, ,1 , ,1

,2

*NeuroAdaptations Group, Max Planck Institute of Psychiatry, Munich, Germany

�Department of Physiology, University College London, London, UK

�Division of Pathology and Neuroscience, University of Dundee and Parkinson’s Disease Society, London, UK

Mis-processing of amyloid precursor protein (APP) andaberrant phosphorylation of the microtubule-associated pro-tein tau are key pathogenic mechanisms in Alzheimer’sdisease (AD). The metabolism of APP and phosphorylationof tau, both highly regulated processes, are intrinsicallylinked to neuronal function and survival. While full-lengthAPP is implicated in normal cell physiology (Reinhard et al.2005), its sequential proteolysis by a, b and c-secretasesresults in the generation of either neuroprotective orneurotoxic peptides. Cleavage by a-secretase yields non-amyloidogenic secreted aAPP (saAPP) and an 83-residueC-terminal fragment (C83); alternative cleavage by b-secre-tase (BACE) generates secreted bAPP (sbAPP) and a 99-residue product (C99); the latter can be further catabolized byc-secretase in the so-called ‘amyloidogenic pathway’ togenerate amyloid b (Ab). While aggregates of Ab give rise tothe characteristic senile plaques found in AD-affected brains,both C99 and soluble Ab have intrinsic neurotoxic properties(Roselli et al. 2005; Yankner et al., 1989).

Substantial evidence suggests that dysregulation of tauphosphorylation is an early event in the cascade of patho-logical events leading to AD (Spillantini and Goedert 1998)

Received December 14, 2007; revised manuscript received April 29,2008; accepted July 25, 2008.Address correspondence and reprint requests to O. F. X. Almeida,

Max Planck Institute of Psychiatry, 80804 Munich, Germany.E-mail: osa@mpipsykl.mpg.de1These authors contributed equally to this study.2The present address of T. M. Michaelidis is the Laboratory of MolecularGenetics, Department of Biological Applications & Technologies,University of Ioannina, and Biomedical Research Institute, Foundation forResearch & Technology-Hellas (FORTH-BRI), Ioannina 45110, Greece.Abbreviations used: AD, Alzheimer’s disease; APP, amyloid precursor

protein; Ab, amyloid beta; C99, 99-residue C-terminal fragment of APP;cdk5, cyclin-dependent kinase 5; DIV, days in vitro; EGFP, enhancedgreen fluorescent protein; GC, glucocorticoid(s); GR, glucocorticoidreceptor; GSK3, glycogen synthase kinase 3; htau, human tau; MTT,3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; Nct,nicastrin; PBS, phosphate-buffered saline; PS1, presenilin 1.

Abstract

Amyloid precursor protein (APP) mis-processing and aberrant

tau hyperphosphorylation are causally related to the patho-

genesis and neurodegenerative processes that characterize

Alzheimer’s disease (AD). Abnormal APP metabolism leads to

the generation of neurotoxic amyloid beta (Ab), whereas tau

hyperphosphorylation culminates in cytoskeletal disturbances,

neuronal dysfunction and death. Many AD patients hyper-

secrete glucocorticoids (GC) while neuronal structure, func-

tion and survival are adversely influenced by elevated GC

levels. We report here that a rat neuronal cell line (PC12)

engineered to express the human ortholog of the tau protein

(PC12-htau) becomes more vulnerable to the toxic effects of

either Ab or GC treatment. Importantly, APP metabolism in

GC-treated PC12-htau cells is selectively shifted towards

increased production of the pro-amyloidogenic peptide C99.

Further, GC treatment results in hyperphosphorylation of

human tau at AD-relevant sites, through the cyclin-dependent

kinase 5 (E.C. 2.7.11.26) and GSK3 (E.C. 2.7.11.22) protein

kinases. Pulse-chase experiments revealed that GC treatment

increased the stability of tau protein rather than its de novo

synthesis. GC treatment also induced accumulation of tran-

siently expressed EGFP-tau in the neuronal perikarya.

Together with previous evidence showing that Ab can activate

cyclin-dependent kinase 5 and GSK3, these results uncover a

potential mechanism through which GC may contribute to AD

neuropathology.

Keywords: Alzheimer’s disease, amyloid precursor protein,

glucocorticoids, PC12 cells, rat neurons, tau.

J. Neurochem. (2008) 107, 385–397.

JOURNAL OF NEUROCHEMISTRY | 2008 | 107 | 385–397 doi: 10.1111/j.1471-4159.2008.05613.x

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397 385

by interfering with cellular homeostasis (Bennett et al. 2005)and destabilization of the cytoskeleton (Alonso et al. 1996).The discovery of mutations in the tau gene that result in thenucleation and aggregation of normal tau in a phosphoryla-tion-dependent manner has provided important clues forunderstanding the role of certain modifications of tau intriggering neuropathology (Chun and Johnson 2007; Wanget al. 2007b). Importantly, among the kinases implicated inAD-like tau hyperphosphorylation, the most significant areglycogen synthase kinase 3 (GSK3; E.C. 2.7.11.22) andcyclin-dependent kinase 5 (cdk5; E.C. 2.7.11.26), which arealso involved in regulating neuronal plasticity (Patrick et al.1999; Jope and Johnson 2004). Interestingly, APP metabo-lism and tau hyperphosphorylation are linked throughintricate regulatory loops. For example, Ab stimulates tauhyperphosphorylation by enhancing GSK3 and cdk5 activ-ities, while these kinases influence APP metabolism (Murrayet al. 1998; Iijima et al. 2000; Phiel et al. 2003). In addition,Ab acts to increase its own generation by influencing APPexpression and metabolism (Davis-Salinas et al. 1995; Yanget al. 1999; Lorenzo et al. 2000; Catania et al. 2007).

Clinical studies have suggested a link between glucocor-ticoids (GC), the primary stress hormones, and the pathogen-esis and/or progression of AD. Hypersecretion of GC isobserved in many AD patients (Hartmann et al. 1997; Weineret al. 1997; Elgh et al. 2006), representing a sensitive markerof cognitive decline in these patients (Weiner et al. 1997).Interestingly, it was recently shown that stress andGCpromoteamyloid deposition and tau accumulation in mouse transgenicmodels of AD (Green et al. 2006; Jeong et al. 2006) andchronic stress was found to trigger the misprocessing of APP(Catania et al. 2007). Other studies have shown that GC renderneurons more vulnerable to a variety of neurotoxic agents,including glutamate and kainate (Sapolsky 2000; Lu et al.2003) and, importantly, Ab (Behl et al. 1997); moreover, GCthemselves can trigger neuronal atrophy and apoptosis(Crochemore et al. 2005; Cerqueira et al. 2007).

Laboratory rodents do not show signs of APP misprocess-ing or tau pathology under normal conditions, althoughneuropathological and behavioral abnormalities can beinduced by transgenesis with human wild-type or mutatedtau and/or APPmolecules (McGowan et al. 2006). In order tofurther analyze the link between GC and AD pathology, thepresent study examined the influence of GC treatment on APPmetabolism and tau hyperphosphorylation in a differentiatedrat neuronal cell line (PC12) that was engineered to stablyexpress human tau (Fath et al. 2002). Our results show that thepresence of human tau in a heterologous neuronal environmentis sufficient to alter the response of the cell to glucocorticoids.More specifically, under these conditions, GC treatmenttriggered significant APP misprocessing and AD-like hyper-phosphorylation accompanied by reduced degradation andaccumulation of tau; in addition, the PC12-htau cells displayedgreater neuronal vulnerability to Ab- and GC-induced death.

Experimental procedures

Cell linesPC12 cells that had been stably transfected with either a human tau

(htau; 0N3R isoform) transgene (PC12-htau) or an empty vector

(courtesy of Dr. R. Brandt; Fath et al. 2002), were grown in

Dulbecco’s modified Eagle’s medium (Invitrogen, Eggenstein,

Germany), supplemented with 0.75% fetal bovine serum and

0.75% horse serum. PC12-htau were additionally supplemented

with geneticin (250 lg/mL; Invitrogen). Cells were differentiated

for 6–7 days with 7S nerve growth factor (NGF; Invitrogen)

showing the morphological characteristics of neuronal cells. For

control purposes, wild-type PC12 cells were used and compared

with PC12 expressing the empty vector; no differences between the

two cell lines were found.

