Article

Preservation of GABA(A) Receptor Function by PTEN Inhibition Protects Against Neuronal Death in Ischemic Stroke

Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Canada.
Stroke (Impact Factor: 5.72). 04/2010; 41(5):1018-26. DOI: 10.1161/STROKEAHA.110.579011
Source: PubMed

ABSTRACT

Downregulation of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is thought to be a novel neuroprotective strategy in ischemic stroke, but the underlying mechanisms remain unclear. In this study, we aimed to validate the use of PTEN regulation of gamma-aminobutyric acid subtype A receptors (GABA(A)Rs) as a molecular target for the treatment of ischemic stroke. Because suppression of GABA(A)Rs contributes to ischemic neuron death, describing the intracellular signaling that interacts with GABA(A)Rs in ischemic neurons would provide a molecular basis for novel stroke therapies.
We measured surface GABA(A)R expression by immunocytochemical labeling and surface protein biotinylation assay. Knockdown and overexpression approaches were used to test the effects of PTEN on the expression and function of GABA(A)Rs. Neuronal death was detected in both in vitro and in vivo stroke models.
The knockdown and overexpression approaches provided the first evidence that PTEN negatively regulated membrane expression and function of GABA(A)Rs in rat hippocampal neurons. Importantly, we demonstrated that a PTEN inhibitor prevented the reduction of surface GABA(A)Rs in injured hippocampal neurons subjected to oxygen-glucose deprivation, an in vitro insult that mimics ischemic injury, whereas a GABA(A)R antagonist significantly reduced this PTEN inhibitor-induced neuroprotection in both the in vitro and in vivo ischemic stroke models.
Our study provides direct evidence that downregulation of PTEN protects against ischemic neuron death by preserving GABA(A)R function. Targeting this pathway may be an effective strategy for development of selective, potent stroke treatments.

