Neuroscience 256 (2014) 360–369

HUMAN APOLIPOPROTEIN E4 MODULATES THE EXPRESSION OF PIN1,SIRTUIN 1, AND PRESENILIN 1 IN BRAIN REGIONS OF TARGETEDREPLACEMENT apoE MICE

F. LATTANZIO, a L. CARBONI, a* D. CARRETTA, a

R. RIMONDINI, b S. CANDELETTI a AND P. ROMUALDI a

aDepartment of Pharmacy and Biotechnology, Alma Mater

Studiorum University of Bologna, via Irnerio 48, 40126 Bologna, Italy

bDepartment of Medical and Clinical Science, Alma Mater

Studiorum University of Bologna, via Irnerio 48, 40126 Bologna, Italy

Abstract—The apolipoprotein E4 (apoE4) allele is consis-

tently associated with increased risk for Alzheimer’s disease

(AD). We investigated the molecular mechanism of this sus-

ceptibility by analyzing the levels of genes involved in AD

pathogenesis in transgenic mice expressing human apoE3

or apoE4 isoforms. mRNA and protein levels of Pin1, Sirtuin

1 (Sirt1), Presenilin 1 (PS1), and pro-Brain-derived Neurotro-

phic Factor (BDNF) were analyzed in brain regions affected

by neuropathological changes in AD. Pin1 mRNA was sig-

nificantly higher in the hippocampus of apoE4 mice than in

apoE3 controls, whereas lower expression was detected in

the entorhinal and parietal cortices. Reduced Pin1 levels

may increase neurofibrillary degeneration and amyloido-

genic processes, while compensatory mechanisms may

take place in the hippocampus to balance spatial memory

deficits. Sirt1 levels were significantly reduced in the frontal

cortex of apoE4 mice. Sirt1 reduction may hinder its protec-

tive role against the formation of plaques and tangles and

diminish its anti-inflammatory actions. Sirt1 decrease may

also play a role in apoE4-associated memory impairments.

Moreover, in apoE4 mice PS1 mRNA levels were lower in

the frontal cortex. Lower PS1 expression may hamper

c-secretase function, thus affecting amyloid precursor

protein processing. Pro-BDNF mRNA levels did not differ

between apoE3 and apoE4 mice in any region analyzed. This

study showed dysregulated expression of Pin1, Sirt1, and

PS1 genes in different cerebral areas of apoE4 mice,

suggesting that these changes may play a role in the

mechanism of AD vulnerability. � 2013 IBRO. Published by

Elsevier Ltd. All rights reserved.

0306-4522/13 $36.00 � 2013 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2013.10.017

*Corresponding author. Address: Department of Pharmacy andBiotechnology, Alma Mater Studiorum University of Bologna, viaIrnerio 48, 40126 Bologna, Italy. Tel: +39-051-2091793; fax: +39-051-2091780.

E-mail address: lucia.carboni4@unibo.it (L. Carboni).Abbreviations: AD, Alzheimer’s disease; apoE, apoliporpotein E; apoETR mice, human apoE3- and apoE4-targeted replacement mice; APP,amyloid precursor protein; Ab, amyloid beta peptide; BDNF, Brain-derived Neurotrophic Factor; DDCt, Delta–Delta Ct; EDTA,ethylenediaminetetraacetic acid; FoxO, Forkhead box protein O; LDL,low-density lipoprotein; NF-jB, Nuclear factor j B; NT, neurofibrillarytangles; PS1, Presenilin 1; RT-qPCR, reverse transcription quantitativereal-time polymerase chain reaction; S.E.M., standard error of themean; Sirt1, Sirtuin 1.

360

Brain-derived Neurotrophic Factor, Pin1, Presenilin 1,

Key words: apolipoprotein E, Alzheimer’s disease,

Sirtuin 1.

INTRODUCTION

Alzheimer’s disease (AD) is themost prevalent form of age-

related dementia in modern society. Current estimates

indicate that about 25–30 million people suffer from AD in

the world, and the number of cases will double during the

next 20 years (Ferri et al., 2005). The principal

pathological hallmarks of AD are abundant extracellular

senile plaques of amyloid beta peptide (Ab) in cerebral

blood vessels and brain parenchyma, deriving from the

cleavage of amyloid precursor protein (APP), and

intraneuronal neurofibrillary tangles (NT), resulting from

aggregation of hyperphosphorylated tau microtubule-

associated protein (Ballard et al., 2011; Selkoe, 2011).

