Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease

*Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy

�Department of Clinical Neurosciences, Brain Repair Centre, University of Cambridge, Cambridge, UK

�Istituto di Genetica e Biofisica ‘‘A. Buzzati Traverso’’, CNR, Napoli, Italy

§Siena Biotech S.p.A., Siena, Italy

Senile amyloid plaques and Tau-containing neurofibrillarytangles (NFTs) are the two major lesions observed in thehippocampus and neocortex of Alzheimer’s disease (AD)patients (Selkoe 2001). Tau inclusions are also characteristicof other neurodegenerative diseases such as progressivesupranuclear palsy, corticobasal degeneration, Pick’s disease,and familial frontotemporal dementia (FTD) and Parkinso-nisms linked to chromosome 17 (Lee et al. 2001). In thesediseases, as in AD, the filamentous protein tau is hyper-phosphorylated at more than 20 different sites, many ofwhich are Ser/Thr-Pro sites. Evidence is accumulating thatglycogen synthase kinase-3b (GSK-3b) is involved in thephosphorylation of tau. GSK-3 is normally constitutivelyactive in all cells, and primarily regulated through inhibition(Doble and Woodgett 2003) via phosphorylation at specificserine residues (serine 9 for GSK-3b and 21 for GSK-3a) atthe N-terminal position. GSK-3b is also regulated by the Wntsignaling pathway (Caricasole et al. 2005) and the possibilitythat Wnt signaling might be involved in the development of

AD was corroborated by the observation that tau hyper-phosphorylation is promoted by GSK-3b activation (Ishiguroet al. 1993). Several members of the Wnt protein family bindto a receptor complex consisting of low-density lipoproteinreceptor-related protein 5/6 (LRP5/6) and a frizzled receptoron the cell membrane (Glinka et al. 1998; Moon et al. 2004).

Received July 10, 2009; revised manuscript received November 9, 2009;accepted December 21, 2009.Address correspondence and reprint requests to Dr Fiorella Casamenti,

Department of Pharmacology, University of Florence, Viale G.Pierac-cini, 650139 Florence, Italy. E-mail: [email protected] Cristina Rosi and Ilaria Luccarini were equally involved in thestudy.Abbreviations used: AD, Alzheimer’s disease; ChAT, choline acetyl-

transferase; DAB, 3,3¢-diaminobenzidine; DKK-1, Dickkopf-1; FTD,frontotemporal dementia; GSK-3, glycogen synthase kinase-3; LRP5/6,lipoprotein receptor-related protein 5/6; NBM, nucleus basalis magno-cellularis; NFTs, tau-containing neurofibrillary tangles; PBS, phosphate-buffered saline; PHF, paired helical filaments; PSEN1, presenilin 1; wt,wild-type.

Abstract

To investigate the role of the Wnt inhibitor Dickkopf-1 (DKK-1)

in the pathophysiology of neurodegenerative diseases, we

analysed DKK-1 expression and localization in transgenic

mouse models expressing familial Alzheimer’s disease

mutations and a frontotemporal dementia mutation. A signifi-

cant increase of DKK-1 expression was found in the diseased

brain areas of all transgenic lines, where it co-localized with

hyperphosphorylated tau-bearing neurons. In TgCRND8

mice, DKK-1 immunoreactivity was detected in neurons sur-

rounding amyloid deposits and within the choline acetyl-

transferase-positive neurons of the basal forebrain. Active

glycogen synthase kinase-3 (GSK-3) was found to co-localize

with DKK-1 and phospho-tau staining. Downstream to GSK-3,

a significant reduction in b-catenin translocation to the nu-

cleus, indicative of impaired Wnt signaling functions, was

found as well. Cumulatively, our findings indicate that DKK-1

expression is associated with events that lead to neuronal

death in neurodegenerative diseases and support a role for

DKK-1 as a key mediator of neurodegeneration with thera-

peutic potential.

Keywords: active glycogen synthase kinase-3, Alzheimer’s

disease, Dickkopf-1, frontotemporal dementia, transgenic

mice, b-catenin.

J. Neurochem. (2010) 112, 1539–1551.

JOURNAL OF NEUROCHEMISTRY | 2010 | 112 | 1539–1551 doi: 10.1111/j.1471-4159.2009.06566.x

� 2010 The AuthorsJournal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1539–1551 1539

Upon activation of the canonical b-catenin-mediated path-way, which is the best characterized of the Wnt signalingpathways, the phosphorylation and proteasome-mediateddegradation of b-catenin by GSK-3b is reduced, resultingin b-catenin accumulation and mediation of its cellularsurvival functions (Nelson and Nusse 2004). Of relevance toneuronal survival and function, the b-catenin-mediated Wntpathway modulates dendritic morphogenesis and synapticplasticity, and is protective toward AD-related neurodegen-eration (Yu and Malenka 2003; Chen et al. 2006). Dickkopf-1 (DKK-1), is an extracellular secreted inhibitor of thecanonical Wnt pathway that binds the co-receptor LRP5/6(Moon et al. 2004). Our and other recent data indicate thatinduction of DKK-1 expression represents a component of asequence of events underlying neuronal death in neurode-generative disorders (De Ferrari and Inestrosa 2000; Carica-sole et al. 2003, 2004; Cappuccio et al. 2005; Scali et al.2006). An induction of DKK-1 expression was found incultured neurons challenged with b-amyloid peptide as wellas in degenerative neurons of the AD brain (Caricasole et al.2004), in neurons subjected to excitotoxic or ischemic insults(Cappuccio et al. 2005) and in cultured human neural stemcells challenged with increasing concentrations of thepurified astrocyte-derived protein S100B (Esposito et al.2008). The induction of DKK-1 expression was described incortical and hippocampal neurons of rats in response tokainate-induced seizures and in degenerating neurons ofpatients affected by mesial temporal lobe epilepsy associatedwith hippocampal sclerosis (Busceti et al. 2007). Recently,Zhang et al. (2008) demonstrated that the neuroprotectiverole of estrogen is largely attributable to its capacity toprevent elevation of DKK-1. Finally, DKK-1 was shown tobe neurotoxic both in vitro and in vivo (Scali et al. 2006).

Here, we employed three different transgenic AD-likemouse models and a tauopathy mouse model and found thatDKK-1 is significantly induced in the brain of all three AD-like mouse models and in the brainstem of mice transgenicfor mutant human P301S tau protein.

Materials and methods

AnimalsTgCRND8 mice: transgenic hemizygous TgCRND8 (tg) mice with a

(C57)/(C57/C3H) genetic background and non-tg hybrid (C57)/

(C57/C3H) wild-type (wt), control littermate male, and female mice,

aged 4, 7, and 12 months, were used. Of note, in this mouse strain

amyloid deposition is already detectable at 3 months of age (Chishti

et al. 2001), becoming robust and widespread at 7 months of age

with no marked differences between 7 and 12 months of age

(Bellucci et al. 2007). TgCRND8 mice encode a double-mutant form

of amyloid precursor protein (APP) 695 (KM670/671NL+V717F)

under the control of the Prion Protein gene promoter (Chishti et al.2001). These mice were obtained from the laboratory of Dr Hyslop

(Center for Research in Neurodegenerative Diseases, Toronto,

Canada) and were bred in the Centre for Laboratory Animals,

University of Florence, Italy. TgCRND8 mice were identified using a

standardized PCR assay on tail DNA. In addition, for immunohis-

tochemical detection of DKK-1 the following two groups of 14-

month-old transgenic and non-transgenic mice were used: (i) Tg2576

mice bred on a C57B6/SJL background and over-expressing the

Swedish mutation of human APP, and age-matched C57B6/SJL non-

transgenic mice which were purchased from Taconic (NY, USA); (ii)

