Diminished Akt phosphorylation in neurons lacking glutathioneperoxidase-1 (Gpx1) leads to increased susceptibility to oxidativestress-induced cell death

Juliet M. Taylor, Ugur Ali, Rocco C. Iannello, Paul Hertzog and Peter J. Crack

Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University,

Melbourne, Australia

Abstract

We have previously identified an increased susceptibility of

glutathione peroxidase-1 (Gpx1)–/– mice to neuronal apop-

tosis following mid-cerebral artery (MCA) occlusion. This

study was designed to elucidate the mechanisms involved in

elevated neuronal cell death arising from an altered

endogenous oxidant state. This was addressed in both an

in vitro and in vivo model of oxidative stress in the form of

exogenous H2O2 and cerebral ischaemia, respectively.

Increased levels of cell death were detected in primary

neurons lacking Gpx1 following the addition of exogenous

H2O2. This increased apoptosis correlated with a down-

regulation in the activation of the phospho-inositide 3-kinase

[PI(3)K]–Akt survival pathway. The importance of this path-

way in protecting against H2O2-induced cell death was

highlighted by the increased susceptibility of wildtype neu-

rons to apoptosis when treated with the PI(3)K inhibitor,

LY294002. The Gpx1–/– mice also demonstrated elevated

neuronal cell death following MCA occlusion. Although Akt

phosphorylation was detected in the Gpx1–/– brains, acti-

vation was not seen in later reperfusion events, as demon-

strated in wildtype brains. Previous studies have highlighted

the importance of Akt phosphorylation in protecting against

neuronal cell death following cerebral ischaemia–reper-

fusion. Our results suggest that the increased susceptibility

of Gpx1–/– neurons to H2O2-induced apoptosis and neur-

onal cell death in vivo following cerebral ischaemia–reper-

fusion injury can be attributed in part to diminished activation

of Akt. Perturbations in key anti-apoptotic mechanisms as a

result of an altered redox state may have implications in the

study of oxidative stress-mediated neuropathologies.

Keywords: Akt, apoptosis, glutathione peroxidase-1, H2O2,

stroke, reactive oxygen species.

J. Neurochem. (2004) 92, 283–293.

Oxidative stress has been implicated in the elevated neuronalcell death associated with a number of neuropathologies,including Alzheimer’s disease (AD), Parkinson’s disease(PD) and cerebral ischaemia–reperfusion injury (Leist andNicotera 1998; Pettmann and Henderson 1998; Schulz et al.2000). Recent studies have shown protective effects ofoverexpressing antioxidant enzymes such as Cu/Zn–super-oxide dismutase (SOD1) or glutathione peroxidase-1 (Gpx1)against neuronal apoptosis following focal cerebral ischaemia(FCI) (Kinouchi et al. 1991; Fujimura et al. 2000). Accord-ingly, mice lacking SOD1 or Gpx1 demonstrate elevatedneuronal cell damage following FCI (Kondo et al. 1997;Crack et al. 2001). Interestingly, both the Gpx1 and SOD1knockouts report high levels of apoptosis when comparedwith their wildtype counterparts. However, while the protect-ive effects of SOD1 have been linked to decreased activationof Bad, a lack of cytochrome c release and reduced caspase-8

Received June 9, 2004; revised manuscript received August 29, 2004;accepted September 1, 2004.Address correspondence and reprint requests to Dr Peter Crack,

Centre for Functional Genomics and Human Disease, Monash Instituteof Reproduction and Development, Monash Medical Centre, MonashUniversity, 246 Clayton Road, Clayton, Victoria 3168, Australia.E-mail: peter.crack@med.monash.edu.auAbbreviations used: AD, Alzheimer’s disease; AFU, arbitrary fluor-

escent units; FCI, focal cerebral ischaemia; Gpx1, glutathione peroxi-dase-1; IHC, immunohistochemistry; MCA, mid-cerebral artery;MCAO, mid-cerebral artery occlusion; PBS, phosphate-buffered saline;MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’sdisease; PI, propridium iodide; PI(3)K, phospho-inositide 3-kinase;ROS, reactive oxygen species; SDS–PAGE, sodium dodecyl sulfate –polyacrylamide gel electrophoresis; SOD1, superoxide dismutase;TUNEL, terminal deoxy nucleotidyl transferase-mediated dUTP biotinnick-end labelling.

Journal of Neurochemistry, 2005, 92, 283–293 doi:10.1111/j.1471-4159.2004.02863.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293 283

activation, the mechanisms by which Gpx1 protects againstneuronal cell death have not been reported. We propose thatthe increased susceptibility of the Gpx1–/– mouse to neuronalcell damage following FCI may be as a result of a down-regulation in pro-survival signalling or, alternatively, becauseof the increased activation of the cell death machinery.

The phospho-inositide 3-kinase [PI(3)K]-Akt pathway is asignalling cascade that is critical for protection againstneuronal cell death (Yao and Cooper 1995; Dudek et al.1997). Although a number of Akt targets have beenidentified, the most widely studied mechanism for its pro-survival actions involve its phosphorylation (and inactiva-tion) of Bad, a pro-apoptotic member of the Bcl-2 family, tosubsequently prevent caspase activation and cell death(Alessi and Cohen 1998). Activation of Akt has beenreported in response to a variety of stimulants, includingH2O2, growth factor withdrawal, heat shock, excitotoxicityand Ab (Yao and Cooper 1995; Wang et al. 2000; Chenget al. 2002; Mearow et al. 2002; Tamagno et al. 2003).Importantly, the neuroprotective effects of active Akt havealso been studied in culture systems with reactive oxygenspecies (ROS)-generating agents such as Ab and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Martin et al.2001; Salinas et al. 2001). Furthermore, Akt phosphorylationhas been reported in a transient focal model of cerebralischaemia–reperfusion (Noshita et al. 2001). Significantly,inhibition of the PI(3)K–Akt pathway with the PI(3)Kinhibitor LY294002 correlated with increased neuronal cellapoptosis. Therefore, the phosphorylation and subsequentactivation of Akt in response to ROS in FCI may be anattempt by a damaged cell to prevent further injury.

In an attempt to understand the mechanism by which theGpx1–/– mouse displays enhanced susceptibility to apoptosisafter mid-cerebral artery (MCA) occlusion (Crack et al.2001), the functionality of the PI(3)K–Akt pathway wasinvestigated. To this end, the PI(3)K–Akt pathway, itsinvolvement in an elevated endogenous ROS state and itsinfluence on neuronal cell survival has not been wellcharacterised. To address these questions, this study aimedto determine the effect of a lack of Gpx1 activity on thePI(3)K–Akt pathway in vitro in primary cultured neurons andin vivo in a model of cerebral ischaemia–reperfusion.

