Báo cáo khoa học: Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer’s disease brain doc

The evolutionarily conserved checkpoint protein kinase, mammalian target of rapamycin mTOR has Keywords 4E-BP1; Alzheimer’s disease; mTOR; translation control; tau Correspondence J.-J..

and eEF2 kinase in relationships with tau in Alzheimer’s disease brain

Xu Li1, Irina Alafuzoff2, Hilkka Soininen3, Bengt Winblad1 and Jin-Jing Pei1

1 Division of Experimental Geriatrics, Department of Neurotec, Karolinska Institutet, Huddinge, Sweden

2 Department of Neuroscience and Neurology, Kuopio University Hospital, Kuopio University, Finland

3 Department of Neurology and Neuroscience and Neurology, Kuopio University Hospital, Kuopio University, Finland

Neurofibrillary tangles (NFTs) is one of the major

neuropathological hallmarks in Alzheimer’s disease

(AD) NFTs are large, nonmembrane-bound bundles

of abnormal fibres that occupy much of the

peri-nuclear cytoplasm of affected neurons These fibres

consist of paired helical filaments (PHFs), composed

of a hyperphosphorylated form of the

microtubule-associated protein tau (PHF-tau) [1–3] Tau plays a

key role in cellular stabilization, however, when tau is

hyperphosphorylated it is less capable of binding to

tubulin, resulting in destabilization of microtubules

and eventually cell death [4]

Although total tau is markedly increased in the hyperphosphorylated form, a significant amount of normal tau still exists in AD brain [5–7] This sugges-ted that tau is continuously produced in order to com-pensate those that have compromised their functions

by abnormal hyperphosphorylation in AD It is known that tau mRNA level is not changed in sporadic AD brains [8,9] However, one can not rule out the possi-bility that translation of tau mRNAs is aberrantly regulated in AD brains

The evolutionarily conserved checkpoint protein kinase, mammalian target of rapamycin (mTOR) has

Keywords

4E-BP1; Alzheimer’s disease; mTOR;

translation control; tau

Correspondence

J.-J Pei, Karolinska Institutet, Department

of Neurotec, Division of Experimental

Geriatrics, KFC Plan 4, Novum, S-141 86,

Huddinge, Sweden

Fax: +46 858583880

Tel: +46 858583751

E-mail: Jin-Jing.Pei@neurotec.ki.se

(Received 21 April 2005, revised 17 June

2005, accepted 24 June 2005)

doi:10.1111/j.1742-4658.2005.04833.x

The pathogenesis of formation of neurofibrillary tangles (NFTs) in Alzhei-mer’s disease (AD) brains is unknown One of the possibilities might be that translation of tau mRNA is aberrantly regulated in AD brains In the current study, levels of various translation control elements including total and phosphorylated (p) forms of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), eukaryotic elon-gation factor 2 (eEF2), and eEF2 kinase were investigated in relationship with tau in homogenates of the medial temporal cortex from 20 AD and

10 control brains We found that levels of p-mTOR (Ser2481), and p-4E-BP1 (Thr70 and Ser65) dramatically increase in AD, and are positively sig-nificantly correlated with total tau and p-tau Levels of p-eEF2K were significantly increased, and total eEF2 significantly decreased in AD, when compared to controls The changes of p-mTOR (2481), p-4E-BP1, and p-eEF2 were immunohistochemically confirmed to be in neurons of AD brains This suggested that there are obvious abnormalities of elements related with translation control in AD brain and their aberrant changes may up-regulate the translation of tau mRNA, contributing to hyperphos-phorylated tau accumulation in NFT-bearing neurons

Abbreviations

AD, Alzheimer’s disease; eEF2, eukaryotic elongation factor 2; eEF2K, eEF2 kinase; eIF4E, eukaryotic initiation factor 4E; ERK, extracellular signal regulated protein kinase; MAPK, mitogen activated protein kinase; mTOR, mammalian target of rapamycin; NFTs, neurofibrillary tangles; PHFs, paired helical filaments; S6K, S6 kinase; 4E-BP, eIF4E-binding protein; 5¢TOP, 5¢-terminal oligopyrimidine tract; 5¢UTR, 5¢ untranslated region.