APP/Presenilin 1 (PSEN1) bigenic mice (line B6C3-Tg(APPswe,

PSEN1dE9)85Dbo/J) were obtained from the Jackson Laboratory,

Bar Harbor, ME, USA. These mice express a chimeric mouse/human

APP gene harboring the Swedish double-mutation (K595N/M596L),

and a human PSEN1 gene with a deletion of exon 9 (Jankowsky

et al. 2004). The line was maintained as double hemizygote by

crossing transgenics to wt B6C3F1/J mice obtained from Charles

River Laboratory, Italy. P301S mice were used as animal model of

frontotemporal dementia: 3- and 5/6-month-old homozygous mice

transgenic for human P301S tau (Allen et al. 2002) and age-matched

C57BL/6J controls were analyzed in immunohistochemical and

western blotting experiments. P301S mice (courtesy of M. Goedert,

Medical Research Council Laboratory of Molecular Biology,

Cambridge, UK) were bred in the animal house facility at the

Department of Clinical Neuroscience, Brain Repair Centre, Univer-

sity of Cambridge, UK. The mice were housed in macrolon cages

with ad libitum food and water, and maintained on a 12 h light/dark

cycle at a room temperature of 23�C. All experiments were carried

out according to the ECC guidelines for animal care (DL 116/92,

application of the European Communities Council Directive 86/609/

EEC) and all efforts were made to minimize animal suffering.

Detailed information on the number, sex, and age of all the mice used

in this study are reported in Table S1.

Processing of animal tissueTgCRND8 mice as well as control mice were killed by cervical

dislocation and brains were rapidly removed and divided sagittally.

One hemibrain was post-fixed in phosphate-buffered 4% parafor-

maldehyde, pH 7.4, at 4�C for 48 h, then rinsed in phosphate-

buffered saline (PBS) and paraffin embedded. Coronal paraffin-

embedded sections were cut at 5 lm and mounted on slides, as

previously described (Bellucci et al. 2006). Cortical and hippocam-

pal samples from the other hemibrain were immediately sectioned,

snap-frozen, and stored at )80�C to be processed for protein

analysis.

Under chloral hydrate (400 mg/kg, intraperitoneally) anesthesia,

transgenic 2576, APP/PSEN1, and P301S mice as well as the

respective control mice were perfused transcardially with 4% ice-

cold paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. As

above, 5 lm coronal and/or sagittal paraffin-embedded sections

were prepared. For western blotting experiments, an additional

group of P301S and wt mice were killed by cervical dislocation and

the parietal cortex, brainstem and spinal cord were rapidly dissected

out on ice, frozen, and stored at )80�C until further processing.

ImmunohistochemistryBriefly, sections were subjected to antigen retrieval by microwave

incubation in 10 mM Na-Citrate buffer (pH 6.0) and then incubated

overnight at 4�C with the primary antibodies at the optimized

working dilution made up in PBS 0.1 M (pH 7.4) with Triton X-100

Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1539–1551� 2010 The Authors

1540 | M. C. Rosi et al.

(0.3%) and bovine serum albumin (5 mg/mL). On the second day,

sections were incubated for 1 h with the secondary antibody at

1 : 1000 dilution made up in PBS 0.1 mM plus bovine serum

albumin (1 mg/mL) and the immunostaining was visualized using

the avidin–biotin system (Vectastain; Vector Laboratories, Burlin-

game, CA, USA) and 3,3¢-diaminobenzidine plus Nickel (DAB Kit;

Vector Laboratories) as the chromogen and observed by light

microscopy (Olympus BX40; Olympus, MI, Italy). For the double

staining with DKK-1 and Ab(1-42) antibodies, the first cycle of

immunohistochemistry was performed with the goat DKK-1

antibody, the correspondent secondary antibody, and DAB Kit

(dark brown). Then, after several washes the sections underwent

another cycle of immunohistochemistry with the rabbit anti Ab(1-42) primary antibody. The second staining was visualized by Vector

NovaRed Kit (Vector Laboratories) (red) that provides excellent

contrast with peroxidase substrates DAB kit. Detailed information

for the primary antibodies used in this work is listed in Table S2.

Secondary biotinylated antibodies (anti-goat and anti-rabbit; Vector

Laboratories) were also used. All antibody concentrations were

titrated to provide optimal staining. For all immunohistochemical

experiments, control sections without primary antibodies were

routinely used and they showed negative staining.

Fluorescence stainingCo-localization experiments were performed as previously reported

(Bellucci et al. 2007) using the appropriate fluorescent secondary

antibodies (monoclonal antimouse Alexa Fluor 594 red conjugated

and polyclonal anti-rabbit or anti-goat Alexa Fluor 488 green

conjugated; Invitrogen, Eugene, OR, USA). Sections were covers-

lipped using Vectashield water-based mounting medium with 4¢,6-diamidino-2-phenylindole (Vector Laboratories). The analysis of

negative controls (omission of primary antibody) was simulta-

neously performed to exclude the presence of non-specific immu-

nofluorescence staining, cross-immunostaining, or fluorescence

bleed-through.

An Olympus BX40 microscope coupled to analySIS^B Imaging

Software (Olympus) or a Zeiss LSM 510-META confocal micro-

scope (Zeiss, Jena, Germany) equipped with argon (488 nm) and

helium/neon (543 nm) excitation lasers were used to acquire

representative images from the examined specimens. For confocal

microscopy analysis, the following excitation laser/emission filter

settings were used: for 488 nm excitation, emission was selected

with a 510–530 nm bandpass filter, whereas for 543 nm excitation

emission was selected using a 560-nm longpass filter. The captured

confocal images were viewed and analysed using LSM 510 META

Imaging software (Zeiss).

Protein extraction and western blotting analysisBrain samples from transgenic and wt mice were homogenized in

ice-cold lysis buffer containing 2 mM sodium pyrophosphate,

4 mM p-nitrophenyl phosphate, 1 mM sodium orthovanadate,

1 mM phenylmethylsulfonyl fluoride, 20 lg/mL leupeptin, 30 ll/mL aprotinin. Equal amounts of protein samples (40 lg) were

applied to sodium dodecyl sulfate–polyacrylamide gels and sub-

jected to electrophoresis as previously described (Bellucci et al.2007). Subcellular fractionation was carried out according to an

established method (Meacci et al. 2003) with minor modifications

(for details on the experimental procedure see Appendix S1 and

Fig. S1). Nuclear and cytoplasmic extracts were used for the

measurement of b-catenin levels by western blotting. Protein bands

were normalized to b-actin or histone H1 levels to provide a control

for equal loading and the data were analyzed using GraphPad Prism

version 4.00 for Windows (GraphPad Prism Software version 4.0;

GraphPad, San Diego, CA, USA).

Data analysisOne-way ANOVA, followed by Bonferroni’s post hoc test, was used toanalyse the differences in DKK-1 levels between TgCRND8 and wt

mice at different ages and between 5/6 month-old P301S and the

age-matched wt mice in different CNS areas. Non-paired Student’s

t-tests were used to analyze the differences in the levels of b-cateninbetween 7-month-old transgenic CRND8 and non-transgenic mice.

The software GraphPad Prism version 4.0 for Windows was used to

perform all calculations and a threshold of p < 0.05 was considered

significant. All the data are expressed as mean ± SEM.

