mTOR/p70S6k signalling alteration by Abeta exposure as well as in APP-PS1 transgenic models and in...

mTOR/p70S6k signalling alteration by Ab exposure as well asin APP-PS1 transgenic models and in patients with Alzheimer’sdisease

Claire Lafay-Chebassier,*,�,1 Marc Paccalin,�,�,1 Guylene Page,� Stephanie Barc-Pain,� MarieChristine Perault-Pochat,*,� Roger Gil,§,� Laurent Pradier¶ and Jacques Hugon§,�

Departments of *Pharmacology, �Geriatrics and §Neurology, Poitiers University Hospital, Poitiers, France

�Research Group on Brain Aging (Equipe associee 3808), University of Poitiers, Poitiers, France

¶Sanofi-Aventis, CRVA Vitry-sur-Seine, France

Abstract

In Alzheimer’s disease, neuropathological hallmarks include

the accumulation of b-amyloid peptides (Ab) in senile plaques,

phosphorylated tau in neurofibrillary tangles and neuronal

death. Ab is the major aetiological agent according to the

amyloid cascade hypothesis. Translational control includes

phosphorylation of the kinases mammalian target of rapa-

mycin (mTOR) and p70S6k which modulate cell growth, pro-

liferation and autophagy. It is mainly part of an anti-apoptotic

cellular signalling. In this study, we analysed modifications of

mTOR/p70S6k signalling in cellular and transgenic models of

Alzheimer’s disease, as well as in lymphocytes of patients and

control individuals. Ab 1–42 produced a rapid and persistent

down-regulation of mTOR/p70S6k phosphorylation in murine

neuroblastoma cells associated with caspase 3 activation.

Using western blottings, we found that phosphorylated forms

of mTOR and p70S6k are decreased in the cortex but not in

the cerebellum (devoid of plaques) of double APP/PS1

transgenic mice compared with control mice. These results

were confirmed by immunohistochemical methods. Finally,

the expression of phosphorylated p70S6k was significantly

reduced in lymphocytes of Alzheimer’s patients, and levels of

phosphorylated p70S6k were statistically correlated with Mini

Mental Status Examination (MMSE) scores. Taken together,

these findings demonstrate that the mainly anti-apoptotic

mTOR/p70S6k signalling is altered in cellular and transgenic

models of Alzheimer’s disease and in peripheral cells of

patients, and could contribute to the pathogenesis of the

disease.

Keywords: Alzheimer, Ab, human lymphocytes, mTOR

signalling, transgenic models, translation.

J. Neurochem. (2005) 94, 215–225.

In eukaryotes, protein translation includes three consecutivephases: initiation, elongation and termination. The initiationphase corresponds to processes associated with the connectionbetweenmRNAand ribosomes. The elongation phase includesthe links between amino acids at the ribosomal level, and isfollowed by the termination phase. These three phases arehighly regulated by proteins, called translation factors, that caninteract directly with mRNAs. In the initiation phase, twomajor factors are involved: eukaryotic initiation factor 2 (eIF2)and eukaryotic initiation factor 4E (eIF4E).

The availability of eIF4E is linked to the binding of specificproteins called 4E-BPs. When these proteins are not phos-phorylated, they have a great affinity for eIF4E, which isunable to bind tomRNAs, leading to a reduction of translation.These proteins are mainly phosphorylated by a kinase calledmTOR (mammalian target of rapamycin) or FKBP12-

Received January 12, 2005; revised manuscript received February 7,2005; accepted February 23, 2005.Address correspondence and reprint requests to Jacques Hugon,

Department of Neurology, CHU Poitiers, rue de la Miletrie, 86000Poitiers, France. E-mail: [email protected] Lafay-Chebassier and Marc Paccalin contributed equally to thiswork.Abbreviations used: Ab, b-amyloid peptides; AD, Alzheimer’s dis-

ease; BDNF, Brain derived neurotrophic factor; eIF2, eukaryotic initi-ation factor 2; eIF4E, eukaryotic initiation factor 4E; ER, endoplasmicreticulum; FRAP, FKBP12-Rapamicyn associated protein; GTP, Gua-nosine triphosphate; LTP, Long Term potentiation; MMSE, mini mentalstate examination; mTOR, mammalian target of rapamycin; PI3K,phosphatidyl Inositol 3 Kinase; PERK, PKR-like endoplasmic reticulumkinase; PKR, double-stranded RNA-dependent protein kinase; RA,all-trans retinoic acid.

Journal of Neurochemistry, 2005, 94, 215–225 doi:10.1111/j.1471-4159.2005.03187.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 215–225 215

rapamicyn associated protein (FRAP) (Schmelzle and Hall2000; Raught et al. 2001). mTOR can also phosphorylatekinase p70S6k that stimulates protein synthesis (Dufner andThomas 1999). The regulation of mTOR activity is importantfor the availability of eIF4E. mTOR is activated by thephosphatidyl Inositol 3 Kinase (PI3K) and Akt pathways. ThemTOR pathway is also activated by IGF1 (Clemens 2001).mTOR signalling is physiologically active and allows proteinand ribosome synthesis.Many studies have shown that cellularstresses can increase the binding of 4E-BPs to the eIF4E factorand can reduce protein translation (Clemens 2001). Recentstudies have shown that two tumour suppressor genes, TSC1andTSC2, and their corresponding proteins are able to regulatemTOR phosphorylation and binding to protein 14-3-3 (Gaoet al. 2002). In addition, reduction of the eIF4E level is notenough to induce cellular apoptosis (Luo et al. 1994) anddouble-stranded RNA dependent protein kinase (PKR) candephosphorylate the protein’s 4EBPs through the activation ofphosphatase 2A, leading to cell apoptosis (Xu and Williams2000; Jeffrey et al. 2002). Recent electrophysiological resultshave also demonstrated that mTOR could play a role inneuronal plasticity and in the process of learning and memory.In brain slices treated with the mTOR inhibitor rapamycin, theauthors observed a decrease in the late phase of long termpotentiation (LTP) induced by synaptic stimulation or brainderived neurotrophic factor (BDNF) exposure (Nguyen 2002;Tang et al. 2002).

