Effects of amyloid beta peptide on glutamine transporter mRNA expression and cell viability in cultured rat cortical cells

R
ScienceAsia 35 (2009): 156–160
ESEARCH ARTICLE
doi: 10.2306/scienceasia1513-1874.2009.35.156
Effects of amyloid-? peptide on glutamine transporter
mRNA expression and cell viability in cultured rat
cortical cells
Doungjai Buntupa, Anek Chayasadomb, Rudee Suraritc, Nuanchan Jutapakdeegula,
Wipawan Thangnipona,?
a Neuro-Behavioural Biology Centre, Institute of Science and Technology for Research and Development,
Mahidol University, Nakorn Pathom 73170, Thailand
b Department of Periodontology, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand
c Department of Physiology and Biochemistry, Faculty of Dentistry, Mahidol University, Bangkok 10400,
Thailand
?Corresponding author, e-mail: grwtn@mahidol.ac.th
Received 11 Nov 2008
Accepted 27 May 2009
ABSTRACT:
Alzheimer’s disease is a major neurodegenerative disorder in which there is an overproduction and
accumulation of amyloid-? (A?) peptides. During the initial stages of the disease, glutamate receptors are dysregulated
by A? accumulation resulting in the disruption of glutamatergic synaptic transmission. We used rat cortical cell cultures
to examine the effects of A?(25–35)-induced neurotoxicity on glutamine transporters involved in the glutamate cycle. In
primary mixed cell cultures prepared from cerebral cortex, incubation with 10 µM A?(25–35) for 12 h, but not for 24 h,
markedly suppressed system A transporter 1 (SAT1) mRNA expression. On the other hand, A?(25–35) had no effect on
SAT1 mRNA level in neuronal cell cultures. Treatment of both types of cell cultures with A?(25–35) resulted in a signi?cant
decrease in cell survival in a concentration and time-dependent manner, as determined by MTT assay. These results indicated
that A? may impair neuronal function and transmitter synthesis and perhaps reduce excitotoxicity through a reduction in
neuronal glutamine uptake.
KEYWORDS: Alzheimer’s disease, amyloid-? peptide, mRNA glutamine transporter
INTRODUCTION
but also to complex physiological processes, such as
memory, learning, plasticity, and neuronal cell death 8.
Alzheimer’s disease is a neurodegenerative disorder
The neurons use glutamine as the main substrate for
characterized by the presence of neuro?brillary tan-
synthesis of glutamate and ?-aminobutyric acid 9, 10.
gles, neuritic plaques, synaptic depletion, and neu-
The release of glutamine from astrocytes and the
ronal cell loss in speci?c cortical and subcortical
uptake of glutamine by neurons are integral steps in
areas of the brain 1. Senile plaques contain amyloid-
the glutamate-glutamine cycle, a major pathway for
? (A?) peptide, produced by cleavage of the amyloid
the replenishment of neuronal glutamate 11.
precursor protein (APP) by ?- and ?-secretases 2. A?
Recently, cDNA encoding three distinct system A
is a peptide of 39–43 amino acids that forms insoluble
amino acid transporters (SAT1 to SAT3) have been
aggregates surrounded by degenerating neuritis and
cloned and functionally identi?ed 12. SAT1 isoform
activated glial cells 3.
Numerous experiments with
is preferentially expressed on the plasma membrane
synthetic A?(1–42) suggest that this peptide is toxic
of glutamatergic neurons and encodes for a highly
to cultured neurons by inducing protein oxidation,
ef?cient glutamine transporter 13, 14. Dolinska et al 15
lipid peroxidation, and oxidative stress in the cellular
reported that SAT1 mRNA is transcribed in neurons
environment in ways that are inhibited by antioxidants
but not in cerebral cortical astrocytes or cerebellar
of free-radicals 4. In culture, A? can directly induce
astrocytes. A malfunction in the glutamate transport
neuronal cell death 5 and can render neurons vulnera-
system can lead to accumulation of excessive glu-
ble to excitotoxicity 6 and oxidative insults 7.
tamate in the synapse, which is harmful to neurons
Glutamate is the major excitatory neurotransmit-
and could result in neurodegeneration 16, 17. However,
ter of the mammalian central nervous system that
the causal relationship between A? and glutamine
contributes not only to fast synaptic neurotransmission
transporters in Alzheimer’s disease is not yet well
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ScienceAsia 35 (2009)
157
de?ned.
mized in order to allow amplicon synthesis within
A small 11-amino acid fragment of the full-
the linear-log phase of ampli?cation, were as follows:
length peptide, A?(25–35), is a convenient alterna-
reverse-transcription at 50 °C for 30 min; inactivation
tive in Alzheimer’s disease investigations as the pep-