Results

DKK-1 expression is increased in cortex and hippocampusof TgCNRD8 miceTo investigate the expression of DKK-1 in the TgCNRD8mouse model of AD, DKK-1 expression was first analyzed bywestern blotting. Qualitative analysis of DKK-1 blotsrevealed intense bands of the appropriate relative molecularweights in the cortex and hippocampus of 7- and 12-month-old TgCRND8 mice as compared with the age-matched wtmice (Fig. 1a). Quantification of the corresponding proteinband intensities (Fig. 1b) demonstrated a statistically signif-icant increase in DKK-1 expression in both the 7- and 12-month-old transgenic mouse brain (*p < 0.001, #p < 0.01). Alow amount of DKK-1 protein was detected in 4-month-old tgand wt mice, both in the cortex and hippocampus, with nodifferences between the two groups (not shown). Therefore,expression of DKK-1 protein is induced in AD-relevant brainregions of the TgCNRD8 mouse model of AD. Next, adetailed immunohistochemical analysis was performed toinvestigate DKK-1 expression at the single cell level in theseareas. Neurons intensely stained by DKK-1 antibody wereconcentrated in the II, III, V, and VI layers of the neocortex(Fig. 2b, d and f for the parietal and piriform neocortices,respectively) and in the hippocampal formation, as shown forthe CA3 subfield (Fig. 2h) of 7-month-old TgCRND8 mice.In these brain areas, DKK-1 immunohistochemistry showednumerous neurons with intense staining in the cytoplasmclose to the cell membrane and apical dendrites (see highermagnification in Fig. 2h). In the neocortex of 7-month-oldTgCRND8 mice, some DKK-1 positive neurons appeared tobe irregularly shaped, displaying either swollen or shrunk cellbodies and distorted processes, as shown for the piriformneocortex (see higher magnification in Fig. 2f). The samescenario of DKK-1 immunoreactivity was observed in the 12-month-old TgCRND8 mouse brain while DKK-1 immuno-

� 2010 The AuthorsJournal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1539–1551

DKK-1 in neurodegenerative diseases | 1541

reactivity was basically absent in the neocortex (Fig. 2a, c ande) and hippocampus (Fig. 2g) of the age-matched wt mice,suggesting that DKK-1 expression is associated with thepathology as modeled in TgCRND8 mice. To verify whetherthe prevalent distribution of DKK-1 immunostaining alongthe cell membrane might be associated with an interactionwith the Wnt co-receptor, double labeling fluorescenceimmunohistochemistry with DKK-1 and LRP5/6 antibodieswas performed. Confocal microscopy analysis demonstratedthat LRP5/6 (Fig. 3a, red) and DKK-1 (Fig. 3b, green)staining overlapped within neurons of the aged (7 months)TgCRND8 mouse brain (Fig. 3c, yellow). In double labelingexperiments with Ab(1-42) and DKK-1 antibodies, DKK-1staining was often observed in close vicinity to Ab(1-42)plaques in the neocortex and the hippocampus, as exemplifiedfor the CA3 area in Fig. 3(d), while in general no DKK-1immunostaining was detected within the amyloid deposits(Fig. 3d). As can be seen, neurons close to Ab(1-42) plaquesoften show swollen cell bodies and dystrophic neurites.Interestingly, DKK-1 staining (Fig. 3g, green) co-localizedwith paired helical filaments (PHF)-1 staining (Fig. 3f, red)indicating that DKK-1 is expressed in phospho-tau bearingneurons as demonstrated by the merging of the two confocalmicroscopy images (Fig. 3h).

Choline acetyltransferase (ChAT)-positive neurons in thenucleus basalis magnocellularis (NBM) of TgCRND8 miceare DKK-1 positiveWe previously reported a significant loss of ChAT-positiveneurons in the nucleus basalis magnocellularis (NBM) ofTgCRND8 mice at 7 months of age (Bellucci et al. 2006). Inthe present study, we explored whether DKK-1 is expressedwithin ChAT-positive neurons in the NBM employing anti-DKK-1 and anti-ChATantibodies. As we already reported, thesurviving ChAT-positive neurons in 7- and 12-month-old

TgCRND8 mice, as exemplified for the 7-month-old tg miceshown in Fig. 4(b), exhibited cell body and dendritic shrinkageas compared with age-matched wt mice (Fig. 4d). NBMsections stained for DKK-1 (Fig. 4a) showed that somemagnocellular neurons in the TgCRND8 mice are DKK-1immunopositive, whereas no DKK-1 staining was revealed inthe NBM of wt mice (Fig. 4c). Double labeling fluorescenceimmunohistochemistrywithDKK-1(Fig. 4e,green)andChAT(Fig. 4f, red) antibodies demonstrated DKK-1 expressionwithin ChAT-positive neurons in the NBM, as demonstratedby themerged confocalmicroscopy images shown inFig. 4(g).

GSK-3 immunohistochemistry in the TgCRND8 mouse brainWe previously demonstrated the occurrence of hyperphosph-orylated tau protein in the TgCRND8 mouse brain (Bellucciet al. 2007). Given the increased expression of DKK-1 in theTgCRND8, further evidence was sought for a modulation(inhibition) of canonical Wnt signaling in the brain of this ADmouse model. A key cytoplasmic relay of canonical Wntsignaling is represented by GSK-3, whose kinase activity on anumber of substrates is indirectly proportional to levels ofcanonical Wnt signaling. On this basis, the expression of theactive form of GSK-3 was investigated by immunohisto-chemical analysis. The GSK-3a/bpTyr279/Tyr216 antibody,which is specific for the active form of the kinase, wasemployed to evaluate the presence of the activated enzyme(which may result from an inhibition of canonical Wntsignaling by DKK-1 for instance) in coronal brain sections oftg and corresponding wt controls. This staining revealed anincreased immunoreactivity in the deep layers of the parietalneocortex (Fig. 5b and c) and in the CA3 subfield (Fig. 5eand f) and dentate gyrus (Fig. 5h and i) of the hippocampus ofTgCRND8 mice at 7 months of age as compared with thecorresponding brain areas of age-matched controls (Fig. 5a, dand g), consistent with an inhibition of canonical Wnt

(a)

(b)

Fig. 1 Dickkopf-1 (DKK-1) protein levels in

the brain of aged TgCRND8 (tg) mice DKK-

1 protein expression was evaluated by

western blotting (a) followed by densito-

metric analysis (b) of the corresponding

bands (38 and 41 kDa). b-actin serves as a

protein loading control (43 kDa bands).

Statistics indicate a significant increase of

DKK-1 levels in the brain of both 7- and 12-

month-old transgenic mice compared with

the age-matched controls. Data are pre-

sented as mean ± SEM normalized to

100% for the controls (n = 3–4 per group).