DrugsTreatments were initiated after 6–7 days in vitro (DIV). Dexameth-

asone (DEX; Fortecortin�,Merck, Darmstadt, Germany) was applied

(10)8 and 10)6 M) for 24–48 h. The glucocorticoid receptor (GR)

antagonist mifepristone (RU 38486 provided by the NHPP, NIDDK;

Torrance, CA, USA)was used at a concentration of 10)5 M. Synthetic

humanAb25-35 (American Peptide Company, Sunnyvale, CA, USA)

was aggregated as previously described (Behl et al. 1997) and used at0.1 or 1 lM. Enzyme inhibitors were used as follows: PNU 112455A

(N4-(6-Aminopyrimidin-4-yl)-sulfanilamide, HCl from Calbiochem,

San Diego, CA, USA) at 1–100 nM (Kicdk5 = 2 lM); and 3-(3-

Carboxy-4-chloroanilino)-4-(3-nitrophenyl) maleimide (Calbio-

chem) at 1–30 lM (IC50GSK3a/b = 26 nM; IC50GSK3b = 5 lM).

RNA extraction and RT-PCRRelative mRNA levels of human and rat tau, APP and of the house-

keeping gene cyclophilin were measured using RT-PCR. Total RNA

was isolated from cells (RNAeasy� Mini Kit, Qiagen, Hilden,

Germany), heat-denatured, and reverse transcribed with the Omni-

script� Reverse Transcriptase Kit (Qiagen) for first-strand cDNA

synthesis. Gene-specific primer pairs (MWG Biotech, Ebersberg,

Germany) were designed on the basis of published rat and human

cDNA sequences: htau: 5¢-CGA AGT GAT GGA AGATCA CG-3

(sense), 5¢-CAT TCT TCA GGT CTG GCA TG-3 (antisense); rattau: 5¢-GGT GAG GGA TGG GGG TGG TA-3 (sense); 5¢-GTGACT GGT TCT CGT GGT A-3 (antisense); APP: 5¢-GAG CGG

ACA CAG ACTATG CTG A-3¢ (sense); 5¢-GAC ATT CTC TCT

CGG TGC TTG-3¢ (antisense); Cyclophilin: 5¢-CTC CTT TGA

GCT GTT TGC AG-3¢ (sense); 5¢-CAC CAC ATG CTT GCC ATC

C-3¢ (antisense). The Taq polymerase was from Sigma, St Louis,

MO, USA. PCR amplification was performed over 28 (human and

rat tau), 30 (APP) or 22 (cyclophilin) cycles, under the following

conditions: denaturation 40 s at 94�C; annealing 1 min at 58�C(human tau), 60�C (rat tau), 56�C (APP) or 56�C (cyclophilin);

primer extension 2 min at 72�C; the linear range of each assay was

determined in pilot optimization experiments. PCR products were

electrophoresed on 1.5% agarose gels and visualized under UV light

after staining with ethidium bromide. The amplified products had

sizes of 565 bp (human tau), 278 bp (rat tau), 562 bp (APP770),

505 bp (APP751), 337 bp (APP695) and 325 bp (cyclophilin).

Control amplifications (omission of either reverse transcriptase or

cDNA template) were run in each assay.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397� 2008 The Authors

386 | I. Sotiropoulos et al.

Transient transfection with htauHuman tau cDNA (0N3R), framed in a pEGFP-C1 host vector

(Clontech, Mountain View, CA, USA), provided by Dr. R. Brandt

(Fath et al. 2002), was used to transiently transfect differentiated

PC12 cells (6 DIV) using Lipofectamine 2000� (Invitrogen).

Transfection efficiency was 3–7% and cells were used 12 h later.

Cells transfected with an empty vector did not differ from those

transfected with htau, as judged by morphology and survival rates.

Western blotting and immunoprecipitationUnless specifically stated, all reagents were purchased from Sigma.

Cells were washed and lysed (100 mM Tris-HCl, 10% glycerol,

1 mM EDTA, 5 mM MgCl2, 250 mM NaCl, 1% NP40, protease

inhibitors and phosphatase inhibitors), sonicated on ice and

centrifuged (14 000 g, 15 min, 4�C). Western blotting and semi-

quantitative analyses were performed as previously described

(Catania et al. 2007). Table S1 (Supporting Information) summa-

rizes the characteristics of the antibodies used to detect proteins and

peptides of interest. Blots were also probed with anti-b-actin(Chemicon, Temecula, CA, USA; 1 : 2000) or anti-tubulin (Sigma;

1 : 2000). When alkaline phosphatase pre-treatment was used in

control experiments, lysates were treated with 15 U of calf intestine

alkaline phosphatase (Roche, Mannheim, Germany) at 37�C for

24 h. For analysis of secreted APP (sAPP), cell culture supernatants

were concentrated by centrifugation (3000 g, 30 min), with Viva-

spin concentrators (Vivascience, Goettingen, Germany) and ana-

lyzed by western blotting, using the G530 antibody that specifically

recognizes the secreted form of sAPPa (Sawamura et al. 2004). Forimmunoprecipitation, cell lysates (750 lg protein) were incubated

with an APP369 antibody (kindly provided by Dr. Sam Gandy,

Philadelphia, PA, USA; Table S1 in Supporting Information) in a

mixture of Protein A- and G-agarose beads (Roche). Immuno-

precipitated proteins were electrophoresed on Tris-Tricine gradient

gels (KMF Laborchemie, Lohmar, Germany), transferred onto

nitrocellulose membranes and incubated with the APP antibodies

OPA1-01132 (Affinity Bioreagents, Golden, CO, USA) or APP369.

Assay of tau protein stabilityDifferentiated PC12 cells were cultured (24 h) in methionine/

cysteine-free medium, supplemented with [35S]-methionine/cyste-

ine (185 lCi/mL; Amersham) (Merrick et al., 1996). Cells were

then washed, exposed to DEX in radioactive-free medium, and lysed

(see above) at various times thereafter. Tau proteins were immuno-

precipitated with a mixture of A- and G-agarose beads (Roche)

using Tau-5 antibody (Table S1 in Supporting Information).

Immunoprecipitates were electrophoresed on sodium dodecyl

sulfate-gels, stained with Coomassie blue, and dried on filter paper

under vacuum. Radioactivity in the dried gels was evaluated

subsequently (Fuji Bio-image 3000 and AIC 7770 analysis software;

Adaptec, Milpitas, CA, USA).

Confocal microscopyCells were cultured on gelatine/poly-D-lysine-coated glass cover-

slips. After washing with phosphate-buffered saline (PBS), they

were fixed in 4% paraformaldehyde/PBS/4% sucrose, incubated in

0.1 mM glycine/PBS, permeabilized in 0.2% TritonX-100/PBS, and

incubated overnight with primary antibodies (Table S1 in Support-

ing Information) and appropriate secondary antibody, followed by

fluorescence-conjugated avidin-streptavidin (RT, 30 min). Cells

were examined by scanning laser confocal microscopy (Zeiss

LSM 510, Carl Zeiss Microimaging, Goettingen, Germany) after

coverslipping under DABCO anti-fade mounting medium (Sigma).

Cell survival assayCell viability was assessed using the 3-(4,5-dimethylthiazole-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay after 6

DIV (cells grown and differentiated at an initial density of 700 cells

per well in 96-well dishes). Briefly, cells were treated with MTT

(Sigma; 455 lg/mL in 0.9% NaCl) and incubated for 4 h. The

formazon product resulting from MTT cleavage was solubilized by

the addition of 0.04 N HCl (in isopropanol) and incubation until

disappearance of formazon crystals; optical density (O.D.) mea-

surements were made using a Dynatech (MR 630) plate reader

(570 nm; reference wavelength = 630 nm). Relative viability (%)

was determined by comparison against O.D. values obtained for

control (no drug) cells. Data shown are derived from six individual

wells in each of three repeats of the experiment.