of reverse transcriptase and activation of Hot start
tide mimics several toxicological and oxidative stress
Taq DNA polymerase at 95 °C for 15 min; 30 cycles
properties of the native full-length peptide 4, 18, 19.
of 30 sec at 94 °C, 1 min at 55 °C (for STAT1) or
Recently, our group demonstrated that treatment of
1 min at 72 °C (for ?-actin); and a ?nal extension
mixed cell cultures with 10 µM A?(25–35) for 12 h
step at 72 °C for 10 min. Amplicons were separated
and 24 h reduces SAT1 protein level as demonstrated
by electrophoresis in 2% agarose gel and stained
by immunocytochemical and quantitative immunore-
with ethidium bromide. Densitometric analysis was
activity analysis 20.
We now report the effects of
performed using QUANTITY ONE 4.5.1 (Bio-Rad).
A?(25–35) on SAT1 expression in rat cortical primary
This method is based on the reduction of 3-
cell cultures.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) tetrazolium salt to a crystalline
MATERIALS AND METHODS
blue formazan product by cellular oxidoreductase 26.
Enriched primary neuronal cell cultures were pre-
Therefore, the amount of formazan produced is
pared from cerebral cortices of 10 embryonic day-18
proportional to the number of viable cells.
The
(E18) Wistar rat foetuses as described previously 21, 22.
culture medium was removed from the treated cells
Cells plated at 5 × 105 cells/ml were cultured in
and replaced with a solution of MTT (0.5 mg/ml) in
poly-L-lysine coated multiwell plates and maintained
phosphate-buffered saline, pH 7.4. After incubation
in serum-free Dulbecco’s modi?ed Eagles medium
for 4 h at 37 °C, the solution was removed and the
(DMEM) supplemented with B27 under a humidi?ed
resulting blue formazan solubilized in 0.1 ml of
atmosphere of 5% CO /95% air at 37 °C for 5 days
0.04 M HCl in isopropanol. Absorbance at 570 nm
2
before incubating with peptide.
was measured at 630 nm using a microplate reader
Primary mixed cell cultures were prepared from
(Bio-Tex EL 311).
cerebral cortices of 10 1-day old Wistar rat neonates
Data were analysed by one-way ANOVA using
as previously described 23.
Cells plated at 5 × 105
LSD for post-hoc comparisons.
Differences were
cells/ml were cultured in poly-L-lysine coated mul-
considered statistically signi?cant if P < 0.05. Each
tiwell plates.
The dissociated cortical cells were
experiment was repeated 3–4 times and results from a
suspended in Neurobasal-A medium containing 2%
representative experiment are shown.
(v/v) B27 and 1% (v/v) GlutaMAX I and maintained
RESULTS
as described for the E18 cells.
The B27-containing medium was removed from
To determine the effect of A?(25–35) on expression
neuronal and mixed cell cultures at day 6.
Cells
of SAT1 in both neuronal and mixed cell cultures,
were then washed twice with DMEM or Neurobasal-
cells were cultured for 5 days and then incubated with
A medium and then incubated for 12 h and 24 h in
A?(25–35) for 12 h and 24 h. SAT1 mRNA levels
the absence or presence of 3 and 10 µM A?(25–35)
were determined relative to those of ?-actin 27.
(Sigma) to produce a sub-lethal neurotoxicity.
The RT-PCR of the mixed cell culture showed
Total RNA was isolated using RNeasy Mini Kit
that 10 µM A?(25–35) signi?cantly suppressed SAT1
(Qiagen) and the yield was determined by spec-
mRNA expression after incubation for 12 h (P <
trophotometry at 260 nm. The Reverse transcriptase
0.001) (Fig. 1). However, SAT1 expression at 24 h
polymerase chain reaction (RT-PCR) was carried out
showed no variation after exposure to 3 and 10 µM
using the Qiagen one step RT-PCR kit.
PCR am-
of A?(25–35). In neuronal cell culture, expression of
pli?cations of SAT1 employed primers 5 -ACAGGC-
SAT1 mRNA did not change after exposure to 3 and
GACATTCTCATCCT-3 (forward) and 5 -GTTTCA-
10 µM of A?(25–35) for 12 and 24 h when compared
GTGGCCTTCACCAT-3 (reverse), giving rise to a
with the control (Fig. 2).
426 base pair amplicon 24, and ?-actin (housekeeping
Neuronal cell cultures showed a signi?cant loss
gene) primers 5 -CCCAGAGCAAGAGAGGCATC-
of cell viability, as determined by MTT method, when
3 (forward) and 5 -CTCAGGAGGAGCAATGATCT-
treated for 24 h with A?(25–35) in a dose-dependent
3 (reverse), giving rise to a 830 base pair amplicon 25.
manner (P < 0.05) (Fig. 3). A signi?cant cell loss
Primers were obtained from the Bioservice Unit of
was also observed after treatment with 10 µM A?(25–
the National Science and Technology Development
35) for 12 h (P < 0.05). Viability of mixed cell
Agency, Thailand. Thermocycling conditions, opti-
cultures was reduced when exposed to 3 and 10 µM
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A)





12 h 24 h


A)



158
Marker 1 2 3 Marker 1 2 3
ScienceAsia 35 (2009)
A)
(a) 12 h 24 h
A)


?

Marker 1 2 3 Marker 1 2 3

(a)
50 12 h 24 h
-actin
0 bp

(a) 12 h 24 h

SAT1

?-actin
Marker 1 2 3 Marker 1 2 3
500 bp
Marker 1 2 3 Marker 1 2 3




SAT1

?-actin

?-actin
500 bp




500 bp

SAT1

SAT1


B)







(b)
B) (b)




1.2
12 h
B) (b)
1.2

24 h
12 h

24 h
B)
1
?
-
1
(b)

?
-actin
1.2
12 h
0.8
**
s.
0.8
24 h
1.2
12 h
0.6
1
24 h
?
-actin
0.6
1
?
-
s.
0.4
r
ession v
0.8

0.4
0.8
0.2
**
e exp
0.6
r
ession v
0.6
0.2
relative expression vs.
actin 0
0.4
Relativ

Control 3 µM A?(25-35) 10 µM A?(25-35)
e exp
0.4
0
0.2
Control 3 µM A?(25-35) 10 µM A?(25-35)
Fig. 1 Expression of SAT1 mRNA in mixed cell culture

0.2

Relativ




exposed to A?(25–35). Mixed cell cultures were incubated
0

Fig. 2 Expression of SAT1 mRNA in the neuronal

cell
relative expression vs.
actin 0

Control 3 µM A?(25-35) 10 µM A?(25-35)
with A?

Contr
(25–35) ol
for
12 3
and µM A
24 ?
h (25-35)
and 10 µM A
subjected to?(2
R5-3
T 5)
-PCR
culture exposed to A?(25–35). Neuronal cell cultures were



for ampli?cation of SAT1 and ?-actin mRNA. (a) Am-
incubated with A?(25–35) for 12 and 24 h and

subjected

plicons separated by gel-electrophoresis and stained with to RT-PCR for ampli?cation of SAT1 and ?-actin mRNA.



ethidium-bromide (b) quantitated bands. Lane 1: control;
(a) Amplicons separated by gel-electrophoresis and stained


lane 2: exposed to 3 µM A?(25–35); lane 3: exposed

to
with ethidium-bromide (b) quantitated bands.
Lane 1:


10 µM A?(25–35). **P < 0.001 compared with control.

control; lane 2: exposed to 3 µM A?(25–35); lane 3:


Columns show mean ± SEM from three independent exper-
exposed to 10 µM A?(25–35). Columns show mean ± SEM

iments.
from three independent experiments.





A?(25–35) for 12 and 24 h (P < 0.05 and P < 0.001

respectively).

an important role in Alzheimer’s disease progression.

However, A?(25–35) had no effect on SAT1 mRNA
DISCUSSION
expression at 24 h in both mixed and neuronal cell

Results from this study indicated a signi?cant de-
cultures.
Tian et al 32 showed that SAT2 mRNA

crease of SAT1 mRNA level after 12 h exposure to
level does not decrease after exposure to a low dose
10 µM A?(25–35) in mixed but not in neuronal cell
of A?(25–35).
This effect may act as a cellular

cultures.
Baron et al 28 suggested that A? exerts
defence against neurodegeneration in pathogenesis of
its toxic effect via activation of transcription factors.
Alzheimer’s disease.
A?(25–35) induces expression of the growth arrest
Viability of mixed and neuronal cell cultures,
and DNA damage-inducible gene (gadd 45) impli-
measured by MTT method, was reduced when ex-
cated in the DNA excision-repair process. Accumula-
posed to A?(25–35) in both a time- and concentration-
tion and oligomerization of A? is also thought to play
dependent manner. In neuron-enriched cultures, A?
a central role in Alzheimer’s disease pathogenesis.
is known to increase vulnerability to excitotoxic-
A? can produce oxidative damage by stimulating
ity 33. Wei et al 34 showed that A?(1–42), in a time
neuroin?ammation, as well as generating reactive
(1–48 h) and concentration (0.01–20 µM)-dependent
oxygen species as a result of binding to mitochondrial
manner, induces toxicity in cultured neurons. Simi-
proteins 29.
Studies of neuron co-cultures showed
larly, Domenici et al 35 demonstrated that while pure
that astrocyte formation of nitric oxide and other
neuronal culture shows a signi?cant cell loss only at
reactive oxygen species does occur in a number of
the highest concentration of A?(25–35), mixed cell
neurodegenerative disorders 30, 31. Thus a mixed cell
culture manifests a toxic effect in a dose-dependent
culture will be more sensitive to toxic effects than the
manner which was signi?cant down to the lowest
neuronal cell culture when exposed to A?. Our data
concentration.
As observed by Pfrieger and Bar-
imply that a decrease of glutamine uptake may play
res 36, neurons co-cultured with astrocytes develop
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ScienceAsia 35 (2009)
159
(a)
12 h
duce excitotoxicity through a reduction in neuronal
24 h
glutamine uptake.
l
)
100
t
r
o
n
Acknowledgements: This work was supported by the
80
c
o
f
o
Thailand Research Fund through the RGJ PhD Programme
60
*
(
%
*
**
(PHD/0230/2545). The animal procedures were approved
**
40
i
l
i
t
y
b
by the committee on ethics in animal research of the Institute
i
a
v
20
of Science and Technology for Research and Development,
e
l
l
C
0
Mahidol University, MUSTA 2006/003.
Control
3 M A (25-35)
10 M A (25-35)
(b)
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