One-way ANOVA plus Bonferroni’s post hoc

test: *p < 0.001, #p < 0.01, tg versus age-

matched wild-type mice.



signaling. A similar pattern of active GSK-3 expression wasobserved at 12 months of age. The staining was mainlylocalized within the neuronal cell bodies and dendrites. Theeffect of GSK-3 activation on one of its targets of relevance toAD, namely tau protein, was next investigated. In fact, severalstudies have reported that tau hyperphosphorylation iscontrolled by a group of serine-threonine kinases, includingGSK-3b (Ishiguro et al. 1993), and it is accepted by manythat GSK-3b plays an important role in the stability of tau andthat inhibition of GSK-3b prevents tau hyperphosphorylation.Double labeling immunohistochemistry with active GSK-3a/b (Fig. 5j, green) and PHF-1 (Fig. 5k, red) antibodiesrevealed active kinase staining within phospho-tau bearing

neurons, as indicated by the merging of the two imagesdepicted in Fig. 5(l). To confirm an association betweenDKK-1 expression and GSK-3 activation, double labelingfluorescence immunohistochemistry was performed withDKK-1 and GSK-3a/b PTyr279/Tyr216 antibodies in 7-month-old TgCRND8 and age-matched wt mice. Co-locali-zation of active GSK-3 (Fig. 5m and p, red) with DKK-1(Fig. 5n and q, green) was found within some cells of theparietal neocortex (Fig. 5o, yellow) and of the dentate gyrusof the hippocampus (Fig. 5r, yellow). To examine whetheractivation of GSK-3 would result in decreased nuclearb-catenin translocation, the expression of b-catenin wasinvestigated by immunohistochemical and western blotting

(a) (c) (e)

(b)

(g) (h)

(d) (f)

Fig. 2 Enhanced Dickkopf-1 (DKK-1) immunoreactivity in the brain of

7-month-old TgCRND8 (tg) mice (a–d): parietal neocortex. DKK-1

immunopositive neurons are shown in the II and III layers (b) as well as

the V and VI (d) layers of the tg parietal cortex. The higher magnifi-

cations (original 40X) of the squared areas in (b) and (d) show neu-

rons whose immunoreactivity is mainly localized in the cytoplasm

close to the cell membrane and in apical dendrites. Control mice do

not display DKK-1 immunoreactivity [see (a) for layers II, III and (c)

for layers V, VI]. (e,f) Piriform neocortex. (e) No DKK-1 immunore-

activity is present in the piriform neocortex of wild-type (wt) mice. (f)

DKK-1-positive neurons in the piriform neocortex of tg mice. A

magnified view (original 40X) of the framed area is reported in the

inset. The higher magnifications in (d) and (f) highlight the occur-

rence of either swollen or shrunk cell bodies and short and distorted

processes among DKK-1 positive neurons. (g–h) CA3 area of the

hippocampus. (h) Numerous DKK-1 immunopositive neurons are

detected in the tg mouse brain. Note in the magnified view (original

40X) of the squared area the cell membrane localization of DKK-1

immunoreactivity within pyramidal neurons. (g) DKK-1 immunoreac-

tivity is weak or absent in the CA3 area of the hippocampus of control

mice. Scale bars: (a–h) 100 lm. Par ctx = parietal cortex; pir

ctx = piriform cortex.



analysis of nuclear extracts prepared from the cortex andhippocampus of 7-month-old TgCRND8 and age-matched wtmice. Immunohistochemistry revealed fewer b-catenin posi-tive cells in the parietal (Fig. 6b) and piriform (Fig. 6d)neocortices and in the CA3 area (Fig. 6f) of the hippocampusof tg mice than in the corresponding brain areas of wt mice(Fig. 6a, c and e). Double labeling immunohistochemistrywith NeuN (Fig. 6g and j, red) and b-catenin (Fig. 6h and k,green) antibodies clearly demonstrated a nuclear translocationof b-catenin in wt mice (Fig. 6i, yellow). In contrast, no co-localization was found between NeuN and b-catenin stainingat the nuclear level in the tg mice (Fig. 6l) and only slight co-localization was revealed at the cytoplasmic level (Fig. 6l,

yellow). In western blotting experiments, the levels of b-catenin were first analysed in total cortical and hippocampalextracts (Fig. 6m) for which no differences were foundbetween wt and tg mice. However, when b-catenin levelswere analysed in nuclear extracts, a significant reduction in b-catenin levels was observed in the hippocampus of tg mice(p < 0.05) and a trend toward a reduction in the cortex ascompared with wt littermates (Fig. 6m and n). Taken together,the immunohistochemical and western blotting results pointto increased GSK-3 activity and reduced nuclear b-cateninlevels in tg mice, which is in accordance with an impairmentof canonical Wnt signaling as a consequence of DKK-1expression.

(a)

(d) (e)

(b) (c)

(f) (g) (h)

Fig. 3 Double labeling immunohistochemistry with Dickkopf-1 (DKK-

1) and lipoprotein receptor-related protein 5/6 (LRP5/6), Ab(1–42) and

PHF-1 antibodies in 7-month-old TgCRND8 (tg) mice (a–c) sections

were double-immunolabeled for LRP5/6 (a, red) and DKK-1 (b, green)

and imaged with the laser scanning confocal microscope. Note in the

merged figure part (c) that LRP5/6 and DKK-1 staining overlap within

neocortical neurons (yellow) in the tg mouse brain. (d and e) Double

labeling immunohistochemistry for DKK-1 and Ab(1–42) in tg (d) and

age-matched control (e) mice. Note DKK-1 positive neurons in close

correspondence of the core plaques (indicated by the asterisks) in the

hippocampus of tg mice. (f–h) Representative confocal images

showing colocalization (h, yellow) between PHF-1 (f, red) and DKK-1

(g, green) antibodies within some cortical neurons of the tg mouse

brain. Scale bars: (a–c) 20 lm, (d and e) 50 lm; (f–h) 20 lm.



DKK-1 expression is increased in Tg2576 and APP/PSEN1mouse brainAs a way to determine the generality of these findings toother mouse AD models, we investigated the expression ofDKK-1 in one exemplary brain area (CA3) in Tg2576 mice(expressing human bAPP with the Swedish mutation; Hsiaoet al. 1996) and in APP/PSEN1 mice (co-expressing the APPSwedish mutation and the exon-9-deleted variant of humanpresenilin 1; Jankowsky et al. 2004). In these two modelsystems, the pathological phenotype develops with compar-atively slower (Tg2576) or faster (APP/PSEN1) progression.To this end, DKK-1 immunoreactivity was analysed incoronal slices of 14-month-old Tg2576 mice and of 14-month-old transgenic APP/PSEN1 mice, and of the respec-tive wt age-matched control mice. Single labeling fluores-cence immunohistochemistry revealed some DKK-1 positiveneurons in the neocortex (not shown) and in the hippocam-pus areas, as shown for the CA3 subfield (Fig. S2a and b) ofTg2576 and a comparatively increased number in APP/PSEN1 mice with no DKK-1 staining in the age-matchedcontrols (not shown).

DKK-1 expression is induced in the CNS in a tauopathymouse model where it is associated with neuronal sufferingAside from mouse models of plaque deposition, miceexpressing human FTD mutations recapitulate some of thetau-related aspects of human neurodegenerative diseases and

are sometimes employed to model tau-associated pheno-types. Such models are particularly interesting as significantneuronal suffering and cell death are evident in selected CNSareas (Allen et al. 2002). We therefore sought to investigateif DKK-1 expression could be associated with degeneratingneurons in a FTD model. As we previously described,homozygous P301S transgenic mice at 5/6 months of agedevelop a neurological phenotype dominated by severeparaparesis and present abundant tau filaments in the brainand spinal cord with the highest number of tau-positive nervecells in the brainstem and spinal cord (Allen et al. 2002).Western blotting analysis (Fig. 7i), carried out on brainhomogenates of 5/6-month-old transgenic P301S and age-matched wt mice, revealed stronger DKK-1 immunopositiveprotein bands in the brainstem of transgenic mouse brain ascompared with wt mouse brain. Protein quantification(Fig. 7j) demonstrated that DKK-1 protein levels weresignificantly (p < 0.01) increased in the brainstem of tgmice, as compared with wt controls. No differences in DKK-1 protein levels were detected in the cortex and spinal cordbetween tg and wt mice. When the analysis was performedby immunofluorescence, numerous cells intensely stained byDKK-1 antibody were found in the brainstem of 5/6-month-old transgenic P301S mice. Figure 7(a) shows that, as in theTgCRND8 mouse brain, DKK-1 staining was mainlylocalized in vicinity to the cell membrane. Few DKK-1positive cells were also detected in the wt age-matched

(a)

(e) (f) (g)

(b) (c) (d)

Fig. 4 Dickkopf-1 (DKK-1) staining within choline acetyltransferase

(ChAT)-positive neurons in the Nucleus Basalis Magnocellularis

(NBM) of 7-month-old TgCRND8 (tg) and wild-type (wt) mice (a–d)

representative 5 lm NBM sections from tg (a,b) and wt (c,d) mice,

stained with DKK-1 (a,c) and ChAT (b,d) antibodies. Note in (a) DKK-1

staining along the plasma membrane and dendrites of some magno-

cellular neurons of tg mice and in (c) the absence of DKK-1 immu-

noreactivity in wt mice. Note in (b) the morphological alterations in the

surviving ChAT-positive neurons in the tg mice as compared with

ChAT-positive neurons in the wt (d) mice. (e–g) Double immunofluo-

rescence of DKK-1 (e, green) and ChAT (f, red) antibodies at confocal

laser microscope in the tg mouse NBM. DKK-1 expression within

ChAT-positive neurons is shown in the merged figure part ‘g’ (yellow).