Full-text

Available from: Lijun Li, Mar 21, 2016
Preservation of GABA
A
Receptor Function by PTEN
Inhibition Protects Against Neuronal Death in
Ischemic Stroke
Baosong Liu, MD, PhD; Lijun Li, MD, PhD; Quanguang Zhang, MD, PhD; Ning Chang, MSc;
Dianshi Wang, MD, PhD; Yuexin Shan, PhD; Lei Li, MD, PhD; Hanbin Wang, MD, PhD;
Hua Feng, MD, PhD; Liang Zhang, MD, PhD; Darrell W. Brann, PhD; Qi Wan, MD, PhD
Background and Purpose—Downregulation of the tumor suppressor, phosphatase and tensin homolog deleted on
chromosome 10 (PTEN), is thought to be a novel neuroprotective strategy in ischemic stroke, but the underlying
mechanisms remain unclear. In this study, we aimed to validate the use of PTEN regulation of
-aminobutyric acid
subtype A receptors (GABA
A
Rs) as a molecular target for the treatment of ischemic stroke. Because suppression of
GABA
A
Rs contributes to ischemic neuron death, describing the intracellular signaling that interacts with GABA
A
Rs in
ischemic neurons would provide a molecular basis for novel stroke therapies.
Methods—We measured surface GABA
A
R expression by immunocytochemical labeling and surface protein biotinylation
assay. Knockdown and overexpression approaches were used to test the effects of PTEN on the expression and function
of GABA
A
Rs. Neuronal death was detected in both in vitro and in vivo stroke models.
Results—The knockdown and overexpression approaches provided the first evidence that PTEN negatively regulated
membrane expression and function of GABA
A
Rs in rat hippocampal neurons. Importantly, we demonstrated that a
PTEN inhibitor prevented the reduction of surface GABA
A
Rs in injured hippocampal neurons subjected to
oxygen-glucose deprivation, an in vitro insult that mimics ischemic injury, whereas a GABA
A
R antagonist significantly
reduced this PTEN inhibitor–induced neuroprotection in both the in vitro and in vivo ischemic stroke models.
Conclusions—Our study provides direct evidence that downregulation of PTEN protects against ischemic neuron death by
preserving GABA
A
R function. Targeting this pathway may be an effective strategy for development of selective, potent
stroke treatments. (Stroke. 2010;41:1018-1026.)
Key Words: GABA
A
receptor
PTEN
ischemia
stroke
neuroprotection
excitotoxicity
T
he tumor suppressor, phosphatase and tensin homolog
deleted on chromosome 10 (PTEN), is a dual-specificity
phosphatase.
1
PTEN is involved in the regulation of several
basic cellular functions, such as cell cycle progression, cell
migration, cell spreading, and cell growth.
2
Recent studies
have shown that suppressing PTEN protects against ischemic
neuron death through both the enhancement of Akt activation
and the inhibition of NR2B subunit– containing N-methyl-
D-
aspartate receptors.
3,4
These data suggest that PTEN may be
a therapeutic target for stroke treatment. However, the cellu-
lar and molecular mechanisms underlying PTEN downregu-
lation–mediated neuroprotection remain largely unknown.
3
Ischemia-induced loss of excitatory and inhibitory equilib-
rium contributes to excitotoxicity-mediated neuronal death in
ischemic stroke. Suppressed function of
-aminobutyric acid
subtype A receptors (GABA
A
Rs) causes neuronal damage
after stroke.
5– 8
Both the GABA uptake inhibitor and the
GABA
A
R agonist are neuroprotective in animal models of
ischemic stroke.
9,10
A recent study has shown that cell surface
GABA
A
Rs are markedly decreased in neurons treated by
oxygen-glucose deprivation (OGD),
11
suggesting that a
change in the number of membrane GABA
A
Rs may be a
crucial process in enhancing ischemic neuron death.
11
How-
ever, use of GABA
A
R agonists as neuroprotective agents has
been disappointing in clinical trials owing to uncontrolled,
global overactivation of the channels.
12
Thus, identifying the
intracellular signaling specifically linked to GABA
A
Rs in
ischemic neurons would aid us to understand the cellular and
Received January 14, 2010; accepted January 27, 2010.
From the Division of Fundamental Neurobiology (B.L., L.J.L., N.C., D.W., Y.S., L.Z., Q.W.), Toronto Western Research Institute, University Health
Network, Toronto, Canada; Developmental Neurobiology Program (Q.Z., D.W.B.), Institute of Molecular Medicine and Genetics, Medical College of
Georgia, Augusta, Ga; Research Institute of Surgery and Daping Hospital (L.L.), Chongqing; Department of Internal Medicine (H.W.), 307 Hospital,
Beijing; and Department of Neurosurgery (H.F.), Southwest Hospital, Chongqing, China; and Department of Physiology and Cell Biology (Q.W.),
University of Nevada School of Medicine, Reno, Nev.
Correspondence to Dr Qi Wan, Department of Physiology and Cell Biology, University of Nevada School of Medicine, 1664 N Virginia St, MS0352,
Reno, NV 89557. E-mail qwan@medicine.nevada.edu; or Dr Darrell W. Brann, Developmental Neurobiology Program, Institute of Molecular Medicine
and Genetics, CA-4004, 1120 15th St, Medical College of Georgia, Augusta, GA 30912. E-mail dbrann@mcg.edu
© 2010 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.110.579011
1018
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molecular mechanisms underlying ischemia-induced neuro-