EIF2 can fix guanosine triphosphate (GTP) and interactwith tRNA carrying methionin. The complex formed canbind to the small 40S subunit of the ribosome, a phase inwhich eIF4E plays also a major role. The availability of thesetwo initiation factors is indispensable for the onset of proteinsynthesis. The control of eIF2 is linked to the state ofphosphorylation on the a subunit on serine 51. Uponphosphorylation, eIF2a is unable to bind to tRNA andtranslation is stopped. Four protein kinases are able tophosphorylate eIF2a: PKR, PERK (PKR-like endoplasmicreticulum kinase), HRI (Heme-regulated kinase) and GCN2(amino acid-regulated kinase). PKR is a ubiquitous proteininvolved in viral infections, but it is also activated duringdifferent cellular stresses such as toxic stress, endoplasmicreticulum (ER) stress, intracellular calcium stress and lack oftrophic factors (Williams 1999). ER stress also leads to theactivation of PERK and the unfolded protein response(Harding et al. 1999). Several studies have revealedincreased PKR expression with ageing (Ladiges et al.2000), in neurodegenerative diseases (Peel et al. 2001;Chang et al. 2002b), or in the activation of eIF2a in cerebralischaemia-reperfusion, hypoxia or zinc toxicity (Frerichset al. 1998; Alirezaei et al. 1999; DeGracia et al. 1999). Ithas been demonstrated that these kinases play an importantrole in the process of cellular apoptosis by interacting withprotein translation and apoptotic factors.

An early study revealed that mRNA translation wasdisturbed in the brain of Alzheimer’s disease (AD) patients(Langstrom et al. 1989). Inhibition of ribosomal translationcould be responsible for modifications of gene expressiondetected in affected brain regions. An increased level ofelongation factor 2was also noted in ADbrains (Johnson et al.1992). We have recently demonstrated an accumulation ofphosphorylated eIF2a in degenerating neurones in AD brainsassociated with increased staining of activated PKR (Changet al. 2002a). This finding was confirmed by another group(Ferrer 2002). In addition, we have shown in vitro that Abcould induce activation of PKR and the phosphorylation ofeIF2a (Chang et al. 2002b). PKR activation was dependentupon caspase 3 and caspase 8 triggering. A reduction in proteinsynthesis has been described during ageing (Rattan 1996), aswell as a decrease in eIF2a and eIF2B and an increase in PKRexpression (Kimball et al. 1992; Ladiges et al. 2000). Veryfew studies have been carried out so far on the involvement ofthemTOR/p70S6k signalling pathway following Ab exposurein cell cultures and in AD models. The results of the presentstudy show that there is a decrease inmTOR/p70S6k signallingin neural cells treated in vitro with Ab, in the cortex oftransgenic mice overexpressing APP and Presenilin 1 muta-tions, and in lymphocytes of patients suffering fromAD.Thesefindings demonstrate that translational control could representa new molecular target for future therapeutic interventionsconcerning Alzheimer’s disease.

Materials and methods

Chemicals

All-trans retinoic acid (RA), peptides Ab 1–42 and Ab 40–1,

paraformaldehyde (PFA), Histopaque 1077, Triton X-100, sodium

fluoride, phenylmethylsulfonyl fluoride, protease and phosphatase

inhibitors, dithiothreitol (DTT) and Caspase 3 substrate Ac-DEVD-

pNA were obtained from Sigma (Saint Quentin Fallavier, France).

Pentobarbital was obtained from Ceva Sante Animale (Libourne

France). Primary antibodies and secondary anti-rabbit IgG antibody

conjugated with horseradish peroxidase were purchased from Cell

Signalling Ozyme (Saint Quentin Yvelines, France) except for anti-

b-tubulin antibody and anti-b-actin antibody which were from

Sigma. The phospho-mTOR antibody was recently used as a marker

for mTOR activation (Bolster et al. 2002). Secondary anti-mouse

IgG antibody conjugated with horseradish peroxidase was obtained

from Amersham (Orsay, France). All reagent-grade chemicals for

buffers were obtained from VWR International (Strasbourg, France).

Cell cultures and Ab treatment

Neuro-2a murine neuroblastoma cell lines were obtained from the

American Type Culture Collection (ATCC). Neuro-2a cells were

cultured with Minimal Essential Medium (MEM) (Gibco-BRL,

Cergy Pontoise, France), supplemented with 10% fetal bovine serum

(FBS) and 1% antibiotics [50 U/mL penicillin + 50 lg/mL

streptomycin (Gibco-BRL)]. The cells were differentiated into

216 C. Lafay-Chebassier et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 215–225

mature neurones by incubating with 20 lM RA for 2 days. Cells

were maintained in a humidified 5% CO2 atmosphere at 37�C.Cells were treated with 20 lM Ab (1–42) or (40–1) from 30 min

to 24 h in serum-free medium. The peptides were incubated at 37�Cfrom 24 to 48 h to form aggregates prior to use. After treatment,

neural cells were lysed in ice-cold lysis buffer containing protease

and phosphatase inhibitor cocktail. The lysate was sonicated and

then centrifuged at 15 000 g for 15 min at 4�C. The protein content

of the supernatant fluid was measured using a protein assay kit

(BioRad, Marnes la Coquette, France); 20 lg protein (from the

supernatant fluid) per lane were used for mTOR analysis, 10 lg for

all other proteins and 60 lg for the caspase activity assay.