Materials and methods

Animals

All animal procedures were approved by the Animal Ethics

Committee of the Monash University Medical Centre and follow

the Australian code of practice for the care and use of animals for

scientific purposes. Mice of a C57/Bl6 strain were bred to obtain a

colony of homozygous Gpx-1–/– (Crack et al. 2001). All proce-dures were carried out using matched litter-mate wildtype mice.

Mice were studied at 6–8 weeks of age (body weight 25–32 g).

Isolation of mixed cortical neurons

Primary cortical neurons were isolated as described previously

with some modifications (Crossthwaite et al. 2002). Cells were

prepared from C57Bl/6 J embryos (day 14–16) and initially

cultured in Neurobasal Media (Gibco-BRL Life Technologies,

Ont., Canada) [containing 10% fetal bovine serum] in poly-L-

lysine-coated plates and allowed to adhere for 4 h before their

media was changed (serum-free media). Half the media was

subsequently replaced every 2 days. After 3 days, cultures were

treated with 2 lM aArabinoside-C (Ara-C) for 24 h to remove

contaminating glial and other proliferative cells. All experiments

were performed on day 6–8 cultures. Purity of cultures was

assessed by immunohistochemistry using NeuN (Chemicon, San

Diego, CA, USA) as a neuronal marker and glial fibrillary acidic

protein (GFAP) (Roche, Mannheim, Germany) to detect glial

contamination with cultures demonstrating greater than 95%

neuronal purity.

Treatment of primary cortical neurons

H2O2 was added to each plate or well to give a final H2O2

concentration in the range from 0 to 200 lM. An untreated control

contained media alone. Cells were harvested 12 h later to assess cell

viability, after 10 min for whole cell extracts and 10 min for

immunohistochemistry and cellular signalling.

Cells were exposed to the PI(3)K inhibitor, LY294002 (Calbio-

chem, La Jolla, CA, USA), for 2 h [or dimethylsulfoxide (DMSO)

for untreated control] before stimulation with nerve growth factor

(NGF) (40 ng/mL) (Sigma, St Louis, MO, USA) or H2O2 (50 lM).Cells were then left for 12 h before a 3-(4,5-dimethyliazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay, terminal deoxy nucleot-

idyl transferase-mediated dUTP biotin nick-end labelling (TUNEL)

staining or flow cytometry was performed to assess cell viability.

Alternatively, cells were immediately harvested to confirm inhibi-

tion of PI(3)K activity by western blot analysis using an antibody

directed against phosphorylated Akt (Ser473) (Cell Signaling,

Beverly, MA, USA).

MTT assay

Cell viability was assessed by measuring the ability of cells to

metabolise MTT as previously described (Behl et al. 1994).

Following an overnight treatment of neurons, MTT [2 mg/mL in

phosphate-buffered saline (PBS)] (Sigma) was added to each well

and the cells incubated for a further 4 h. The media containing MTT

was then removed, 200 lL of DMSO added to each well and

absorbances read at 595 nm. Each treated sample was compared

with an untreated control.

ROS measurements

Levels of ROS were assessed by loading cells with 10 lMdihydrorhodamine 123 (DHR123, Molecular Probes, Eugene,

OR, USA) as described previously (Cai and Jones 1998). Briefly,

cells growing in a 24-well plate were exposed to a dose–response

curve of H2O2 (0–80 lM) and then incubated with 10 lMDHR123 for 30 min before being washed three times with PBS.

DHR123 was assayed by a Fluorostar fluorescence plate reader

with an excitation wavelength of 488 nm and an emission

wavelength of 520 nm. Results are expressed as arbitrary

fluorescent units.

284 J. M. Taylor et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

Flow cytometry

To determine levels of cells undergoing apoptosis, cells were

harvested and fixed in 90% ethanol for at least 24 h before staining

with propridium iodide (PI) (Sigma) for 3 h and read by flow

cytometry.

TUNEL assay

TUNEL-positive cells were detected using an Apoptag� Fluoresc-

ein In situ Apoptosis Detection Kit (Intergen, Carlsbad, CA, USA).

After treatment of cells for 12 or 24 h, cells were fixed in 1% (w/v)

paraformaldehyde before being post-fixed with methanol : acetic

acid (2 : 1). Cells were incubated for 1 h at 37�C with working-

strength TdT enzyme. A negative control was performed in the

absence of TdT enzyme. The reaction was terminated with stop

buffer with the signal detected using a fluorescein anti-digoxigenin

conjugate [30 min at room temperature (21�C)]. Cells were further

stained with Hoechst 33342 (2 lg/mL) (Sigma) for 10 min to

determine nuclear integrity. To quantitate, three fields of view were

assessed with the number of TUNEL-positive cells counted and

expressed as a percentage of the number of cells counted (as

identified by the corresponding Hoechst staining).

Cleaved (activated) caspase-3 staining by flow cytometry

Cells were fixed in 0.5% (w/v) paraformaldehyde for 10 min at

37�C before being fixed overnight at )20�C in 90% (v/v) methanol.

Neurons were incubated for 30 min with 1/200 dilution of cleaved

caspase-3 antibody (Cell Signaling) at room temperature and then

incubated for 30 min at room temperature with a 1/400 dilution of

FITC-conjugated goat anti-rabbit secondary antibody (Molecular

Probes). Cells were re-suspended in 0.5% bovine serum albumin

(BSA) (in PBS) and analysed by flow cytometry.

Induction of focal cerebral ischaemia

Focal cerebral ischaemia was produced by occlusion of the MCA.

The MCA occlusion technique was based on that described

previously for the mouse (Crack et al. 2001). Male mice were

anaesthetised using a 300-lL mixture of ketamine (10 mg/mL)

and xylazine (0.5 mg/mL). Because repeated blood withdrawal

from mice was not compatible with further ischaemia experi-

ments, the samples for arterial pO2, pCO2 and pH were taken

from a separate group of animals (n ¼ 5 each). After ligation of

the right proximal common carotid artery, a 6-0 nylon monofil-

ament (Dermalon, Davis and Geck, Wayne, NJ, USA) with a

heat-blunted tip was introduced into the distal internal carotid

artery and was advanced � 11–12 mm distal to the carotid

bifurcation. The filament occluded the mid-cerebral artery where

it junctions off the Circle of Willis. The wound was closed and

the animal returned to its cage. To induce transient focal cerebral

ischaemia, the suture was withdrawn from the carotid artery under

anaesthesia from the carotid artery 2 h after insertion. Blood flow

was monitored via a Perimed PX5010 laser Doppler (Sweden).

