasting

aversive

effects

of

drug

withdrawal

-LSB-

-RSB-

These

functions

of

the

gene

product

and

its

dendritic

expression

in

the

cerebral

cortex

-LSB-

-RSB-

are

of

special

interest

in

relation

to

our

finding

that

immunoreactivity

was

also

occasionally

present

in

the

cortical

neuropil

where

it

had

a

ramified

appearance

reminiscent

of

dendritic

arbors

There

is

limited

knowledge

on

the

pathway

-LRB-

Fig.

-RRB-

Known

physiological

functions

of

include

a

role

in

the

induction

of

AMPA

receptor

clustering

-LSB-

-RSB-

and

the

subunit

of

AMPA

receptors

are

all

upregulated

in

our

dataset

All

other

AMPA

subunits

and

the

receptor

for

NPTXR

are

downregulated

Two

additional

genes

whose

functions

may

be

associated

with

AMPA

receptors

in

dopaminergic

neurons

are

also

highly

dysregulated

in

PD

NSF

-LRB-

N-ethylmaleimide

sensitive

fusion

protein

P

E

DElog

-RRB-

and

Calcyon

-LRB-

dopamine

receptor

interacting

protein

P

E

DElog

-RRB-

NSF

binds

the

AMPA

receptor

subunit

and

acts

to

disrupt

interactions

-LSB-

-RSB-

it

may

play

a

role

in

AMPA

receptor

trafficking

and

stabilization

Calcyon

is

co-expressed

in

a

number

of

dopamine

receptor-positive

neurons

in

brain

and

like

dopamine

receptors

is

found

in

dendritic

spines

-LSB-

-RSB-

We

also

found

in

glial

cells

possibly

including

microglia

However

staining

of

glia

was

only

present

in

a

subpopulation

of

cells

while

neuronal

cell

processes

appeared

to

be

consistently

labelled

Why

exactly

accumulates

in

Lewy

bodies

remains

unclear

at

present

However

it

is

conceivable

that

is

involved

in

the

uptake

of

synaptic

material

Synaptic

dysfunction

in

PD

is

very

significant

as

suggested

by

our

microarray

experiments

-LRB-

unpublished

data

-RRB-

The

presence

of

in

only

a

subset

of

Lewy

bodies

and

its

association

with

their

crenellated

surface

shown

in

Fig.

as

well

as

immunoreactive

bridges

between

Lewy

bodies

might

indicate

a

role

of

in

Lewy

body

growth

Fig.

pathway

obtained

by

means

of

the

Resnet

eukaryotic

database

of

molecular

interactions

-LRB-

Ariadne

-RRB-

overlayed

with

microarray

expression

values

Changes

of

PD

relative

to

normal

are

indicated

in

red

-LRB-

increased

in

PD

-RRB-

and

blue

-LRB-

downregulated

-RRB-

respectively

Abbreviations

AMPA

acid

glutamate

receptor

ionotropic

AMPA

glutamate

receptor

ionotropic

AMPA

glutamate

receptor

ionotrophic

AMPA

glutamate

receptor

ionotrophic

AMPA

neuronal

pentraxin

I

neuronal

pentraxin

II

NPTXR

neuronal

pentraxin

receptor

Green

line

regulation

purple

line

binding

The

dotted

grey

lines

connect

to

a

cell

process

whereas

the

solid

lines

connect

to

a

molecule

The

grey

colour

indicates

that

the

effect

and/or

mechanism

are

unknown

References

for

all

protein

interactions

shown

are

provided

in

the

Electronic

Supplement

to

this

figure

Many

PD

patients

also

show

non-motor

symptoms

such

as

hallucinations

depression

autonomic

dysfunction

and

sleep-disturbances

All

these

symptoms

are

present

in

narcolepsy

where

there

is

loss

of

orexin

-LRB-

hypocretin

-RRB-

producing

nerve

cells

in

the

hypothalamus

-LSB-

-RSB-

It

is

noteworthy

therefore

that

the

same

population

of

neurons

shows

massive

cell

loss

in

PD

as

shown

recently

-LSB-

-RSB-

In

addition

Thannickal

et

al.