emerged as a major effector of cell growth and

prolif-eration via the regulation of protein synthesis mTOR

controls protein synthesis through a number of

down-stream targets Several components of the ribosome

recruitment machinery as well as ribosomal

compo-nents are either direct or indirect targets of mTOR,

such as eukaryotic initiation factor 4E (eIF4E) and its

repressor eIF4E-binding protein (4E-BP), S6 kinase

(S6K) and its target ribosomal protein S6, and

eukar-yotic elongation factor 2 (eEF2)

Previously, we have found increased levels of

phos-phorylated (p) p70S6K [6] and p-eIF4E [10] in AD

brains In the current study (Table 1), levels of total

and p-forms of mTOR, 4E-BP1, eEF2, and eEF2K

were investigated in relationship with tau in

homogen-ates of the medial temporal cortex from 20 AD and 10

control brains

Results

Levels of total and phosphorylated mTOR

in AD and control brains Levels of total mTOR did not show a significant change between AD and control (Fig 1A) Levels of mTOR phosphorylated at the Ser2481 were signifi-cantly increased (about threefold) in AD brains, as compared with controls No change was found for p-mTOR (Ser2448) levels in AD brains Consistently, a significant correlation was only seen between p-mTOR (Ser2481) levels and the progression of neurofibrillary degeneration according to Braak and Braak criteria (Fig 1B) Antibodies to total mTOR and p-mTOR (Ser2448, Ser2481) only showed one band correspond-ing to its molecular mass at 289 kDa in AD homogen-ates (60 lg per lane), suggesting these antibodies are specific (Fig 1)

Levels of p-mTOR (Ser2481) showed a significant positive correlation with total tau (r¼ 0.517 and P ¼ 0.003 for Tau2, r¼ 0.619 and P ¼ 0.000 for R134d), PHF-tau (r¼ 0.631 and P ¼ 0.000 for AT8; r ¼ 0.445 and P¼ 0.014 for PHF-1) (Table 2) In contrast, a negative but significant correlation was seen between levels of p-mTOR (Ser2481) and nonphosphorylated tau labeled by Tau1 (r¼ –0.474, P ¼ 0.008) (Table 2) Levels of p-mTOR (Ser2448) only showed a significant correlation with PHF-tau (r¼ 0.056 and P ¼ 0.001 for AT8; r¼ 0.523 and P ¼ 0.003 for PHF-1), and total tau labeled by R134d (r¼ 0.394, P ¼ 0.031) Total mTOR only showed a significant correlation with PHF-tau labeled by AT8 (r¼ 0.417, P ¼ 0.022)

Levels of total and phosphorylated 4E-BP1

in AD and control brains

As shown in panel A of Fig 2, levels of total 4E-BP1 were about 50% decreased in AD brains, as compared with controls A significantly negative correlation was observed between levels of total 4E-BP1 and the pro-gression of neurofibrillary degeneration according to Braak and Braak criteria (Fig 2B) In contrast, levels

of 4E-BP1 phosphorylated at the Thr70 (
20%) or Ser65 (
70%) site but not at Thr37 ⁄ 46 sites were sig-nificantly increased However, a sigsig-nificantly positive correlation was observed only between levels of 4E-BP1 phosphorylated at the Ser65 site but not at the Thr70 site and the progression of neurofibrillary degeneration according to Braak and Braak criteria (Fig 2B) Levels of p-4E-BP1 (Thr37⁄ 46) did not show significant correlation with the progression of neuro-fibrillary degeneration according to Braak and Braak

Table 1 Detailed information of cases used in this study.

F, female; M, male; NA, not available; PMD, postmortem delay;

AD, Alzheimer’s disease Average age (years): control 82.40 ±

9.30; AD 81.50 ± 10.83 (P ¼ 0.824) Average postmortem delay

(h): control 6.50 ± 2.88; AD 5.95 ± 1.85 (P ¼ 0.592).