Protein expression analysis

Whole cell lysatesFollowing treatments, wildtype or Gpx1–/– neurons were harvested

in ice-cold PBS. A 20-lL aliquot was then removed for protein

estimation using the Bradford Protein Assay Reagent (Bio-Rad,

Richmond, CA, USA) before the addition of an equal volume of

2 · sodium dodecyl sulfate – polyacrylamide gel electrophoresis

(SDS–PAGE) sample buffer.

Brain extractsFollowing MCA occlusion and reperfusion, brains were perfused

with PBS, pH 7.4, removed from the skull, and contralateral and

ipsilateral sides isolated. Cortex and striatum were dissected and

homogenised in 10 mM Tris-HCl containing a protease inhibitor

cocktail tablet (Roche, Mannheim, Germany). The sample was

centrifuged at 10 000 g for 20 min at 4�C before an aliquot was

taken for protein estimation by the Bradford method and an equal

volume of 2 · SDS–PAGE sample buffer was added to the

supernatant.

Western blot analysis

Western blot analysis was performed with 10–20 lg total protein

extract separated on 10% SDS–PAGE gels that were subsequently

transferred to polyvinylidene difluoride (PVDF) membrane (Immo-

bilon) (Millipore, Billerica, MA, USA). Blocking of membranes,

washes (PBS) and secondary antibody [goat anti-rabbit and goat

anti-mouse, 1/1000 dilution (Dako, Carpinteria, CA, USA)] incu-

bations were all performed at room temperature, while primary

antibodies were allowed to incubate overnight at 4�C [anti-Bad, 1/

1000; anti-Bad-P (Ser155) 1/500; anti-Akt, 1/1000 dilution and anti-

Akt-P (Ser473), 1/500 dilution (Cell Signaling); anti-PI(3)K, 1/300

dilution (Upstate, Waltham, MA, USA); antib-tubulin, 1/1000

(Dako)]. Signals were developed using the Supersignal Chemilu-

minescence Substrate system (Pierce, Rockford, IL, USA), with

filters exposed to X-ray film (Kodak Scientific, Rochester, NY,

USA).

Immunohistochemistry

Cultured primary neuronsCells were plated onto glass coverslips into 6-well plates and

treatments performed as outlined above. Media was removed and

cells rinsed with PBS before an overnight incubation at 4�C in

4% (w/v) paraformaldehyde. Cells were permeabilised with 2 : 1

methanol : acetic acid at )20�C for 5 min before being incubated

at room temperature for 30 min with Cas-block (Zymed, South

San Francisco, CA, USA). After an overnight incubation with 1/

100 dilution of Akt-P (Ser473) [immunohistochemistry (IHC)

specific] (Cell Signaling) and/or 1/1000 dilution of NeuN

(Chemicon) antibody at 4�C, cells were washed with PBS and

incubated for 1 h at room temperature with 1/400 FITC-

conjugated goat anti-rabbit antibody (Molecular Probes) (for

Akt-P) and/or 1/400 Texas Red-conjugated goat anti-mouse

antibody (Molecular Probes) (for NeuN). Cells were post-stained

with Hoechst 33342 (2 lg/mL) (Sigma) for 10 min, washed with

PBS and mounted onto slides with Anti-fade (Bio-Rad).

Brain sectionsFollowing 1-, 4- and 12-h artery occlusion, brains were processed as

previously described (Crack et al. 2001). Briefly, brains were

perfusion fixed with 4% (w/v) paraformaldehyde then removed and

post-fixed for 12 h in 4% paraformaldehyde before being placed in

20% (w/v) sucrose/PBS overnight. Following embedding in

Tissuetek� OCT media (Sakura Finetek, Torrance, CA, USA),

10-lM cryosections were processed for immunostaining for

PI(3)-kinase/Akt signalling in Gpx1–/– neurons 285

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

phosphorylated Akt, NeuN or cleaved caspase-3 (1/100 dilution,

Cell Signaling). Sections were also post-stained with

Hoechst 33342.

Statistical analysis

A Student’s unpaired t-test was used to compare control and treated

groups where p < 0.05 was considered significant. ANOVA was used

when multigroup comparisons were needed. All experiments were

performed at least three times with similar results, with values

plotted using GraphPad Prism Version 3.0 (GraphPad Software Inc.,

San Diego, CA, USA). Graphs correspond to the mean with error

bars indicating standard error (SEM).

Results

Gpx1–/– neurons demonstrate an increased susceptibility

to cell death induced by H2O2

Mixed cortical neurons were isolated and allowed toculture for 6–8 days before being exposed to increasingamounts of H2O2. Cell viability was then assessed by MTTassay. Treatment of wildtype and Gpx1–/– neurons withH2O2 resulted in a dose-dependent decrease in cell viability(Fig. 1a). Gpx1–/– neurons were far more susceptible thantheir wildtype counterparts, with greater than 50% celldeath after a 12-h treatment with 20 lM H2O2. At thisH2O2 concentration, cell viability of wildtype cells was stillgreater than 80%, with 50% cell death not seen until H2O2

concentrations in excess of 80 lM. Significantly, theGpx1–/– neurons demonstrated elevated levels of celldeath even in the absence of H2O2 (60% of wildtype).Phase-contrast images of these neurons showed cells ofpoor morphology, which was exacerbated upon H2O2

treatment (Fig. 1b). Loading of the cells with DHR123, amarker of oxidative stress (Cai and Jones 1998), showsthat the Gpx1–/– neurons were under a higher level ofROS, which correlates with their decreased viability(Fig. 1c).