Caspase 3 assay

In a reaction volume of 100 lL, lysate buffer supernatant fluid was

added to assay buffer [312.5 mM HEPES (pH 7.5), 31.25% w/v

sucrose, 0.3125% w/v CHAPS] with 100 mM DTT and the caspase-

3 substrate Ac-DEVD-pNA (200 lM), and incubated at 37�C.Release of p-nitroaniline was monitored by recording the OD405nm

at 15 min intervals.

Western blot analysis

Protein (20 lg per sample) was separated on 6% or 10% Tris-

glycine polyacrylamide gels and transferred to polyvinylidene

difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford,

MA, USA). Immunoblots were blocked for 2 h in Tris-buffered

saline Tween-20 (TBST, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5,

0.05% Tween 20) containing 5% non-fat milk and 0.21% sodium

fluoride. The blots were incubated with primary antibody in

blocking buffer at 4�C overnight. Membranes were washed twice

with TBST and then incubated with the secondary antibody : per-

oxidase-conjugated anti-rabbit IgG (1 : 1000 dilution) for 1 h at

25�C. Membranes were washed again and then developed with

Enhanced Chemiluminescence (ECL) plus western blotting (Amer-

sham Biosciences), followed by apposition of the membrane to

autoradiographic films (Hyperfilm ECL, Amersham Biosciences).

After two washes with TBST, membranes were probed with a

monoclonal antibody against tubulin (1 : 1000, Sigma) or b-actin(1 : 1000, Sigma) for 2 h at 25�C. They were then washed with

TBST, incubated with anti-mouse peroxidase-conjugated secondary

antibody for 1 h and developed. Band intensities were analysed with

a gel documentation system (BioRad). mTor and p70S6k expres-

sions were adjusted to tubulin expression. The protein levels were

expressed as densitometry and percentage of controls.

Transgenic mice

The different transgenic mouse lines were generated at the Research

Centre of Paris, Sanofi Aventis, in Neurodegenerative Disease Group

(Vitry sur Seine, France). All themicewere female and all experiments

were performed in compliance with, and following approval of the

Aventis Animal Care and Use Committee, in accordance with the

standards of the Guide for the Care and Use of Laboratory Animals

(CNRS ILAR), and with respect to French and European Community

rules. The genetic background of the mice was mixed CBA/C57BL/6.

The following groups of mice were examined: 24-month-old

control mice [n ¼ 5, body weight (BW) ¼ 36.14 ± 4.49 g];

24-month-old transgenic mice (n ¼ 5, BW ¼ 28.78 ± 1.84 g)

expressing human mutant APP751 (APP HE) (carrying the

Swedish and London mutations KM670/671NL and V717l, Thy1

promoter); 12-month-old control mice (C57BL/6, n ¼ 8, BW ¼34.58 ± 2.22 g); 12-month-old transgenic mice expressing single

human mutant presenilin-1 (APP/PS1 WT/HE) (PS-1 M146L,

HMG promoter, n ¼ 6, BW ¼ 34.06 ± 3.05 g), 12-month-old

transgenic mice expressing human mutant APP751 SL and human

mutant presenilin-1 M146L (APP/PS1 HE/HE, n ¼ 4, BW ¼28.85 ± 0.48 g). APP/PS-1 double-transgenic mice were generated

by crossing PS-1 (HMG PS-1 M146L) homozygous mice with

hemizygous APP (Thy1 APP751 SL) transgenic mice. A detailed

description of these transgenic mice has been given previously

(Wirths et al. 2001; Blanchard et al. 2003). APP mice were

analysed at 24 months of age and APP-PS1 mice were assessed at

12 months of age because they revealed comparable levels of

amyloid plaques in the brain at these respective ages.

Brain tissue preparation

For western blot analysis, mice were anaesthetized with pentobarbital

(40 mg/kg, i.p.) and killed. Brains were removed and cortex and

cerebellum dissected. These cerebral regions were homogenized by

10 up-and-down strokes of a pre-chilled Teflon-glass homogenizer in

20 volumes of lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM

EDTA, pH 7.4) and supplementedwith 50 mMsodiumfluoride, 1 mM

phenylmethylsulfonyl fluoride, protease and phosphatase inhibitor

cocktails (50 lL/g tissue and 10 lL/mL lysis buffer, respectively).

Lysates were sonicated and centrifuged at 15 000 g for 15 min at 4�C.The resulting supernatant fluids were collected for BioRad protein

assay and western blot analysis as described above.

The brain tissue preparation for immunohistochemistry required

an intracardial perfusion with Phosphate-Buffered Saline (PBS)

(154 mM NaCl, 1.54 mM KH2PO4, 2.7 mM Na2HPO4.7H2O,

pH 7.2) followed by 4% (v/v) paraformaldehyde (PFA) in PBS.

Brains were removed and post-fixed for 24 h in 4% PFA at 4�C.They were then rinsed in PBS, dehydrated, and embedded in

paraffin for sectioning (4 lm in thickness).

Immunohistochemistry

Sections were de-waxed for 2 · 10 min with Histosol (Shandon,

Cergy Pontoise, France) and rehydrated by 5 min incubations in each

of 100, 95 and 75% ethanol. Sections were rinsed twice for 5 min in

ultra-high quality water (H2OUHQ) and washed twice in PBS

(2 · 5 min). To unmask antigens, the slides were immersed in citrate

buffer (10 mM sodium citrate, pH 6) and microwaved for 2 · 10 min

(with 650 W). For amyloid peptide (Ab) immunostaining, sections

were incubated in 10 mM sodium citrate buffer, pH 9, for 40 min in a

98�C water bath. Slides were allowed to reach room temperature

before two 5 min incubations in H2OUHQ. For rabbit polyclonal

primary antibody anti-phospho-mTOR (Ser2448), dilution 1/20, the

EnVision System (DakoCytomation, Trappes, France) was used. This

protocol includes four steps: (i) a 5 min peroxidase block to quench

endogenous peroxidase activity; (ii) an incubation for 30 min at room

temperaturewith diluted primary antibody in TBST (50 mMTris-HCl,

150 mM NaCl pH 7,6 with 0.1% Tween) containing 1% of bovine

serum albumin; (iii) an incubation for 30 min at room temperature

with the peroxidase-labelled polymer; (iv) staining (8 min incubation)