PMD (Hours)

Clinical Diagnosis

Braak’s Neurofibrillary Staging

criteria Antibodies to total and p-4E-BP1 only showed

one band corresponding to its molecular mass at the

range of 15–20 kDa in AD homogenates (100 lg per

lane), suggesting these antibodies are specific (Fig 2)

Levels of p-4E-BP1 but not total 4E-BP1 had

significant positive correlation with total tau as

follows: r¼ 0.578 and P¼ 0.001 for p-4E-BP1

(Ser65)⁄ Tau2; r ¼ 0.396 and P ¼ 0.030 for p-4E-BP1

(Ser65)⁄ R134d; r ¼ 0.426 and P ¼ 0.019 for

p-4E-BP1 (Thr70)⁄ Tau2; r ¼ 0.528 and P ¼ 0.003 for

p-4E-BP1 (Thr70)⁄ R134d (Table 2) Levels of

p-4E-BP1 at both Thr70 and Thr37⁄ 46 sites not the Ser65

site were significantly correlated to PHF-tau labelled

by AT8 or PHF-1 However, only p-4E-BP1 at the

Ser65 site showed a negative but significant correla-tion with tau nonphosphorylated at Tau1 sites

Total and phosphorylated levels of eEF2K and eEF2 in AD and control brains

Levels of p-eEF2K were significantly increased (
40%) in AD brains, as compared with controls (Fig 3A) A similar tendency of increase without signi-ficance was observed for p-eEF2 (Thr56) and total eEF2K Levels of total eEF2 were significantly decreased in AD as compared with control In general, the decrease of total eEF2 was significantly correlated with the progression of neurofibrillary degeneration

Table 2 Relationships of 4E-BP1, mTOR, eEF2K, and eEF2 with tau in AD and control brains.

Fig 1 Levels of total and phosphorylated (p) mTOR in AD and control brains (A) When compared with controls, levels of p-mTOR (Ser2481) significantly increased in AD brain by dot blot Levels of total mTOR and p-mTOR (Ser2448) did not show any change between

AD and control Filled bar, AD; unfilled bar, control (B) Significantly increased trend of p-mTOR (Ser2481) with the progression of Braak neu-rofibrillary stagings (C) Antibodies to total mTOR and p-mTOR (Ser2448, Ser2481) only showed one band in AD homogenates (60 lg per lane), respectively, corresponding to its molecular mass by western blots (6% SDS ⁄ PAGE) *P < 0.05; **P < 0.001.

according to Braak and Braak criteria, more dramatic

in brains with stages from IV to VI, as compared with

brains from stages I to III (Fig 3B) Levels of eEF2

(Thr56) and levels of total and p-eEF2K (Ser366) (not

shown) did not show significant correlation with the

progression of neurofibrillary degeneration Antibodies

to eEF2K and eEF2 showed only one band

corres-ponding to the molecular mass at 105 kDa and

100 kDa, respectively, in AD homogenates (eEF2K:

100 lg per lane; total eEF2: 60 lg per lane; p-eEF2:

12 lg per lane), suggesting these antibodies are specific (Fig 3)

Immunohistochemistry of AD and control brains with antibodies to p-mTOR, p-4E-BP1, and p-eEF2

We used phosphospecific antibodies to p-mTOR, p-4E-BP1, p-eEF2 and PHF-tau (PHF-1) to stain the sec-tions of the medial temporal cortex from AD and control brains We observed that the stainings were

Fig 2 Levels of total and phosphorylated (p) 4E-BP1 in AD and control brains (A) When compared with controls, levels of total 4E-BP1 sig-nificantly decreased in AD brain, while p-4E-BP1 (Thr70 and Ser65) sigsig-nificantly increased in AD brain by dot blot Filled bar, AD; unfilled bar, control (B) A decreased trend of total 4E-BP1 with the progression of Braak neurofibrillary staging, and a significantly increased trend of p-4E-BP1 (Ser65) are shown (C) Antibodies to total and p-4E-BP1 only showed one band in AD homogenates (100 lg per lane), respectively, corresponding to its molecular mass by western blots (15% SDS ⁄ PAGE) *P < 0.05; **P < 0.001.