Scale bars: (a–d) 50 lm, (e–g) 20 lm.



controls (Fig. 7b). However, in the brainstem region ofcontrols, the staining appears homogenous in the somaticcytoplasm and overall lighter than in the transgenic P301Smice. Also, in the brainstem of 3-month-old transgenicP301S and wt mice, DKK-1 immunoreactivity appeared lightand homogenous in the entire cytoplasm with no differencesin DKK-1 staining intensity and distribution between trans-genic and wt mice (data not shown). Taken together, thesedata suggest that DKK-1 undergoes distinct cytoplasmic re-localization along the cell membrane in diseased brain. Wenext assessed the subcellular localization of DKK-1. Doublelabeling experiments with NeuN (Fig. 7c, red) and DKK-1

(Fig. 7d, green) antibodies demonstrated DKK-1 immunore-activity (Fig. 7e, green) in the cytoplasm close to the cellmembrane of NeuN-positive (Fig. 7e, red) neurons. More-over, double labeling experiments with AT8 and DKK-1antibodies revealed DKK-1 staining (Fig. 7g, green) in thecell membrane of phospho-tau-positive neurons (Fig. 7f, red)as demonstrated by merging of two images (Fig. 7h) and inthe cytoplasm of some AT8 positive neurons (Fig. 7h,yellow). The immunohistochemical analysis of DKK-1 wasalso performed in the spinal cord, the other CNS area mainlyaffected by tau-pathology in this FTD model. Although nodifferences in DKK-1 protein levels were detected between

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)

Fig. 5 Glycogen synthase kinase-3 (GSK-

3)a/bpTyr279/Tyr216 expression in the 7-

month-old TgCRND8 (tg) mouse brain.

GSK-3apT279/b-pT216 immunopositive

cells are present in the deep layers of the

parietal neocortex (b,c), in the CA3 subfield

(e,f) and in the polymorph layer of the

dentate gyrus (h,i) of the hippocampus in 7-

month-old tg mice. (a,d and g) GSK 3a/b

immunostaining of the same brain areas in

wild-type control mice. (j–l) Double labeling

immunofluorescence with PHF-1 (k, red)

and GSK 3a/b (j, green) antibodies showing

co-localization of the two staining (l, yellow)

within cortical neurons of tg mice. (m–r)

Double labeling immunofluorescence with

active GSK-3 (m,p, red) and DKK-1 (n,q,

green) antibodies in the parietal neocortex

(m–o) and in the dentate gyrus of the hip-

pocampus (p–r). Note the co-localization

between the two staining within some neu-

rons (o,r yellow). Scale bars: (a,b,d,e,g,h)

100 lm; (c,f,i) 50 lm; (j–l) 30 lm; (m–r)

50 lm. Par ctx = parietal cortex.



tg and wt mice, likely a consequence of the massive neuronalloss occurring in the former (Allen et al. 2002), strong DKK-1 immunoreactivity was revealed along the cell membraneand processes of motor neurons in 5/6-month-old P301Smice (Fig. S3a). In age-matched controls, DKK-1 immuno-

reactivity was light and diffusely distributed over the entirecytoplasm of the cells (Fig. S3b). Double labeling immuno-histochemistry (Fig. S3c–h) demonstrated that DKK-1immunoreactivity (Fig. S3d and g) in the spinal cord ofP301S mice was localized along the cell membrane of

(a) (b) (m)

(n)

(c) (d)

(e) (f)

(g) (h) (i)

(j) (k) (l)

Fig. 6 b-catenin immunohistochemistry and western blotting in 7-

month-old TgCRND8 (tg) mice (a,c,e) and (b,d,f): single b-catenin

immunohistochemistry in the wild-type (wt) and tg mouse brain,

respectively. Transgenic mice show fewer b-catenin positive cells

than wt mice in the parietal (b vs. a) and piriform (d vs. c) neocortex

and in the CA3 area (f vs. e) of the hippocampus. (g–l) Confocal

images of wt (g–i) and tg (j–l) brain sections are immunolabeled with

NeuN (g,j, red) and b-catenin (h,k, green) antibodies. In the wt mice,

both cytoplasmic and nuclear localization of b-catenin can be clearly

seen within NeuN-positive neocortical neurons, as demonstrated by

the yellow color in the merged figure part (i). In the tg mice, b-

catenin staining is revealed mostly in the cytoplasm close to the cell

membrane of NeuN-positive neurons and no b-catenin staining is

present at the nuclear level (k and l). Scale bars: (a–d) 100 lm; (e,f)

50 lm; (g–l) 20 lm. (m) Qualitative immunoblot analysis of total and

nuclear extracts from cortex and hippocampus of wt and tg mice

probed with anti-b-catenin antibody (92 kDa band). b-actin (43 kDa

band) and Histone H1 (30 kDa band) are shown as controls of total

and nuclear protein loading, respectively. (n) Densitometric quanti-

fication of b-catenin against histone H1 in nuclear extracts from

cortex and hippocampus of tg and wt mice shows a statistically

significant decrease in protein levels in the hippocampus of tg mice

compared with controls. Data are presented as mean ± SEM nor-

malized to 100% for the controls (n = 3–4 per group). Two-tailed

Student’s t-test: *p < 0.05; n.s.: not significant. b-cat = b-catenin,

hist H1 = histone H1.



neurons (NeuN staining Fig. S3c, merged Fig. S3e), most ofwhich containing tau (AT8 staining Fig. S3f, mergedFig. S3h).