with 3,3¢-diaminobenzidine (DAB) substrate-chromogen which

results in a brown-coloured precipitate at the antigen site. The

monoclonal mouse primary antibody anti-human b-amyloid

mTOR/p70S6k signalling alteration by Ab 217


(1–40/42) C-terminal, dilution 1/25 (Calbiochem, Strasbourg,

France), was immunostained with the DakoCytomation DAKO

ARK system. The diluted primary antibody was labelled using

the biotinylation reagent, a monovalent biotinylated anti-mouse

immunoglobulin. Tissue sections were incubated for 15 min at room

temperature (RT) with the biotin-labelled primary antibody and then

incubated, also for 15 min at RT, with a streptavidin-alcaline

phosphatase conjugate. Staining was completed with fast red

substrate-chromogen, resulting in a red end product at the site of the

target antigen. Mayer’s haematoxylin or methyl green was eventually

used as counterstain.

Selection of patients and cognitive tests

Twenty-six patients (18 female, 8 male) (mean age: 76.3 ± 6.5 years,

range: 60–89 years) suffering from AD were selected from the

department of Neurology (Poitiers University Hospital), according to

the NINCDS-ADRDA clinical criteria, and included in the study

along with 28 control subjects (21 female, 7 male) (mean age:

77.4 ± 7.2 years, range: 64–94 years) without neurological disor-

ders. All patients and controls gave their written informed consent for

the study. All control individuals had a Mini Mental Status

Examination (MMSE) above 25. AD patients had a MMSE below

26. Blood samples (20 mL) were drawn at the same time as MMSE

was performed. This study was approved by the regional ethics

committee of the Region Poitou-Charentes (France) (CCPPRB).

Preparation of lymphocyte cells

Peripheral venous blood was obtained from healthy volunteer donors

andAlzheimer patients.Mononuclear cells were isolated using Ficoll-

Histopaque 1077 (Sigma, Saint Quentin Fallavier, France) density

gradient centrifugation at 700 g for 20 min. The lymphocyte cells

obtained were then washed in RPMI 1640 (Gibco-BRL) and

re-suspended in 200 lL lysis buffer [50 mM Tris-HCl, 50 mM NaCl,

pH 6.8, 1% Triton X-100, 1% (v/v) protease inhibitor cocktail, 1%

(v/v) phosphatase inhibitor cocktail]. After homogenization, lysates

were sonicated for 2 min and centrifuged at 15 000 g for 15 min at

4�C. The supernatant fluid was analysed for protein content using a

protein assay kit (BioRad). Aliquots of 2 lg protein/lLwere frozen at

) 80�C until further analysis. As internal control, we used the same

protein sample obtained frommurine neuroblastoma cells (Neuro-2a).

Statistical analysis

Results are expressed as means with SEM. Data for multiple

variable comparisons were analysed by one-way analysis of

variance (ANOVA) or the non-parametric Kruskal–Wallis test. For

comparison of significance, Bonferroni’s test or Dunns’s test was

used as a post hoc test according to the statistical program GraphPad

Instat. For comparison of significance between two groups,

Student’s t-test was conducted. The level of significance was

p < 0.05. The Pearson correlation and linear regression between

p70S6k levels and MMSE scores was also analysed.

Results

Caspase 3 activation induced by 1–42 AbFigure 1a shows the results of caspase 3 activation inNeuro-2acells after exposure to Ab. After 8 h, there is a significant

Control

30m

in 2h 4h 8h 16h

24h 8h

0

50

100

150

***

1-42 40-1

Ph

osp

ho

-mT

OR

/mTO

R(%

ofc

on

tro

l)Neuro-2a

Neuro-2(a)

(b)

(c)

a

Neuro-2a

289 kDa

55 kDa

289 kDa

55 kDa

Phospho-mTOR

Tubulin

Tubulin

Total mTOR

289 kDa

55 kDa

289 kDa

55 kDa

30min 2h 4h 8h 16h 24h 8hContro

l

4h 8h0

100

200

300

400

1-42

*

Cas

pas

e-3

acti

vity

(% o

f co

ntr

ol)

Fig.1 (a) Neuro-2a cells were treated with Ab (20 lM) for 4 and 8 h

and caspase 3 activity analysed. The results are expressed as a

percentage of their corresponding control at each time (treated cells

and corresponding time controls). Results were obtained from three

independent experiments; *p < 0.05. (b) Detection of total and phos-

phorylated mTOR in differentiated murine Neuro-2a cells exposed to

Ab 1–42 peptide. After differentiation by 20 lM all-trans retinoic acid

for 2 days and treatment by 20 lM Ab 1–42 from 30 min to 24 h, cells

were lysed and subjected to western blotting. Untreated cells served

as controls. The first lane shown in the western blots corresponds to a

control culture at time 0. (c) Histograms represent the ratio between

the phosphorylated and the total forms of mTOR in Neuro-2a taken

from three independent experiments. Data are shown as fold of the

corresponding time control. The results in all treated cell cultures were

compared with the results in control cell cultures at the same time

(30 min and 2, 4, 8, 16 and 24 h); *p < 0.05 compared with the

respective control by one-way ANOVA followed by a Bonferroni’s test for

multiple comparisons. There is a clear decrease in phosphorylated

mTOR expression induced by Ab.



increase in active caspase 3 in treated cells compared withcontrol cells, indicating that apoptosis pathways are triggeredby Ab in Neuro-2a cells. The activity of caspase 3 is aug-mented (174% of control) 4 h after Ab exposure. After 8 h, theactivity is 267% of controls and at the same time, inactivationof mTOR is around 70% of controls in Neuro-2a cells.