Fig 3 Levels of total and phosphorylated (p) forms of eEF2K and eEF2 in AD and control brains (A) When compared with controls, level of p-eEF2K (Ser366) significantly increased, and levels of total eEF2 significantly decreased in AD brains Filled bar, AD; unfilled bar, control (B) Only at the late stages V and VI, level of eEF2 showed an apparent decrease (C) Antibodies to eEF2K and eEF2 showed only one band in

AD homogenates (eEF2K: 100 lg per lane; total eEF2: 60 lg per lane; p-eEF2: 12 lg per lane), respectively, corresponding to its molecular mass by western blots (6% SDS ⁄ PAGE) *P < 0.05; **P < 0.001.

mainly found in neuronal cell bodies For the antibody

to p-mTOR at Ser2448, there was no big difference

between AD and control cases (Fig 4B,C) This

antibody strongly stained the pyramidal neurons of the hippocampal CA1, CA2 and CA3 sectors, the subicu-lum, and entorhinal regions In the adjacent temporal

Fig 4 Immunostainings of

paraffin-embed-ded sections from the medial temporal

cor-tex of AD and control brains using

antibodies to phosphorylated (p) mTOR,

p-4E-BP1, p-eEF2, and PHF-tau p-mTOR

(Ser2481) in AD brain (A); p-mTOR

(Ser2448) in AD (B) and and control (C);

p-eEF2 (Ser56) in AD (D) and control (E);

p-4E-BP1 (Thr70) in AD brain (F) and

phos-phorylated tau ⁄ PHF-1 in AD brain (G) The

scale bar indicated 20 lm.

cortex, intensive staings were also found in the

pyram-idal cell bodies of layers III and V CA4 sector and

granule layer of the dentate gyrus were moderately

stained by antibody to mTOR (Ser2448) The pattern

of immunostainings for p-mTOR at Ser2481 was

sim-ilar to that of Ser2448, but the immunoreactivities in

AD (Fig 4A) were much stronger than controls (not

shown) The antibody against p-4E-BP1 intensively

stained the pyramidal neurons in the hippocampal

CA1, CA2 sectors, and the entorhinal regions,

especi-ally in some tangel-like neurons in AD brains This

antibody moderately stained the pyramidal neurons of

the CA3 and CA4 sectors, and the granule layer of the

dentate gyrus (Fig 4F), while in the control sections

processed in parallel, the corresponding stainings

were weak in all of these areas as compared with the

AD brain sections (data not shown) In AD brain,

immunostainings with the antibody against p-eEF2 at

Thr56 showed moderate to strong staining in the

pyramidal neurons of the hippocampal CA1, CA2,

CA3, CA4 sectors, and the entorhinal and temporal

cortice (Fig 4D), but weak staining in the granule

layers of the dentate gyrus (not shown) In contrast, in

control brain slices there was weak stainings in the

pyramidal neurons of the CA1, CA2, CA3 and the

entorhinal cortex (Fig 4E), but relatively more

pro-nounced immunostainings were seen in the granule

layer of the dentate gyrus (not shown)

Discussion

Depositions of PHF-tau form the pathological

hall-mark NFTs in AD brains Although extensive studies

have been carried out to understand the pathogenesis

of the NFTs in AD in the past two decades, none has

ever analysed the possible changes of various

control-ling factors of protein translation such as mTOR,

4E-BP1, eEF2K, and eEF2 in relationship with tau in

AD brains

mTOR acts as a sensor for ATP and amino acids,

balancing the availability of positive signals to p70S6K,

and participates in the inactivation of the eIF4E

inhi-bitor 4E-BP1 mTOR is autophosphorylated at the

Ser2481 site and phosphorylated at the Ser2448 site via

phosphoinositide-3 kinase (PI3K) signalling pathway

[13] In the current study, we found: (a) an approx

threefold increase of mTOR autophosphorylation

(Ser2481) in AD homogenates as compared with the

controls; (b) a clearly increased tendency of mTOR

autophosphorylation (Ser2481) following the

progres-sion of neurofibrillary degeneration according to Braak

and Braak criteria; (c) a significant correlation of

mTOR autophosphorylation (Ser2481) with PHF-tau

labelled by AT8 (r¼ 0.631; P ¼ 0.000) and PHF-1 (r¼ 0.445, P ¼ 0.014) These data suggested an exclu-sive increase of mTOR autophosphorylation, which correlates with PHF-tau associated pathologies Levels

of mTOR phosphorylated at the Ser2448 site neither showed significant change between AD and control homogenates, nor did any significant correlation with neurofibrillary degeneration according to Braak and Braak criteria However, a significant correlation was seen between levels of p-mTOR (Ser2448) and PHF-tau labelled by AT8 (r¼ 0.560, P ¼ 0.001) and PHF-1 (r¼ 0.523, P¼ 0.003) Most recently, levels of p-mTOR (Ser2448) was obviously increased in 6 of 9 cases of the medial temporal cortex staged at IV, V and

