Silencing

α-Synuclein

Gene

Expression

Enhances

Tyrosine

Hydroxylase

Activity

in

Cells

α-Synuclein

has

been

implicated

in

the

pathogenesis

of

Parkinson

's

disease

-LRB-

PD

-RRB-

Previous

studies

have

shown

that

α-synuclein

is

involved

in

the

regulation

of

dopamine

-LRB-

DA

-RRB-

metabolism

possibly

by

down-regulating

the

expression

of

tyrosine

hydroxylase

-LRB-

TH

-RRB-

the

rate-limiting

enzyme

in

DA

biosynthesis

In

this

study

we

constructed

α-synuclein

stably

silenced

cells

by

vector

mediated

RNA

interference

and

examined

its

effects

on

DA

metabolism

We

found

that

there

were

no

significant

differences

in

TH

protein

and

mRNA

levels

between

and

cells

suggesting

that

silencing

α-synuclein

expression

does

not

affect

TH

gene

expression

However

significant

increases

in

phosphorylated

TH

cytosolic

-LRB-

l-DOPA

-RRB-

and

DA

levels

were

observed

in

cells

Our

data

show

that

TH

activity

and

DA

biosynthesis

were

enhanced

by

down-regulation

of

α-synuclein

suggesting

that

α-synuclein

may

act

as

a

negative

regulator

of

cytosolic

DA

With

respect

to

PD

pathology

a

loss

of

functional

α-synuclein

may

result

in

increased

DA

levels

in

neurons

that

may

lead

to

cell

injury

or

even

death

Introduction

α-Synuclein

is

a

acid

protein

which

has

been

implicated

in

the

pathogenesis

of

Parkinson

's

disease

-LRB-

PD

-RRB-

-LSB-

-RSB-

Previous

studies

have

shown

that

α-synuclein

mutations

-LRB-

-RRB-

are

associated

with

some

autosomal-dominant

PD

-LSB-

-RSB-

and

that

aggregated

α-synuclein

is

the

major

component

of

Lewy

bodies

and

Lewy

neurites

pathological

hallmarks

of

PD

-LSB-

-RSB-

Although

the

normal

function

of

α-synuclein

and

its

role

in

the

pathogenesis

of

PD

remain

unclear

several

hypotheses

have

been

proposed

based

on

its

physical

properties

or

interacting

partners

-LSB-

-RSB-

One

theory

suggested

that

α-synuclein

might

be

linked

to

PD

via

the

regulation

of

dopamine

-LRB-

DA

-RRB-

homeostasis

-LSB-

-RSB-

Several

studies

have

shown

that

α-synuclein

may

be

involved

in

regulating

the

biosynthesis

vesicular

storage

and

release

as

well

as

reuptake

of

DA

-LSB-

-RSB-

However

some

of

these

findings

remain

controversial

It

has

also

been

reported

that

α-synuclein

may

down-regulate

the

gene

expression

of

tyrosine

hydroxylase

-LRB-

TH

-RRB-

the

rate-limiting

enzyme

involved

in

DA

biosynthesis

-LSB-

-RSB-

Others

report

that

α-synuclein

regulates

DA

biosynthesis

by

reducing

TH

activity

-LSB-

-RSB-

Another

study

showed

that

the

tyrosine-mediated

enhancement

of

phosphorylated

TH

in

nigrostriatal

DA

neurons

was

inhibited

in

α-synuclein

knockout

mice

suggesting

that

α-synuclein

may

either

enhance

TH

phosphorylation

or

hinder

TH

inactivation

during

accelerated

neuronal

activity

-LSB-

-RSB-

Although

it

has

been

established

that

α-synuclein

plays

an

important

role

in

enhancing

TH

activity

conflicting

reports

indicate

that

further

studies

are

required

to

examine

the

role

of

α-synuclein

in

regulating

DA

metabolism

RNA

interference

-LRB-

RNAi

-RRB-

is

a

new

strategy

for

silencing

gene

expression

that

uses

short

double-stranded

-LRB-

ds

-RRB-

RNA

to

mediate

the

degradation

of

sequence

specific

mRNA

-LSB-

-RSB-

It

has

been

reported

that

introducing

small

interfering

RNAs

-LRB-

siRNAs

-RRB-

or

short

hairpin

RNA

-LRB-

shRNA

-RRB-

into

mammalian

cells

could

specifically

silence

the

expression

of

target

genes

-LSB-

-RSB-

The

high

specificity

efficiency

and

convenience

of

this

technique

means

that

it

is

a

powerful

tool

for

studying

gene

function

In

the

present

study

we

silenced

the

expression

of

α-synuclein

using

vector

mediated

RNAi

in

dopaminergic

cells

and

examined

the

effect

of

down-regulation

of

α-synuclein

on

TH

and

DA

biosynthesis

Materials

and

Methods

Construction

of

shRNA

Expression

Plasmids

Short

hairpin

RNA

were

designed

to

target

specific

regions

of

mouse

α-synuclein

mRNA

-LRB-

GenBank

accession

no

-RRB-

We

identified

two

stretches

within

the

coding

region

of

the

α-synuclein

gene

that

were

GC

rich

located

within

one

exon

and

unique

within

the

mouse

genome

A

nine-nucleotide

loop

structure

was

designed

to

form

the

hairpin

We

designed

three

sets

of

oligonucleotides

as

instructed

by

the

BLOCK-iT

Inducible

RNAi

Entry

Vector

Kit

-LRB-

Invitrogen

Carlsbad

CA

USA

-RRB-

CACCGAGCAAGTGACAAATTTGTTCAAGAGACAACATTTGTCACTTGCTC-3

-LRB-

forward

-RRB-

AAAAGAGCAAGTGACAAATGTTGTCTCTTGAACAACATTTGTCACTTG

-LRB-

reverse

-RRB-

CACCAGGCTACCAAGACTATGAGTTCAAGAG

-LRB-

forward

-RRB-

AAAAGGCTACCAAGACTATGA

-LRB-

reverse

-RRB-

and

SH/CON

CACCGGATCGCCAGAACAAGTATTTCAAGAGAATACTTGTTCTGGCGATCC-3

-LRB-

forward

-RRB-

AAAAGGATCGCCAGAACAAGTATTCTCTTGAAATACTTGTTCTGG

-LRB-

reverse

-RRB-

Single-stranded

-LRB-

ss

-RRB-

DNA

oligos

-LRB-

BioSia

Co.

Ltd

Shanghai

China

-RRB-

were

synthesized

Equal

amounts

of

each

pair

of

ss

oligos

were

annealed

to

generate

ds

oligos

The

ds

oligos

were

inserted

into

the

vector

-LRB-

Invitrogen

-RRB-

using

DNA

ligase

and

transformed

into

One

Shot

chemically

competent

Escherichiacoli

-LRB-

Invitrogen

-RRB-

The

transformants

were

verified

by

PCR

and

sequencing

The

positive

plasmids

were

named

and

pSH/CON

respectively

Cell

Culture

The

dopaminergic

cell

line

was

generated

by

fusion

of

rostral

mesencephalic

neurons

from

embryonic

-LRB-

embryonic

day

-RRB-

mice

with

neuroblastoma

cells

-LSB-

-RSB-

-LRB-

gift

from

Dr

Bastian

Hengerer

Novartis

AG

-RRB-

Cells

were

cultured

in

media

supplemented

with

neonatal

calf

serum

-LRB-

GIBCO

BRL

Grand

Island

NY

USA

pH

-RRB-

and

incubated

at

°C

in

an

atmosphere

of

cells

endogenously

express

α-synuclein

and

TH

and

produce

measurable

levels

of

DA

Transfection

and

Selection

cells

were

transfected

with

pSH/CON

or

containing

human

wild-type

-LRB-

WT

-RRB-

α-synuclein

cDNA

-LRB-

constructed

in

our

laboratory

-RRB-

For

stable

transfection

of

and

pSH/CON

cells

were

seeded

in

tissue

culture

plates

pre-coated

with

poly-l-lysine

-LRB-

Sigma

St

Louis

MO

USA

-RRB-

at

confluency

Transfections

were

performed

using

Lipofectamine

-LRB-

Invitrogen

-RRB-

according

to

the

manufacturer

's

instructions

Four

to

six

hours

after

transfection

the

media

was

replaced

with

fresh

growth

media

The

following

day

cells

were

trypsinized

and

replated

into

a

larger

sized

tissue

culture

format

Stably

transfected

cells

were

selected

using

μg/ml

Zeocin

-LRB-

Invitrogen

-RRB-

for

days

The

Zeocin

resistant

clones

were

then

selected

and

expanded

Cells

were

maintained

with

media

containing

μg/ml

Zeocin

Seventy-two

hours

post-transfection

cells

transfected

with

containing

human

WT

α-synuclein

cDNA

were

collected

and

named

RNA

Isolation

RT-PCR

and

Real-time

RT-PCR

Total

RNA

was

extracted

from

and

pSH/CON

stably

transfected

cells

and

cells

using

Trizol

reagent

First-strand

cDNA

was

synthesized

from

μg

of

total

RNA

using

the

SuperScript

First-strand

synthesis

System

for

RT-PCR

-LRB-

Invitrogen

-RRB-

The

reaction

mixture

for

PCR

consisted

of

μl

of

cDNA

template

μl

of

sterile

and

pmol

-LRB-

μl

-RRB-

of

each

specific

primer

The

following

primers

were

used

α-synuclein

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

As

an

internal

control

β-actin

cDNA

was

co-amplified

using

the

following

primer

sequences

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

Each

PCR

was

started

by

predenaturation

at

°C

for

min

Each

PCR

cycle

consisted

of

°C

for

s

°C

for

s

and

°C

for

s

followed

by

a

final

min

extension

at

°C

PCR

amplification

was

carried

out

for

cycles

for

α-synuclein

and

cycles

for

β-actin

using

a

Perkin

Elmer

DNA

thermal

cycler

-LRB-

Applied

Biosystems

Foster

City

CA

USA

-RRB-

The

reaction

mixture

-LRB-

μl

-RRB-

for

real-time

PCR

consisted

of

μl

SYBR

GREEN

PCR

Master

Mix

-LRB-

Applied

Biosystems

-RRB-

μl

of

cDNA

template

μl

of

each

specific

primer

-LRB-

μM

-RRB-

and

μl

of

sterile

The

following

primers

were

used

α-synuclein

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

TH

-LRB-

forward

-RRB-

and

TACGGGTC

-LRB-

reverse

-RRB-

and

β-actin

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

Quantitative

PCR

was

performed

in

duplicate

or

triplicate

using

a

real-time

PCR

thermal

cycler

-LRB-

Applied

Biosystems

-RRB-

Each

reaction

was

started

by

°C

for

min

°C

for

min

and

°C

for

min

followed

by

cycles

of

°C

for

s

°C

for

s

and

°C

for

s.