Inactivation of mTOR in neural cells induced by Abpeptide 1–42

Expression of mTOR was examined using polyclonalantibodies against its total and phosphorylated forms.Figure 1b shows a marked decrease in mTOR phosphoryla-tion when the differentiated murine neuroblastoma cells weretreated with 20 lM aggregated Ab 1–42 for various times.The ratio between the phosphorylated and the total forms isalready reduced (Fig. 1c) 30 min after the beginning ofexposure. This inactivation augments with time and is around75% in Neuro-2a cells after 24 h (*p < 0.05 compared withthe respective control). As a control, the non-toxic homo-logue Ab 40–1 did not enhance mTOR inactivation in cells.

Western blots also depicted the effects of Ab 1–42exposure on phosphorylated (Thr389 controlled by mTORsignalling) p70S6k expression in neural cells (Figs 2a and b).Analysis revealed that Ab 1–42 induced a progressivereduction of the ratio between the phosphorylated and thetotal forms of p70S6k. This inactivation is significant after24 h (*p < 0.05 compared with the respective control). As acontrol, the reverse peptide Ab 40–1 did not induce p70S6kinactivation in neural cells.

mTOR and p70S6k expression in transgenic mice

Antibody against p70S6k phosphorylated at Thr389 showedtwo positive bands corresponding to phosphorylated p70/p85 S6 kinases that are the cytosolic and nuclear isoforms,respectively. In mouse homogenates, we analysed only thesignal band at 70 kDa (Fig. 4). There was no difference inthe activation of mTOR and p70S6k (corresponding to theratio between phosphorylated and total forms of the kinases)between transgenic mice expressing human mutant APP751and control mice in cortex homogenates (Figs 3a and 4a). Incerebellum homogenates, an increased level of phosphoryl-ated mTOR and p70S6k was observed but was onlysignificant for p70S6k (increase: 34%, p < 0.01) (Figs 4band d). A significant decrease in mTOR activation wasobserved in the cortex (decrease: 34%) of APP/PS-1 double-transgenic mice expressing human mutant APP751 SL andhuman mutant presenilin-1 M146L compared with12-month-old control mice (Figs 3a and c). As shown inFig. 5, APP/PS-1 double-transgenic mice developed in thecortical regions extracellular Ab deposits (Fig. 5a) with veryintensively Ab-immunostained neurones (Fig. 5b). A reduc-tion in phosphorylated mTOR immunostaining was observedin cortical neurones of APP/PS-1 double-transgenic mice(Fig. 5d) compared with cortical neurones of control mice

showing mainly cytoplasmic localizations (Fig. 5c). In thecortex of APP/PS-1 double-transgenic mice, the inactivationof mTOR detected by western blots was associated with asignificant inactivation of p70S6k (decrease: 31%, Figs 4aand c). On the other hand, a large increase in mTORactivation was observed in cerebellum homogenates of APP/PS-1 double-transgenic mice (increase: 250% of the ratiophospho-mTOR/mTOR, p < 0.01) (Figs 3b and d) with anon-significant increase in the ratio phospho-p70S6k/p70S6k(Figs 4b and d). Morphological results confirmed thatphospho-mTOR immunostaining was clearly marked in thecerebellum of APP/PS1 transgenic mice (Fig. 5f).

Expression and phosphorylation of p70S6 kinase in

lymphocytes of Alzheimer patients

The expression and phosphorylation of p70S6 kinase(Thr389) was analysed by western blotting in lymphocytesof 28 control individuals and 26 Alzheimer patients (Figs 6a,

Contro

l

Phospho-p70S6K(T389)

Tubulin

Tubulin

30min 2h 4h 8h 16h 24h 8h

(a)

(b)

70 kDa

55 kDa

70 kDa

55 kDa

Total p70S6K

Contro

l

Neuro-2a

Neuro-2a

Control

30m

in 2h 4h 8h 16h

24h 8h

0

50

100

150

200

1-42 40-1

*

Ph

osp

ho

-p70

S6K

/p70

S6K

(% o

f co

ntr

ol)

Fig.2 (a) Detection of total and phosphorylated (Thr389 controlled by

mTOR signalling) p70S6k in differentiated murine Neuro-2a cells

exposed to Ab 1–42 peptide. After differentiation by 20 lM all-trans

retinoic acid for 2 days and treatment with 20 lM Ab 1–42 for various

times, cells were lysed and subjected to western blotting. Untreated

cells served as controls. The first lane shown in the western blots

corresponds to a control culture at time 0. (b) Histograms represent

the ratio between phosphorylated and total forms of p70S6k in Neuro-

2a and result from three independent experiments. Data are shown as

fold of the corresponding time control. The results in all treated cell

cultures were compared with the results in control cell cultures at the

same time (30 min, 2, 4, 8, 16 and 24 h). There is a clear decrease in

phosphorylated p70S6k expression induced by Ab.



b and c). Phosphorylation site (Thr389) is the specific site ofp70S6k phosphorylation by kinase mTOR. Data areexpressed as a percentage of internal control taken fromneuroblastoma cells. Individual results and means aredepicted in Fig. 6(b) and indicate that p70S6k activation,

measured by the ratio phospho-p70S6k/p70S6k, was signi-ficantly decreased (51%, p < 0.0005) in lymphocytes ofAlzheimer patients compared with control individuals (ADpatients: 241.8 ± 30.2, controls: 490.2 ± 52.6). The sensi-tivity of the results is 88.4% and the specificity is 64.2%.