observed

a

significant

correlation

with

the

clinical

stage

of

PD

-LSB-

-RSB-

It

is

intriguing

that

orexin

neurons

of

the

hypothalamus

demonstrate

robust

expression

of

Narp

in

the

rat

-LSB-

-RSB-

The

relationship

between

and

cell

death

deserves

further

study

All

subunits

of

the

NMDA

AMPA

and

kainate

classes

of

glutamate

receptors

and

a

set

of

NMDA

receptor-associated

intracellular

proteins

are

expressed

in

the

human

substantia

nigra

pars

compacta

and

may

play

a

role

in

glutamate

regulation

of

dopaminergic

activity

and/or

release

-LSB-

-RSB-

AMPA

in

turn

has

been

discovered

to

be

a

mediator

of

selective

non-apoptotic

cell

death

in

dopaminergic

neurons

-LSB-

-RSB-

The

latter

differs

ultrastructurally

from

both

neuronal

apoptosis

and

typical

excitotoxicity

Importantly

this

mode

of

cell

death

is

independent

of

caspase

activity

and

does

not

have

the

morphological

appearance

of

classical

apoptosis

The

findings

are

thus

in

keeping

with

our

old

postulate

that

nigral

neurons

are

likely

to

die

of

a

mechanism

other

than

classical

apoptosis

and

that

aposklesis

-LRB-

withering

-RRB-

may

be

a

more

appropriate

term

-LSB-

-RSB-

The

involvement

of

in

cell

death

mechanisms

is

additionally

supported

by

studies

on

the

effect

of

ceramide

which

activates

TNF-pathways

-LSB-

-RSB-

Therefore

has

to

be

considered

a

new

important

gene

in

PD

dysregulation

which

may

be

linked

to

both

motor

dysfunction

caused

by

nigral

dopaminergic

nerve

cell

death

in

the

midbrain

and

dementia

due

to

synaptic

changes

in

the

cerebral

cortex

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Supplemental

Figure

Cluster

analysis

is

a

method

to

organize

genes

in

a

microarray

dataset

into

groups

-LRB-

clusters

-RRB-

of

similar

expression

values

We

have

used

ArrayAssist

software

-LRB-

Stratagene

-RRB-

to

produce

this

image

which

shows

that

is

not

the

only

gene

that

is

significantly

up-regulated

in

the

Parkinsonian

substantia

nigra

compared

to

control

and

whose

expression

varies

within

the

cohort

The

latter

is

important

because

unlike

a

number

of

other

highly

dysregulated

genes

which

show

greater

similarity

across

cases

and

therefore

higher

p

values

fits

better

with

both

the

clinical

and

neuromorphological

notion

of

different

disease

stages

in

different

cases

There

was

a

good

correlation

between

expression

values

and

immunocytochemical

results

-LRB-

Table

-RRB-

Abbreviations

CON

control

LN

lateral

nigra

MN

medial

nigra

PD

Parkinson

's

disease

Codes

relate

to

study

cases

respectively

-LRB-

Supplemental

Table

-RRB-

The

colour

range

of

expression

values

represents

a

logarithmic

scale

The

correlation

of

expression

between

different

cases

or

the

distance

between

rows

i.e.

their

similarity

measure

was

determined

as

follows

a

hierarchical

clustering

algorithm

was

applied

with

Pearson

centred

as

the

distance

function

the

centroid

linkage

rule

was

used

and

clustering

was

performed

on

the

whole

nigral

transcriptome

-LRB-

JPG

mb

-RRB-

Supplement

to

Fig.

-LRB-

PDF

kb

-RRB-

Supplemental

Table

Cases

used

in

the

study

-LRB-

PDF

kb

-RRB-

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

expresssion profiling
orexin/hypocretin system