50 lM H2O2 induces increased levels of apoptosis in

Gpx1–/– neurons compared with wildtype cells

To determine whether the type of cell death identified byMTT assay in the Gpx1–/– cells was through an apoptoticprocess, several markers of apoptosis were looked at. Firstly,wildtype and Gpx1–/– neurons were TUNEL stained. Asshown in Fig. 2(a), an increased number of TUNEL-positivecells were identified in the Gpx1–/– cells (panel i) comparedwith the wildtype cells (panel ii), even in the absence ofH2O2. When this level of TUNEL staining was quantitated,there was a greater than twofold increase in the percentage ofGpx1–/– neurons that were TUNEL positive compared withtheir wildtype counterparts (Fig. 2b). Following a 12-h50 lM H2O2 treatment (Fig. 2a), TUNEL-positive cells weredetected in the wildtype neurons (panel vi), however, therewas a greater number of positive staining cells in the treated

Gpx1–/– neurons (panel viii). Apoptosis after 12-h 50 lMH2O2 exposure was quantitated by flow cytometry using anantibody against cleaved (activated) caspase-3. As shown in

(a)

(b)

(c)

Fig. 1 Neurons isolated from Gpx1–/– mice demonstrate decreased

cell viability induced by H2O2. (a) Wildtype and Gpx1–/– primary

neurons were treated for 12 h with varying concentrations of H2O2

before cell viability was assessed by MTT assay. Data represents

mean ± SEM of six independent experiments where **p < 0.01. (b)

Phase-contrast images of wildtype (panel i) and Gpx1–/– (panel ii)

neurons treated for 12 h with 50 lM H2O2 (panels iii and iv, respect-

ively). Scale bar ¼ 100 lm. (c) Levels of ROS generation correlate

with decreased cellular viability. Wildtype and Gpx1–/– primary neu-

rons were treated with varying concentrations of H2O2 as in (a) and

then labelled with 10 lM DHR123. Results are expressed as arbitrary

fluorescent units (AFU) and data represents mean ± SEM of four

independent experiments where **p < 0.01.

286 J. M. Taylor et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

Fig. 2(c), Gpx1–/– neurons treated with 50 lM H2O2 (27%)demonstrated greater cleaved caspase-3 staining than wild-type cells (13%). Furthermore, Gpx1–/– cells demonstratedhigher levels of PI staining than their wildtype counterparts(Fig. 2d). Together, this data suggests that Gpx1–/– neuronsare more sensitive to apoptosis induced by H2O2.

Activation of the PI(3)K–Akt pathway occurs in wildtype

but not Gpx1–/– neurons following NGF or H2O2

treatment

The increased susceptibility to apoptosis suggested that theGpx1–/– neurons may have perturbations in either pro- oranti-apoptotic signalling networks. The PI(3)K–Akt pathwayhas been widely implicated in providing protection againstcell death induced by H2O2 and indeed this pathway isreported to be activated by both growth factors and H2O2 inneuronal cultures (Yao and Cooper 1995; Crossthwaite et al.2002). Western blot analysis did confirm a dose-dependentincrease in phosphorylated levels in wildtype cells upontreatment with NGF with no significant Akt phosphorylationdetected in the Gpx1–/– neurons (Fig. 3a). Similarly, min-imal and often non-detectable activation of Akt was evidentin Gpx1–/– cells following treatment with the addition of200 lM H2O2 (Fig. 3b) and contrasts with that seen inwildtype neurons. Total levels of Akt were unaltered betweenwildtype and Gpx1–/– cells following either NGF or H2O2

treatment. Interestingly, it also appeared that total levels ofthe upstream PI(3)K (p85 subunit) were decreased inGpx1–/– neurons (Fig. 3a). Immunohistochemistry with anantibody directed against Akt-P (Ser473) also confirmed alack of Akt phosphorylation in the cytoplasm of Gpx1–/–neurons in response to a 10-min dose of 200 lM H2O2

(Fig. 3c). In contrast, wildtype neurons demonstrated bothcytoplasmic and nuclear localisation of phosphorylated Akt

(a)

(b)

(c)

(d)

(i) (ii)

(iii) (iv)

(v) (vi)

(vii) (viii)

Fig. 2 (a, b) Gpx1–/– neurons demonstrate increased TUNEL staining

induced byH2O2. (a)Wildtype (panel ii) andGpx1–/– (panel iv) neurons

were treated for 12 h with 50 lM H2O2 (panels vi and viii, respectively)

before being TUNEL stained (panels ii, iv, vi and viii). Cells were also

stained with Hoechst 33342 to identify nuclei (panels i, iii, iv and vii).

Scale bar ¼ 100 lm. High magnification inserts show apoptotic mor-

phology in panel vii (Hoechst) and panel viii (TUNEL). (b) Quantification

of TUNEL staining of wildtype and Gpx1–/– cells treated with 50 lM

H2O2. TUNEL-positive cells were expressed as a percentage of total

cells where **p < 0.01. **significance relates to wildtype phenotype,

#significance relates to untreated neurons. (c, d) Gpx1–/– neurons

demonstrate increased cleaved caspase-3 and PI staining following

treatment with H2O2. Wildtype and Gpx1–/– neurons were treated for

12 h with 50 lM H2O2. (c) Cells were fixed in 0.5% paraformaldehyde

and stained with a cleaved (activated) caspase-3 antibody. A FITC-

conjugated secondary antibody allowed the cells to be counted by flow

cytometry. (d) Neurons were stained with propridium iodide and apop-

totic cells sorted by flow cytometry. Data represents themean ± SEMof

three independent experiments where **p < 0.01. **significance relates

to wildtype phenotype, #significance relates to untreated neurons.

PI(3)-kinase/Akt signalling in Gpx1–/– neurons 287

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

following H2O2 stimulation (panel ii). It was thereforepossible that the diminished activity of the PI(3)K–Aktpathway may be contributing to the increased apoptosis ofthe Gpx1–/– cells when exposed to H2O2.

Inhibition of the PI(3)K–Akt pathway in wildtype

neurons increases their susceptibility to H2O2

To identify the importance of Akt phosphorylation inproviding neuroprotection against oxidative insult, wildtypeand Gpx1–/– cells were treated with the PI(3)K inhibitor,LY294002. Western blot analysis confirmed inhibition of Aktphosphorylation induced by H2O2 through a 2-h pre-treatment with 25 lM LY294002 (Fig. 4a). Slight Aktphosphorylation was still detected with only 10 lMLY294002. Pre-treatment with LY294002 resulted in adecrease in cell viability of wildtype cells following a 12-hexposure to 50 lM H2O2 (Fig. 4b). This cell viability of 65%was comparable with that induced in Gpx1–/– neuronstreated only with 50 lM H2O2. Inhibition of Akt phosphory-lation was shown to be detrimental to cells in terms ofincreased caspase-3 activation coupled with decreased cellviability.