VI according to Braak and Braak criteria as compared with 7 cases staged at 0, I and II [18] Taken together, aberrant regulation of mTOR phosphorylation in AD brain might be not only predominantly mediated by its autophosphorylation at the Ser2481 site, but also medi-ated by its phosphorylation at the Ser2448 regulmedi-ated by PI3K pathway, the aberrant activation of which exists

in AD [6,7,14–18] The implication of the disordered mTOR phosphorylation, in particular its autophospho-rylation, remains to be clarified

Induction of 4E-BP1 hyperphosphorylation is medi-ated primarily by rapamycin-sensitive mTOR-depend-ent pathway, while some evidence also suggested an extracellular signal regulated protein kinase (ERK)-dependent modulation of its phosphorylation at the Ser65 site [19,20] Phosphorylation at Ser65 and Thr70 sites showed a relatively higher degree of rapamycin sensitivity than Thr37 and Thr46 suggested that mTOR plays a more important regulatory role in the phosphorylation of Ser65 and Thr70 sites [21–24] Interestingly, in AD brain, the significant increase of 4E-BP1 phosphorylated at Ser65 and Thr70 but not at Thr37⁄ 46 sites coincided with the significant increase

of mTOR autophosphorylation The data suggested that regulation of 4E-BP1 phosphorylation at Ser65 and Thr70 sites might most likely be mediated by mTOR autophosphorylation-dependent pathway Fur-ther studies need to be carried out to understand the cause and effect of the decreased total 4E-BP1 in AD

as compared with control The 4E-BP1 binds to eIF4E, and sequesters eIF4E function Phosphorylation of a critical set of specific serine and threonine residues of 4E–BP1 abrogates this interaction, and releases eIF4E, the phosphorylated state of which promotes cap-dependent translation An elevated eIF4E phosphory-lation has been recently found in AD brain [10] The up-regulation of PI3K, mitogen activated protein kinase (MAPK), and rapamycin-dependent pathways [6,7,14–18,25] in PHF-tau associated pathologies

suggested a systematic disorder of protein translation

regulation occurs in AD brains

One of the elongation controlling proteins is eEF2,

whose mRNA contains a 5¢-terminal oligopyrimidine

tract (5¢TOP) Both the synthesis and activity of eEF2

are regulated by rapamycin-sensitive pathway

Activa-tion of Ca2+⁄ calmodulin-dependent eEF2K (CamKIII)

could phosphorylate eEF2 in its N-terminus (Thr56)

that subsequently blocks eEF2 binding to ribosomes,

resulting in decreased rates of protein synthesis

Dephosphorylation of eEF2 relieves its translational

restriction and accelerates the translation rate of

rapa-mycin-sensitive proteins In the current study, level of

eEF2K phosphorylated at the Ser366 site was

dramatic-ally increased in AD brain that coincided with the

phos-phorylation of p70S6K [6] This suggested that p70S6K

could phosphorylate eEF2K at the Ser366 site, leading

to the inactivation of eEF2K in AD brain, which may

subsequently facilitate the dephosphorylation of eEF2,

and thus may promote translation ([26], Fig 5)

Recently, we have found that tau synthesis is

rapa-mycin-sensitive in rat primary hippocampal neuronal

cultures and SH-SY5Y neuroblastoma cells [6,25]

Strikingly, alignments of the 5¢ untranslated region

(5¢UTR) structures of tau mRNA with the known 5¢-TOP mRNAs that generally encode ribosomal pro-teins, revealed a similar 5¢TOP motif (not published) Previously, we found a significant correlation between levels of p-p70S6K (Thr421⁄ Ser424) and p-eIF4E, and total tau in AD and control brains [6,10] In the cur-rent study, a significant correlation was seen between phosphorylation of 4E-BP1 at both Ser65 and Thr70 sites, and mTOR at both Ser2481 and Ser2448 sites with total tau level As tau mRNA level did not change in AD brain [8,9], the continuous production

of tau in neurons during the progression of neuronal degeneration [6,25] might be regulated by mTOR-dependent pathway via p70S6K, 4E-BP1, and eIF4E in