Statistical analysisNumerical data are expressed as group means ± SEM. All data sets

were subjected to 1-way ANOVA, before application of appropriate

post hoc pair-wise comparisons (SigmaStat, Systat, San Jose, CA,

USA). Differences were considered to be significant if p < 0.05.

Results

Human tau renders PC12 cells more vulnerable toglucocorticoids and amyloid b (Ab) peptideTau plays an undisputed role in human neurodegenerativeprocesses, including AD (Rapoport et al. 2002; Robersonet al. 2007), but the paucity of data implicating it inneurodegeneration in animals prompted us to examinewhether the human form of tau specifically confers vulner-ability to GC. To this end, the current experiments wereconducted on a differentiated rat pheochromocytoma-derivedneuronal (PC12) cell line that had been stably transfectedwith human tau (PC12-htau; Fath et al. 2002). Survival ratesof PC12 and PC12-htau cells did not differ under basalconditions. However, as compared to wild type PC12 cells,PC12-htau cells showed a higher vulnerability to the death-inducing effects of a synthetic GC (dexamethasone; 10)6 M)(p < 0.05; Fig. 1a). In line with our previous findings, lowerdoses of this GC (10)8–10)7 M) did not influence cellviability (Crochemore et al. 2005) The toxic effects werefound to be mediated by GC receptors (GR) since, in allcases, the GR antagonist, mifepristone (RU 38486; 10)5M)blocked the actions of the GC (Fig. 1b).

In view of our hypothesis that htau protein may becritically involved in GC-induced toxicity, and given that tauprotein seems essential for the manifestation of the toxicactions of stimuli such as kainate and Ab (Rapoport et al.2002; Roberson et al. 2007), we sought to compare the

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397

Glucocorticoids, human tau and Alzheimer’s disease | 387

neurotoxic effects of Ab in PC12 and PC12-htau cells. Asshown in Fig. 2a, exposure to 1 lM of Ab25-35 peptideresulted in significantly reduced viability of PC12-htau cellsversus PC12 cells (p < 0.05). Furthermore, as previousstudies showed that GC can potentiate the neurotoxic actionsof Ab (Behl et al. 1997) and amplify AD-like neuropatho-logy in transgenic mouse models of AD (Green et al. 2006;Jeong et al. 2006), it was of interest to analyze whetherPC12-htau cells were more sensitive to the combinedtreatment of GC and Ab, as compared to normal PC12 cells.Consistent with the results depicted in Fig. 1, GC treatmentalone compromised cell survival (p < 0.05) to a greaterextent in PC12-htau (Fig. 2b, cf. bars 5 and 7) than in PC12

cells (Fig. 2b, cf. bars 1 and 3). Importantly, PC12-htau cellswere also more sensitive to a low dose of Ab (0.1 lM)(Fig. 2b, cf. bars 5 and 6), compared to PC12 cells (Fig. 2b,cf. bars 1 and 2), and the GC treatment further potentiated theneurotoxic actions of Ab in PC12-htau cells (p < 0.05;Fig. 2b, cf. bars 6 and 8), but not in PC12 cells (Fig. 2b, cf.bars 2 and 4).

Glucocorticoids induce tau hyperphosphorylation in acdk5- and GSK3-dependent mannerGlucocorticoids are implicated in the initiation and/ormaintenance of AD neuropathology (Hartmann et al. 1997;Weiner et al. 1997; Elgh et al. 2006; Green et al. 2006;Jeong et al. 2006). Since aberrant tau hyperphosphoryla-tion is considered a key pathogenic factor in ADneuropathology (Fath et al. 2002; Geschwind 2003;Alonso et al. 2006), we examined the influence of GC

120

% C

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PC12-htauPC12

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*

NS

– + + – –

– – + + –

– – + + +

(a)

(b)

Fig. 1 Enhanced sensitivity of PC12-htau cells to glucocorticoids

(GC). Data representing means ± SEM (n = 6 individual measure-

ments) show that the viability (measured by MTT assay) of PC12-htau

cells is further reduced as compared to normal PC12 cells after

exposure to the synthetic glucocorticoid dexamethasone (a) (10)6 M;

48 h; *p £ 0.05) and that the GC actions are GR-mediated (b).

0

20

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PC12 cells

*

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0.1 µM 1 µM CON

*

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(b)

Fig. 2 GC treatment potentiates the neurotoxic actions of Ab in PC12-

htau cells. Cell viability was assessed by the MTT assay after Ab (a) or

co-administration of dexamethasone (10)6 M, 48 h) with Ab (0.1 lM)

to both PC12 and PC12-htau cells (b). All results represent

mean ± SEM (n = 8; *p £ 0.05 between indicated treatment pairs).

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397� 2008 The Authors

388 | I. Sotiropoulos et al.

treatment on the pattern of tau phosphorylation. Use of apanel of phosphorylation-dependent tau antibodies (seeTable S1 in Supporting Information) in western blotanalyses revealed that exposure of PC12-htau, but not ofnormal PC12, cells to GC (48 h) leads to marked increasesin the hyperphosphorylation of tau at specific epitopes(recognized by AT8, CP9 and PHF1) implicated in ADneuropathology (p < 0.01; Fig. 3a). As expected, theendogenous ‘big tau’ (the largest tau isoform) was detectedin both PC12 and PC12-htau cells; however, the phos-phorylation levels of this isoform were not altered by GCtreatment. Confocal microscopy on cells stained with thephosphorylation-dependent antibodies AT8 and PHF-1(Fig. 3b) confirmed the results obtained by westernblotting. Strikingly, increased levels of immunoreactivitywere also observed in GC-treated PC12-htau cells afterstaining with the conformation-dependent antisera, MC1and Alz50 (Fig. 3b) which recognize aberrant forms of tauin AD brains. The above-described effects were shown tobe mediated by glucocorticoid receptors (GR); thus, GC-induced tau hyperphosphorylation was blocked when cellswere co-treated with the GR antagonist mifepristone (RU38486) (Fig. 3c).

Tau is known to be a substrate for several proteinkinases (Shahani and Brandt 2002), the most prominent inthe context of AD being cdk5 and GSK3b. Thus, weexamined both total and active levels of these kinases inGC-treated PC12-htau cells. As shown in Fig. 4(a), GCapplication resulted in significant (p < 0.05) increases inthe levels of the activated forms of the protein kinasescdk5 (phosphoTyr15-cdk5) and GSK3a/b (phospho-Tyr216/279-GSK3a/b); on the contrary, total levels ofcdk5 and GSK3a/b as well as of inactive phosphoSer9-GSK3b were unaffected by GC exposure.

In order to corroborate the involvement of these kinasesin GC actions, we treated PC12-htau cells with GC in thepresence of selective kinase inhibitors and subsequentlymonitored cell viability and the tau phosphorylationprofile. As shown in Fig. 4, cdk5 and GSK3 inhibitorsdose-dependently attenuated (p < 0.05) GC-inducedelevation of tau phosphorylation, as monitored by thePHF-1 and CP9 tau antibodies. Interestingly, however, theGC-induced cell death was attenuated by the cdk5inhibitor PNU 112455A, but not by the GSK3inhibitor [3-(3-carboxy-4-chloroanilino)-4-(3-nitrophenyl)maleimide].

(a)

(c)

(b)

GC

CON

AT8 PHF1 MC1 TubAlz50

AT8 PHF1 MC1 TubAlz50

PC12-htauPC12

PHF1AT8 CP9

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CP9AT8

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GCRU

0

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200

250

Fig. 3 Dexamethasone, acting through

glucocorticoid receptors (GR), induces tau

hyperphosphorylation at specific AD-rele-

vant epitopes in PC12-htau cells. Cells

were treated with dexamethasone (GC;

10)6 M; 48 h) and quantitative results from

immunoblots, normalized against total tau

levels (CON, set at 100%, dotted line) are

shown in (a) and (c). Results are shown as

mean ± SEM values (n = 6–8; *p £ 0.05

untreated vs. GC-treated cells) of relative

O.D. units; a representative western blot is

shown alongside the numerical results. The

effects of GC on tau phosphorylation in

PC12 and PC12-htau cells are shown in

Panel (a). Panel (b) illustrates examples of

untreated (CON) and GC-treated PC12-

htau cells examined by confocal micros-

copy (· 60) after immunostaining with

phosphorylation-dependent (AT8 and PHF-

1) and conformation-dependent (Alz50 and

MC1) antibodies, confirming the AT8 and

PHF1 results obtained by western blotting.