To determine whether this elevated cell death in theGpx1–/– neurons was as a result of apoptosis, levels ofcleaved (activated) caspase-3 were evaluated. This wasassessed firstly by flow cytometry as shown in Fig. 4(c). Aspreviously highlighted, prior to treatment with LY294002,Gpx1–/– neurons demonstrated a greater than twofoldincrease in the number of cleaved caspase-3 positive cellsupon exposure to 50 lM H2O2 for 12 h. Following treatmentwith 25 lM LY294002 for 2 h prior to stimulation with50 lM H2O2 for 14 h, there was no significant differencebetween the percentage of cleaved caspase-3 staining ofGpx1–/– and wildtype neurons (Fig. 4c). Significantly, whilethere was greater than threefold increase in H2O2-inducedcaspase-3 staining in the wildtype cells when treated withLY24002, this increase was only approximately twofold inthe corresponding Gpx1–/– neurons.

Increased H2O2-induced apoptosis as a result of PI(3)Kinhibition was also confirmed by increased TUNEL staining(Fig. 4d). Treatment of wildtype cells with LY294002resulted in an increased number of TUNEL-positive cellscompared with cells treated with H2O2 alone. In contrast,PI(3)K inhibition had no significant effect on the percentageof Gpx1–/– cells that were TUNEL positive following H2O2

treatment. Furthermore, there was no significant differencesin TUNEL staining between the LY294002/H2O2 treatedwildtype cells and the H2O2 Gpx1–/– neurons that alreadyhad diminished PI(3)K–Akt signalling.

Phosphorylated Bad at Ser155 was diminished in

Gpx1–/– neurons but not in wildtype neurons after

H2O2 treatment

Phosphorylated Bad immunoreactivity was evident as asingle band with a molecular mass of 23 kDa (Fig. 5). Badphosphorylation was found to be decreased in Gpx1–/–primary cultured neurons in response to 50 lM H2O2

treatment when compared with wildtype neurons exposedto the same treatment. This result is consistent with the

(a)

(b)

(c)

(i) (ii) (iii)

(iv) (v) (vi)

Fig. 3 (a, b) Gpx1–/– neurons demonstrate decreased levels of Akt

phosphorylation compared with wildtype cells following stimulation

with NGF or H2O2. Wildtype and Gpx1–/– neurons were exposed to (a)

the indicated concentrations of NGF for 10 min or (b) 200 lM H2O2 for

the indicated times. Western blot analysis was performed with an

antibody directed against phosphorylated Akt (Ser473). Total levels of

Akt, PI(3)K and b-tubulin were also determined. (c) Wildtype neurons

demonstrate increased cytoplasmic and nuclear localisation of phos-

phorylated Akt following treatment with H2O2. Wildtype (panel i) and

Gpx1–/– (panel iv) neurons were treated for 10 min with 200 lM H2O2

(panels ii and v, respectively) before being fixed in 4% paraformal-

dehyde. Immunohistochemistry was performed using an antibody

directed against phosphorylated Akt (Ser473) (IHC specific). Negative

controls (without primary antibody) were also performed (panels iii and

vi). Staining with Hoechst 33342 identified nuclei. Scale bar ¼100 lm.

288 J. M. Taylor et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

inability of the Gpx1–/– neurons to phosphorylate Akt andhighlights the lack of downstream functionality of this part ofthe PI(3)K–Akt pathway in the Gpx1–/– neurons. Thedecreased level of Bad activation may help to explain theincreased levels of apoptosis that are found in the Gpx1–/–neurons after 50 lM H2O2 treatment.

Cerebral infarction and physiological responses

Physiological studies for blood gases and blood pH for boththe wildtype and the Gpx-1–/– were found to be consistentboth before and after MCA stroke surgery, suggesting thatthese parameters had little impact on the outcome of theMCA surgery. The pre-MCA surgery values were 7.3 ± 0.2pH, 110 ± 11.4 PaO2, 45 ± 3.5 PaCO2 for the wildtype and7.3 ± 0.15 pH, 108 ± 10.5 PaO2, 42 ± 4.1 PaCO2 forthe Gpx1 knockout (values were calculated as mean ± SE;

n ¼ 4). There was no change in these values after MCAsurgery. The progression and development of the infarct inboth the wildtype and the knockout mirrored the findings ofCrack et al. (2001) (data not shown).

Akt is phosphorylated (activated) in wildtype and

Gpx1–/– brains following MCA occlusion

Activation of the PI(3)K–Akt pathway has been shown toplay an important role in protecting neurons from oxidativeinsult in vitro. Significantly, Akt phosphorylation has alsobeen detected following cerebral ischaemia–reperfusion, withthis response critical for neuronal survival (Noshita et al.2001). We have previously reported the increased suscepti-bility of Gpx1–/– mice to neuronal cell damage followingcerebral ischaemia (Crack et al. 2001). To investigate the

Fig. 5 Wildtype neurons demonstrate increased levels of phosphor-

ylated Bad following treatment with H2O2. Wildtype and Gpx1–/–

neurons were treated with 50 lM H2O2 for 12 h. Western blot analysis

demonstrated increased levels of phosphorylated Bad (Ser155) in

wildtype neurons compared with a negligible increase in the Gpx1–/–

cells following exposure to H2O2. b-Tubulin levels were also deter-

mined as loading control.

Fig. 4 (a, b) Treatment of wildtype neurons with the PI(3)K inhibitor,

LY294002, increases their susceptibility to H2O2-induced cell death.

Wildtype and Gpx1–/– neurons were pre-treated with LY294002

(25 lM) for 2 h before being exposed to 50 lM H2O2 for 12 h. (a)

Western blot analysis demonstrating an inhibition of H2O2-induced Akt

phosphorylation by a 2-h LY294002 (25 lM) pre-treatment. (b) Cell

viability was assessed by MTT assay identifying increased cell death

in wildtype cells in response to H2O2 when PI(3)K activity was inhib-

ited. Data represents the mean ± SEM of six independent experi-

ments where *p < 0.05. (c, d) Wildtype and Gpx1–/– neurons

demonstrate increased cleaved caspase-3 and TUNEL staining

induced by H2O2 when treated with LY294002. Wildtype and Gpx1–/–

neurons were treated with the PI(3)K inhibitor LY294002 (25 lM) for

2 h before being exposed to 50 lM H2O2 for 12 h. (c) Cells were

harvested and flow cytometry was performed using an antibody

directed against cleaved caspase-3. The percentage of wildtype and

Gpx1–/– cells positive for cleaved caspase-3 was determined by

comparing with their untreated counterparts. Data represents the

mean ± SEM of three independent experiments where **p < 0.01. (d)

Cells were fixed in 1% paraformaldehyde and TUNEL stained. The

number of TUNEL-positive cells was expressed as a percentage of the

total number of cells as determined by the corresponding Hoe-

chst 33342 staining where *p < 0.05.