CORTEX

Phospho-p70S6K

Total p70S6K

Tubulin

Tubulin

70 kDa

70 kDa

55 kDa

55 kDa

(a) (b)

(d)(c)

Total p70S6K

Phospho-p70S6K

Tubulin

Tubulin

APP

HE

APP

HE

APP

/PS-

1W

T/H

EA

PP/P

S-1H

E/H

E

APP

/PS-

1W

T/H

E

APP

/PS-

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HE

Con

trol

(24-

mon

th)

Con

trol

(24-

mon

th)

Con

trol

(12-

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th)

Con

trol

(12-

mon

th)

CEREBELLUM

contro

l (24

-month

)

APPHE

contro

l (12

-month

)

APP/PS1 W

T/HE

APP/PS1 HE/H

E0

25

50

75

100

125

150

*

ph

osp

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-p70

S6K

/p70

S6K

(%o

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contro

l (24

-month

)

APPHE

contro

l (12

-month

)

APP/PS1 W

T/HE

APP/PS1 HE/H

E0

25

50

75

100

125

150**

ph

osp

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-p70

S6K

/p70

S6K

(%o

fco

ntr

ol)

Fig. 4 Western blot analysis of phosphor-

ylated (Thr389) and total forms of p70S6k in

cortex (a) and cerebellum (b) in different

transgenic mice as described in Fig. 3. An

aliquot of 30 lg protein of cortex and cere-

bellum homogenates prepared as des-

cribed in Materials and methods was run in

each lane. Tubulin was used as control.

Three mice in each group were studied and

results are summarized in bar charts for

cortex (c) and cerebellum (d). Data repre-

sent the ratios of phospho p70S6k and are

the mean ± SEM expressed as percentage

of respective control. Statistical analysis

was carried out using an ANOVA (Kruskal–

Wallis test) followed by Dunn’s test. There

is a clear decrease of the ratio in the cortex

of APP/PS1 mice.

CORT

(a) (b)

(d)(c)EX CEREBELLUM

Phospho-mTOR

Total mTOR

Tubulin

Tubulin

289 kDa

289 kDa

55 kDa

55 kDa

Phospho-mTOR

Total mTOR

Tubulin

Tubulin

APP

HE

APP

HE

APP

/PS-

1W

T/H

EA

PP/P

S-1H

E/H

E

APP

/PS-

1W

T/H

EA

PP/P

S-1H

E/H

E

Con

trol

(24-

mon

th)

Con

trol

(24-

mon

th)

Con

trol

(12-

mon

th)

Con

trol

(12-

mon

th)

contro

l (24

-mon

th)

APPHE

contro

l (12

-month

)

APP/PS1 W

T/HE

APP/PS1 HE/H

E0

25

50

75

100

125

150

*

ph

osp

ho

-mT

OR

/mT

OR

(%o

fco

ntr

ol)

contro

l (24

-month

)

APPHE

contro

l (12

-month

)

APP/PS1

WT/H

E

APP/PS1 HE/H

E0

100

200

300

400**

ph

osp

ho

-mT

OR

/mT

OR

(%o

fco

ntr

ol)

Fig. 3 Representative immunnoblots show immunoreactivity of total

and phosphorylated forms of mTOR in cortex (a) and cerebellum (b) in

different transgenic mice: 24-month-old control mice, 24-month-old

hemizygous transgenic mice expressing human mutant APP751 (APP

HE), 12-month-old control mice, 12-month-old hemizygous trans-

genic mice expressing single human mutant presenilin-1 (APP/PS-1

WT/HE), 12-month-old hemizygous double transgenic mice expres-

sing human mutant APP751 SL and human mutant presenilin-

1 M146L (APP/PS-1 HE/HE). An aliquot of 30 lg protein from cortex

and cerebellum homogenates prepared as described in Materials and

methods was run in each lane. Tubulin was used as control. Histo-

grams in (c) and (d) represent the ratio between phosphorylated and

total forms of mTOR in cortex (c) and cerebellum (d), respectively.

Data expressed as percentage of respective control are the mean ±

SEM. Three mice in each group were studied. Statistical analysis was

carried out using an ANOVA (Kruskal–Wallis test) followed by Dunn’s

test. There is a clear decrease of the ratio in the cortex of APP/PS1

mice and a clear increase in the cerebellum of these mice.



This clear-cut decrease was due to a significant reduction inthe phosphorylated form of p70S6k. The scattergram(Fig. 6c) shows that the mean value of phospho-p70S6k(Thr389) expression alone was significantly decreased by74% in lymphocytes of Alzheimer patients compared withcontrol lymphocytes (AD patients: 49.2 ± 10.4; controls:188.0 ± 17.2; p < 0.0005).

Correlation between p70S6 kinase activation

in lymphocytes of Alzheimer patients and MMSE scores

Correlation and regression analyses were carried out toinvestigate the relations between p70S6k activation inlymphocytes of Alzheimer patients and MMSE scores. Thephospho-p70S6k/p70S6k ratio measured in Alzheimer

patients lymphocytes showed a significant correlation withMMSE scores (r ¼ 0.726; p < 0.0001; Fig. 7).

Discussion

Our results demonstrate that Ab exposure induces a sustainedreduction in mTOR/p70S6k signalling in neuroblastoma cell

d

(d)

(b)(a)

c

(f)(e)

(c)

Fig. 5 Immunohistochemical staining of paraffin-embedded mouse

brains showing expression of Ab (cortex: 5a and 5b) and phosphor-

ylated mTOR (cortex: 5c and 5d, cerebellum: 5e and 5f) using DAKO

ARK and EnVision systems, respectively, as described in Materials

and methods. (a) The extracellular accumulation of Ab deposits in

the brain of Thy-APP751 SL X PS-1M146L transgenic line (APP/PS-1

HE/HE). (b) Intense Ab staining in a cortical neurone of these double

transgenic mice. In (c) and (d), respectively, the expression of phos-

phorylated mTOR in the cortex of control mice (12 m) and an APP/PS-

1 HE/HE mouse is illustrated. A reduction of phosphorylated mTOR

staining was observed in double transgenic mice (d) compared with

control mice (c) where the expression of active mTOR is localized near

the nuclear membrane and in the plasma membrane of cortical neu-

rones indicated by arrowheads. In (e) and (f), respectively, immuno-

staining of phosphorylated mTOR in the cerebellum of a control mouse

and of an APP/PS-1 HE/HE mouse is shown. Horizontal bar in (a) ¼40 lm; (b), (c) and (d) ¼ 10 lm; (e) and (f) ¼ 20 lm.