As shown in Panel (c), GC effects on tau

phosphorylation (AT8 and CP9 epitopes) in

PC12-htau cells are attenuated by the GR

antagonist mifepristone (RU; RU38486

10)5 M).

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397

Glucocorticoids, human tau and Alzheimer’s disease | 389

Glucocorticoids alter tau trafficking and stabilitySince GC activate transcription after binding to GR, it was ofinterest to examine whether the modulation of tau by GCoccurs at the transcriptional level. Exposure of PC12-htaucells to GC did not increase the steady-state levels of taumRNA despite a significant increase in total tau proteinlevels (Fig. 5a). Additionally, inhibition of de novo proteinsynthesis by cycloheximide did not interfere with the GC-induced increases in total tau protein levels (not shown),suggesting rather that GC influence the stability of tauprotein.

Given that the hyperphosphorylation of tau is coupled toits degradation (Litersky and Johnson 1992), we monitoredthe stability of tau protein directly in a pulse-chase exper-iment in which newly-synthesized proteins were labeled with[35S]-methionine/cysteine for 24 h prior to the treatment

with GC (10)6 M), for varying times. Immunoprecipitationand quantification of the residual [35S]-labeled tau proteinlevels (Fig. 5b) revealed that exposure to GC leads to asignificant decrease in the rate of loss of [35S]-labeled tauprotein. This finding is in accordance with the data shown inFig. 5a, indicating an increased stability of tau protein inGC-treated cells.

Previous studies have shown that disruption of the normalphosphorylation pattern of tau results in tau malfunction andredistribution of this protein into the somatodendritic com-partment of the neuron (Spillantini and Goedert 1998;Geschwind 2003). Consistently, we observed that GCtreatment leads to accumulation of htau in the somata ofPC12 cells transiently transfected with an EGFP-htau fusionprotein (Fig. 5c). Importantly, such changes were notobserved after GC treatment of cells that had been transfected

(b)

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CO N

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cdk5 p-cdk5

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GC CON

pSer9-GSK3β β p-G

SK3α α p-G

SK3β β

CON

0

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400 (a)

p-cdk5

*

* *

pSer9-

GSK3β β

Fig. 4 Protein kinases involved in GC-in-

duced hyperphosphorylation of tau. Quan-

titative evaluations of western blots probed

with anti-phospho-cdk5 and anti-phospho-

GSK3 are shown (a) alongside represen-

tative western blots. Samples were

obtained from PC12-htau cells under

untreated control conditions (CON, shown

as a dotted line at 100%) and after expo-

sure to GC (dexamethasone, 10)6 M, 48 h).

Results were first normalized with respect

to total levels of the corresponding kinase.

GC treatment did not alter levels of another

tau kinase, CaMKII (not shown). Both the

cdk5 inhibitor and the GSK3b inhibitor

attenuate GC-induced tau hyperphosph-

orylation (b and c), as detected by immu-

noblotting with PHF-1 and CP9 antibodies.

GC-induced cell death is blocked by a cdk5

inhibitor, but not by an inhibitor of GSK3b (d

and e). All numerical results represent

mean ± SEM (n = 6–8; *p < 0.05 untreated

vs. GC-treated cells).

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397� 2008 The Authors

390 | I. Sotiropoulos et al.

with EGFP only, indicating that the effects observed in thisstudy are tau-specific and not a general response to the over-expression of a foreign protein.

Glucocorticoids influence the production and processing ofAPPSince both Ab (Busciglio et al. 1995) and GC (presentstudy) can trigger tau hyperphosphorylation, and GC caninduce tau pathology by stimulating the production of Ab(Green et al. 2006; Jeong et al. 2006), we investigatedwhether GC influence the synthesis and/or processing ofAPP in the presence of htau. Application of GC to PC12 cellscaused a significant increase in APP levels (p < 0.05;Fig. 6a). In contrast, the same treatment resulted in signif-icantly reduced levels of APP in PC12-htau cells (p < 0.05;Fig. 6a). These changes in APP protein levels were accom-panied by corresponding alterations in the levels of themRNAs encoding for the APP770, APP751 and APP695isoforms (visualized as three major bands of 562 bp, 505 bpand 337 bp, respectively) in GC-treated PC12 and PC12-htaucells (Fig. 6b).

Next, we examined the effects of GC on APP processing,initially focusing on the amyloidogenic pathway byanalyzing the levels of the C99 fragment as well as ofBACE, and two major components of the c-secretasecomplex, the proteins presenilin 1 (PS1) and nicastrin (Nct)(Fig. 7a). Exposure of PC12-htau cells to GC resulted in aselective and highly significant increase of C99 levels inPC12-htau, but not in PC12 cells (Fig. 7b; p < 0.001).Consistently, we observed concomitant selective elevationsof BACE and Nct as well as of total PS1 protein levels inthe PC12-htau cells only (p < 0.05; Fig. 7b). Notably, thestrong induction of the PS1 holoprotein by GC treatmentwas not accompanied by a similar elevation of the CTFfragment, possibly reflecting the tight regulation of theendoproteolytic fragments of PS1 at a fairly constant level(cf. Thinakaran et al. 1996). In addition, GC treatment didnot significantly influence 83-residue C-terminal fragmentand sAPPa levels, in either cell line (Fig. 7b). The abovedata collectively suggest that the GC effects on APPprocessing in PC12-htau cells can occur via the amyloido-genic pathway.

(b)

[35S

]-m

et-T

au0

20

40

60

80

100

120

12 h 24 h 48 h0

*

GC

CON*

(c)

(a) Tau protein (WB) Tau mRNA (RT-PCR)

Tau5

Actin

Actin

CON GC*

100

200

Rel

ativ

e O

.D.

0CON GC

Tau5

PC

12-

hta

uP

C12

CON GC

htau (565 bp)

Cycl (325 bp)

rtau (278 bp)

CON GC 0

100

200

Rel

ativ

e O

.D.

CON GC

Fig. 5 Reduced turnover and accumulation

of tau in PC12-htau cells after exposure to

GC. Western blotting and RT-PCR analy-

ses of GC-treated (dexamethasone,

10)6 M, 48 h) PC12-htau cells show (a) GC

treatment leading to elevated levels of tau

protein that are not matched by concomitant

increases in the steady-state levels of tau

mRNA. Values from densitometry of wes-

tern blot and RT-PCR films are shown as

mean ± SEM (n = 6–8) (CON cells set at

100%). The same GC treatment results in a

retardation of the rate of tau degradation in

PC12-htau cells, as assessed by immuno-

precipitation of labeled tau protein in a

pulse-chase study with [35S]-methionine

(mean ± SEM, n = 6). (b) Pairs of immuno-

fluorescence and phase-contrast images

(· 40) of PC12 cells transiently transfected

with htau-EGFP under basal conditions

(CON) and after exposure to GC (10)6 M,

48 h) are also shown (c) to illustrate the

accumulation of EGFP-htau in the perikarya

of GC-treated cells. *p £ 0.05 untreated vs.

GC-treated cells.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397

Glucocorticoids, human tau and Alzheimer’s disease | 391

Discussion

The mis-processing of APP into a 99-amino acid C-terminalfragment (C99) and Ab, together with the abnormalhyperphosphorylation of the cytoskeletal protein tau, areconsidered to be the core pathogenic mechanisms underly-ing AD neurodegeneration. While the current consensus isthat APP mis-processing precedes tau hyperphosphoryla-tion, evidence from animal studies indicates that eitherprocess can independently contribute to the neurodegener-ation and behavioral disruption that characterize the disease(Gotz et al. 2004; Hardy 2006). Although laboratoryrodents produce both APP and tau, they do not normallydevelop AD neuropathology unless engineered to expresshuman forms of these proteins (McGowan et al. 2006).Recent studies in such transgenic models have described theexacerbating role of stress or exogenous GC on AD-likepathology (Green et al. 2006; Jeong et al. 2006). These

observations are consistent with the results of clinicalstudies that suggest that GC hypersecretion may have acausal or maintenance role in AD (Hartmann et al. 1997;Wilson et al. 2005; Elgh et al. 2006). As an initial attemptto address the link between abnormal tau hyperphosphory-lation and APP misprocessing, we here employed adifferentiated rat neuronal cell line that was stably trans-fected with human tau (PC12-htau); the suitability of thismodel for studying tau-mediated AD-like neuropathologyhas been demonstrated previously (Fath et al. 2002; Shah-ani et al. 2006).