AD brain

Changes of immunoreactivities of mTOR, p-mTOR, 4E-BP1, p-4E-BP1, eEF2 and p-eEF2 were studied by dot blot Significant correlations observed between lev-els of p-mTOR, p-4E-BP1, and p-eEF2, and total and phosphorylated taus suggested that these changes are localized in neurons due to that tau is primarily a neuronal proteins Data from immunohistochemistry using phosphospecific antibodies to p-mTOR and p-4E-BP1 proved that the immunoreactivities of phos-phorylated forms of mTOR and 4E-BP1 are increased

in neurons, while levels of p-eEF2 did not show any significant correlation with tau by dot blot, a relatively more intensive immunostainings of antibody to p-eEF2 were observed in neurons of AD brains as compared to controls

Rapamycin has been recently shown to enhance clearance of cytosolic aggregate-prone proteins with either polyglutamine or polyalanine expansions in Huntington disease, Huntington related diseases, and various forms of a-synuclein associated with Parkinson disease and synucleinopathies [27] Taken together with the findings that mTOR phosphorylated at the Ser2481 site has a significant positive correlation with total tau,

it provided clues for our hypothesis that mTOR abnor-mality may play an important role in the pathogenesis

of NFTs in AD (Fig 5)

In general, our data drew an outline of aberrant changes of protein translation control system in AD brains The abnormality of translation control may be relatively specifically involved in the increased level of tau in AD brains, contributing to hyperphosphorylated tau accumulation in NFT-bearing neurons Further studies need to be carried out to see whether or not other funtional proteins are dis-regulated by mTOR pathway in AD, and whether or not the positive corre-lations between aberrant changes of protein translation control system and tau are also seen in other neuro-degenerative tauopathies

Fig 5 A hypothetical scheme showing aberrant regulation of tau

translation by mTOR via 4E-BP1 and p70S6K-dependent pathways

in AD brain mTOR is phosphorylated at serine 2448 via the PI3

kin-ase ⁄ Akt signalling pathway and autophosphorylated at serine 2481.

In AD brain, high levels of phosphorylated mTOR phosphorylate

p70S6K and transmit a positive signal to the p70S6K pathway, and

phosphorylate and disassociate the eIF4E inhibitor 4E-BP1 from

eIF4E, resulting activation of the eIF4E On the other hand, p70S6K

can phosphorylate eEF2K, leading to the inactivation of eEF2K,

which facilitates the dephosporylation of eEF2, and thus promotes

protein translation All above events may contribute to up-regulation

of the translation of specific mRNA subpopulations such as tau

protein.

Experimental procedures

Materials

Affinity-purified rabbit polyclonal antibodies against total

and p-forms of 4E-BP1, mTOR, eEF2, and eEF2K were

purchased from the Cell Signaling Technology (Beverly,

MA) Mouse mAb AT8 was from Innogenetics (anti-human

PHF-tau; Zwujndrecht, Belgium), and mAb Tau2 from

Sigma-Aldrich (St Louis, MO, USA) mAb PHF-1 was a

gift from Dr Peter Davies (Albert Einstein College of

Medi-cine, Bronx, NY, USA), and mAb Tau1 from L Binder

(North-Western University, Chicago, IL, USA) Rabbit

antiserum R134d to the longest isoform of recombinant

human tau were gifted from K Iqbal (New York State

Institute for Basic Research in Developmental Disabilities,

Staten Island, NY, USA)

Homogenate preparation and protein

measurement

Tissue blocks of the medial temporal cortex of 20 AD and

10 control brains were from Kuopio Brain bank (Table 1)