controls alzheimer0

250

500

750

1000

1250

(a)

(b)

(c)

ph

osp

ho

-p70

S6K

/ p

70S

6K(%

of

inte

rnal

co

ntr

ol)

controls alzheimer0

100

200

300

400

ph

osp

ho

-p70

S6K

(T

389)

(% o

f in

tern

al c

on

tro

l)

phospho-p70S6K

actin

70 kDa

Alzheimer Controls

Alzheimer Controls

actin

total p70S6K

42 kDa

70 kDa

42 kDa

Internalcontrol

Internalcontrol

Fig. 6 Western blot analysis of p70S6k activation in lymphocytes of

control individuals and Alzheimer patients. The internal control cor-

responds to the result obtained in neuroblastoma cell cultures. Rep-

resentative immunnoblots of phospho-p70S6k (Thr389) and total

p70S6k expression is shown in lymphocytes of three control individ-

uals and three Alzheimer patients (a). The antibodies recognize spe-

cific bands of 70 kDa and 42 kDa for phospho-p70S6k (Thr389), total

p70S6k and actin, respectively. The bands were quantified by densi-

tometry and the results were expressed as percentage of internal

control (Neuro-2a cells). p70S6k activation, measured by the ratio

phospho-p70S6k/total p70S6k, is summarized as a scattergram (b).

Results of phospho-p70S6k (Thr 89) expression alone are shown in

(c). In (b) and (c), data are means ± SEM of 28 control individuals and

26 Alzheimer patients. The symbols represent the means of the group.

***p < 0.0005 statistical difference from control individuals (Student’s

t-test). (j) Control individuals; (m) Alzheimer patients.



cultures, marked by a progressive decrease in phosphorylatedmTOR and phosphorylated p70S6k. In addition, mTORsignalling is reduced in the cortex but not in the cerebellumof APP/PS-1 mutant transgenic mice and in lymphocytes ofAD patients compared with control individuals. Takentogether, these findings suggest that the inactivation ofmTOR/p70S6k pathway, which regulates cell growth andproliferation, protein translation and autophagy and isthought to transfer anti-apoptotic properties, could be afactor contributing to the degenerative process detected in thebrains of AD patients.

mTOR is known to phosphorylate p70S6k at Thr389, butother kinases such as ERK1/2 are able to phosphorylatep70S6k at Thr421/Ser424 (Dennis et al. 1998). In addition, arecent report showed that p70S6k could be activated not by alinear pathway from mTOR but by two separate pathwaystriggered by mTOR and PI3 kinase (Raught et al. 2001).This could explain the different activation profile observedfor mTOR and p70S6k dephosphorylation. This dephospho-rylation is not very rapid and complete as it was observedafter energy depletion in another cell culture system (Inokiet al. 2003). This difference is possibly linked to the slowtoxicity induced by Ab. We have previously demonstratedthat Ab 1–42 activates PKR, a kinase known to down-regulate protein translation, acting prior to mTOR intranslational control and leading to phosphorylation of theeukaryotic initiation factor 2a (eIF2a) and cellular apoptosis(Suen et al. 2003). Increased PKR signalling and reducedmTOR/p70S6k signalling are the combined consequences ofAb 1–42 exposure in neural cells. Previous reports haverevealed an increase in intracellular calcium following Abexposure (Mattson and Chan 2003). The role of calcium inPKR activation was also demonstrated in our previous study(Suen et al. 2003) and could be involved in the transient riseof p70S6k phosphorylation observed at 2 h because this

enzyme is known to be sensitive to variations in intracellularcalcium (Hannan et al. 2003). It is now known that mTOR isan oncogenic protein participating in cell growth andproliferation but with probable anti- and pro-apoptoticproperties that have been recently reviewed (Castedo et al.2002). In our neural cell system, the reduction in mTORphosphorylation occurred as early as 30 min to 2 h after Abexposure and was amplified over the following 24 h. Thisfinding could contribute to the neurotoxic and apoptoticproperties of Ab previously described in neural cells (Changet al. 2002b).

The immunohistochemical results showed a large numberof Ab)positive plaques in different regions of the centralnervous system including the cortex, as well as an increase inexpression of Ab inside neurones. As previously described,almost no neuronal degeneration was detected in the brainsof the transgenic mice at the opposite of what was in thebrain of AD patients. The changes in mTOR/p70S6ksignalling were only detected in APP/PS1 transgenic miceand not in transgenic mice expressing only human mutantAPP. Since both types of transgenic mice expressed Abplaques, it is possible to postulate that the augmentedexpression of Ab can only be partly responsible for thereduced mTOR/p70S6k signalling in the same regions.Presenilin (Culvenor et al. 1997) and mTOR (Drenan et al.2004) have been localized in the ER–Golgi network andmutated PS1 could contribute, together with Ab accumula-tion, to the reduced activity of mTOR/p70S6k in the brains ofthese APP/PS1 mice. A recent report has also noted thatmutated human PS1 down-regulates the PI3K/Akt pathwaywhich controls mTOR activity (Baki et al. 2004). We haveobserved a significant modification of mTOR phosphoryla-tion in the cerebellum of transgenic mice overexpressing thedouble APP/PS1 human mutations. In addition, usinghistological methods, we did not observe plaques or overtpositive Ab immunostaining in the cerebellum of theseanimals; this observation concurs with previously publisheddata assessing the same mice (Blanchard et al. 2003). Theincreased levels of phosphorylated mTOR observed in thecerebellum with western blots in APP-PS1 mice is associatedwith a lack of Ab accumulation. The reasons for thesebiochemical and histological findings are not known but itcan be assumed that specific molecular pathways in thecerebellum might prevent major extracellular and intracellu-lar Ab accumulations. Further studies will be necessary todetermine whether these two observations are linked and areimplicated in specific neuronal susceptibility of the corticalversus the cerebellar areas. In AD brains, only diffuseplaques and very few senile plaques have been described inthe cerebellum of patients with a faint morphological reactionin their microenvironment (Joachim et al. 1989). A recentreport has revealed that positive staining for phosphorylatedp70S6k on two sites (Thr389, Thr421/Ser424) was detectedin neurones of AD patients with increased brain levels. Only