(a)

(b)

(c)

(d)

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� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

role of this pathway in the elevated neuronal apoptosis in theGpx1–/– brains following MCA occlusion, immunohisto-chemistry was performed using an antibody directed againstphosphorylated Akt (Ser473) (IHC specific). In wildtypebrains, no detectable staining was evident in sham-operatedanimals (Fig. 6a). However, following 4-h reperfusion,phosphorylated Akt staining was detected in the wildtypebrains (Fig. 6, panels iii). In contrast, brains from Gpx1–/–mice exhibited reduced Akt-P staining at 4 h after reper-fusion (Fig. 6, panel iv). Little detectable staining was seenin either wildtype or Gpx1–/– brains following 12-hreperfusion (data not shown).

As identified by co-staining with the neuronal markerNeuN, those cells positive for Akt-P in wildtype brainsfollowing MCA occlusion and 4-h reperfusion were neurons(Fig. 7, panel iii). Interestingly, the pattern of stainingseemed to differ between wildtype and Gpx1–/– brains. Inthe wildtype mice, Akt-P staining was both cytoplasmic andnuclear in localisation, as identified by co-staining withHoechst 33342 (Fig. 7, panel ix). The cells positive forphosphorylated Akt demonstrated intact nuclei (althoughchromatin condensation was beginning to occur in a numberof these cells) while no staining was detected in those cellswith pyknotic nuclei. In contrast, the staining pattern of Akt-P in the Gpx1–/– brains was predominantly nuclear as seenfollowing reperfusion (Fig. 8).

Following MCA occlusion and reperfusion events of 1, 4and 12 h, total cell extracts were prepared from the cortex andstriatum of wildtype and Gpx1–/– brains. Western blotanalysis identified a slight induction of phosphorylated Akt

in wildtype brains in both the cortex and striatum of theipsilateral side, while no activation was seen in the Gpx1–/–brains (Fig. 9). Phosphorylation of Akt was not detected inthe contralateral side of the brains from wildtype or theGpx1–/– mice. Following 4-h reperfusion, there was asignificant increase in the activation of Akt in the cortex ofthe ischaemic side of the wildtype brain, while no Akt-P wasdetected in the Gpx1–/– samples. By 12 h, phosphorylatedAkt could not be detected in either of the wildtype or Gpx1–/–brain samples (data not shown). This data is consistent withwhat was found in the immunohistochemistry studies.

Discussion

A balanced redox environment has been shown to be criticalfor neuronal survival, and cell death induced by elevatedROS has been associated with a number of neuropathologiesincluding AD, PD and cerebral ischaemia (Leist and Nicotera1998; Pettmann and Henderson 1998; Schulz et al. 2000). Inthis study, the effect of a loss of Gpx1 on neuronal cellsurvival has been investigated. Our data demonstrates theconcept that an imbalance in the antioxidant defence system,resulting in elevated levels of H2O2, is an importantdeterminant in neural injury and can seriously affect theability of signal transduction pathways to co-ordinate pro-survival cellular responses.

Gpx1–/– primary cultured neurons were found to be moresusceptible to cell death than wildtype neurons, even in theabsence of a cell death inducing stimulus. Not surprisingly, inthe presence of H2O2, cell death was also elevated in theGpx1–/– neurons. This increase in cell death was attributedto elevated apoptosis as identified by activation of cleavedcaspase-3 and increased PI staining. The increased suscep-tibility of Gpx1–/– neurons in vitro to apoptosis in conjunc-tion with previous findings from this lab, that Gpx1–/– micedemonstrate elevated neuronal cell death following mid-cerebral artery occlusion (Crack et al. 2001), suggested that alack of Gpx1 is detrimental to neuronal cell survival. It washypothesised that a cellular change in a key anti-apoptoticpathway may lead to this increased cell death. The PI(3)K–Akt pathway was an attractive target as a result of previousreports highlighting its importance in protecting againstneuronal cell death, in various models both in vitro and invivo (Williams and Doherty 1999; Kaplan and Miller 2000)and recent reports investigating this pathway’s response tooxidative stress (Weihl et al. 1999; Salinas et al. 2001;Crossthwaite et al. 2002).

The diminished levels of Akt phosphorylation in theGpx1–/– neurons in response to NGF and H2O2, suggest thata lack of activation of the PI(3)K–Akt pathway in these cellsmay be contributing to their increased susceptibility to celldeath. Activation of the PI(3)K–Akt pathway has beenshown to be neuroprotective in vitro against other ROS-inducing agents, such as Ab peptides and MPTP, while the

Fig. 6 Akt phosphorylation in wildtype and Gpx1–/– brain cerebral

ischaemia–reperfusion injury. Wildtype and Gpx1–/– mice were sub-

jected to MCA occlusion for 2 h before reperfusion events of 1 or 4 h.

Immunohistochemistry was performed using an antibody directed

against Akt-P (Ser473). Minimal activation was detected in the ipsi-

lateral hemispheres of either the wildtype or Gpx1–/– sham-operated

animals (panels a and b). After 4 hours reperfusion, phosphorylated

Akt was present in the wildtype cortex (panel c), while staining was

decreased in the Gpx–/– brains (panel d). Scale bar ¼ 50 lm.

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� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

down-regulation in Akt1 by mutant presenilin-1 has beenassociated with a concurrent induction of apoptosis (Weihlet al. 1999; Martin et al. 2001; Salinas et al. 2001). Takentogether, this data suggests that the PI(3)K–Akt pathway isactivated in in vitro models of neuropathologies such as AD

and PD to provide protection against neuronal cell death.Indeed, this was supported in this study by the increasedsusceptibility of wildtype cells to H2O2-induced apoptosiswhen this pathway was inhibited with elevated TUNEL, PIand cleaved caspase-3 staining.

Fig. 7 Wildtype neurons demonstrate increased phosphorylated Akt

staining following MCA occlusion (4-h reperfusion). Wildtype mice

were subjected to MCA occlusion for 2 h before reperfusion events of

4 h. Immunohistochemistry was performed with an antibody directed

against Akt-P (Ser473) (a) or the neuronal marker, NeuN (panel b).