Discussion

DKK-1 is a secreted negative modulator of the canonicalWnt/b-catenin pathway (Caricasole et al. 2003; Moon et al.2004) and a transcriptional target of p53 whose increasedexpression is associated with a variety of neurodegenerativeconditions (Caricasole et al. 2004; Cappuccio et al. 2005;Busceti et al. 2007; Zhang et al. 2008; Mastroiacovo et al.2009). The induction of DKK-1 expression and concomitantdecrease in Wnt/b-catenin signaling are thought to beresulted from Aß neurotoxicity and resulting cell cycleevents (Caricasole et al. 2004; Scali et al. 2006). Several

lines of evidence demonstrate the involvement of up-regulation of DKK-1 and its antagonistic effect on Wnt/b-catenin pathway in the pathological cascade underlying theonset and development of AD (De Ferrari and Inestrosa2000; Mudher et al. 2001; Caricasole et al. 2003; Boonenet al. 2009). A study reporting the association between LRP6variants and late-onset AD further supports the hypothesisthat altered Wnt/b-catenin signaling may be involved in thisneurodegenerative disease (De Ferrari et al. 2007). In threedifferent transgenic mouse AD-like models, which reproduceseveral features of AD in humans, and in a transgenic mousemodel of a human tauopathy, DKK-1 expression was foundto be increased in diseased brain areas. In the transgenicmouse AD-like models, increased DKK-1 expressionoccurred in brain areas most affected by amyloid depositionand NFT in AD, such as the II and III, and Vand VI layers of

(a)

(c) (d) (e)

(f)

(i) (j)

(g) (h)

(b)

Fig. 7 Dickkopf-1 (DKK-1) immunostaining

in the brainstem of 5- to 6-month-old P301S

mice. DKK-1 immunoreactivity in the trans-

genic P301S mouse (a) and in the age-

matched control (b). Note in the higher

magnification (original 40X) of the squared

area in (a) that DKK-1 staining in the

transgenic mice is mainly localized close to

the cell membrane of neurons. (c–h) Double

labeling immunofluorescence with DKK-1

(d,g, green) and NeuN (c, red) or AT8 (f,

red) antibodies, showing that DKK-1 is

localized along to the cell membrane of

NeuN positive neurons (e, merged), and

extended into the cytoplasm of some AT8

positive cells [arrow in (g) and merged (h),

yellow]. Scale bars: (a,b) 100 lm; (c–h)

25 lm. (i) Qualitative western blotting

analysis of DKK-1 (38 and 41 kDa) levels in

5/6-month-old transgenic and age-matched

wt mice. b-actin was used as a protein

loading control (43 kDa band). (j) Quanti-

tative analysis of protein levels showing a

statistically significant increase of DKK-1

expression in the brainstem of tg mice

compared with age-matched controls. Data

are presented as mean ± SEM (n = 4 per

group). One-way ANOVA plus Bonferroni’s

post hoc test: *p < 0.01.



the neocortex, in the hippocampus and in the basal forebrain.Double labeling experiments demonstrated the occurrence ofDKK-1 immunoreactivity within the ChAT-positive neuronsin the NBM of aged TgCRND8 mice, within PHF-positiveneurons in the cortex and hippocampus of TgCRND8 miceand within AT8-positive neurons in the brainstem and spinalcord of 5- to 6-month-old P301S mice. This is in accordancewith published data indicating that DKK-1 directly inducesneuronal death, tau phosphorylation, and reactive astrocyto-sis (Caricasole et al. 2004; Scali et al. 2006; Esposito et al.2008). Furthermore, double labeling experiments with Ab(1-42) and DKK-1 antibodies revealed DKK-1 staining in thecell body and processes of neurons surrounding Ab depositsof aged TgCRND8 mouse brain. DKK-1 staining was mainlydetected in the proximity of the cell membrane of neurons,consistent with an interaction with the Wnt co-receptorLRP5/6 as also demonstrated in TgCRND8 mouse brain. Noor only light and diffuse DKK-1 staining was detected in thecytoplasm in age-matched controls and in non-diseased 3-month-old transgenic P301S mice. Taken together theseresults indicate that DKK-1 undergoes distinct cytoplasm re-localization in diseased brain and suggest that the expressionof DKK-1 is associated with upstream disease events,including Ab production/accumulation/aggregation, and withdownstream events associated with tau hyperphosphorylationand neuronal death. Furthermore, in the diseased brain areasof TgCRND8 mice, immunoreactivity of active GSK-3 wasincreased and decreased nuclear translocation of b-cateninoccurred. In these brain areas, active GSK-3 was found to co-localize with DKK-1 and phospho-tau staining. Altogether,these data corroborate previous studies indicating impairedcanonical Wnt signaling as a component of AD pathology(Caruso et al. 2006; De Ferrari et al. 2007).

In the neocortex of TgCRND8 mice, DKK-1 positiveneurons were found in the Ab surroundings in specificneocortical areas, hippocampus, and basal forebrain areas,which are the most affected by amyloid deposition at7 months of age (Bellucci et al. 2006), suggesting that theAb deposition and the Ab-associated inflammatory reactionand nitrosative stress (Bellucci et al. 2006, 2007) may triggerthe induction of DKK-1. Moreover, DKK-1 expression is notexclusive for this tg mouse line since a few DKK-1-positiveneurons were also detected in the hippocampus and thecortex of Tg2576 and APP/PSEN1 mouse lines at 14-monthsof age, when Ab deposition – although to a lesser extent thanin TgCRND8 mice – and Ab-associated inflammation arealready present (Apelt et al. 2004; Noda-Saita et al. 2004).In the neocortex and hippocampus of aged TgCRND8 mice,both enlarged and atrophic DKK-1-positive neurons wereobserved and DKK-1 staining was detected within neuronsintensely stained by PHF-1 antibody, indicating that DKK-1expression occurs in phospho-tau bearing neurons.

We previously reported a significant loss of ChAT-positiveneurons in the NBM as well as a significant reduction in

cortical extracellular acetylcholine levels in 7-month-oldTgCRND8mouse brain (Bellucci et al. 2006). Here, we showthat in these transgenic mice, DKK-1 is expressed withinsome magnocellular ChAT-positive neurons of the NBM.Overall, these data indicate that a disruption of the canonicalWnt pathway might be involved in the degeneration of basalforebrain cholinergic neurons seen in AD brains.

Also in the 5/6-month-old P301S tg mice, in which no Abis present, DKK-1 positive neurons were abundant in thosebrain areas most affected by nerve cell degeneration. In thesetg mice, DKK-1 protein levels were significantly increased inthe brainstem, and the cell membranes of abnormallyenlarged neurons in the brainstem and in the spinal cordwere intensely immunoreactive for DKK-1 antibody, furtherpointing to a dysfunction of Wnt/b-catenin underlyingneurodegeneration. The fact that in the spinal cord ofP301S mice, the intense DKK-1 immunoreactivity was notparalleled by increased DKK-1 protein levels might be due tothe significant 49% reduction in the number of motorneurons, which was already previously reported for this tgmice (Allen et al.2002). Finally, our data demonstrate thatDKK-1 immunoreactivity along the cell membrane wasparticularly evident in the early onset and more aggressiveTgCRND8 and P301S mice models of neurodegenerativediseases than in Tg2576 and APP/PSEN1 mice, in whichneuropathological changes develop later. Taken together,these findings indicate that DKK-1 membrane re-localizationprogresses in parallel with Ab and tau deposition as part ofthe pathological cascade.

It is widely accepted that activation of the canonical Wntpathway leads to GSK-3b inhibition and prevents tauhyperphosphorylation (Moon et al. 2004; Balaraman et al.2006). In AD, disruption of the Wnt pathway leads to GSK-3b activation and tau hyperphosphorylation (Kaytor and Orr2002). It was also demonstrated that the Wnt pathway isdown-regulated in response to Ab and that inhibition ofGSK-3b protects neurons from Ab-induced damage (Inest-rosa et al. 2002). In addition, it was suggested that neuronsthat do not immediately die in response to Ab might developNFTs as a consequence of inhibition of Wnt signaling(Caricasole et al. 2003). In a fly model of human tauexpression, GSK-3b was shown to co-localize with PHF andto enhance neurodegeneration – via tau hyperphosphoryla-tion and PHF formation (Jackson et al. 2002). Furthermore,inducible over-expression of GSK-3b in Ca2+/Calmodulin-Dependent Protein Kinase II positive areas leads to decreasednuclear b-catenin levels, neurodegeneration and tau phos-phorylation (Lucas et al. 2001). In accordance, here we findthat active GSK-3 co-localizes with PHF-1 and DKK-1 inbrains of aged TgCRND8 mice. Activation of GSK-3b hasalso been suggested to play a role in the loss of thecholinergic system seen in AD (Hoshi et al. 1996). IncreasedGSK-3b activity is hypothesized to affect axonal transportthrough tau phosphorylation and, in turn, APP metabolism