Abnormal tau hyperphosphorylation is an early event inAD disease (Braak et al. 1994). In this study we show thatGC treatment of PC12-htau cells results in the hyperphosph-orylation of tau at Ser202/205, Thr231 and Ser396/404(recognized by the antibodies AT8, CP9 and PHF1, respec-tively). These sites are hyperphosphorylated in AD and theantibodies that recognize these sites constitute importantdiagnostic tools which are associated with the severity andprogression of AD pathology (Holzer et al. 1994; Augusti-nack et al. 2002; Hampel et al. 2005). Importantly, some ofthe phospho-epitopes which showed increased immunoreac-tivity after GC treatment of PC12-htau cells have beenimplicated in cellular mechanisms leading to neurodegener-ation. For example, phosphorylation of Thr231 results inreduced microtubule stability by disturbing microtubule-taubinding capacity (Johnson and Stoothoff 2004) and isinvolved in tau self-assembly into paired helical filaments(PHF) (Wang et al. 2007b). In AD, hyperphosphorylated tauis associated with disrupted axonal transport and/ordecreased tau turnover (Litersky and Johnson 1992) as wellas cytoplasmic accumulation of tau protein (Mandelkow1999). The present study shows that rather than increasingtau synthesis, GC reduce the rate at which tau is degraded.Interestingly, increased total tau protein levels withoutconcomitant increases in tau mRNA levels are found inAD patients and experimental animal models (Goedert et al.1989; Busciglio et al. 1995; Le et al. 1997). Indeed, tauhyperphosphorylation inhibits its own degradation (Poppeket al., 2006) and can also modulate proteasome activity (Renet al. 2007). Clinical studies have shown that the activity ofthe proteasome is decreased in AD brains (Keller et al.2000), correlating with increased neurofibrillary tangles(Smith et al. 1994); these findings suggest that inhibitionor dysfunction of the proteasome system may represent amechanism for neurodegeneration in AD (Keck et al. 2003).Furthermore, our confocal analysis revealed an accumulationof tau in the perikarya of cells exposed to GC. Previousstudies have suggested that cells which show this pattern ofstaining and which otherwise have a normal appearance, maybe destined to contain neurofibrillary tangles and, therefore,may be at the pre-tangle stage (see Kimura et al. 1996; Terry1998; Mandelkow 1999). Together, the above findingssuggest that GC can be an aggravating factor during early

(a)

(b) APP mRNA

CON GC CON GC

PC12

APP770 APP751

APP695

PC12-htau

Cyclophilin

0

50

100

150 *

*

Rel

ativ

e O

.D.

Full-length APP

PC12 PC12-htau

CON

GC

Rel

ativ

e O

.D.

PC12 PC12-htau 0

50

100

150

CON

* * *

APP695

APP751

APP770

* *

Fig. 6 Differential effects of GC on APP levels in PC12 and PC12-

htau cells. Both PC12 and PC12-htau cells were treated with GC

(10)6 M, 24 h) before processing for measurements of APP content by

western blotting (a) and steady-state levels of mRNA encoding the

three isoforms of APP (APP695, APP751 and APP770) by RT-PCR (b).

All numerical results represent mean ± SEM (n = 6–8; CON, set at

100% and shown as dotted line; *p £ 0.05 untreated vs. GC-treated

cells).

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397� 2008 The Authors

392 | I. Sotiropoulos et al.

stages of tau pathology, eventually promoting dissociation oftau molecules from their natural niche and favoring theformation of tau pre-tangles.

Our initial analysis involving measurements of steady-state tau mRNA levels and total tau protein levels suggestedthe involvement of two mechanisms in the manifestation ofGC effects on tau metabolism: on the one hand, GC increaseintracellular levels of tau protein and, on the other, theysupport the activity of protein kinases which have beenstrongly associated with the characteristic pattern of abnor-mal hyperphosphorylation in AD brains (Shahani and Brandt2002). Using pharmacological approaches, we show herethat both GSK3b and cdk5 contribute to GC-induced tauhyperphosphorylation. Interestingly, selectivity was observedinsofar as the effects of cdk5 inhibition were morepronounced on the phosphorylation of CP9 than on thePHF-1 epitope; in contrast, GSK3b inhibition affected thePHF-1 epitope more than the CP9 epitope. These observa-tions most probably reflect the sequential phosphorylation oftau at multiple sites; for example, cdk5 promotes subsequentphosphorylation of tau by GSK3b (Shahani and Brandt 2002;Wang et al. 2007a). Additionally, as both kinases havenumerous cellular substrates and are known to be involved inmany cellular pathways (e.g. the Wnt and PI3K/Aktpathways – Shulman and Feany 2003), we can not exclude

the possibility of indirect effects of these kinases on thephosphorylation of tau. However, it is interesting to note that,in contrast to GSK3 inhibition, cdk5 inhibition blocks bothGC-induced cell death and tau hyperphosphorylation, indi-cating a direct involvement of cdk5 in the manifestation ofGC effect on these cells.

Notably, GSK3b and cdk5 are known to be activated byAb (Murray et al. 1998) and to influence the processing(Phiel et al. 2003) and intracellular distribution (Iijimaet al. 2000; Chang et al. 2006; Rockenstein et al. 2007) ofAPP. Additionally, it has been reported that manipulationof the GC milieu influences APP levels (Islam et al. 1998;Budas et al. 1999). These considerations, together with theobservation that GC can trigger APP misprocessing (Greenet al. 2006; Catania et al. 2007), prompted us to investi-gate the effects of GC exposure on amyloidogenesis inPC12-htau cells. Firstly, we showed that GC treatmentresults in increased levels of BACE, an enzyme respon-sible for initiating the amyloidogenic pathway through theproduction of C99. The observed GC-induced increase inBACE is consistent with reports of glucocorticoid-respon-sive sites within the BACE gene promoter (Sambamurtiet al. 2004). Although the increase in BACE following GCtreatment was relatively small, it is conceivable thatmoderate increases in the level of this enzyme can lead

(a)

(b)

γ-secretase (Nct, PS1)

A β β

β-secretase (BACE-1)

APP

α-secretase

PC12-htau PC12 CON GC CON GC

BACE

Total APP

CTF-PS1

Actin

holo PS1

Nct m i

m i

C99 C99 C83

OPA1

APP 369

CON

BACE Nct APP C99 0

50

100

150 R

elat

ive

O.D

. un

its

PS1 C83

* PC12-htau PC12

200

C99 sAPPβ

sAPPα C83

sAPPα G530

* * * *

Mat

ure

Imm

atu

re *

ho

lo P

S1

CT

-PS

1 C

T-P

S1

Mat

ure

Imm

atu

re

*

ho

lo P

S1

sAPPa

Fig. 7 GC treatment drives APP processing along the amyloidogenic

pathway in PC12-htau cells. A schematic representation of the suc-

cessive enzymatic cleavage of APP into amyloidogenic and non-

amyloidogenic peptides is shown (a) above quantitative results from

western blots, (b) to compare the influence of GC (dexamethasone;

10)6 M, 24 h) on the levels of full-length APP, C99, C83, BACE,

Presenilin 1 (PS1) and nicastrin (Nct) in PC12 and PC12-htau cells,

using antibodies listed in Table S1 (Supporting Information). The pro-

amyloidogenic effects of GC are demonstrated by increases in the

levels of C99, BACE, Nct and PS1, and concomitant reductions in total

full-length APP in PC12-htau cells. No significant changes were found

in the levels of non-amyloidogenic sAPPa in the culture medium of

PC12-htau cells that were treated with dexamethasone (10)6 M,

24 h). All results represent mean ± SEM (n = 6–8; CON, set at 100%).