All cases were pathologically staged according to Braak

and Braak criteria in Kuopio Brain Bank [11] All

demen-ted subjects fulfilled the AD neuropathological criteria

according to CERAD [12] Grey matter was separated from

white matter and homogenized in 50 mm Tris⁄ HCl buffer,

pH 7.0, containing 2.5 mm EDTA, 2.5 mm EGTA, 2 mm

benzamidine, 0.5 mm PMSF, 1% 2-mercaptoethanol,

20 mm b-glycerophosphate, 2 mm sodium vanadate, 50 mm

NaF, 2.5% SDS and 0.1% (v⁄ v) protease-inhibitor cocktail

(Sigma-Aldrich, Stockholm, Sweden) at 4
C Protein

con-centration was determined by the BCA assay kit

(Sigma-Aldrich)

Dot and western blots and indirect ELISA

Following the procedures described previously [10],

homo-genates from AD and control cases were spotted on the

squares in triplicates (3 lg protein per square), which were

then incubated with primary antibodies overnight at 4
C,

followed by secondary antibodies linked with horseradish

peroxidase (Amersham Biosciences AB, Uppsala, Sweden)

at room temperature for 1 h The primary antibodies used

in dot blots included antibodies against total and p-forms

of mTOR, 4E-BP1, eEF2 and eEF2K Immunoreactive

proteins were detected according to the enhanced

chemi-luminescence protocol (Amersham Biosciences AB)

Intensi-ties of spots were quantified with The Discovery Series

Quantity One 1-D Analysis Software (Bio-Rad

Laborator-ies, Inc.) To check the specificities of antibodLaborator-ies, AD

homo-genates were separated in 6% SDS⁄ polyacrylamide gel for

antibodies to total and p-forms of mTOR, eEF2K and

eEF2, and in 15% SDS⁄ polyacrylamide gel for total and

p-4E-BP1 After transferring, the procedures were followed the same as the dot blot Levels of total tau labeled by R134d and Tau2, PHF-tau labeled by AT8 and PHF-1, and unphosphorylated tau labeled by Tau1 were previously measured by indirect ELISA in the same set of tissues as dot blots [6]

Immunohistochemistry Immunohistochemical staining was performed on 20-lM formalin-fixed frozen sections (two AD cases, and two control cases) or 6 lm formalin-fixed paraffin-embedded sections of the medial temporal cortex from pathologic-ally verified AD (three cases) and control (two) cases (Braak and Braak neurofibrillary stages 5–6) (Kupio Brain Bank) The sections were incubated at 4
C for

48 h with rabbit antibodies to p-mTOR (Ser2448), p-mTOR (Ser2481) at 1 : 12.5 dilution, p-4E-BP-1 (Thr70) at 1 : 50, p-eEF2 (Thr56) at 1 : 25, and mAb PHF-1 at 1 : 100 dilution after antigen retrieval at 80
C

9 min in citrate-buffered saline This was followed by incubation with biotinylated anti-mouse IgM or anti-rab-bit IgG at 1 : 300 dilution for 2 h, and by visualization with the avidin-biotin-peroxidase complex kit (Vector, Burlingame, CA, USA) with 3–3¢-diaminobenzidine-4 HCl⁄ H2O2 (DAB; Sigma) as a substrate The images were taken by LEICA DC480 microscope using the LE-ICA IM50 image manager software (Leica Microsystems

AG, Heerbrugg, Germany)

Statistical analysis The average age and postmortem delay between AD and control cases were compared by nonpaired Student t-test Levels of total and p-forms of mTOR, 4E-BP1, eEF2, and eEF2K in homogenates between AD and control brains were also compared by nonpaired Student t-test, and in different stages of neurofibrillary degeneration according to Braak and Braak criteria by one-way anova Relationships between levels of various translation control elements, and levels of tau were analyzed using Bivariate Pearson correlations The significance level was set at P < 0.05

Acknowledgements

We thank Dr Lars Tjernberg for his helpful comments

on the manuscript This study was supported by Alzheimerfonden, Gamla Tja¨narinnor Foundation, Gun och Bertil Stohnes Stiftelse, Loo and Hans Oster-mans Foundation, SADF (Insamlingsstiftelsen fo¨r Alzheimer- och Demensforskning), Socialstyrelsens Stiftelser, Stiftelsen fo¨r A˚lderssjukdomar, and Svenska La¨karesa¨llskapet

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