and processing (Takashima et al. 1995). The functionsexerted by GSK-3a/b depend on the state of phosphorylation(Cole et al. 2004). GSK-3a/b are inactivated by phosphor-ylation at Ser21 and Ser9, respectively (Doble and Woodgett2003; Cohen and Goedert 2004). GSK-3b-catalyzed phos-phorylation of tau is regulated by other kinases and,probably, several phosphatases as well (Ferrer et al. 2005).In AD brain, both inactive and active forms of GSK-3b havebeen found in neurons with NFT and dystrophic neuritissurrounding b-amyloid plaques (Ferrer et al. 2002). How-ever, the role of GSK-3 in the pathological progression ofneurodegeneration in humans as well as in animal models isstill a matter of debate, which is very complex and notcompletely understood. Our immunohistochemistry resultsclearly demonstrate that in TgCRND8 mouse brain GSK-3(active form) staining is increased in the cell body anddendrites of PHF-1 and DKK-1-positive neurons in thosebrain areas which are the most affected in AD. As a‘downstream’ consequence of GSK-3 activation an increasedrate of phosphorylation and degradation of b-catenin occurs,which is thus no longer available for nuclear translocationand transcriptional regulation (Levina et al. 2004). Theresults of our work demonstrate a substantial neuronalreduction of b-catenin translocation to the nucleus in agedTgCRND8 mouse brain, indicating that neurons are deprivedof the trophic support provided by the gene programtriggered by b-catenin. Overall, these results associate theactivation of GSK-3 and the increased expression of DKK-1with an impaired Wnt/b-catenin function.

In conclusion, our findings demonstrate for the first timethat DKK-1 is expressed in transgenic AD-like mousemodels as well as in a transgenic model of tau aggregation.The brain areas where DKK-1 is expressed are those mostaffected by neuronal death in AD and frontotemporaldementia with P301S tau mutation, indicating DKK-1expression as a common marker of neuronal death inneurodegenerative diseases. Furthermore, DKK-1 co-local-izes with hyperphosphorylated tau and active GSK-3 and, inaddition, is present in the Ab surroundings in the AD-liketransgenic mouse model, thus indicating that DKK-1expression is associated with upstream and downstreamevents that lead to neuronal death in neurodegenerativediseases. Our results support that disruption of Wnt signalingis a critical pathological event in neurodegenerative diseasesand point to the Wnt inhibitor DKK-1 as a key mediator ofneurodegeneration with therapeutic potential.

Acknowledgements

This work was supported by a grant from the European Community

(ADIT, project LSHB-CT-2005-511977) and the UK Alzheimer’s

Research Trust (MGS). I.L., C.G., and A.F. are PhD students with an

ADIT fellowship. The authors would like to thank Dr P.St.G.

Hyslop and Dr D. Westaway for supplying TgCRND8 mice, Dr P.

Davies for kindly providing PHF-1 antibody, and Dr R. Roncarati

for her precious assistance in confocal microscopy.

Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Table S1. Mice used in the study.

Table S2. Antibodies employed in the study.

Figure S1. Histone H1 qualitative immunoblot analysis of

subcellular fractions from cortex and hippocampus of 7-month-old

wt and tg mice.

Figure S2. Representative photomicrographs showing DKK-1

staining in the hippocampus of Tg2576 and APP/PSEN1 transgenic

mice.

Figure S3. DKK-1 immunostaining in the spinal cord of 5-6-

month-old P301S mice.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such materials are

peer-reviewed and may be re-organized for online delivery, but are

not copy-edited or typeset. Technical support issues arising from

supporting information (other than missing files) should be

addressed to the authors.

References

Allen B., Ingram E., Takao M. et al. (2002) Abundant tau filaments andnonapoptotic neurodegeneration in transgenic mice expressinghuman P301S tau protein. J. Neurosci. 22, 9340–9351.

Apelt J., Bigl M., Wunderlich P. and Schliebs R. (2004) Aging-relatedincrease in oxidative stress correlates with developmental patternof beta-secretase activity and beta-amyloid plaque formation intransgenic Tg2576 mice with Alzheimer-like pathology. Int. J. Dev.Neurosci. 22, 475–484.

Balaraman Y., Limaye A. R., Levey A. I. and Srinivasan S. (2006)Glycogen synthase kinase 3beta and Alzheimer’s disease: patho-physiological and therapeutic significance. Cell. Mol. Life Sci. 63,1226–1235.

Bellucci A., Luccarini I., Scali C., Prosperi C., Giovannini M. G., Ca-samenti F. and Pepeu G. (2006) Cholinergic dysfunction, neuronaldamage and axonal loss in TgCRND8 mice. Neurobiol. Dis. 165,1643–1652.

Bellucci A., Rosi M. C., Grossi C., Fiorentini A., Luccarini I. and Ca-samenti F. (2007) Abnormal processing of tau in the brain of agedTgCRND8 mice. Neurobiol. Dis. 27, 328–338.

Boonen R. A., van Tijn P. and Zivkovic D. (2009) Wnt signaling inAlzheimer’s disease: up or down, that is the question. Ageing Res.Rev. 8, 71–82.

Busceti C. L., Biagioni F., Aronica E. et al. (2007) Induction of the Wntinhibitor, Dickkopf-1, is associated with neurodegeneration relatedto temporal lobe epilepsy. Epilepsia 48, 694–705.

Cappuccio I., Calderone A., Busceti C. L. et al. (2005) Induction ofDickkopf-1, a negative modulator of the Wnt pathway, is requiredfor the development of ischemic neuronal death. J. Neurosci. 25,2647–2657.

Caricasole A., Copani A., CarusoA., Caraci F., Iacovelli L., SortinoM.A.,TerstappenG.C. andNicoletti F. (2003) TheWnt pathway, cell-cycleactivation and beta-amyloid: novel therapeutic strategies in Alzhei-mer’s disease? Trends Pharmacol. Sci. 24, 233–238.

Caricasole A., Copani A., Caraci F., Aronica E., Rozemuller A. J.,Caruso A., Storto M., Gaviraghi G., Terstappen G. C. and Nicoletti



F. (2004) Induction of Dickkopf-1, a negative modulator of theWnt pathway, is associated with neuronal degeneration in Alz-heimer’s brain. J. Neurosci. 24, 6021–6027.

Caricasole A., Bakker A., Copani A., Nicoletti F., Gaviraghi G. andTerstappenG.C. (2005) Two sides of the same coin:Wnt signaling inneurodegeneration and neuro-oncology. Biosci. Rep. 25, 309–327.

Caruso A., Motolese M., Iacovelli L., Caraci F., Copani A., Nicoletti F.,Terstappen G. C., Gaviraghi G. and Caricasole A. (2006) Inhibitionof the canonical Wnt signaling pathway by apolipoprotein E4 inPC12 cells. J. Neurochem. 98, 364–371.

Chen Y., Stump R. J., Lovicu F. J. and McAvoy J. W. (2006) A role forWnt/planar cell polarity signaling during lens fiber cell differenti-ation? Semin. Cell Dev. Biol. 17, 712–725.

Chishti M. A., Yang D. S., Janus C. et al. (2001) Early-onset amyloiddeposition and cognitive deficits in transgenic mice expressing adouble mutant form of amyloid precursor protein 695. J. Biol.Chem. 276, 21562–21570.