NS, non-significant and *p £ 0.05 untreated vs. GC-treated cells.

Insets are representative immunoblots.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397

Glucocorticoids, human tau and Alzheimer’s disease | 393

to substantial increases in the generation of amyloidogenicproducts, including Ab (Li et al. 2006). Indeed, ourexperiments show that GC exposure stimulates the gener-ation of C99. This peptide exerts deleterious effects onneuronal function and survival (Yankner et al. 1989;Nalbantoglu et al. 1997) and, in addition, can be subse-quently cleaved to generate Ab, the prototypic trigger ofAD-associated neurodegeneration. The latter cleavagedepends on c-secretase activity. Thus, our finding thatGC increase the levels of Nct and PS1, two criticalcomponents of the c-secretase complex (cf. De Strooper2005), is strongly indicative of increased production ofAb; unequivocal proof for this interpretation cannot beprovided here, however, because of difficulties encounteredin the detection of rat Ab in PC12 cells. Interestingly,although total PS1 levels were significantly increased afterGC treatment, those of CTF-PS1 were unchanged. In fact,in contrast to the PS holoproteins, NTF- and CTF-PS1levels are known to be tightly regulated at a rather (high)constant level (Thinakaran et al. 1996; Steiner et al. 1998);the disposal of excess PS1-CTF is regulated through itsphosphorylation by GSK3b and, importantly, this eventdoes not interfere with the generation of Ab (Kirschen-baum et al. 2001a,b). Thus, a likely explanation for ourfinding is that, whereas PS1 levels are elevated after GCtreatment of PC12-htau cells, surplus PS1-CTF endopro-teolytic fragments are cleared through the mediation ofGSK-3b.

An important finding of the present study was that GC-induced changes in APP processing and the tau phosphor-ylation profile was restricted to PC12 cells that had beenmanipulated to express htau. As GC-induced tau hyper-phosphorylation was also found in differentiated humanneuroblastoma (SHSY5Y) cells endogenously expressing thesame isoform of htau (unpublished observations), it isunlikely that the difference in the total amounts of tau proteinin PC12-htau and PC12 cells is responsible for the tauhyperphosphorylation pattern observed after GC treatment.Taken together, the fact that human and rat tau proteins differin their phosphorylation profiles (Busciglio et al. 1995; Leet al. 1997) and that there are species differences in themanifestation of Ab-induced neurodegeneration andthe extent of abnormal tau phosphorylation suggests thatthe role of tau in the etiopathogenesis of AD may be species-specific (Geula et al. 1998). Indeed, brains from agedlaboratory rodents show little or no AD-like neuropathologyunless they express AD-related human genes (McGowanet al. 2006). Further investigations into the importance of thedifferent species-specific tau proteins in neurodegenerationare warranted.

Interestingly, our study demonstrates that, as compared toPC12 cells, PC12-htau cells are more sensitive to the death-inducing effects of both GC and Ab. Accordingly, wesuggest that changes in APP processing and human tau

phosphorylation are likely mediatory mechanisms thatunderlie the neurotoxic effects of GC. Our observationsthat GC exacerbate Ab-induced neurotoxicity to agreater extent in PC12-htau cells than in PC12 cellsindicate that the presence of htau pre-disposes neurons toAb- and GC-induced cell death. While it is widely heldthat tau hyperphosphorylation occurs secondary to Abproduction and activation of tau kinases, the present datahighlight the importance of htau availability in the regula-tion of Ab production and vulnerability to neurotoxicinsults. Notably, several studies indicate that tau serves as astructural link between several key proteins, such as APP,GSK3b and presenilin, which ultimately contribute to ADneurodegeneration (Smith et al. 1995; Takashima et al.1998; Lucas et al. 2001; Baki et al. 2004).Clinical studies suggest that chronic stress, accompanied

by GC hypersecretion, can be a risk factor for AD (Hartmannet al. 1997; Miller et al. 1998; Wilson et al. 2005; Elgh et al.2006). The results of the present work add new insights intothe role of stress and GC in the pathogenesis and/ormaintenance of AD pathology; they complement other recentreports that implicated GC in the progression of AD (Greenet al. 2006; Jeong et al. 2006). We have shown that GC cantrigger APP mis-processing and tau hyperphosphorylationand accumulation, while increasing the vulnerability ofneurons to the toxic actions of Ab. Importantly, thesepathological processes seem to specifically depend on thepresence of htau.

Acknowledgements

We thank Dieter Fischer, Rainer Stoffel and Jutta Waldherr for

excellent technical assistance and Carola Hetzel for administrative

support, and various colleagues for insightful comments. We are

grateful to Dr. R. Brandt for kindly providing the various PC12 cell

lines, to Drs. P. Davies, S. Gandy, A. Takashima, T. Suzuki, C. Behl

for antibodies, and Dr. M. Duchen for providing access to confocal

facilities at University College London (UCL). This work was partly

supported by grants from the Deutsche Forschungsgemeinschaft

(SPP 1085) and the European Commission (CRESCENDO, FP6

Contract LSHM-CT-2005-018652). I.S. and C.C. were supported by

the Max Planck Society and EU Marie Curie Training Site Host

Fellowships.

Supporting information

Additional supporting information may be found in the online

version of this article.

Table S1 Antibodies used in the present study, with sources and

dilutions used for western blotting (W.B.) and immunocytochem-

istry (ICC).

Please note: Blackwell Publishing are not responsible for the

content or functionality of any supporting materials supplied by the

authors. Any queries (other than missing material) should be

directed to the corresponding author for the article.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397� 2008 The Authors

394 | I. Sotiropoulos et al.

References

Alonso A. C., Grundke-Iqbal I. and Iqbal K. (1996) Alzheimer’s diseasehyperphosphorylated tau sequesters normal tau into tangles offilaments and disassembles microtubules. Nat. Med. 2, 783–787.

Alonso A. C., Li B., Grundke-Iqbal I. and Iqbal K. (2006) Polymeri-zation of hyperphosphorylated tau into filaments eliminates itsinhibitory activity. Proc. Natl Acad. Sci. USA 103, 8864–8869.

Augustinack J. C., Schneider A., Mandelkow E. M. and Hyman B. T.(2002) Specific tau phospho-rylation sites correlate with severity ofneuronal cytopathology in Alzheimer’s disease. Acta Neuropathol.(Berl) 103, 26–35.

Baki L., Shioi J., Wen P., Shao Z., Schwarzman A., Gama-Sosa M.,Neve R. and Robakis N. K. (2004) PS1 activates PI3K thusinhibiting GSK-3 activity and tau overphosphorylation: effects ofFAD mutations. EMBO J. 23, 2586–2596.

Behl C., Lezoualch F., Trapp T., WidmannM., Skutella T. and Holsboer F.(1997) Glucocorticoids enhance oxidative stress-induced cell deathin hippocampal neurons in vitro. Endocrinology 138, 101–106.

Bennett E. J., Bence N. F., Jayakumar R. and Kopito R. R. (2005) Globalimpairment of the ubiquitin-proteasome system by nuclear orcytoplasmic protein aggregates precedes inclusion body formation.Mol. Cell 17, 351–365.

Braak H., Braak E. and Strothjohann M. (1994) Abnormally phos-phorylated tau protein related to the formation of neurofibrillarytangles and neuropil threads in the cerebral cortex of sheep andgoat. Neurosci. Lett. 171, 1–4.

Budas G., Coughlan C. M., Seckl J. R. and Breen K. C. (1999) Theeffect of corticosteroids on amyloid beta precursor protein/amyloidprecursor-like protein expression and processing in vivo. Neurosci.Lett. 276, 61–64.

Busciglio J., Lorenzo A., Yeh J. and Yankner B. A. (1995) Beta-amyloidfibrils induce tau phosphorylation and loss of microtubule binding.Neuron 14, 879–888.

Catania C., Sotiropoulos I., Silva R., Onofri C., Breen K. C., Sousa N.and Almeida O. F. X. (2007) The amyloidogenic potential andbehavioral correlates of stress. Mol. Psychiatry (in press).