Immunofluorescence

Staining

Cells

were

washed

with

M

PBS

fixed

in

paraformaldehyde

-LRB-

min

-RRB-

permealized

with

Triton-X

-LRB-

min

-RRB-

and

blocked

in

normal

goat

serum

-LRB-

min

-RRB-

Samples

were

incubated

with

a

specific

monoclonal

antibody

against

α-synuclein

-LRB-

-RRB-

-LRB-

gift

from

Dr

Shun

Yu

Xuanwu

Hospital

Capital

Medical

University

Beijing

China

-RRB-

overnight

at

°C

Cells

were

washed

with

M

PBS

and

incubated

with

goat

anti-mouse

IgG

conjugated

to

cyanine

-LRB-

Sigma

-RRB-

at

°C

for

min

After

a

final

wash

cells

were

examined

by

fluorescence

microscopy

Cell

Viability

Assay

Cell

viability

was

assessed

using

a

MTT

Kit

-LRB-

Promega

WI

USA

-RRB-

Briefly

the

transfected

cells

were

plated

into

plates

-LRB-

cells

per

well

-RRB-

and

cultured

for

h

prior

to

the

assay

Sixteen

wells

were

measured

for

each

stably

transfected

cell

line

Western

Blot

Analysis

Lysates

of

cells

were

prepared

as

previously

described

-LSB-

-RSB-

Protein

concentrations

were

determined

using

BCA

relative

to

BSA

protein

standards

according

to

the

manufacturer

-LRB-

Pierce

Rockford

IL

USA

-RRB-

Whole

cell

protein

extracts

were

resolved

by

SDS-PAGE

and

electrophoretically

transferred

to

nitrocellulose

membrane

Prestained

protein

standards

were

used

to

determine

the

relative

molecular

mass

of

proteins

The

membranes

were

blocked

in

nonfat

dry

milk

in

Tris-buffered

saline

then

incubated

with

the

TH

specific

monoclonal

antibody

-LRB-

Sigma

-RRB-

or

α-synuclein

specific

monoclonal

antibody

-LRB-

-RRB-

at

room

temperature

for

h

followed

by

a

incubation

with

a

peroxidase-conjugated

secondary

antibody

-LRB-

ZhongShan

Beijing

China

-RRB-

at

room

temperature

The

blots

were

washed

and

immunodetection

was

carried

out

by

chemiluminescence

using

SuperSignal

-LRB-

Pierce

-RRB-

Blots

were

then

stripped

and

re-probed

with

an

antibody

against

β-actin

-LRB-

Zhongshan

-RRB-

Blots

were

scanned

using

Kodak

Image

Station

-LRB-

CF

Kodak

Rochester

MN

USA

-RRB-

and

the

optical

densities

of

the

bands

relative

to

β-actin

within

each

lane

were

obtained

HPLC

for

Assaying

DA

and

-LRB-

l-DOPA

-RRB-

Levels

cells

were

collected

and

re-suspended

in

HPLC

buffer

consisting

of

N

perchloric

acid

mM

EDTA

and

l-lysine

Cells

were

freeze-thawed

three

times

then

centrifugated

at

g

for

min

at

°C

to

remove

particulates

The

supernatants

were

collected

and

stored

at

°C

until

analysis

Briefly

the

supernatant

samples

-LRB-

μl

-RRB-

were

injected

onto

a

Phase

II

Column

-LRB-

ODS

mm

cartridge

Φ3

μm

Bioanalytical

Systems

Inc

USA

-RRB-

The

mobile

phase

consisted

of

M

M

citric

acid

mM

sodium

octyl

sulfate

mM

TEA

mM

and

methanol

-LRB-

v/v

-RRB-

pH

The

mobile

phase

was

pumped

through

the

system

at

ml/min

using

a

pump

-LRB-

Bioanalytical

Systems

Inc

-RRB-

Compounds

were

detected

and

quantified

with

an

detector

-LRB-

Bioanalytical

Systems

Inc

-RRB-

The

limit

of

detection

for

DA

was

μl

Peaks

were

identified

by

retention

times

set

to

known

standards

Tyrosine

hydroxylase

activity

was

assessed

by

measuring

the

accumulation

of

l-DOPA

within

cells

treated

with

the

aromatic

l-amino

acid

decarboxylase

-LRB-

AADC

-RRB-

inhibitor

n-hydroxybenzylhydrazine

dihydrochloride

-LRB-

-RRB-

and

measured

by

HPLC

as

described

above

Briefly

cells

were

cultured

in

tissue

culture

plates

washed

twice

in

artificial

CSF

-LRB-

ACSF

containing

mM

NaCl

mM

KCl

mM

and

mM

-RRB-

and

equilibrated

for

min

at

°C

before

addition

of

μM

in

ACSF

for

min

ACSF

samples

were

collected

and

assayed

by

HPLC

as

described

above

Statistical

Analysis

ANOVA

followed

by

Bonferroni

post

hoc

multiple

comparisons

were

used

to

examine

the

significance

between

differences

among

the

experimental

groups

A

value

of

p

was

considered

to

be

statistically

significant

All

data

are

expressed

as

mean

SE

Results

Construction

of

the

shRNA

Expression

Plasmids

After

synthesis

each

pair

of

the

ss

oligos

was

annealed

to

generate

ds

oligos

The

annealing

efficiency

was

analyzed

by

agarose

gel

electrophoresis

Each

ds

oligo

annealing

reactant

showed

a

clearly

detectable

molecular

weight

band

around

bp

as

expected

for

the

length

of

the

designed

ds

oligos

The

bp

bands

were

brighter

than

the

corresponding

lower

molecular

weight

band

which

represented

the

unannealed

ss

oligos

in

the

annealing

reactant

These

data

indicate

that

each

pair

of

ss

oligos

annealed

effectively

to

form

ds

oligos

The

ds

oligos

were

ligated

with

linearized

pENTR

vectors

and

the

positive

clones

were

expanded

and

verified

by

PCR

Bands

near

bp

were

found

in

all

PCR

reactants

indicating

that

the

ds

oligos

were

ligated

into

the

vectors

Further

analysis

by

sequencing

confirmed

that

the

orientation

and

sequence

of

the

inserted

ds

oligos

were

correct

α-Synuclein

Expression

in

Transfected

Cells

The

gene

expression

of

α-synuclein

in

the

transfected

cells

was

assayed

by

RT-PCR

real-time

RT-PCR

immunofluorescence

and

Western

blot

RT-PCR

analysis

revealed

that

α-synuclein

mRNA

levels

were

decreased

in

cells

compared

with

and

pSH/CON-transfected

cells

-LRB-

Fig.

-RRB-

However

α-synuclein

mRNA

levels

in

cells

were

not

significantly

different

to

levels

observed

in

control

and

pSH/CON-transfected

cells

-LRB-

Fig.

-RRB-

These

data

suggest

that

α-synuclein

expression

may

be

inhibited

by

transfection

with

In

addition

real-time

RT-PCR

revealed

that

α-synuclein

mRNA

levels

in

cells

-LRB-

-RRB-

were

significantly

lower

than

in

and

-LRB-

-RRB-

cells

-LRB-

Fig.

-RRB-

The

immunofluorescence

data

also

indicated

that

α-synuclein

gene

expression

was

down-regulated

in

cells

-LRB-

Fig.

-RRB-

Weak

immunofluorescence

staining

was

observed

in

cells

compared

to

strong

α-synuclein

staining

in

control

pSH/CON-transfected

and

cells

Furthermore

western

blot

analysis

revealed

that

α-synuclein

protein

levels

were

significantly

lower

in

cells

-LRB-

-RRB-

than

in

cells

-LRB-

-RRB-

-LRB-

Fig.

e

-RRB-

Thus

α-synuclein

expression

was

inhibited

after

transfection

of

cells

with

These

data

suggest

that

was

an

effective

siRNA

sequence

and

that

α-synuclein

expression

was

stably

silenced

in

cells

The

and

pSH/CON

stably

transfected

cells

were

then

named

and

respectively

Fig.

of

α-synuclein

expression

in

cells

-LRB-

a

-RRB-

α-Synuclein

mRNA

levels

were

measured

by

RT-PCR

Lane

cells

Lane

cells

Lane

pSH/CON-transfected

cells

Lane

cells

Lane

bp

DNA

ladder

-LRB-

b

-RRB-

Statistical

analysis

of

α-synuclein

gene

expression

by

real-time

RT-PCR

α-Synuclein

mRNA

levels

were

decreased

by

approximately

in

cells

compared

with

normal

cells

-LSB-

n

F

df

-LRB-

total

-RRB-

p

compared

with

cells

p

compared

with

cells

-RSB-

α-Synuclein

mRNA

levels

were

not

significantly

different

in

cells

compared

with

and

cells

-LRB-

c

-RRB-

Immunofluorescence

staining

of

normal

cells

-LRB-

a

-RRB-

and

cells

transfected

with

-LRB-

b

-RRB-

pSH/CON

-LRB-

c

-RRB-

and

-LRB-

d

-RRB-

using

an

antibody

against

α-synuclein

Strong

α-synuclein

staining

was

observed

in

and

pSH/CON-transfected

cells

whereas

weak

staining

was

observed

in

cells

Bar

μm

-LRB-

d

-RRB-

Western

blot

analysis

of

α-synuclein

protein

levels

Lane

cells

Lane

cells

Lane

cells

-LRB-

e

-RRB-

Statistical

analysis

of

α-synuclein

protein

levels

in

and

cells

using

the

linear

density

ratio

of

α-synuclein/β-actin

α-Synuclein

protein

levels

were

significantly

decreased

in

cells

-LRB-

-RRB-

compared

with

-LRB-

-RRB-

and

-LRB-

-RRB-

cells

-LSB-

n

F

df

-LRB-

total

-RRB-

p

compared

with

cells

p

compared

with

cells

-RSB-

Effects

of

Silencing

α-Synuclein

Expression

on

Cell

Viability

No

significant

differences

in

cell

morphology

were

observed

between

the

three

groups

of

cells

-LRB-

data

not

shown

-RRB-

As

shown

in

Fig.

the

cell

viability

of

cells

was

significantly

decreased

compared

with

control

and

cells

-LSB-

n

F

df

-LRB-

total

-RRB-

p

compared

with

cells

p

compared

with

cells

-RSB-

Cell

viability

was

decreased

by

and

in

cells

-LRB-

-RRB-

compared

with

-LRB-

-RRB-

and

-LRB-

-RRB-

cells

respectively

-LRB-

Fig.