Cohen P. and Goedert M. (2004) GSK3 inhibitors: development andtherapeutic potential. Nat. Rev. Drug Discov. 3, 479–487.

Cole A., Frame S. and Cohen P. (2004) Further evidence that the tyrosinephosphorylation of glycogen synthase kinase-3 (GSK3) in mam-malian cells is an autophosphorylation event. Biochem. J. 377,249–255.

De Ferrari G. V. and Inestrosa N. C. (2000) Wnt signaling function inAlzheimer’s disease. Brain Res. Brain Res. Rev. 33, 1–12.

De Ferrari G. V., Papassotiropoulos A., Biechele T. et al. (2007) Com-mon genetic variation within the Low-Density LipoproteinReceptor-Related Protein 6 and late-onset Alzheimer’s disease.Proc. Natl Acad. Sci. USA 104, 9434–9439.

Doble B. W. and Woodgett J. R. (2003) GSK-3: tricks of the trade for amulti-tasking kinase. J. Cell Sci. 116, 1175–1186.

Esposito G., Scuderi C., Lu J., Savani C., De Filippis D., Iuvone T.,Steardo L., Jr, Sheen V. and Steardo L. (2008) S100B induces tauprotein hyperphosphorylation via Dickopff-1 up-regulation anddisrupts the Wnt pathway in human neural stem cells. J. Cell Mol.Med. 12, 914–927.

Ferrer I., Barrachina M. and Puig B. (2002) Glycogen synthase kinase-3is associated with neuronal and glial hyperphosphorylated taudeposits in Alzheimer’s disease, Pick’s disease, progressivesupranuclear palsy and corticobasal degeneration. Acta Neuropa-thol. 104, 583–591.

Ferrer I., Gomez-Isla T., Puig B., Freixes M., Ribe E., Dalfo E. and AvilaJ. (2005) Current advances on different kinases involved in tauphosphorylation, and implications in Alzheimer’s disease andtauopathies. Curr. Alzheimer Res. 2, 3–18.

Glinka A., Wu W., Delius H., Monaghan A. P., Blumenstock C. andNiehrs C. (1998) Dickkopf-1 is a member of a new family ofsecreted proteins and functions in head induction. Nature 391,357–362.

Hoshi M., Takashima A., Noguchi K., Murayama M., Sato M., KondoS., Saitoh Y., Ishiguro K., Hoshino T. and Imahori K. (1996)Regulation of mitochondrial pyruvate dehydrogenase activity bytau protein kinase I/glycogen synthase kinase 3beta in brain. Proc.Natl Acad. Sci. USA 93, 2719–2723.

HsiaoK.,ChapmanP.,NilsenS.,EckmanC.,HarigayaY.,YounkinS.,YangF. andColeG. (1996)Correlativememory deficits, Ab elevation, andamyloid plaques in transgenic mice. Science 274, 99–106.

Inestrosa N., De Ferrari G. V., Garrido J. L., Alvarez A., Olivares G. H.,Barria M. I., Bronfman M. and Chacon M. A. (2002) Wnt sig-naling involvement in beta-amyloid-dependent neurodegeneration.Neurochem. Int. 41, 341–344.

Ishiguro K., Shiratsuchi A., Sato S., Omori A., Arioka M., Kobayashi S.,Uchida T. and Imahori K. (1993) Glycogen synthase kinase 3 beta

is identical to tau protein kinase I generating several epitopes ofpaired helical filaments. FEBS Lett. 325, 167–172.

Jackson G. R., Wiedau-Pazos M., Sang T. K., Wagle N., Brown C. A.,Massachi S. and Geschwind D. H. (2002) Human wild-type tauinteracts with wingless pathway components and produces neuro-fibrillary pathology in Drosophila. Neuron 34, 509–519.

Jankowsky J. L., Slunt H. H., Gonzales V., Jenkins N. A., Copeland N.G. and Borchelt D. R. (2004) APP processing and amyloid depo-sition in mice haplo-insufficient for presenilin 1. Neurobiol. Aging25, 885–892.

Kaytor M. D. and Orr H. T. (2002) The GSK3 beta signaling cascade andneurodegenerative disease. Curr. Opin. Neurobiol. 12, 275–278.

Lee V. M., Goedert M. and Trojanowski J. Q. (2001) Neurodegenerativetauopathies. Annu. Rev. Neurosci. 24, 1121–1159.

Levina E., Oren M. and Ben Ze’ev A. (2004) Downregulation of beta-catenin by p53 involves changes in the rate of beta-catenin phos-phorylation and Axin dynamics. Oncogene 23, 4444–4453.

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

Mastroiacovo F., Busceti C. L., Biagioni F., Moyanova S. G., MeislerM. H., Battaglia G., Caricasole A., Bruno V. and Nicoletti F. (2009)Induction of the Wnt antagonist, Dickkopf-1, contributes to thedevelopment of neuronal death in models of brain focal ischemia.J. Cereb. Blood Flow Metab. 29, 264–276.

Meacci E., Nuti F., Catarzi S., Vasta V., Donati C., Bourgoin S., Bruni P.,Moss J. and Vaughan M. (2003) Activation of phospholipase D bybradykinin and sphingosine 1-phosphate in A549 human lung ade-nocarcinoma cells via different GTP-binding proteins and proteinkinase C delta signaling pathways. Biochemistry 42, 284–292.

Moon R. T., Kohn A. D., De Ferrari G. V. and Kaykas A. (2004) WNTand beta-catenin signalling: diseases and therapies. Nat. Rev.Genet. 5, 691–701.

Mudher A., Chapman S., Richardson J. et al. (2001) Dishevelled regu-lates the metabolism of amyloid precursor protein via protein ki-nase C/mitogen-activated protein kinase and c-Jun terminal kinase.J. Neurosci. 21, 4987–4995.

Nelson W. J. and Nusse R. (2004) Convergence of Wnt, beta-catenin,and cadherin pathways. Science 303, 1483–1487.

Noda-Saita K., Terai K., Iwai A., Tsukamoto M., Shitaka Y., KawabataS., Okada M. and Yamaguchi T. (2004) Exclusive association andsimultaneous appearance of congophilic plaques and AT8-positivedystrophic neurites in Tg2576 mice suggest a mechanism of senileplaque formation and progression of neuritic dystrophy in Alz-heimer’s disease. Acta Neuropathol. 108, 435–442.

Scali C., Caraci F., Gianfriddo M. et al. (2006) Inhibition of Wnt sig-naling, modulation of Tau phosphorylation and induction of neu-ronal cell death by DKK1. Neurobiol. Dis. 24, 254–265.

Selkoe D. J. (2001) Alzheimer’s disease results from the cerebralaccumulation and cytotoxicity of amyloid beta-protein. J. Alzhei-mer’s Dis. 3, 75–80.

Takashima A., Yamaguchi H., Noguchi K., Michel G., Ishiguro K., SatoK., Hoshino T., Hoshi M. and Imahori K. (1995) Amyloid betapeptide induces cytoplasmic accumulation of amyloid proteinprecursor via tau protein kinase I/glycogen synthase kinase-3 betain rat hippocampal neurons. Neurosci. Lett. 198, 83–86.

Yu X. and Malenka R. C. (2003) Beta-catenin is critical for dendriticmorphogenesis. Nat. Neurosci. 6, 1169–1177.

Zhang Q. G., Wang R., Khan M., Mahesh V. and Brann D. W. (2008)Role of Dickkopf-1, an antagonist of the Wnt/beta-catenin sig-naling pathway, in estrogen-induced neuroprotection and attenua-tion of tau phosphorylation. J. Neurosci. 28, 8430–8441.



Documents

Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease