Synaptic localization of acylpeptide hydrolase in adult rat telencephalon.

Abstract

Acylpeptide hydrolase (ACPH), a serine protease present in the central nervous system (CNS), is believed to have a function in modulating synaptic plasticity, cleavage of beta amyloid peptide and degradation of aggregated oxidized proteins. In this report, we demonstrate for the first time the presence of ACPH in the synapse and its preferential localization at the pre-synaptic side. We isolated subcellular fractions from the rat telencephalon enriched in pre- versus post-synaptic components by using differential centrifugation steps to evaluate ACPH catalytic activity and expression level. Relative ACPH levels were determined by Western blot techniques while antibodies against synaptophysin and PSD-95 were used as positive pre- and post-synaptic markers, respectively. Our results show that ACPH protein levels are significantly increased at the synapse, which correlates with a 56% increase in ACPH activity. Furthermore, Western blot experiments show that ACPH is preferentially located at the pre-synaptic side and this is consistent with the increase of its enzymatic activity in fractions enriched in pre-synaptic components. These results give new insights regarding the localization and a putative role of ACPH in the CNS.

Full text

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Neuroscience
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520 (2012) 98–
103
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Synaptic
localization
of
acylpeptide
hydrolase
in
adult
rat
telencephalon
Rodrigo
Soledad
Sandovala, Sebastián
Navarrob, Gonzalo
García-Rojoa, Rodrigo
Calderónc,
Andrea
Pedreroa,
Sandovald,
Ursula
Wynekend,
Floria
Pancettia,∗
aLaboratory
bFaculty
cLaboratory
dLaboratory
of Environmental
Neurotoxicology,
Department
of
Biomedical
Sciences,
Faculty
of
Medicine,
Universidad
Católica
del
Norte,
Larrondo
1281,
Coquimbo,
Chile
of
Medicine,
Universidad
Católica
del
Norte,
Larrondo
1281,
Coquimbo,
Chile
of Physiology
and
High
Altitude
Medicine,
Department
of
Biomedical
Sciences,
Faculty
of
Medicine,
Universidad
Católica
del
Norte,
Larrondo
1281,
Coquimbo,
Chile
of Neurosciences,
Faculty
of
Medicine,
Universidad
de Los
Andes,
San
Carlos
de Apoquindo
2200,
Santiago,
Chile
h
i
g
h
l
i
g
h
t
s
? Synaptic
? Pre-
? ACPH
? We
? We
localization
of
ACPH
was
studied.
and
post-synaptic
components
were
isolated
from
rat
brain.
activity
and
protein
levels
were
measured.
demonstrated
the
presence
of active
ACPH
in
pre-synaptic
components.
provide
new
insights
on
the
localization
and
the
role
of ACPH
in
the
CNS.
a
r
t
i
c
l
e

i
n
f
o
Article
Received
Received
Accepted
history:
23
March
2012
in revised
form
7 May
2012
10 May
2012
Keywords:
Acylpeptide
Excitatory
Synaptic
Rat
hydrolase
synapse
localization
hippocampus
a
b
s
t
r
a
c
t
Acylpeptide
to
aggregated
synapse
the
tion
by
pre-
increased
blot
with
give
hydrolase
(ACPH),
a
serine
protease
present
in
the
central
nervous
system
(CNS),
is believed
have
a
function
in
modulating
synaptic
plasticity,
cleavage
of
beta
amyloid
peptide
and
degradation
of
oxidized
proteins.
In
this
report,
we
demonstrate
for
the
first
time
the
presence
of
ACPH
in
the
and
its
preferential
localization
at
the
pre-synaptic
side.
We
isolated
subcellular
fractions
from
rat
telencephalon
enriched
in
pre-
versus
post-synaptic
components
by
using
differential
centrifuga-
steps
to
evaluate
ACPH
catalytic
activity
and
expression
level.
Relative
ACPH
levels
were
determined
Western
blot
techniques
while
antibodies
against
synaptophysin
and
PSD-95
were
used
as
positive
and
post-synaptic
markers,
respectively.
Our
results
show
that
ACPH
protein
levels
are
significantly
at
the
synapse,
which
correlates
with
a
56%
increase
in
ACPH
activity.
Furthermore,
Western
experiments
show
that
ACPH
is preferentially
located
at
the
pre-synaptic
side
and
this
is
consistent
the
increase
of
its
enzymatic
activity
in
fractions
enriched
in
pre-synaptic
components.
These
results
new
insights
regarding
the
localization
and
a
putative
role
© 2012 Elsevier Ireland Ltd. All rights reserved.
of
ACPH
in
the
CNS.
1.
Introduction
Acylpeptide
the
chimotrypsin-type
activities
brain,
it
hydrolase
(ACPH,
EC 3.4.19.1),
a member
of
prolyl-oligopeptidase
family
[28], is a serine
protease
with
endopeptidase
and
N-acylpeptide
exopeptidase
[9,11].
It is found
in several
types
of
tissues
including
the
muscle,
erythrocytes
and
gastro-intestinal
tract
[7,12]
where
functions
as
an
exopeptidase
catalyzing
the
hydrolysis
of
peptides
Abbreviations:
acetylcholinesterase;
zoate;
nAChR,
protein.
∗Corresponding
E-mail
(U.
ACPH,
acylpeptide
hydrolase;
A?,
amyloid
beta
peptide;
AChE,
AANA,
acetyl-alanyl-p-nitroanilide;
DTNB,
dithiobisnitroben-
DDVP,
2,2-dichlorovinyl
dimethyl
phosphate;
LTP,
long-term
potentiation;
nicotinic
acetylcholine
receptor;
PSD-95,
95 kDa
postsynaptic
density
author.
Tel.:
+56
51 209852;
fax:
+56
51 209837.
addresses:
rsandoval@ucn.cl
(R.
Sandoval),
uwyneken@uandes.cl
Wyneken),
pancetti@ucn.cl
(F.
Pancetti).
containing
lated
displays
and
Previous
ical
as
Surprisingly,
functional
(AChE)
one
(DDVP,
significantly
dependent
synapses
involves
vation
N-acylated
amino
acids
[3],
thereby
releasing
the
acy-
amino
acid
and
a free
N-terminal
peptide
[28]. In vitro,
ACPH
endopeptidase
activity,
cleaving
oxidized
proteins
[7–11]
the
beta
amyloid
peptide
1–40
(A?1–40) [37,38].
reports
have
shown
that
ACPH
is
a
pharmacolog-
target
of
some
chemical
compounds
classically
described
“anticholinesterasics”
or acetylcholinesterase
inhibitors
[23].
its
affinity
for
these
compounds,
as
well
as
its
inhibition,
is
higher
than
that
of
acetylcholinesterase
[26,27].
Results
from
our
laboratory
have
shown
that
of these
compounds,
2,2-dichlorovinyl
dimethyl
phosphate
dichlorvos),
applied
acutely
to rat
hippocampal
slices
can
increase
long-term
potentiation
(LTP),
an
activity-
model
of
synaptic
plasticity
measured
at glutamatergic
in the
rat
hippocampus.
The
increase
of
LTP
by
DDVP
inhibition
of
ACPH
instead
of
AChE
activity
and
the
acti-
of post-synaptic
?7 nicotine
receptors
(?7-nAChR)
[18].
0304-3940/$
http://dx.doi.org/10.1016/j.neulet.2012.05.041
– see
front
matter ©