-RRB-

These

data

suggest

that

α-synuclein

may

be

important

for

the

viability

of

cells

Fig.

of

silencing

α-synuclein

expression

on

cell

viability

Cell

viability

was

measured

using

an

MTT

assay

Cell

viability

was

decreased

in

cells

compared

with

and

cells

-LSB-

n

F

df

-LRB-

total

-RRB-

p

compared

with

cells

p

compared

with

cells

-RSB-

TH

Expression

and

Activity

in

Transfected

Cells

Previous

studies

have

reported

that

TH

expression

may

be

down-regulated

by

over-expression

of

α-synuclein

in

and

cells

-LSB-

-RSB-

Here

we

examined

whether

a

similar

effect

occurred

after

the

silencing

of

α-synuclein

expression

in

cells

TH

mRNA

protein

and

phosphorylation

levels

were

measured

by

real-time

RT-PCR

and

Western

blot

analysis

respectively

Real-time

RT-PCR

showed

that

there

were

no

significant

differences

in

TH

mRNA

levels

between

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

cells

-LRB-

Fig.

-RRB-

These

observations

show

that

silencing

α-synuclein

expression

did

not

alter

TH

gene

expression

suggesting

that

α-synuclein

does

not

regulate

expression

of

TH

Similar

TH

protein

levels

were

observed

in

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

cells

by

Western

blot

analysis

-LRB-

Fig.

c

-RRB-

However

TH

phosphorylation

levels

were

significantly

increased

in

-LRB-

-RRB-

cells

compared

with

and

-LRB-

-RRB-

cells

and

significantly

decreased

in

cells

-LRB-

-RRB-

-LRB-

Fig.

d

-RRB-

To

directly

assess

TH

activity

we

used

to

inhibit

AADC

thereby

blocking

the

conversion

of

l-DOPA

to

DA

We

found

that

l-DOPA

levels

were

increased

in

cells

-LRB-

nmol/l

-RRB-

compared

with

-LRB-

nmol/l

-RRB-

and

-LRB-

nmol/l

-RRB-

cells

-LRB-

Fig.

-RRB-

In

contrast

a

significant

decrease

in

l-DOPA

levels

was

observed

in

cells

-LRB-

nmol/l

-RRB-

Since

accumulation

of

l-DOPA

could

only

occur

if

TH

actively

converted

tyrosine

to

l-DOPA

these

data

suggest

that

TH

activity

was

increased

in

cells

and

diminished

in

cells

over-expressing

α-synuclein

These

data

support

the

hypothesis

that

silencing

α-synuclein

expression

does

not

affect

TH

gene

expression

but

can

enhance

TH

activity

by

increasing

TH

phosphorylation

Fig.

of

silencing

α-synuclein

expression

on

TH

and

levels

-LRB-

a

-RRB-

Statistical

analysis

of

TH

gene

expression

by

real-time

RT-PCR

Differences

in

TH

mRNA

levels

between

the

transfected

cells

were

not

statistically

significant

-LRB-

n

-RRB-

-LRB-

b

-RRB-

Western

blot

analysis

of

TH

and

β-actin

protein

levels

Lane

normal

Lane

Lane

Lane

cells

-LRB-

c

-RRB-

Statistical

analysis

of

TH

protein

levels

in

and

cells

using

the

linear

density

percent

of

TH/β-actin

No

significant

differences

in

TH

protein

levels

were

observed

between

the

transfected

cells

-LRB-

n

-RRB-

-LRB-

d

-RRB-

Statistical

analysis

of

protein

levels

in

and

cells

using

the

linear

density

percent

of

TH

protein

levels

were

significantly

increased

in

cells

and

significantly

decreased

in

cells

compared

with

and

cells

-LSB-

n

F

df

-LRB-

total

-RRB-

p

cells

compared

with

cells

p

cells

compared

with

cells

p

cells

compared

with

cells

p

cells

compared

with

cells

-RSB-

Fig.

of

silencing

α-synuclein

expression

on

l-DOPA

levels

Cell

lysates

were

prepared

from

transfected

cells

incubated

in

the

presence

of

μM

for

min

l-DOPA

levels

were

measured

by

HPLC

l-DOPA

levels

were

significantly

increased

in

cells

and

significantly

decreased

in

cells

compared

with

and

cells

-LSB-

n

F

df

-LRB-

total

-RRB-

p

cells

compared

with

cells

p

cells

compared

with

cells

p

cells

compared

with

cells

p

cells

compared

with

cells

-RSB-

Effects

of

Silencing

α-Synuclein

Expression

on

DA

Levels

To

assess

differences

in

cytosolic

DA

levels

between

the

transfected

cells

we

prepared

cell

extracts

and

measured

DA

content

by

HPLC

with

electrochemical

detection

As

shown

in

Fig.

DA

levels

were

significantly

increased

in

cells

compared

with

and

cells

The

DA

content

was

approximately

higher

in

cells

-LRB-

nmol/l

-RRB-

than

in

normal

cells

-LRB-

nmol/l

-RRB-

and

higher

than

in

cells

-LRB-

nmol/l

-RRB-

No

significant

differences

in

DA

content

were

observed

between

and

cells

-LRB-

Fig.

-RRB-

These

results

indicate

that

silencing

α-synuclein

expression

leads

to

an

increase

in

DA

levels

in

cells

Fig.

of

silencing

α-synuclein

expression

on

DA

levels

Changes

in

DA

levels

were

detected

by

HPLC

DA

levels

were

significantly

increased

in

cells

compared

with

and

cells

-LSB-

n

F

df

-LRB-

total

-RRB-

p

compared

with

cells

p

compared

with

cells

-RSB-

Differences

in

DA

levels

between

the

and

cells

were

not

statistically

significant

Discussion

In

the

present

study

we

silenced

the

expression

of

α-synuclein

in

dopaminergic

cells

by

vector

mediated

RNAi

using

the

BLOCK-iT

inducible

RNAi

entry

vector

This

vector

was

selected

for

several

reasons

Firstly

the

vector

contains

a

kanamycin

resistance

gene

for

selection

in

E.coli

and

a

ZeocinTM

resistance

marker

to

allow

generation

of

stable

cell

lines

that

express

the

shRNA

of

interest

Secondly

the

cloning

site

of

the

vector

contains

a

overhang

on

the

end

of

each

DNA

strand

allowing

directional

cloning

of

the

shRNA

of

interest

Thirdly

the

promoter

of

the

vector

is

recognized

by

RNA

polymerase

III

resulting

in

high-level

constitutive

expression

of

shRNA

in

most

mammalian

cell

types

We

identified

an

effective

targeting

sequence

for

RNAi

which

localized

in

the

C-terminal

coding

sequence

of

the

mouse

α-synuclein

gene

and

used

this

to

generate

cells

in

which

the

expression

of

α-synuclein

was

stably

silenced

Due

to

the

endogenous

expression

of

α-synuclein

and

the

dopaminergic

characteristics

of

cells

is

a

good

cellular

model

for

studying

the

normal

function

of

α-synuclein

and

examining

its

role

in

PD

pathogenesis

We

found

that

the

cell

viability

of

cells

was

significantly

decreased

compared

with

and

cells

suggesting

that

loss

of

α-synuclein

may

induce

cell

injury

directly

or

indirectly

Based

on

subsequent

observations

we

propose

that

a

marked

increase

in

cytosolic

DA

levels

may

account

for

decreased

cell

viability

DA

can

readily

oxidize

to

generate

hydrogen

peroxide

superoxide

and

reactive

DA

quinones

-LSB-

-RSB-

which

are

cytotoxic

due

to

their

inhibitory

effects

on

the

mitochondrial

respiratory

system

-LSB-

-RSB-

In

addition

superfluous

DA

itself

may

be

cytotoxic

via

inhibition

of

mitochondrial

complex

I

activity

-LSB-

-RSB-

Thus

it

is

also

possible

that

increased

cytosolic

DA

levels

induced

mitochondrial

dysfunction

resulting

in

decreased

viability

in

cells

α-Synuclein

may

be

involved

in

the

regulation

of

TH

the

rate-limiting

enzyme

of

DA

biosynthesis

Several

studies

have

shown

that

α-synuclein

regulates

TH

gene

expression

In

α-synuclein

over-expressing

dopaminergic

cells

TH

mRNA

and

protein

levels

were

significantly

reduced

-LSB-

-RSB-

A

similar

effect

was

observed

in

dopaminergic

cells

transfected

with

WT

α-synuclein

-LSB-

-RSB-

However

in

the

current

study

no

significant

differences

in

TH

mRNA

and

protein

levels

were

observed

between

the

four

groups

of

cells

Interestingly

a

previous

study

using

cells

reported

that

α-synuclein

over-expression

did

not

diminish

endogenous

TH

levels

-LSB-

-RSB-

which

is

consistent

with

our

results

We

propose

that

the

difference

in

TH

levels

between

the

present

α-synuclein

silenced

system

and

the

previously

described

α-synuclein

over-expression

system

is

mainly

due

to

the

different

cell

types

and

not

due

to

the

different

ways

in

which

the

cells

were

manipulated

Although

TH

protein

levels

remained

unchanged

in

cells

phosphorylation

of

TH

at

its

site

was

significantly

enhanced

Furthermore

l-DOPA

levels

were

significantly

increased

after

treatments

These

results

suggest

that

TH

activity

was

enhanced

in

cells

which

is

consistent

with

previous

reports

-LSB-

-RSB-

In

addition

we

also

found

that

cytosolic

DA

levels

were

significantly

increased

in

cells

Our

data

show

that

TH

activity

was

enhanced

in

α-synuclein-silenced

cells

and

that

this

was

associated

with

an

increase

in

DA

biosynthesis

followed

by

increased

cytosolic

DA

levels

The

mechanisms

by

which

α-synuclein

may

regulate

TH

activity

have

not

been

elucidated

It

is

known

that

only

the

phosphorylated

form

of

TH

is

active

and

that

the

phosphorylation

and

dephosphorylation

of

TH

are

important

in

regulating

DA

biosynthesis

-LSB-

-RSB-

Furthermore

phosphorylation

of

TH

at

results

in

the

most

prominent

activation

of

TH

-LSB-

-RSB-

Thus

it

has

been

suggested

that

α-synuclein

regulates

TH

activity

by

regulating

the

phosphorylation

of

TH

-LSB-

-RSB-

Protein

phosphatase

-LRB-

-RRB-

an

important

enzyme

required

for

dephosphorylation

of

TH

can

be

activated

by

α-synuclein

resulting

in

a

significant

increase

in

TH

dephosphorylation

and

reduction

in

TH

activity

-LSB-

-RSB-

In

addition

other

factors

may

also

be

involved

in

the

regulation

of

TH

phosphorylation

by

α-synuclein

For

example

enhances

TH

activity

by

binding

directly

to

phosphorylated

TH

α-Synuclein

may

directly

interact

with

-LSB-

-RSB-

thereby

accelerating

the

dissociation

of

from

phosphorylated

TH

α-Synuclein

may

also

affect

TH

activity

by

directly

interacting

with

TH

-LSB-

-RSB-

In

conclusion

our

data

demonstrate

that

in

PD

pathology

silencing

of

α-synuclein

expression

results

in

enhanced

TH

activity

and

increased

DA

levels

in

neurons

We

propose

that

in

cells

that

unable

to

remove

excess

DA

from

the

cytosol

DA

itself

and

its

oxidized

products

including

reactive

oxygen

species

and

reactive

DA

quinones

may

lead

to

cell

injury

or

even

death

Further

understanding

of

the

normal

function

of

α-synuclein

in

maintaining

DA

homeostasis

may

help

to

identify

novel

preventative

or

therapeutic

strategies

for

PD

O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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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Correlation of quantitative MRI and neuropathology in epilepsy surgical resection specimens -- T2 correlates with neuronal tissue in gray matter Newer MRI methods can detect cerebral abnormalities not identified on routine imaging in patients with focal epilepsy . Correlation of MRI with histopathology is necessary to understand the basis of MRI abnormalities and subsequently predict histopathology from in vivo MRI . The aim of this study was to determine if particular quantitative MR parameters were associated with particular histological features . Introduction The structural basis of focal epilepsy has a major effect on the natural history of the condition in an individual patient -LRB- Semah et al. , 1998 -RRB- . In 30 % of patients , epilepsy is refractory to current drug treatments . In patients with drug-resistant focal epilepsy , surgical resection may be curative , and the nature of the underlying lesion influences post-operative outcome -LRB- McIntosh et al. , 2001 ; Luders and Schuele , 2006 -RRB- . MRI has greatly facilitated assessment for epilepsy surgery by revealing underlying structural cerebral abnormalities . When optimal MRI and post-operative neuropathological examination of the resection specimen do not identify an underlying structural abnormality , seizure outcome from surgery is less likely to be good -LRB- Blume et al. , 2004 ; Jeha et al. , 2006 -RRB- , reflecting the absence of a discrete , removed abnormality or one that is currently undetectable by imaging or pathology . This is to some extent a self-fulfilling strategy , such that patients in whom no structural abnormality is identified may not be considered for surgery -LRB- Spencer et al. , 2005 -RRB- . Even optimal MRI , however , may not reveal known underlying pathological substrates such as focal cortical dysplasia and mild malformations of cortical development -LRB- Palmini et al. , 2004 -RRB- , which are evident on histopathological examination of the resected tissue -LRB- Lee et al. , 2005 ; Widdess-Walsh et al. , 2006 -RRB- . Thus , there is a pressing need for further developments in MRI , such as higher field strengths to improve signal-to-noise ratio , and newer sequences that may better pick out culpable neuropathological lesions in the neocortex . The correlations between hippocampal volumes and T2 measures with glial and neuronal densities were established more than a decade ago -LRB- Cascino et al. , 1991 ; Van Paesschen et al. , 1995 -RRB- . For the more recently developed MR contrasts , the correlation between MR parameters and the underlying neocortical structure is not well understood and it is important for this to be clarified if these contrasts are to contribute to presurgical evaluation . Quantitative histopathology has developed in parallel with MRI and led to more accurate and sophisticated interpretation of microscopic tissue structure -LRB- Thom et al. , 2005 -RRB- . We have previously shown that temporal lobe resection specimens and their resultant histological slides can be aligned with pre-operative volumetric MRI data , allowing comparison of advanced MRI data sets in regions of interest to the equivalent areas on histopathological slides -LRB- Eriksson et al. , 2005 -RRB- . Thus , quantitative MRI data and quantitative histopathological data can be directly compared . In this study we compared the data obtained using two quantitative MRI contrasts , T2 relaxation time maps -LRB- T2 -RRB- ; and magnetization transfer ratio -LRB- MTR -RRB- , with quantitative neuropathological data from the resected material . The aim was to explore the clinical utility of T2 and MTR and determine if particular quantitative MR parameters were associated with particular histological features . Methods Subjects We studied nine consecutive patients for temporal lobe epilepsy surgery at the National Hospital for Neurology and Neurosurgery who had had pre-operative advanced MRI and in whom resection specimens were processed according to our protocol -LRB- Eriksson et al. , 2005 -RRB- . The study was approved by the Joint Research Ethics Committee of the National Hospital for Neurology and Neurosurgery and Institute of Neurology , UCL . All patient and control subjects gave written informed consent to participate in the study . The age range of subjects was 31 -- 46 years -LRB- median 36 years -RRB- and 4 were male . Seven had a right anterior temporal lobe resection , and two had surgery on the left . The surgical procedure was selected after our standard presurgical work-up , including EEG-video telemetry , conventional MRI -LRB- T1 - and T2-weighted , proton density and FLAIR -RRB- , psychological and psychiatric assessments and multi-disciplinary case conference . Control groups for the advanced MRI consisted of healthy volunteers with no history of neurological disease and normal conventional MRI and were the same subjects as reported previously -LRB- Salmenpera et al. , 2007 -RRB- . Twenty-nine volunteers served as a control population for the analysis of T2 maps and 26 volunteers for MTR . MR acquisition All scans were acquired on the same 1.5 T GE Signa MR scanner -LRB- GE Medical Systems , Milwaukee , WI -RRB- . The T1-weighted volume sequence from the conventional scan protocol for all subjects was required for the MR : pathology correlation . The sequence parameters were : TE/TR/TI / NEX , 4.2 / 15/450/1 ; flip angle 20 ° ; acquisition matrix 256 × 192 ; field of view 24 × 18 cm ; 3/4 phase FOV , 124 contiguous 1.5 mm slices , giving a voxel size of 0.94 × 1.25 × 1.5 mm . Subjects had two additional scans to generate quantitative MR data : T2 mapping Dual-echo CSF suppressed , FFT2 -LRB- fast FLAIR T2 -RRB- data sets were acquired using TR/TI/TE1 ,2 = 5000/1638/15 ,120 ms. Twenty-eight contiguous axial slices were obtained with an acquisition matrix of 256 × 256 and a 24 cm in-plane field of view , giving a voxel size of 0.94 × 0.94 × 5 mm . T2 signal intensity was calculated pixel-by-pixel using the dual-echo data sets , one set from the early echo -LRB- TE1 -RRB- and one from the late echo -LRB- TE2 -RRB- , according to the equation : T2 signal = -LRB- TE2 − TE1 -RRB- / ln -LRB- S1/S2 -RRB- where S1 and S2 represent the signal intensities for the early and late echoes respectively -LRB- Rugg-Gunn et al. , 2005 -RRB- . Total acquisition time was 13 min and 50 s. Magnetization transfer imaging Magnetization transfer -LRB- MT -RRB- - weighted and non-MT-weighted three-dimensional data sets were acquired using TR/TE = 22.6 / 5.4 ms. Volume matrix was 256 × 256 -LRB- in-plane -RRB- × 124 over a 240 × 240 × 180 mm field of view , giving a voxel size of 0.94 × 0.94 × 1.5 mm . MT-weighted images were collected after the application of a pre-pulse to saturate the broad resonance of immobile macromolecule protons . The MT radiofrequency pulse used was a three-lobe Hamming apodized sinc pulse with a duration of 6.4 ms and of 5.1 μT , applied 2 kHz off-resonance -LRB- Rugg-Gunn et al. , 2003 -RRB- . Total acquisition time was 13 min and 54 s. MRI quantitation Voxel-by-voxel statistical comparisons were performed on each of the two image contrasts . This process used SPM99 -LRB- Wellcome Dept. of Imaging Neuroscience , Institute of Neurology , London , UK -RRB- to produce intensity probability maps that were normalized to a standard space defined by a template of all the controls -LRB- Salmenpera et al. , 2007 -RRB- . The normalized T2 maps were smoothed with a 4 mm and MTR maps an 8 mm isotropic Gaussian kernel to improve signal to noise ratio , to allow the images to conform more closely to a Gaussian field model and to increase the validity of statistical inference . A standard univariate t-test was applied using SPM99 to every voxel in the image in order to create a map from which statistical inference was drawn . Significant increases or decreases in MR contrast signal intensity were detected at an individual voxel threshold of p < 0.001 . The theory of Gaussian fields was used to calculate a corrected multiple comparison p value of 0.05 . We tested for regional differences in intensity between each patient and the control group . Further details of the image processing are given in a previous paper -LRB- Salmenpera et al. , 2007 -RRB- . Quantitative MR values were obtained by overlaying a region of interest on the quantitative MR data . The mean intensity value of all voxels within the region of interest was calculated . MR : pathology visual correlation All subjects had a standard anterior temporal lobe resection performed by the same surgeon . During and after resection , specimens were processed according to standard histopathological and co-registration protocols and digital images of the tissue blocks were correlated with the pre-operative volumetric MRI data -LRB- Eriksson et al. , 2005 -RRB- . Briefly , after fixation the resected temporal lobe was sliced in 5 mm blocks perpendicular to the maximum linear extent of the superior temporal sulcus . The MR volume data were rotated and reformatted into an oblique coronal plane that matched the orientation of these blocks . These oblique coronal images were visually inspected in comparison to the pathological blocks . Using standardized criteria , the best fit MRI slice for any one pathological block was identified and matches for adjacent blocks of tissue ensued . Two raters -LRB- SF and SE -RRB- performed this task separately and reached a consensus in any case with discrepancy . All subjects had post-operative MRI scans to indicate the extent of the resection . Histopathology Standard laboratory protocols were used to prepare resected tissue in sections stained for Nissl -LRB- cresyl violet/LFB -RRB- , glial fibrillary acidic protein -LRB- GFAP ; Dako , Cambridge , UK ; polyclonal 1:1500 -RRB- and neuronal nuclear antigen -LRB- NeuN ; Chemicon , Temecula , CA , USA , monoclonal 1:2000 -RRB- . An experienced epilepsy neuropathologist -LRB- MT -RRB- made a qualitative tissue assessment for clinical purposes . For quantitative histopathology the following sections were used : the standard 7 μm GFAP sections ; additional 20 μm sections stained with NeuN ; 20 μm sections stained for microtubule associated protein -LRB- MAP2 ; SIGMA , Saint Louis , MO , USA ; 1:2000 -RRB- ; 7 μm sections stained with anti-synaptophysin antibody -LRB- Dako , Cambridge , UK ; 1:100 -RRB- . All sections were processed in the same batch to ensure uniform immunostaining , which is important when assessing the field fraction , which relies on the intensity of staining . A commercial image analysis system -LRB- Histometrix , Kinetic Imaging , Liverpool , UK -RRB- , attached to a Zeiss Axioskop microscope , was used to generate quantitative histopathology measures . Two regions of tissue were assessed in each subject : ROI1 in the deep white matter of the middle temporal gyrus -LRB- a region identified from MRI-directed correlation , see below -RRB- ; and ROI2 , in the cortex in the gyral crown of the middle temporal gyrus . Quantitative measures in ROI1 were : stereological counts of neurons ; field fraction estimates of immunostaining for GFAP , MAP2 and synaptophysin . Quantitative measures in ROI2 were stereological counts of neurons and field fraction estimates of staining for NeuN and GFAP . The stereological neuronal counts were performed as previously described -LRB- Eriksson et al. , 2006 -RRB- . For field fraction estimates the ROI was outlined on the computer screen of the image analysis system at × 2.5 magnification . The number of fields within the ROI to be analyzed was chosen -LRB- 70 -- 90 in our study -RRB- and the computer software randomly chose a starting point and then a field spacing to cover the whole ROI at the relevant sampling rate . All cortical layers were included in the analysis . Magnification was then increased to × 40 and each field displayed on the computer screen . In the first field for analysis , light intensity parameters were optimized to visualize the immunopositive pixels and the RGB -LRB- red-green-blue -RRB- parameters representing immunopositive pixels were set . Light intensity and RGB parameters were kept constant for all subsequent fields . The software program then automatically estimated the number of immunopositive pixels as a proportion of the entire field . An average of field fraction for all fields was calculated , thus reporting a single field fraction value for each ROI in a given patient . RGB parameters were defined uniquely for each case before analyses as signal intensity can vary slightly between cases . An example of images used for field fraction analyses in one patient is shown in Fig. 1 . Measures in ROI1 were made by an experienced epilepsy neuropathologist -LRB- MT -RRB- and measures in ROI2 by a neurologist -LRB- SE -RRB- with extensive experience of quantitative neuropathology . Histopathology and MRI correlation MRI-directed Abnormal regions in the SPM results for the MRI data were observed for several subjects -LRB- Table 1 -RRB- . To correlate MRI data with histopathological analyses in the same area in all cases , region of interest analyses were performed . A region that had abnormal T2 signal on voxel based analysis in one subject that was within the deep white matter of the middle temporal gyrus was selected and designated ROI1 . This region was of sufficient size to provide robust histopathological data -LRB- Eriksson et al. , 2006 -RRB- and was present in tissue which had been resected in all 9 subjects . Since ROI1 and all quantitative MRI data used for the SPM analyses were normalized to SPM space , quantitative MTR and T2 data could be achieved by superimposing ROI1 on each case 's normalized MTR and T2 data . To achieve quantitative histopathological data , the ROI was aligned with the volume MRI of each patient by a sequence of two normalizations as follows -LRB- Fig. 2 -RRB- . Each subject 's volume MRI -LRB- cropped and tilted as used for the initial MR : pathology correlation -RRB- was normalized to the T1 image template in SPM99 using the default normalization protocol in SPM99 -LRB- Fig. 2 -RRB- . The T1 image template was in the same image space as ROI1 , thus ROI1 could be associated with each subject 's normalized MRI . The normalized volume was then back normalized to the volume MRI in each subject 's native image space . The parameters used for the back-normalization were also used to transform ROI1 from T1 space back to the native space -LRB- Fig. 2 -RRB- . This ROI could then be transferred to the equivalent pathological space by visual inspection of the previously determined MRI : pathology correlation -LRB- Fig. 2 -RRB- . Thus ROI1 was located on the pathology slices of each subject . This region , directly drawn onto the pathology slides of interest , was used for subsequent quantitative histopathology . An example of the images of ROI1 for one subject is shown in Fig. 3 . We also defined a region in the pathological data and determined its MR characteristics . To achieve this , the MRI volume data were segmented in subject space using intensity thresholding and manual drawing in Analyze AVW 5.0 -LRB- BIR , Mayo Clinic , Minnesota -RRB- . The entire resection volume and the middle temporal gyrus within the resection volume were extracted by manual drawing guided by visual inspection of the previously determined MR : pathology correlation . Intensity thresholding and visual inspection were used to separate gray and white matter . A final manual segmentation step was used to separate the gray matter of the gyral crown -LRB- ROI2 -RRB- , from the gray matter in the rest of the middle temporal gyrus . This step used similar criteria to characterize the limits of the gyral crown as were used by the neuropathologist in the quantitative neuropathology . The data from the initial MR : pathology correlation were used to determine which MR slices within the middle temporal gyrus in each subject corresponded to ROI2 . Each subject 's MR volume data were registered to the advanced MR image data in the native subject space using SPM99 and the default registration option . Because of the different qualities of the T2 maps and MTR image data , different registration was performed and different templates used that more closely resembled the two data sets . For the MTR images , the segmented gray and white matter -LRB- summed -RRB- were used , while the MR volume data were registered to the proton density image which was acquired simultaneously with the T2 data , thus effectively registering the MR volume to the T2 data . Once optimal registration parameters were identified , these were used to register ROI2 in the MR volume data to the T2 maps and MTR data sets -LRB- Fig. 4 -RRB- . Thus , for example , gray matter in the middle temporal gyrus could be identified on the T2 image and T2 values determined for the gray matter in that region . An example of the images of ROI2 for one subject is shown in Fig. 5 . Quantitative MR and histopathology measures were compared using SPSS -LRB- version 11.0.0 -RRB- . Results Careful correlation of abnormal regions in the SPM data and the resected tissue indicated that regions that were abnormal on SPM analysis were physically present in the resected tissue in four patients -LRB- Table 1 -RRB- . In one patient -LRB- patient 3 -RRB- qualitative neuropathology identified a hamartia -LRB- a subtle malformation of cortical development -RRB- in the white matter approximately 3 mm in diameter -LRB- Table 2 -RRB- . This was not detected using the SPM analysis and was not a part of ROI1 or ROI2 . The abnormal region in one patient -LRB- T2 region in patient 5 -RRB- was in the white matter of the middle temporal gyrus and was labeled as the region of interest , ROI1 . The quantitative histopathology and quantitative MR data for this region for the nine patients are given in Table 3 . Spearman Rho correlation did not reveal any significant correlations between any of these pathological and MR measures . This region had an abnormal average T2 value in one other patient -LRB- patient 1 -RRB- but MT values were within the normal range for all subjects . Clustering analysis of the histopathological data could not separate the two patients with abnormal T2 values from those subjects with normal T2 values in ROI1 . Quantitative histopathology was determined for the gray matter in the gyral crown of the middle temporal gyrus . The registration process enabled us to determine quantitative MR data for the same region -LRB- ROI2 -RRB- -LRB- Table 4 -RRB- . There was a significant Spearman Rho negative correlation between the field fraction measure of NeuN and the T2 value in ROI2 -LRB- correlation coefficient − 0.72 , p = 0.028 -RRB- . Discussion We have previously shown that in vivo MRI and pathological blocks may be aligned using visual inspection and a standardized protocol -LRB- Eriksson et al. , 2005 -RRB- . We have now shown that , with additional registration protocols , quantitative parameters from MRI and histopathology can be directly compared . There was a negative correlation between the histopathological field fraction measure of NeuN immunopositivity and the MR measure of T2 relaxation time in the gray matter of the middle temporal gyrus . This is the first correlation between a quantitative MR parameter and histopathological data from neocortex in patients with epilepsy . We wished to compare the same area in all patients because it is not known how much histopathological measures and MR parameters differ as a function of location within cerebral tissue . Although some of the histopathological features of interest were readily quantified with a semi-automated protocol , stereological measures require significant expert time input . In consequence , we restricted this study to two regions of interest . The first region investigated corresponded to a region of abnormal T2 signal on SPM analysis in one patient and was chosen since it was of sufficient size to provide robust histopathological data and was available in all resections . Our second region was identified in each subject 's histopathological sections , the gray matter of the crown of the middle temporal gyrus overlying ROI1 , giving us data in adjacent gray matter . The limitation of the initial MR and pathology matching is that the matching was visual and was guided by anatomy and was to some extent subjective . Our previous study , however , showed that the protocol gave consistent , reliable and accurate registrations within the structural limitations of the histopathological data -LRB- Eriksson et al. , 2005 -RRB- . The subsequent matching of specific MR or pathology regions with their respective pathology or MR regions was carried out using computational normalization and registration , both of which were entirely repeatable . This may seem a complex procedure when , for example , the middle temporal gyrus is generally readily identified on anatomical MRI . However , the advanced quantitative MR sequences are optimized for the appropriate MR parameter and not for visualization of anatomy and it can therefore be difficult to confidently identify anatomical features using these contrasts . Identifying the structure of interest in a high quality anatomical scan and then using registration and/or normalization protocols reduced the number of subjective steps -LRB- only one definition of the tissue structure was required rather than one in each MR scan -RRB- and therefore reduced computation time and increased repeatability . The T2 signal in MRI is dependent upon the relationship between bound and free water in a tissue , which is itself dependent upon the macromolecular environment . Disruption of tissue integrity can result in an increase in the free water in tissue and an increase in T2 relaxation time . Changes in T2 in the cerebrum have been related to , among other processes , gliosis , demyelination , edema , neuronal loss and infarction . In an earlier study from our group , significant increases in T2 signal were observed in 51 % of patients with cryptogenic focal epilepsy -LRB- Rugg-Gunn et al. , 2005 -RRB- . The MTR signal is dependent upon the exchange of magnetization between free and bound protons in tissue and this depends upon the integrity of the macromolecular environment . Changes in MTR have been related to histopathologic changes of axonal loss , gliosis , demyelination and edema -LRB- Lexa et al. , 1994 ; Brochet and Dousset , 1999 -RRB- . Our earlier study found reductions of MTR in acquired lesions , malformations of cortical development and in regions that were normal on conventional MRI -LRB- Rugg-Gunn et al. , 2003 -RRB- . In the present study ROI1 was defined by an abnormal T2 value in one subject . Additional analysis revealed that this region also had an abnormal T2 signal in one other subject . MTR values were normal in this region in all subjects . There were no significant linear correlations between the MR values and the histopathological values in ROI1 in our group . The two subjects with abnormal T2 values did not differ from the other seven subjects in relation to their standard or quantitative histopathological data . The T2 and MTR values were all normal in ROI2 . There was a significant negative correlation between the T2 relaxation time and the field fraction estimate of NeuN staining in the cortex of the middle temporal gyrus . A decrease in NeuN staining could suggest a reduction in the number of mature neurons in the tissue , altered packing density or alterations in cell size or NeuN immunoreactivity . An increase in T2 signal has been correlated with neuronal loss and increased gliosis -LRB- Van Paesschen et al. , 1997 ; Rugg-Gunn et al. , 2005 -RRB- . Increased gliosis may further reduce the relative concentration of neurons in a region , although sometimes , tissue sclerosis or gliosis causes reduction in tissue volume which could paradoxically increase packing density . We did not observe any relationship between T2 value and GFAP field fraction . Thus , the correlation detected may reflect a consistent biological relationship focused on neuronal loss . However , one subject had neocortical neuronal loss observed during routine qualitative neuropathology -LRB- Case 4 -RRB- . This subject had the lowest value of neuronal count in the gray matter of ROI2 -LRB- the crown of the middle temporal gyrus -RRB- but also the lowest value of T2 in ROI2 which appears contrary to the correlation observed in the whole group . The reason for this is not clear . The cortical NeuN field fraction in this case was 0.11 -LRB- range 0.08 -- 0.13 -RRB- . Gray matter neuronal counts did not correlate with the field fraction estimate of NeuN . The gray matter neuronal counts are a count for each neuron observed in the region of interest expressed as a proportion of the total area assessed . The field fraction estimate of NeuN is an estimate of the area occupied by tissue immunopositive for NeuN as a proportion of the total area assessed . This tissue is predominantly neuronal cell bodies with some axons and dendrites . Thus 5 large neurons will occupy a greater area of immunopositivity than 5 small neurons . The neuronal count would be the same in each case but the field fraction would be different . This may explain the lack of correlation between the field fraction and the neuronal count . The relation of these two measures with the T2 relaxation value suggests that the T2 relaxation is dependent on more than just the number of neurons , also on their size or complexity , which may reflect their function . Further staining protocols and analyses of neuronal size are necessary to elucidate further this difference . The lack of any other correlations between the MR and the histopathology measures could be due to a number of factors . A real correlation may exist and not have been detected , possibly because of the limited number of subjects in the study . A change of MR scanner curtailed data collection for this series and we are now recruiting more subjects imaged using a new 3 T scanner . We could also have missed a true correlation because the registration between the MR region and the pathological region was inadequate as discussed previously . Alternatively , there may not be any other true correlations between the MRI sequences and pathological measures studied . The pathological measures characterize real physical features -- for example , the actual number of neurons and the proportion of tissue affected by gliosis . However , the underlying physical significance of the MR data is not yet well characterized . We may have chosen pathological measures which do not relate to the features characterized by the MR signals . Myelin content , for example , might influence both T2 and MTR . We did not think it likely that myelin studies would be of relevance in epilepsy in the context of this paper , but additional histopathological analyses , such as LFB for myelin or myelin basic protein , may lead to further useful data in future studies . There was also limited tissue specificity for absolute T2 and MTR values between white and gray matter -LRB- see Tables 3 and 4 for details -RRB- . Thus the MR measures and the histopathology measures may be measuring different features of the tissue and would not be expected to correlate . A hamartia , seen as a cluster of neurons in the white matter microscopically , was not detected as an abnormal MR region with either MTR or T2 using SPM . There are new histopathological techniques in development and some of these may prove a better match to the MR signal changes . MR acquisition on a 3 T scanner will produce data with better signal-to-noise qualities and improved spatial resolution and may increase the sensitivity of quantitative MR measures . Further correlations of quantitative histopathology and quantitative MRI measures may lead to derivation of MRI measures that are most sensitive to pathological changes . Our protocol for matching MR and pathology has demonstrated how we can investigate both the histopathology directed by an MR finding and the MR data directed by a histopathological finding . We identified a biologically plausible correlation between an increased T2 relaxation time and a reduction in neuronal labeling in the cortex of the middle temporal gyrus . If newer , quantitative MRI sequences are to be adopted more widely in routine clinical practice , further research exploring their underlying biological and pathological substrates will be necessary and may ultimately lead to a reduction in the proportion of patients with drug-resistant focal epilepsy whose optimal MRI is said to be `` normal '' .

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