Cerqueira J. J., Taipa R., Uylings H. B., Almeida O. F. X. and Sousa N.(2007) Specific configuration of dendritic degeneration in pyra-midal neurons of the medial prefrontal cortex induced by differingcorticosteroid regimens. Cereb. Cortex 17, 1998–2006.

Chang K. A., Kim H. S., Ha T. Y. et al. (2006) Phosphorylation ofamyloid precursor protein (APP) at Thr668 regulates the nucleartranslocation of the APP intracellular domain and inducesneurodegeneration. Mol. Cell. Biol. 26, 4327–4338.

Chun W. and Johnson G. V. (2007) The role of tau phosphorylation andcleavage in neuronal cell death. Front. Biosci. 12, 733–756.

Crochemore C., Lu J., Wu Y., Liposits Z., Sousa N., Holsboer F. andAlmeida O. F. X. (2005) Direct targeting of hippocampal neuronsfor apoptosis by glucocorticoids is reversible by mineralocorticoidreceptor activation. Mol. Psychiatry 10, 790–798.

Davis-Salinas J., Saporito-Irwin S. M., Cotman C. W. and Van NostrandW. E. (1995) Amyloid beta protein induces its own production incultured degenerating cerebrovascular smooth muscle cells.J. Neurochem. 65, 931–934.

De Strooper B. (2005) Nicastrin: gatekeeper of the gamma-secretasecomplex. Cell 122, 318–320.

Elgh E., Lindqvist Astot A., Fagerlund M., Eriksson S., Olsson T. andNasman B. (2006) Cognitive dysfunction, hippocampal atrophyand glucocorticoid feedback in Alzheimer’s disease. Biol. Psychi-atry 59, 155–161.

Fath T., Eidenmuller J. and Brandt R. (2002) Tau-mediated cytotoxicityin a pseudohyperphosphorylation model of Alzheimer’s disease.J. Neurosci. 22, 9733–9741.

Geschwind D. H. (2003) Tau phosphorylation, tangles, and neurode-generation: the chicken or the egg? Neuron 40, 457–460.

Geula C., Wu C. K., Saroff D., Lorenzo A., Yuan M. and Yankner B. A.(1998) Aging renders the brain vulnerable to amyloid beta-proteinneurotoxicity. Nat. Med. 4, 827–831.

Goedert M., Spillantini M. G., Potier M. C., Ulrich J. and CrowtherR. A. (1989) Cloning and sequencing of the cDNA encoding anisoform of microtubule-associated protein tau containing fourtandem repeats: differential expression of tau protein mRNAs inhuman brain. EMBO J. 8, 393–399.

Gotz J., Schild A., Hoerndli F. and Pennanen L. (2004) Amyloid-inducedneurofibrillary tangle formation in Alzheimer’s disease: insightfrom transgenic mouse and tissue-culture models. Int. J. Dev.Neurosci. 22, 453–465.

Green K. N., Billings L. M., Roozendaal B., McGaugh J. L. and LaFerlaF. M. (2006) Glucocorticoids increase amyloid-beta and taupathology in a mouse model of Alzheimer’s disease. J. Neurosci.26, 9047–9056.

Hampel H., Burger K., Pruessner J. C. et al. (2005) Correlation ofcerebrospinal fluid levels of tau protein phosphorylated at threo-nine 231 with rates of hippocampal atrophy in Alzheimer disease.Arch. Neurol. 62, 770–773.

Hardy J. (2006) Has the amyloid cascade hypothesis for Alzheimer’sdisease been proved? Curr. Alzheimer Res. 3, 71–73.

Hartmann A., Veldhuis J. D., Deuschle M., Standhardt H. and Heuser I.(1997) Twenty-four hour cortisol release profiles in patients withAlzheimer’s and Parkinson’s disease compared to normal controls:ultradian secretory pulsatility and diurnal variation. Neurobiol.Aging 18, 285–289.

Holzer M., Holzapfel H. P., Zedlick D., Bruckner M. K. and Arendt T.(1994) Abnormally phosphorylated tau protein in Alzheimer’sdisease: heterogeneity of individual regional distribution andrelationship to clinical severity. Neuroscience 63, 499–516.

Iijima K., Ando K., Takeda S., Satoh Y., Seki T., Itohara S., GreengardP., Kirino Y., Nairn A. C. and Suzuki T. (2000) Neuron-specificphosphorylation of Alzheimer’s beta-amyloid precursor protein bycyclin-dependent kinase 5. J. Neurochem. 75, 1085–1091.

Islam A., Kalaria R. N., Winblad B. and Adem A. (1998) Enhancedlocalization of amyloid beta precursor protein in the rat hippo-campus following long-term adrenalectomy. Brain Res. 806, 108–112.

Jeong Y. H., Park C. H., Yoo J., Shin K. Y., Ahn S. M., Kim H. S., LeeS. H., Emson P. C. and Suh Y. H. (2006) Chronic stress accelerateslearning and memory impairments and increases amyloid deposi-tion in APPV717I-CT100 transgenic mice, an Alzheimer’s diseasemodel. FASEB J. 20, 729–731.

Johnson G. V. and Stoothoff W. H. (2004) Tau phosphorylation inneuronal cell function and dysfunction. J. Cell Sci. 117, 5721–5729.

Jope R. S. and Johnson G. V. (2004) The glamour and gloom of gly-cogen synthase kinase-3. Trends Biochem. Sci. 29, 95–102.

Keck S., Nitsch R., Grune T. and Ullrich O. (2003) Proteasome inhibi-tion by paired helical filament-tau in brains of patients withAlzheimer’s disease. J. Neurochem. 85, 115–122.

Keller J. N., Hanni K. B. and Markesbery W. R. (2000) Impairedproteasome function in Alzheimer’s disease. J. Neurochem. 75,436–439.

Kimura T., Ono T., Takamatsu J., Yamamoto H., Ikegami K., Kondo A.,Hasegawa M., Ihara Y., Miyamoto E. and Miyakawa T. (1996)Sequential changes of tau-site-specific phosphorylation duringdevelopment of paired helical filaments. Dementia 7, 177–181.

Kirschenbaum F., Hsu S. C., Cordell B. and McCarthy J. V. (2001a)Glycogen synthase kinase-3beta regulates presenilin 1 C-terminalfragment levels. J. Biol. Chem. 276, 30701–30707.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397

Glucocorticoids, human tau and Alzheimer’s disease | 395

Kirschenbaum F., Hsu S. C., Cordell B. and McCarthy J. V. (2001b)Substitution of a glycogen synthase kinase-3beta phosphorylationsite in presenilin 1 separates presenilin function from beta-cateninsignaling. J. Biol. Chem. 276, 7366–7375.

Le W. D., Xie W. J., Kong R. and Appel S. H. (1997) Beta-amyloid-induced neurotoxicity of a hybrid septal cell line associated withincreased tau phosphorylation and expression of beta-amyloidprecursor protein. J. Neurochem. 69, 978–985.

Li Y., Zhou W., Tong Y., He G. and Song W. (2006) Control of APPprocessing and Abeta generation level by BACE1 enzymaticactivity and transcription. FASEB J. 20, 285–292.

Litersky J. M. and Johnson G. V. (1992) Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain.J. Biol. Chem. 267, 1563–1568.

Lorenzo A., Yuan M., Zhang Z., Paganetti P. A., Sturchler-Pierrat C.,Staufenbiel M., Mautino J., Vigo F. S., Sommer B. and YanknerB. A. (2000) Amyloid beta interacts with the amyloid precursorprotein: a potential toxic mechanism in Alzheimer’s disease. Nat.Neurosci. 3, 460–464.

Lu J., Goula D., Sousa N. and Almeida O. F. X. (2003) Ionotropicand metabotropic glutamate receptor mediation of glucocorticoid-induced apoptosis in hippocampal cells and the neuroprotectiverole of synaptic N-methyl-D-aspartate receptors. Neuroscience 121,123–131.