2012 Elsevier Ireland Ltd. All rights reserved.

Page 2

R. Sandoval
et
al.
/ Neuroscience
Letters
520 (2012) 98–
103
99
The
strength
trations
In the
been
structures
as
tion
post-synaptic
more
of
enriched
cal
and
results
tic
data
described
above
suggest
that
ACPH
modulates
synaptic
at
brain
synapses
by
regulating
the
extracellular
concen-
of
neuropeptides
[18,37].
central
nervous
system
(CNS),
the
presence
of
ACPH
has
observed
in brain
homogenates
[18,27]
and
in certain
brain
such
as
the
prefrontal
cortex
and
the
cerebellum
[38],
well
as
in neuroblastoma
cell
lines
[36]. However,
the
localiza-
of
ACPH
in the
synapse
and
its
distribution
between
pre
and
components
is unclear.
Therefore,
in order
to achieve
insight
on
this
point,
we
studied
the
synaptic
distribution
ACPH
in synaptic
fractions
obtained
from
the
rat
telencephalon
in pre
and
post-synaptic
components,
using
biochemi-
approaches
such
as
subcellular
fractionation,
immunodetection
quantification
of
ACPH
and
AChE
enzymatic
activities.
The
obtained
in this
study
help
to further
propose
specific
synap-
roles
for
ACPH
in the
CNS.
2.
Materials
and
methods
2.1.
Animals,
materials
and
drugs
Experimental
rats
approved
and
Health.
trobenzoate
USA).
Switzerland).
(Bubendorf,
Thermo
purchased
95
Synaptophysin
Horseradish
Inc.
Healthcare
procedures
using
adult
male
Sprague–Dawley
(250–400
g) were
performed
in accordance
with
protocols
by
Institutional
Committees
and
the
Guide
for
the
Care
Use
of
Laboratory
Animals
from
the
National
Institutes
of
Salts,
buffers,
acetylthiocholine
iodide
and
dithiobisni-
(DTNB)
were
purchased
from
Sigma
(St.
Louis,
MO,
Protease
inhibitor
cocktail
was
purchased
from
Roche
(Basel,
N-acetyl-l-alanine
p-nitroanilide
was
from
Bachem
Switzerland).
Bicinchoninic
acid
was
provided
by
Fisher
Scientific
(Waltham,
MA,
USA).
ACPH
antibody
was
from
LifeSpan
Biosciences
(Seattle,
WA,
USA).
PSD-
antibody
was
purchased
from
UC
Davis
(California,
CA,
USA).
was
purchased
from
BD Biosciences
(NJ,
USA).
peroxidase
secondary
antibody
was
provided
by
KPL
(Gaithersburg,
MD,
USA).
ECL
detection
kit
was
provided
by GE
Bio-Sciences
(NJ,
USA).
2.2.
Sub
cellular
fractionation
For
synaptic
fractionations,
we
used
a modified
version
of
the
method
tioned,
et
tion
whereas
observed.
in
pre-
sive
pre-synaptic
tex
were
homogenization
consisted
plus
as
tion
at
while
was
in
synaptosomes
interphase.
tons
described
by
Carlin
et
al.
[4].
All
buffers,
unless
men-
were
prepared
at both
pH
8.1
and
6.8.
According
to Phillips
al.
[21], the
synaptic
fractionation
at pH 8.1
allows
the
purifica-
of
synaptic
junctions
enriched
in post-synaptic
components
at pH 6.8,
pre-
as
well
as
post-synaptic
enrichment
is
Briefly,
these
authors
showed
that
filaments
involved
cellular
adhesion
(N-cadherin,
protocadherin-?, CASK)
connect
and
post-synaptic
components
at low
pH (6.8)
and
progres-
elevation
of
pH to 8.1
disrupts
this
interaction
and
solubilize
components.
Rat
telencephalon
(consisting
of
cor-
and
hippocampus)
of
at
least
3
male
Sprague–Dawley
rats
minced
and
homogenized
using
a teflon-glass
homogenizer
in
buffer
at 5 ml/g
wet
tissue.
Homogenization
buffer
of
0.32
M sucrose,
5 mM
HEPES,
0.5
mM
EGTA,
pH 7.4,
protease
inhibitors
cocktail.
Synaptic
fractions
were
collected
described
previously
[32]. Following
homogenate
centrifuga-
at
1000
× g for
10
min,
the
supernatant
(S1)
was
centrifuged
12,000
× g for
20 min.
The
resulting
supernatant
(S2)
was
stored
the
pellet
(P2),
that
corresponds
to crude
membrane
fraction,
further
fractionated
by
centrifuging
at 200,000
×g for
60 min
a
discontinuous
(1/1.2
M)
sucrose
gradient
in order
to obtain
(Syn)
by
collecting
them
at the
1/1.2
M
sucrose
Syn
fraction
corresponds
to re-sealed
synaptic
but-
(i.e.,
re-sealed
pre-
and
post-synaptic
membranes
containing
intracellular
cellular
30
EGTA,
resulting
(1/1.2
correspond
ated
mainly
apparatus,
others
60
method
vesicles
and
mitochondria).
To
eliminate
the
intra-
components,
the
synaptosomal
fraction
was
lysed
for
min
in 10
volumes
of
an
ice-cold
hypo-osmotic
solution
(0.5
mM
5 mM
Tris–Cl)
and
centrifuged
at 33,000
× g for
30 min.
The
pellet
was
subjected
to a second
discontinuous
sucrose
M)
gradient
in order
to obtain
synaptic
junctions
(SJ)
that
to pre-
and
post-synaptic
membranes
and
their
associ-
proteins.
Finally,
a
microsomal
fraction
(MIC)
corresponding
to intracellular
membrane
vesicles
derived
from
the
Golgi
endoplasmic
reticulum,
trafficking
organelles
among
was
obtained
by
the
centrifugation
of S2 at 200,000
× g for
min.
Protein
content
was
determined
by
the
bicinchoninic
acid
[31].
2.3.
Acylpeptide
hydrolase
activity
assays
ACPH
activity
was
performed
as
described
previously
[20].
Briefly,
the
Each
tion,
buffer
ume
20
reaction
a
lytik
absorbance
activity
expressed
tions.
ACPH
activity
was
assayed
monitoring
the
hydrolysis
of
synthetic
substrate
N-acetyl-l-alanine
p-nitroanilide
(AANA).
one
of the
different
samples
(homogenates,
microsomal
frac-
synaptosomes
and
synaptic
junctions)
was
mixed
(1:40)
with
A (200
mM
Tris–HCl,
1 mM
DTT
pH 7.4)
in 1 ml
final
vol-
and
incubated
at 37◦C.
The
reaction
was
initiated
by adding
?l of substrate
(4 ?mol)
dissolved
in dimethylsulfoxide.
The
product
p-nitroaniline
was
determined
quantitatively
in
Specord
205
temperature-regulated
spectrophotometer
(Ana-
Jena
Aktiengesellschaft,
Jena,
Germany)
by
measuring
the
at
405
nm using
ε405= 7530
M−1cm−1. The
enzymatic
was
normalized
to the
protein
content
in the
assay
and
as
the
mean
± SEM
of
at least
5 independent
prepara-
2.4.
Acetylcholinesterase
activity
assays
AChE
activity
present
in homogenates,
microsomal
fraction,
synaptosomes
metrically
at
iodide
mixed
DTNB,
by
by
thio-2-nitrobenzoate
at
and
synaptic
junctions
was
determined
spectropho-
monitoring
the
hydrolysis
of
S-acetylthiocholine
iodide
30◦C as
described
by
Ellman
et
al.
[6],
using
acetylthiocholine
as
a synthetic
substrate
for
AChE.
Briefly,
samples
(7 ?l) were
with
1 ml
of
50
mM
phosphate
buffer
containing
0.25
mM
pH
7.9,
and
incubated
at
30◦C.
The
reaction
was
initiated
adding
30
?l of
substrate
(0.03
?mol).
The
thiocholine
released
substrate
hydrolysis
reacts
in the
presence
of DTNB,
yielding
5-
(ε406= 13,300
M−1cm−1) that
was
quantified
406
nm and
normalized
as
described
above.
2.5.
Western
blotting
Twenty
micrograms
of protein
of
each
sample,
dissolved
at
1
dodecyl
10%
polyvinylidene
incubated
incubation
gated
was
quantification
gram
expressed
dent
mg/ml
in Laemmli
loading
buffer,
were
separated
by sodium
sulfate–polyacrylamide
gelsunder
electrophoresis
conditions
(SDS–PAGE)
transferred
on
onto

reducing

and

fluoride
(PVDF)
membranes.
Membranes
were
overnight
at 4◦C with
primary
antibodies
followed
by
at
room
temperature
with
secondary
antibody
conju-
with
horseradish
peroxidase
for
60
min.
Immunoreactivity
visualized
using
the
ECL
detection
system.
Densitometric
was
performed
using
the
image
processing
pro-
ImageJ
1.44o
(National
Institute
of
Health,
USA).
Data
were
as
fold
change
from
homogenate
for at
least
4 indepen-
preparations
and
mean
±
SEM
for
each
fraction
was
calculated.
2.6.
Statistical
analysis
Enzymatic
ferroni
activities
were
compared
(one
way
ANOVA
test,
Bon-
post
hoc)
using
GraphPad
Prism
5 analysis
software
(La
Jolla,

Page 3

100
R.
Sandoval
et
al.
/ Neuroscience
Letters
520 (2012) 98–
103
Fig.
junctions
activity
1.
Enzymatic
activity
profile
of ACPH
and
AChE:
(A)
ACPH
specific
activity
in rat
forebrain
homogenate
(H),
microsomal
fraction
(MIC),
synaptosomes
(Syn)
and
synaptic
(SJ)
at pH 8.1
(white
bars)
and
6.8
(black
bars).
(B)
ACPH
activity
remaining
after
Syn
purification
and
isolation
of
SJ
fractions
at pH 8.1
and
6.8.
(C)
AChE
specific
in rat
forebrain
from
the
same
fractions
described
in (A).
*p < 0.05,
**p
<
0.01,
***p
< 0.001,
and###p < 0.001.
CA,
Student’s
ered
USA)
and
densitometric
quantifications
were
compared
using
t-test
with
Welch’s
correction.
Differences
were
consid-
significantly
different
at
p < 0.05.
3.
Results
3.1.
ACPH
and
AChE
enzymatic
activities
in rat
brain
fractions
The
distribution
of
ACPH
enzymatic
activity
in the
CNS
was
studied
somes
telencephalon
In
activity
homogenate
ACPH
overall
obtained
increase
homogenate
ACPH
p
ingly,
at
homogenate
tively.
in homogenates
(H),
microsomal
fraction
(MIC),
synapto-
(Syn)
and
synaptic
junctions
(SJ)
obtained
from
adult
rat
and
obtained
at pHs
6.8
and
8.1
(see
Section
2).
the
Syn
fraction
purified
at pH 8.1,
we
found
that
the
specific
of
ACPH
(sA)
was
increased
by
43%
as
compared
to the
(ACPH
sAH= 0.014
sASyn= 0.020
± 0.002
?mol
min−1mg
prot−1;
F(3, 4)= 19.9
± 0.001
?mol
min−1mg
prot−1;
ANOVA;
p < 0.05,
Fig.
1A,
white
bars).
Similar
results
were
in the
Syn
fraction
purified
at
pH 6.8,
where
a 53%
in ACPH
activity
was
observed
when
compared
with
the
(ACPH
sASyn= 0.020
± 0.001
?mol
min−1mg
prot−1;

sAH= 0.013
± 0.001
?mol
min−1mg
prot−1;
F(3, 4)= 10.9,
= 0.003
overall
ANOVA;
p < 0.01,
Fig.
1A,
black
bars).
Interest-
the
ACPH
enzymatic
activity
in the
SJ fraction
obtained
pH 8.1 was
significantly
decreased
when
compared
to both
and
Syn
fractions
(51%
and
66%
of decrease,
respec-
ACPH
sASJ= 0.007
± 0.001
?mol
min−1mg
prot−1, p < 0.01
for homogenate
to
obtained
matic
Syn
85.4%
results
the
Results
reported
some
determine
shown
either
pHs
sASyn= 0.017
?mol
p
for
sAH= 0.017
?mol
F(3, 4)= 17.96
sA
MIC
intracellular
homogeneously
[30,35]
and
Syn
fraction,
Fig.
1A,
white
bars).
Contrary
the
results
observed
at pH 8.1,
ACPH
activity
in SJ fractions
at pH 6.8
did
not
show
a significant
change
in enzy-
specific
activity
as
compared
to both
homogenate
and
fractions
(ACPH
sASJ= 0.017
±
0.004
?mol
min−1mg
prot−1,
of
ACPH
sASyn, p = 0.51;
Fig.
1A black
bars
and
B).
These
strongly
suggest
that
ACPH
activity
is mainly
confined
to
synapse
and
preferentially
located
at
the
pre-synaptic
side.
from
our
laboratory
and
others
[18,27]
have
previously
that
ACPH
is a more
sensitive
target
than
AChE
for
anticholinesterase
drugs.
Therefore,
our
next
step
was
to
the
activity
profile
of
AChE
in the
same
samples.
As
in Fig.
1C,
AChE
enzymatic
activity
was
not
enriched
in
the
Syn
or SJ
fractions
as
compared
to homogenates
at both
(at
pH 8.1:
AChE
sAH= 0.01
± 0.004
?mol
min−1mg
sASJ= 0.014
overall
pH
sASyn= 0.015
prot−1,
±
0.006
?mol
min−1mg
F(3, 4)= 11.25
p = 0.55
prot−1, ±
ANOVA;
AChE
0.006
min−1mg
prot−1
Syn;

=
1
for

SJ;

at
6.8:

± 0.004
?mol
min−1mg
prot−1, ± 0.004
min−1mg
prot−1, sASJ= 0.017
± 0.004
?mol
min−1mg
prot−1;
overall
ANOVA;
p = 1 for
Syn
and
SJ).
However,
AChE
was
enriched
in the
MIC
fraction
at
both
pHs
(p < 0.05).
The
fraction
was
composed
mainly
of
membranes
coming
from
organelles
and
cell
membrane,
and
since
AChE
is
distributed
along
the
neuron
plasma
membrane
this
result
is not
surprising.
These
results
demonstrate
that

Page 4

R. Sandoval
et
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/ Neuroscience
Letters
520 (2012) 98–
103
101
Fig.
the
protein
in
tification
(white
**p
2.
ACPH
protein
amount
in synaptic
fractions.
(A)
Representative
blots
of ACPH,
pre-synaptic
protein
synaptophysin
(Synph)
and
the
excitatory
post-synaptic
PSD-95
in H,
Syn
and
SJ
fractions
at pH
8.1
and
6.8.
All
the
proteins
shown
the
figure
correspond
to the
bands
revealed
from
the
same
membrane.
(B)
Quan-
of
ACPH
relative
amount
with
respect
to H in Syn
and
SJ fractions
at pH
8.1
bars)
and
6.8
(black
bars)
obtained
from
at least
3 independent
preparations.
< 0.01,
***p < 0.001,
and##p < 0.01.
AChE
to
displays
a more
diffuse
pattern
of
distribution
as
compared
ACPH.
3.2.
Distribution
of
ACPH
in rat
brain
fractions
In
order
to confirm
the
profile
for
the
distribution
of
ACPH
activity
experiments.
synaptic
scaffold
respectively.
As
amount
1.81
6.8:
for
1.60
p < 0.01
SJ,
to the
physin
(1.54
Also,
Syn
This
With
was
compared
1.72
for
icantly
(1.00
in rat
brain
fractions,
we
performed
Western
blot
Furthermore,
we
also
used
antibodies
against
the
vesicle
protein
synaptophysin
and
the
post-synaptic
protein
PSD-95
as
pre-
and
post-synaptic
biomarkers,
seen
in Fig.
2A,
Syn
and
SJ synaptic
fractions
display
a higher
of
PSD-95
(at
pH 8.1:
1.45
± 0.04
fold
change
for
Syn
and
±
0.07
fold
change
for
SJ,
p < 0.01
for
both
fractions,
at pH
1.40
± 0.05
fold
change
for
Syn
and
1.66
± 0.04
fold
change
SJ,
p < 0.01
for
both
fractions)
and
synaptophysin
(at
pH 8.1:
± 0.02
fold
change
for
Syn
and
1.54
± 0.04
fold
change
for
SJ,
for
both,
at
pH 6.8:
1.63
± 0.05
for
Syn
and
2.3
± 0.13
for
p < 0.01
for
Syn
and
p < 0.05
for
SJ)
at both
pHs
as
compared
homogenate.
As
we
expected,
a higher
amount
of
synapto-
was
found
in the
SJ fraction
at pH 6.8
as
compared
to pH 8.1
±
0.04
at pH 8.1
compared
to 2.3
± 0.13
at
pH 6.8,
p < 0.05).
we
found
higher
amounts
of
PSD-95
in the
SJ than
in the
fraction
at both
pHs
(p < 0.01
at pH 8.1
and
p < 0.05
at
pH 6.8).
is consistent
with
results
shown
in the
literature
[4,21,36].
regard
to ACPH
distribution,
the
data
demonstrated
that
ACPH
significantly
enriched
in the
Syn
fraction
at
both
pHs
when
to the
homogenate
(fold
change
from
homogenate:
±
0.22,
p <
0.05
at
pH 8.1
and
1.69
± 0.2,
p <
0.05
at
pH 6.8,
n
= 4
each
value,
Fig.
2A and
B).
ACPH
protein
level
decreased
signif-
in the
SJ
fraction
at
pH 8.1
as
compared
to the
Syn
fraction
±
0.11,
p < 0.05,
Fig.
2A and
B white
bars).
These
data
support
the
ing
of
compared
fraction
Fig.
analysis
localization
results
derived
from
enzymatic
activity
(see
Fig.
1A),
suggest-
that
ACPH
is not
enriched
in postsynaptic
structures.
The
levels
ACPH
in the
SJ
fractions
obtained
at pH 6.8
were
increased
as
to pH 8.1
and
reached
similar
levels
to those
in the
Syn
at pH
6.8
(1.70
±
0.09,
p < 0.05
compared
to homogenate;
2A and
B,
black
bars).
These
results
correlate
with
enzymatic
of synaptic
fractions,
strongly
suggesting
a pre-synaptic
for
ACPH
in CNS
synapses.
4.
Discussion
In
our
study,
we
show
for
the
first
time
that
catalytically
active
ACPH
tion
Previously,
ied
patients
was
in
partmentalization
described
organophosphates
the
organophosphates
and
laboratory
pal
[18,19].
was
in synaptic
telencephalon
in
undetermined
An
the
obtain
this
Therefore,
it
synapses.
vious
we did
pal
determine
the
Regarding
[37,38]
erentially
hand,
DDVP
tinic
a
together
that
of
synaptic
the
As
than
[16,22,24,27,28].
ity
several
lum,
AChE
tions,
is located
at CNS
synapses
with
a
preferential
distribu-
in subcellular
fractions
enriched
in pre-synaptic
components.
the
expression
of the
gene
encoding
for
ACPH
was
stud-
in the
CNS
of post-mortem
samples
from
Alzheimer’s
disease
and
their
age-matched
controls
[37,38].
ACPH
expression
found
in the
prefrontal,
frontal
and
temporal
cortex,
as
well
as
the
cerebellum,
but
no attempt
was
made
to identify
its
com-
at the
synapse.
To date,
only
two
reports
have
the
presence
of
catalytically
active
ACPH
sensitive
to
in brain
tissue:
Richards
et al.
[27]
showed
for
first
time
the
presence
of ACPH
as
a high-sensitive
target
for
in cytosol
preparations
obtained
from
porcine
rat
whole
brain
homogenates
and
recently,
results
from
our
showed
that
ACPH
activity
measured
in rat
hippocam-
homogenates
were
sensitive
to the
inhibitory
effect
of
DDVP
However,
until
now,
its
localization
in synaptic
structures
unclear.
Our
data
provide
insight
into
a
possible
role
for
ACPH
function,
although
its
histological
distribution
in the
and
sub-cortical
structures,
as
well
as its
presence
different
cell
types
of
the
CNS,
such
as
in glial
cells,
remains
and
needs
further
investigations.
important
issue
regarding
the
results
reported
here
is that
method
for
synaptic
fractionation
that
we
used
allowed
us to
fractions
mainly
composed
of excitatory
synapses,
due
to
method
allows
the
isolation
of
Gray
synapse
type
I [4,14,32,39].
our
data
corresponds
to a
mean
excitatory
synapse
and
is
still
unknown
whether
ACPH
is also
present
at
inhibitory
However,
some
outlines
can
be obtained
from
our
pre-
experiments
using
electrophysiological
techniques,
in which
not
find
a role
of
ACPH
in modulating
inhibitory
hippocam-
synapses
[18]. Furthermore,
it would
be
also
interesting
to
a
possible
ACPH
association
to cytomatrix
structures
of
active
zone
at
the
presynaptic
site
[33].
the
function
of
ACPH
at
the
synapse,
Yamin
et al.
reported
that
this
enzyme
was
able
to cleave
A?1–40pref-
as
dimer
and
trimer
oligomers
in vitro.
On
the
other
as
already
mentioned
above,
acute
inhibition
of
ACPH
by
at
doses
unable
to inhibit
AChE
facilitated
LTP
in an ?7nico-
receptor-dependent
manner
[18], indicating
that
ACPH
plays
role
in modulating
synaptic
strength.
Therefore,
these
results
with
the
data
reported
in this
paper
strongly
suggest
pre-synaptic
ACPH
could
be
able
to regulate
the
concentration
postsynaptic
receptor
modulators,
which
in turn
could
modify
strength
and,
therefore,
this
enzyme
could
be
a target
for
treatment
of
diseases
involving
cognitive
decline.
previously
reported,
ACPH
is
a
more
sensitive
target
AChE
to the
action
of
some
organophosphorous
substances
Our
results
show
an
enrichment
of
AChE
activ-
in the
MIC
fraction,
which
contains
membrane
vesicles
from
subcellular
compartments
such
as
endoplasmic
reticu-
Golgi
apparatus
and
the
cell
membrane
[2].
On
the
contrary,
does
not
display
enrichment
in synaptic
membrane
frac-
as
ACPH
does.
In fact,
AChE
is homogeneously
distributed

Page 5

102
R.
Sandoval
et
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/ Neuroscience
Letters
520 (2012) 98–
103
in the
innervating
can
anchored
synaptic
cleft
ticides
the
[1,5,13,15,34]). These
the
erative
been
6–10
chlorpyrifos-methyl
[18,25,27]. Taken
necessary
and
be
or activators.
Finally,
to the
pharmacological
CNS,
localized
in both
cholinergic
cell
bodies
and
axons
the
cortex
from
the
basal
forebrain
[30]. This
enzyme
be found
at
three
locations
in the
cholinergic
synapses:
(1)
to the
pre-synaptic
membrane,
(2)
inserted
at the
post-
membrane,
and
(3)
as
a soluble
form
in the
synaptic
[3].
Organophosphorous
substances
are
mainly
used
as
insec-
and
their
toxicity
is caused
by
inhibition
of
AChE
and
increase
of
acetylcholine
in the
synaptic
cleft
(reviewed
in
kind
of
compounds
have
also
been
used
in
treatment
of
cognitive
impairment
associated
to neurodegen-
diseases
such
Alzheimer’s
disease
[17,29].
However,
it has
described
that
erythrocyte
and
brain
isoforms
of
ACPH
are
times
more
sensitive
to some
organophosphates
including
oxon,
diisopropylfluorophosphate
and
DDVP
together,
these
data
reinforce
the
idea
that
it is
to accurately
determine
the
relationship
between
AChE
ACPH
activities
regarding
to their
physiological
effects
that
can
modulated
by
inhibitors
(such
as
organophosphorus
substances)
we
expect
that
the
results
reported
here
can
contribute
comprehension
of
ACPH
function
in the
CNS
and
its
use
as
a
target
for
synaptic
activity
modulation.
5.
Conclusion
We demonstrate
synapse
tial
required
(i.e.,
for
the
first
time
the
presence
of
ACPH
in the
and
our
results
suggest
that
ACPH
would
have
a
preferen-
localization
at the
pre-synaptic
side.
Further
experiments
are
in order
to accurately
confirm
its
pre-synaptic
localization
electron
microscopy
studies).
Acknowledgments
The
authors
are
indebted
to the
following
funding
grants:
Pro-
grama
F.P.;
R.S.
to U.W.
Bicentenario
en
Ciencia
y Tecnología
(No
PSD-11)
to R.S.
and
VRIDT
funding
grant
from
Universidad
Católica
del
Norte
to
and
F.P.;
Fondecyt
No 1100322
and
Proyecto
Anillo
ACT09-06
Fondef
D09I1057
to R.S
and
F.P.
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