0 5 10 15 20 25 300

250

500

750

1000

MMSE score

ph

osp

ho

-p70

S6K

/ p

70S

6K(%

of

inte

rnal

co

ntr

ol) r = .726

p< .0001

Fig. 7 p70S6 kinase activation in lymphocytes of Alzheimer patients

and correlation with Mini Mental Status Examination (MMSE) scores.

Expression of phospho-p70S6 kinase (Thr389) and total p70S6 kinase

was analysed by western blot in lymphocytes of 26 Alzheimer patients.

p70S6k activation measured by the ratio phospho-p70S6k/total

p70S6k was expressed as percentage of internal control and was

correlated with MMSE score of Alzheimer patients. p < 0.0001

(Pearson correlation). (m) Alzheimer patients.



activated p70S6k expression at the Thr421/Ser424 site wascorrelated with levels of total tau and phosphorylated tau (Anet al. 2003). The phosphorylation of p70S6k on Thr421/Ser424 is linked to ERK1/ERK2 triggering and not to mTORactivation (Dufner and Thomas 1999). The molecularmechanisms leading to increased expression of phosphoryl-ated p70S6k on Thr389 in specific AD neurones could becomparable with those leading to the transient increasedactivity induced in vitro by Ab exposure in our cell culturesystem. Whether or not the staining of activated p70S6k(Thr389) in AD brains corresponds to specific modulation ofmTOR signalling activity or to another contributory factor inthese neurones remains to be determined.

We analysed the expression of phosphorylated p70S6k(Thr389) and total p70S6k in lymphocytes of patients andcontrol individuals. There was a clear reduction in the meanlevel of the ratio between phosphorylated p70S6k versustotal p70S6k. This difference was also detected for phos-phorylated p70S6k alone in AD patients as compared withcontrol subjects. This finding demonstrates that peripheralcells are marked by a decrease in the initiation phase ofprotein translation in patients suffering from AD. Manyprevious studies have demonstrated various biological dis-turbances in lymphocytes and other peripheral cells in ADpatients, including increased cytosolic free calcium levels(Gibson et al. 1987; Adunsky et al. 1991; Eckert et al. 1997;Palotas et al. 2002), an impairment of mitogenic proliferation(Stieler et al. 2001), an enhanced sensitivity to apoptosis(Eckert et al. 2001) and the presence of increased reactiveoxygen species (Mecocci et al. 2002). In line with the resultsof the present study, several of these observations could beeither the cause or the consequence of reduced translationalcontrol. Recent data have revealed that lymphocytes of ADpatients are less sensitive to the action of rapamycin, a potentantagonist of mTOR signalling, on cell cycle kinesis andproliferation (Nagy et al. 2002). We suggest that if themTOR/p70S6k signalling is already down-regulated in ADlymphocytes, the availability of the remaining activatedmTOR/p70S6k proteins is insufficient for proper rapamycinaction. The other interesting finding is that the reduction oftranslational control assessed by the ratio between activatedp70S6k versus total p70S6k is correlated with the cognitivedecline of patients evaluated by MMSE scores. In this study,we have only included AD patients with a MMSE scoreranging from 25 to 10; in these patients, memory anomaliesin the first phase of the disease are very often at the forefrontof clinical signs. mTOR has been experimentally associatedwith molecular mechanisms of long-term potentiation (LTP),and learning and memory (Cammalleri et al. 2003). In fact,rapamycin can experimentally modify these cognitive pro-cesses. If the translational control is altered in AD brains, thismodification could contribute to the onset of memoryimpairment observed in AD. As mentioned previously,protein synthesis appears to be reduced in AD brains. This

finding was also observed in experimental transient cerebralischaemia associated with a decrease in mTOR/p70S6ksignalling marked by reduced activated p70S6k expression(Mengesdorf et al. 2002).

We have previously shown that PKR levels are altered inhippocampal neurones of AD patients and are marked byincreased expression of phosphorylated PKR, suggestingthat protein translation is reduced in these neurones (Changet al. 2002a). It would be interesting to find out whether ornot the modifications of mTOR/p70S6k signalling inperipheral lymphocytes can reflect biological neuronaldisturbances associated with memory decline in ADpatients, and it can be assumed that a general alterationof translational control implicates various cell types in ADmodels and in the cells of AD patients, including neuronesand peripheral lymphocytes.

In summary, it is not yet known whether the down-regulation of mTOR/p70S6k signalling observed in lympho-cytes is also present in the brains and neurones of ADpatients. Cellular and transgenic AD models have revealeddown-regulated mTOR/p70S6k signalling, and further stud-ies will have to determine the contribution of this disturbedsignalling to neuronal degeneration. The mTOR/p70S6kpathway, which is already at the centre of active anti-cancerresearch (Bjornsti and Houghton 2004), could also representa new target for future therapeutic approaches and neuro-protection in AD, as well as in other neurodegenerativedisorders.

Acknowledgements

The authors would like to thank Raymond Poncharaud and Brigitte

Vidal for technical help, Claudette Pluchon, Bernard Fauconneau

and Alain Piriou for useful advice, and Andre Brizard for scientific

support. This study was supported by the CHU de Poitiers with a

grant from the French Ministry of Health (PHRC) and by the

University of Poitiers and the French Ministry of Education and

Research, with a grant to the Research Unit EA 3808.

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