Lucas J. J., Hernandez F., Gomez-Ramos P., Moran M. A., Hen R. andAvila J. (2001) Decreased nuclear beta-catenin, tau hyperphospho-rylation and neurodegeneration in GSK-3beta conditionaltransgenic mice. EMBO J. 20, 27–39.

Mandelkow E. (1999) Alzheimer’s disease. The tangled tale of tau.Nature 402, 588–589.

McGowan E., Eriksen J. and Hutton M. (2006) A decade of modelingAlzheimer’s disease in transgenic mice. Trends Genet. 22, 281–289.

Merrick S. E., Demoise D. C. and Lee V. M. (1996) Site-specificdephosphorylation of tau protein at Ser202/Thr205 in response tomicrotubule depolymerization in cultured human neurons involvesprotein phosphatase 2A. J. Biol. Chem. 271, 5589–5594.

Miller T. P., Taylor J., Rogerson S., Mauricio M., Kennedy Q., SchatzbergA., Tinklenberg J. and Yesavage J. (1998) Cognitive and noncog-nitive symptoms in dementia patients: relationship to cortisol anddehydroepiandrosterone. Int. Psychogeriatr. 10, 85–96.

Murray M. J., Merritt D. J., Brand A. H. and Whitington P. M. (1998)In vivo dynamics of axon path-finding in the Drosophilia CNS: atime-lapse study of an identified motorneuron. J. Neurobiol. 37,607–621.

Nalbantoglu J., Tirado-Santiago G., Lahsaini A. et al. (1997) Impairedlearning and LTP in mice expressing the carboxy terminus of theAlzheimer amyloid precursor protein. Nature 387, 500–505.

Patrick G. N., Zukerberg L., Nikolic M., de la Monte S., Dikkes P. andTsai L. H. (1999) Conversion of p35 to p25 deregulates Cdk5activity and promotes neurodegeneration. Nature 402, 615–622.

Phiel C. J., Wilson C. A., Lee V. M. and Klein P. S. (2003) GSK-3alpharegulates production of Alzheimer’s disease amyloid-beta peptides.Nature 423, 435–439.

Poppek D., Keck S., Ermak G., Jung T., Stolzing A., Ullrich O., DaviesK. J. and Grune T. (2006) Phosphorylation inhibits turnover of thetau protein by the proteasome: influence of RCAN1 and oxidativestress. Biochem. J. 400, 511–520.

Rapoport M., Dawson H. N., Binder L. I., Vitek M. P. and Ferreira A.(2002) Tau is essential to beta-amyloid-induced neurotoxicity.Proc. Natl Acad. Sci. USA 99, 6364–6369.

Reinhard C., Hebert S. S. and De Strooper B. (2005) The amyloid-betaprecursor protein: integrating structure with biological function.EMBO J. 24, 3996–4006.

Ren Q. G., Liao X. M., Chen X. Q., Liu G. P. and Wang J. Z. (2007)Effects of tau phosphorylation on proteasome activity. FEBS Lett.581, 1521–1528.

Roberson E. D., Scearce-Levie K., Palop J. J., Yan F., Cheng I. H., WuT., Gerstein H., Yu G. Q. and Mucke L. (2007) Reducing endo-genous tau ameliorates amyloid beta-induced deficits in anAlzheimer’s disease mouse model. Science 316, 750–754.

Rockenstein E., Torrance M., Adame A., Mante M., Bar-on P., RoseJ. B., Crews L. and Masliah E. (2007) Neuroprotective effects ofregulators of the glycogen synthase kinase-3beta signaling pathwayin a transgenic model of Alzheimer’s disease are associated withreduced amyloid precursor protein phosphorylation. J. Neurosci.27, 1981–1991.

Roselli F., Tirard M., Lu J., Hutzler P., Lamberti P., Livrea P.,Morabito M. and Almeida O. F. X. (2005) Soluble H-Amyloid1-40 induces NMDA-dependent degradation of postsynapticdensity-95 at glutam-atergic synapses. J. Neurosci. 25, 11061–11070.

Sambamurti K., Kinsey R., Maloney B., Ge Y. W. and Lahiri D. K.(2004) Gene structure and organization of the human beta-secretase(BACE) promoter. FASEB J. 18, 1034–1036.

Sapolsky R. M. (2000) The possibility of neurotoxicity in the hippo-campus in major depression: a primer on neuron death. Biol.Psychiatry 48, 755–765.

Sawamura N., Ko M., Yu W., Zou K., Hanada K., Suzuki T., Gong J. S.,Yanagisawa K. and Michikawa M. (2004) Modulation of amyloidprecursor protein cleavage by cellular sphingolipids. J. Biol. Chem.279, 11984–11991.

Shahani N. and Brandt R. (2002) Functions and malfunctions of the tauproteins. Cell. Mol. Life Sci. 59, 1668–1680.

Shahani N., Subramaniam S., Wolf T., Tackenberg C. and Brandt R.(2006) Tau aggregation and progressive neuronal degeneration inthe absence of changes in spine density and morphology aftertargeted expression of Alzheimer’s disease-relevant tau con-structs in organotypic hippocampal slices. J. Neurosci. 26, 6103–6114.

Shulman J. M. and Feany M. B. (2003) Genetic modifiers of tauopathyin Drosophila. Genetics 165, 1233–1242.

Smith M. A., Taneda S., Richey P. L. et al. (1994) Advanced Maillardreaction end products are associated with Alzheimer diseasepathology. Proc. Natl Acad. Sci. USA 91, 5710–5714.

Smith M. A., Siedlak S. L., Richey P. L. et al. (1995) Tau proteindirectly interacts with the amyloid beta-protein precursor: impli-cations for Alzheimer’s disease. Nat. Med. 1, 365–369.

Spillantini M. G. and Goedert M. (1998) Tau protein pathology inneurodegenerative diseases. Trends Neurosci. 21, 428–433.

Steiner H., Capell A., Pesold B. et al. (1998) Expression of Alzhei-mer’s disease-associated presenilin-1 is controlled by proteolyticdegradation and complex formation. J. Biol. Chem. 273,32322–32331.

Takashima A., Murayama M., Murayama O. et al. (1998) Presenilin 1associates with glycogen synthase kinase-3beta and its substratetau. Proc. Natl Acad. Sci. USA 95, 9637–9641.

Terry R. D. (1998) The cytoskeleton in Alzheimer disease. J. NeuralTransm. Suppl. 53, 141–145.

Thinakaran G., Borchelt D. R., Lee M. K. et al. (1996) Endoproteolysisof presenilin 1 and accumulation of processed derivatives in vivo.Neuron 17, 181–190.

Wang J. Z., Grundke-Iqbal I. and Iqbal K. (2007a) Kinases and phos-phatases and tau sites involved in Alzheimer neurofibrillarydegeneration. Eur. J. Neurosci. 25, 59–68.

Wang Y. P., Biernat J., Pickhardt M., Mandelkow E. and MandelkowE. M. (2007b) Stepwise proteolysis liberates tau fragments thatnucleate the Alzheimer-like aggregation of full-length tau in a

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397� 2008 The Authors

396 | I. Sotiropoulos et al.

neuronal cell model. Proc. Natl Acad. Sci. USA 104, 10252–10257.

Weiner M. F., Vobach S., Olsson K., Svetlik D. and Risser R. C. (1997)Cortisol secretion and Alzheimer’s disease progression. Biol.Psychiatry 42, 1030–1038.

Wilson R. S., Barnes L. L., Bennett D. A., Li Y., Bienias J. L., Mendesde Leon C. F. and Evans D. A. (2005) Proneness to psychologicaldistress and risk of Alzheimer disease in a biracial community.Neurology 64, 380–382.

Yang A. J., Chandswangbhuvana D., Shu T., Henschen A. and GlabeC. G. (1999) Intracellular accumulation of insoluble, newly syn-thesized abetan-42 in amyloid precursor protein transfected cellsthat have been treated with Abeta1-42. J. Biol. Chem. 274,20650–20656.

Yankner B. A., Dawes L. R., Fisher S., Villa-Komaroff L., Oster-GraniteM. L. and Neve R. L. (1989) Neurotoxicity of a fragment of theamyloid precursor associated with Alzheimer’s disease. Science245, 417–420.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 385–397

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