Panel c depicts an overlayed image of panels a and b, demonstrating

wildtype neurons positive for phosphorylated Akt. Negative control

sections for the polyclonal (panel d) and monoclonal (panel e) anti-

bodies were also performed which were also stained with Hoe-

chst 33342 (panel f). Cells positive for Akt-P (panel g) were also

stained with Hoechst 33342 (panel h). Overlayed images (panel i)

demonstrated cells positive for phosphorylated Akt displayed im-

proved nuclear morphology (shown by c) compared with other cells

highlighted by . Scale bar ¼ 50 lm.

Fig. 8 Localisation of Akt phosphorylation in wildtype and Gpx1–/–

brains after cerebral ischaemia–reperfusion injury. Wildtype and

Gpx1–/– mice were subjected to MCA occlusion for 2 h before rep-

erfusion events of 1 or 4 h. Immunohistochemistry was performed

using an antibody directed against Akt-P (Ser473). Wildtype mice

displayed Akt-P staining in both the cytoplasm and nucleus (panel a),

whereas Gpx1–/– mice display Akt-P staining predominantly in the

nucleus after MCA (panel d). Panels b and e show Hoechst-stained

nuclei for wildtype and Gpx1–/–, respectively. Double labelling of Akt-P

and Hoechst confirm Akt-P staining in both the cytoplasm and nucleus

for the wildtype (panel c) and nuclear staining for the Gpx1–/– (panel

f). Scale bar ¼ 5 lm.

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� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

Activation of the PI(3)K–Akt pathway has previouslybeen reported in models of both transient and permanentFCI (Noshita et al. 2001; Shibata et al. 2002). Further-more, Akt phosphorylation was detected in neurons in theouter area of the MCA territory where caspase-3 activationwas absent (in contrast with the central MCA area)(Shibata et al. 2002). This suggested that activation ofthe pro-survival Akt occurred prior to the cell deathmachinery, a response that may be crucial for neuronal cellsurvival. This was supported by evidence demonstratingincreased DNA fragmentation in brains following FCI byinhibition of Akt phosphorylation by LY294002 (Noshitaet al. 2001). In light of the Gpx1–/– neurons demonstratingdiminished Akt phosphorylation following H2O2 exposurein vitro, and elevated levels of apoptosis, it was reasonedthat a possible perturbation in the PI(3)K–Akt pathwaymay be a major contributor to the elevated neuronal celldeath and apoptosis seen in Gpx1–/– brains followingMCA occlusion (Crack et al. 2003).

Analysis of the PI(3)K–Akt pathway identified that,in vivo, the Gpx1–/– mouse does display Akt phosphoryla-tion following MCA occlusion. However, the response wasearlier (1-h reperfusion) with a different localisation than inthe wildtype mice (at 4-h reperfusion). Therefore, althoughAkt could be phosphorylated following cerebral ischaemia–reperfusion in the Gpx1–/– mouse, the pro-survival responseof this pathway seemed to be concluding within 1 h.Furthermore, activated Akt was situated in the nucleus ofthese cells. Nuclear translocation has been associated withthe neuroprotective actions of phosphorylated Akt where itinteracts and phosphorylates transcription factors such asthose of the Forkhead family (Neri et al. 2002; Shibata et al.2002). However, western blot analysis failed to detectincreased phosphorylated Akt levels in the Gpx1–/– at 4 h,with a minor amount detected at 1 h. The combined datasuggests that the accelerated activation and translocation ofAkt seen in the Gpx1–/– may provide some initial neuro-protection against the oxidative insult, but the failure to

further activate the PI(3)K–Akt pathway is detrimental to cellsurvival. This correlates with earlier findings from ourlaboratory that the Gpx1–/– mice exhibit accelerated activa-tion of caspase-3 and increased lipid peroxidation afterMCAO (Crack et al. 2001).

The neuroprotective actions of Akt have also been shown tobe through its phosphorylation and inhibition of the pro-apoptotic Bcl-2 family member, Bad (Alessi and Cohen1998). Phosphorylated Akt interacts with Bad in the cyto-plasm, thus preventing it from forming homodimers and alsoheterodimers with Bcl-2. This prevents cytochrome c releasefrom the mitochondria, and consequently the activation ofcaspases does not occur. The lack of Akt phosphorylation inthe cytoplasm of Gpx1–/– neurons at 4-h reperfusion (such asthat previously reported and seen in the wildtype mice)suggests that inactivation of Bad would not occur. Thiscorrelates with the earlier activation of caspase-3 in theGpx1–/– mouse reported by Crack et al. (2001), and that Badphosphorylation was not detected in the Gpx1–/– neurons.

The effects of altered redox balance on the activation ofthe PI(3)K–Akt pathway following cerebral ischaemia–reperfusion injury have recently been reported. Noshita et al.(2001) demonstrated an earlier activation of Akt (1 hcompared with 4 h) in SOD1 overexpressing mice followingtransient focal cerebral ischaemia. However, they suggestthat the neuroprotective actions of SOD1 can be, in part,attributed to the earlier activation of the PI(3)K–Aktpathway. The early activation of Akt in the SOD1 overex-pressing mice could possibly be through the enhanceddetoxification of superoxide to H2O2, to stimulate the pro-survival pathway. Similarly, in this study, the lack of Gpx1 toeliminate H2O2 may also result in earlier activation of Akt.The importance of Gpx1 in the neuroprotective actions ofSOD1 has been previously highlighted (Crack et al. 2003),and indeed the detoxification of H2O2 by Gpx1 seemsparamount in not only providing a balanced redox environ-ment but also in controlling aberrant signalling pathwaysfollowing cerebral ischaemia.

The implications of an altered redox state on anti-apoptoticsignalling pathways following oxidative insult have beenexplored in this study to identify a mechanism for theincreased susceptibility of the Gpx1–/– mouse to neuronalapoptosis. It was identified that increased endogenous ROScan down-regulate the key anti-apoptotic PI(3)K–Akt path-way, a response detrimental to neuronal cell survival followingexogenous insult. This study has highlighted the importance ofa balanced redox environment in the cellular response tooxidant injury and may have implications in the study ofneurodegenerative disorders involving oxidative stress.

References

Alessi D. R. and Cohen P. (1998) Mechanism of activation and functionof protein kinase B. Curr. Opin. Genet. Dev. 8, 55–62.

Fig. 9 Western blot analysis demonstrating increased Akt phos-

phorylation in wildtype brains following cerebral ischaemia–reper-

fusion injury. Wildtype and Gpx1–/– mice were subjected to MCA

occlusion and reperfusion events of either 1 or 4 h. Brains were re-

moved and total cell extracts prepared from the cortex (C) and striatum

(S) of both the contralateral (contra) and ipsilateral (ipsi) hemispheres.

Western blot analysis was performed with an Akt-P (Ser473) antibody

with total Akt levels also determined.

292 J. M. Taylor et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293

Behl C., Davis J. B., Lesley R. and Schubert D. (1994) Hydrogen per-oxide mediates amyloid b protein toxicity. Cell 77, 817–827.

Cai J. and Jones D. P. (1998) Superoxide in apoptosis. Mitochondrialgeneration triggered by cytochrome c loss. J. Biol. Chem. 273,11 401–11 404.

Cheng G., Yu Z., Zhou D. and Mattson M. P. (2002) Phosphatidy-linositol-3-kinase-Akt kinase and p42/p44 mitogen-activated pro-tein kinases mediate neurotrophic and excitoprotective actions of asecreted form of amyloid precursor protein. Exp. Neurol. 175, 407–414.

Crack P. J., Taylor J. M., Flentjar N. J., de Haan J., Hertzog P., IannelloR. C. and Kola I. (2001) Increased infarct size and exacerbatedapoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mousebrain in response to ischemia/reperfusion injury. J. Neurochem. 78,1389–1399.

Crack P. J., Taylor J. M., de Haan J. B., Kola I., Hertzog P. and IannelloR. C. (2003) Glutathione peroxidase-1 contributes to the neuro-protection seen in the superoxide dismutase-1 transgenic mouse inresponse to ischemia/reperfusion injury. J. Cereb. Blood FlowMetab. 23, 19–22.

Crossthwaite A. J., Hasan S. and Williams R. J. (2002) Hydrogen per-oxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK incortical neurones: dependence on Ca(2+) and PI3-kinase.J. Neurochem. 80, 24–35.

Dudek H., Datta S. R., Franke T. F., Birnbaum M. J., Yao R., Cooper G.M., Segal R. A., Kaplan D. R. and Greenberg M. E. (1997)Regulation of neuronal survival by the serine-threonine proteinkinase Akt. Science 275, 661–665.

Fujimura M., Morita-Fujimura Y., Noshita N., Sugawara T., Kawase M.and Chan P. H. (2000) The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrialcytochrome c in ischemic brain after transient focal cerebral isc-hemia in mice. J. Neurosci. 20, 2817–2824.

Kaplan D. R. and Miller F. D. (2000) Neurotrophin signal transduction inthe nervous system. Curr. Opin. Neurobiol. 10, 381–391.

Kinouchi H., Epstein C. J., Mizui T., Carlson E., Chen S. F. and Chan P.H. (1991) Attenuation of focal cerebral ischemic injury in trans-genic mice overexpressing CuZn superoxide dismutase. Proc. NatlAcad. Sci. USA 88, 11 158–11 162.

Kondo T., Reaume A. G., Huang T. T., Carlson E., Murakami K., ChenS. F., Hoffman E. K., Scott R. W., Epstein C. J. and Chan P. H.(1997) Reduction of CuZn-superoxide dismutase activity exacer-bates neuronal cell injury and edema formation after transient focalcerebral ischemia. J. Neurosci. 17, 4180–4189.

Leist M. and Nicotera P. (1998) Apoptosis, excitotoxicity, and neuro-pathology. Exp. Cell Res. 239, 183–201.

Martin D., Salinas M., Lopez-Valdaliso R., Serrano E., Recuero M. andCuadrado A. (2001) Effect of the Alzheimer amyloid fragmentAb(25–35) on Akt/PKB kinase and survival of PC12 cells.J. Neurochem. 78, 1000–1008.

Mearow K. M., Dodge M. E., Rahimtula M. and Yegappan C. (2002)Stress-mediated signaling in PC12 cells – the role of the small heatshock protein, Hsp27, and Akt in protecting cells from heatstress and nerve growth factor withdrawal. J. Neurochem. 83, 452–462.

Neri L. M., Borgatti P., Capitani S. and Martelli A. M. (2002) Thenuclear phosphoinositide 3-kinase/AKT pathway: a new secondmessenger system. Biochim. Biophys. Acta 1584, 73–80.

Noshita N., Lewen A., Sugawara T. and Chan P. H. (2001) Evidence ofphosphorylation of Akt and neuronal survival after transient focalcerebral ischemia in mice. J. Cereb. Blood Flow Metab. 21, 1442–1450.

Pettmann B. and Henderson C. E. (1998) Neuronal cell death. Neuron20, 633–647.

Salinas M., Martin D., Alvarez A. and Cuadrado A. (2001) Akt1/PKBaprotects PC12 cells against the parkinsonism-inducing neurotoxin1-methyl-4-phenylpyridinium and reduces the levels of oxygen-free radicals. Mol. Cell Neurosci. 17, 67–77.

Schulz J. B., Lindenau J., Seyfried J. and Dichgans J. (2000) Glutathi-one, oxidative stress and neurodegeneration. Eur. J. Biochem. 267,4904–4911.

Shibata M., Yamawaki T., Sasaki T., Hattori H., Hamada J., Fukuuchi Y.,Okano H. and Miura M. (2002) Upregulation of Akt phosphory-lation at the early stage of middle cerebral artery occlusion in mice.Brain Res. 942, 1–10.

Tamagno E., Robino G., Obbili A., Bardini P., Aragno M., Parola M. andDanni O. (2003) H2O2 and 4-hydroxynonenal mediate amyloidb-induced neuronal apoptosis by activating JNKs and p38MAPK.Exp. Neurol. 180, 144–155.

Wang X., McCullough K. D., Franke T. F. and Holbrook N. J. (2000)Epidermal growth factor receptor-dependent Akt activation byoxidative stress enhances cell survival. J. Biol. Chem. 275,14 624–14 631.

Weihl C. C., Ghadge G. D., Kennedy S. G., Hay N., Miller R. J. andRoos R. P. (1999) Mutant presenilin-1 induces apoptosis anddownregulates Akt/PKB. J. Neurosci. 19, 5360–5369.

Williams E. J. and Doherty P. (1999) Evidence for and against a pivotalrole of PI 3-kinase in a neuronal cell survival pathway. Mol. CellNeurosci. 13, 272–280.

Yao R. and Cooper G. M. (1995) Requirement for phosphatidylinositol-3kinase in the prevention of apoptosis by nerve growth factor. Sci-ence 267, 2003–2006.

PI(3)-kinase/Akt signalling in Gpx1–/– neurons 293

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 283–293