nal death, which may ultimately lead to the development of
selective, potent therapies for stroke patients.
GABA is the major inhibitory neurotransmitter in the
mammalian central nervous system. Inhibitory synaptic trans-
mission is mediated by postsynaptic GABA
A
Rs, and the most
abundant population of GABA
A
Rs in the mammalian brain is
the
1
2
2 combination.
13
Recent studies indicate that traf-
ficking of GABA
A
Rs to and from the membrane surface
plays an important role in posttranslational signaling mecha-
nisms that modulate the stability, density, and function of the
channels
14,15
and that the
2 subunit is required for the
trafficking of GABA
A
Rs to the postsynaptic membrane.
14,15
Interestingly, a recent study has shown that GABA
A
Rs are
upregulated by activation of phosphatidyl inositol 3-kinase/
Akt– dependent signaling.
16
Because Akt activation is nega-
tively regulated by PTEN,
17
we hypothesized that GABA
A
Rs
may be upregulated by PTEN inhibition.
Given the involvement of PTEN and GABA
A
Rs in ische
-
mic neuron damage, the present study tested the effect of
PTEN downregulation on GABA
A
R expression and function
in the in vitro OGD and in vivo ischemic stroke models. We
provide direct evidence that PTEN downregulation protects
against ischemic neuron death by preventing GABA
A
R sup
-
pression in ischemic neurons.
Materials and Methods
Hippocampal Neuron Culture and
OGD Treatment
To create an in vitro ischemia-like injury model, hippocampal
neuron cultures were treated by OGD. The cultures were prepared
from Sprague-Dawley rats at gestation day 18, and detailed methods
are described in our previous study.
18
To initiate the OGD challenge,
cells were transferred to deoxygenated, glucose-free, extracellular
solution (in mmol/L: 116 NaCl, 5.4 KCl, 0.8 MgSO
4
, 1.0 NaH
2
PO
4
,
1.8 CaCl
2
, and 26 NaHCO
3
); introduced into a specialized, humid
-
ified chamber (Plas-Labs, Lansing, Mich); and maintained at 37°C in
85% N
2
/10% H
2
/5% CO
2
for 40 minutes.
18
For “sham” treatment,
cultures were transferred to another extracellular solution
(in mmol/L: 116 NaCl, 5.4 KCl, 0.8 MgSO
4
, 1.0 NaH
2
PO
4
, 1.8
CaCl
2
, 26 NaHCO
3
, and 33 glucose); introduced into the humidified
chamber; and maintained at 37°C for 40 minutes in 95% O
2
/5% CO
2
.
Immunofluorescence Labeling, Image Acquisition,
and Analysis
To measure the surface expression of GABA
A
R
2 subunits in
control and OGD-treated neurons,
18,19
unpermeabilized cells were
labeled with rabbit anti-
2 primary antibody against the extracellular
domain of the
2 subunit (Millipore Corp, Billerica, Mass), and
Alexa Fluor 594 (red fluorescence) secondary antibody (Invitrogen
Canada Inc, Burlington, Canada). To examine PTEN expression,
neurons were permeabilized by treatment with 4% paraformaldehyde
in phosphate-buffered saline for 20 minutes and then with 0.3%
Triton X-100 for 10 minutes. Rabbit anti-PTEN primary antibody
(Cell Signaling Technology Inc, Danvers, Mass) and Alexa Fluor
594 (red fluorescence) secondary antibody (Invitrogen Canada Inc)
were used for labeling.
Fluorescence-labeled neurons were imaged with a Zeiss LSM 510
META confocal microscope (Carl Zeiss, Germany) and analyzed as
described previously.
18,19
Images were acquired with a Zeiss Axio-
Cam digital camera in the linear range with constant settings. Each
image was a z-series of 6 to 13 images, taken at 0.75-
m-depth
intervals. The resultant stack was “flattened” into a single image by
using a maximum projection. For all experiments, we analyzed the
fluorescence signal in regions of interest by measuring the average
fluorescence intensity per unit area. All images in all experiments
were analyzed according to identical acquisition parameters. During
data acquisition and analysis, the investigator was blinded to treat-
ment group assignment. In each experiment, neurons were selected
randomly under bright-field optics, and fluorescence images of each
neuron acquired from a single plane were transferred for analysis.
The cells in control and OGD groups from the same culture
preparation were processed and imaged in parallel. Three fields were
randomly selected in each culture. The fluorescence density was
analyzed by Image J software (National Institutes of Health, Be-
thesda, Md).
18
The expression of surface receptors and whole-cell
proteins represented by labeled fluorescence densities in treated
groups was normalized versus that in control groups. The n value
refers to the number of cells analyzed.
Surface Protein Biotinylation and Western
Blot Assays
To quantify the surface expression of GABA
A
Rs, surface protein
biotinylation assays were performed. The neuronal membrane pro-
Figure 1. OGD decreases surface GABA
A
R
2 subunit expres
-
sion. A, Left, Representative images showing that surface
expression of membrane GABA
A
R
2 subunits is reduced in cul
-
tured rat hippocampal neurons after OGD insult. Right, Bar
graph showing expression of
2 subunits in the OGD vs control
group (n91 cells for controls and n79 cells for the OGD
group; *P0.05, Student t test). Three independent experiments
were performed, and a minimum of 25 randomly selected cells
per