AD is a multifactorial disease characterized by the

interaction between genetic and environmental risk factors.

The cholesterol-carrying apolipoprotein E4 (apoE4) allele is

the strongest and most consistently associated risk gene

for late-onset AD, also confirmed in genome-wide

association studies (Harold et al., 2009). The most

common allele apoE3 is associated with intermediate risk

of developing AD; in contrast, apoE4 frequency correlates

with increased risk and earlier onset of AD (Liu et al.,

2013). ApoE4 carriers experience increased Ab deposition,

decreased Ab clearance, enhanced AD symptoms,

accelerated age-dependent cognitive decline, and worse

memory performances (Corder et al., 1993; Liu et al., 2013).

The molecular mechanism of the association between

apoE4 and AD has not been yet elucidated. Several

hypotheses have been put forward, involving differential

effects on synaptic plasticity and synaptogenesis,

contribution to conversion of Ab to fibrillar forms, and

increased tau phosphorylation (Hauser et al., 2011).

The human apoE3- and apoE4-targeted replacement

(apoE TR) mice have been generated to study the role

of apoE genotypes in neurodegenerative processes

related to AD. In apoE TR mice, mouse apoE coding

sequences were replaced by those encoding for human

apoE3 or apoE4 isoforms, under the control of mice

regulatory sequences (Sullivan et al., 1997).

Deregulation of protein phosphorylation may contribute

to age-related pathologies, including AD. Recent studies

suggest that dysfunctions of the ubiquitous protein

peptidyl-prolyl cis/trans isomerase Pin1 may play a role

d.

F. Lattanzio et al. / Neuroscience 256 (2014) 360–369 361

in neurodegenerative diseases (Pastorino et al., 2006).

Pin1 interacts with phosphorylated serine or threonine

preceding proline motifs (pSer/Thr-Pro), promoting cis/

trans isomerization of the peptide bond and increasing

the accessibility for dephosphorylation by phosphatases.

This conformational change modulates catalytic activity,

phosphorylation status, stability, and localization of

several proteins. Therefore, Pin1 plays a regulatory role

in many biological processes, such as cell cycle, cell

growth, protein degradation, and neuronal differentiation

(Liou et al., 2011). Growing evidence supports the

notion that Pin1 provides an important contribution to

the development of AD. In healthy neurons, Pin1 binds

to phosphorylated tau and APP, accelerating their cis to

trans isomerization that facilitates tau dephosphorylation

and promotes non-amyloidogenic pathways of APP

processing. Reduced Pin1 levels or activity may reverse

these pathways, causing accumulation of

phosphorylated tau that may lead to NT formation, and

promoting the amyloidogenic pathway of APP that may

enhance plaque pathology (Balastik et al., 2007).

Sirtuin 1 (Sirt1) is a Nicotinamide Adenine

Dinucleotide+-dependent histone deacetylase involved in

the regulation of many cellular processes, such as cellular

stress resistance, genomic stability, tumorigenesis, and

energy metabolism (Finkel et al., 2009). Recent data

support a protective role of Sirt1 in aging and AD, possibly

through attenuation of APP amyloidogenic processing

(Bonda et al., 2011) and reduced Ab deposition (Donmez

et al., 2010). Furthermore, lower Sirt1 mRNA and protein

levels were detected in the parietal cortex of AD patients,

suggesting association with disease progression (Julien

et al., 2009). Sirt1 may also exert protective effects by

preventing inflammation. In fact, Sirt1 activation protected

cultured neurons against Ab toxicity by inhibiting

Ab-stimulated nuclear factor jB (NF-jB), signaling in

microglia (Chen et al., 2005). Although Sirt1 activation

exerts a protective role against neurodegeneration, it has

been shown that also Sirt1 inhibition seems to have

neuroprotective effects (Li et al., 2008).

Presenilin 1 (PS1) is the active catalytic component of

the c-secretase complex responsible for APP cleavage

into Abs of different lengths (De Strooper et al., 1998).

Mutations in PS1 have been associated to most cases

of familial AD. Disease-associated PS1 mutations

increased the ratio of Ab42 peptides, shown to be more

prone to amyloid fibril formation with respect to Ab40(Tanzi and Bertram, 2005). Reduced PS1 protein levels

have been reported in hippocampus and brain cortex of

AD patients (Davidsson et al., 2001).

In addition to their role in brain development and

differentiation, neurotrophins are also involved in

synaptic plasticity by translating synaptic activity signals

into structural changes in neurons. At the same time,

synaptic dysfunction possibly produced by Ab is an

early event in AD, appearing before senile plaques and

NT. Indeed, accumulating evidence suggests an

association between decreased levels of the

neurotrophin Brain-derived Neurotrophic Factor (BDNF)

and AD pathogenesis (Arancio and Chao, 2007;

Tapia-Arancibia et al., 2008).

The objective of this work was to study the molecular

mechanisms of apoE4 predisposing effects to AD. To this

aim, the apoE TR mice were investigated, since this

model allows a comparison of the consequences of

bearing the human high-risk allele versus the normal,

more diffused isoform in a background of physiological

localization. We assessed the expression levels of Pin1,

Sirt1, PS1, and pro-BDNF, as these molecules are

believed to play relevant roles in AD pathophysiology.

mRNA and protein levels were analyzed in brain

regions affected by neuropathological changes in AD

(Dickerson and Sperling, 2009; Selkoe, 2011; Jacobs

et al., 2012).

EXPERIMENTAL PROCEDURES

Animals

Human apoE TR transgenic mice were purchased by

Taconic Farms (Hudson, NY, USA). Mice were housed

in groups of six in ventilated cages (Tecniplast, Varese,

Italy) with free access to water and food in controlled

conditions of light (from 7.00 a.m. to 7.00 p.m.),

temperature (22 ± 2 �C) and humidity (65%). The

studies were performed in 18-month-old male mice.

Experiments were carried out in accordance with the

European Communities Council Directive of 24

November 1986 (86/609/EEC) and National Ministry of

Health laws and policies (authorization no. 139/2012-B)

in the Department of Pharmacy and Biotechnology of

the University of Bologna. Procedures received approval

by the local Ethics Committee and animal comfort was

monitored by the University Veterinary Service. All

efforts were made to reduce the number of animals and

minimize animal suffering.

Sample preparation

Mice were rapidly killed by cervical dislocation. Brains

were removed and hippocampus, entorhinal, parietal,

and frontal cortices were quickly dissected out, frozen in

dry ice and stored at �80 �C until use for RNA or

protein extraction.

Reverse transcription quantitative real-timepolymerase chain reaction (RT-qPCR)

Total RNA was extracted according to the method

described by Chomczynski and Sacchi (1987) with

10 vol of TRI Reagent solution containing phenol and

guanidine thiocyanate (Ambion, Life Technologies Italia,

Monza, Italy). Total RNA was dissolved in 25 ll of

RNase-free water, digested with RNase-free DNase, and

quantified by absorbance. RNA purity was confirmed by

a ratio value OD260/OD280 > 2 and integrity was

assessed by 1% agarose electrophoresis. RNA samples

were converted to cDNA with the GeneAmp RNA PCR

kit (Applied Biosystems, Foster City, CA, USA) by using

random hexamers (0.45 lg total RNA in 20 ll final

reaction volume). cDNAs were subsequently threefold

diluted in Nuclease-free water. Relative abundance of

each mRNA species was assessed by real-time RT-PCR

362 F. Lattanzio et al. / Neuroscience 256 (2014) 360–369

in 2 ll of the diluted cDNA samples in 20 ll final volume,

using TaqMan Gene expression Master Mix (Taqman

probes: Pin1: Mm00777269_mH; Sirt1: Mm00490758_m1;

PS1 Mm00501184_m1; GAPDH: Mm99999915_g1;

Applied Biosystems, Foster City, CA, USA) or SYBR

Green PCR (pro-BDNF: forward primer: GCGGCAGATA

AAAAGACTGC; reverse primer CCTATGAATCGCCAG

CCAAT; GAPDH: forward primer: AACTTTGGCATTG

TGGAAGG; reverse primer: ACACATTGGGGGTAG

GAACA; primers from Eurofins, Italy) on a StepOne

Detection System (Applied Biosystems). To provide

quantification, the point of product accumulation in the

early logarithmic phase of the amplification plot was

defined by assigning a fluorescence threshold above the

background, defined as the threshold cycle (Ct) number.

Differences in threshold cycle numbers were used to

quantify the relative amount of PCR target contained

within each well. Relative expression of different gene

transcripts was calculated by the Delta–Delta Ct (DDCt)

method and converted to relative expression ratio

(2�DDCt) for statistical analysis (Livak and Schmittgen,

2001). All data were normalized to the endogenous

reference gene glyceraldehyde-3-phosphate dehydro-

genase (GAPDH) expression. In SYBR Green PCRs,

a dissociation curve was built in the 60–95 �C range

to evaluate amplification product specificity. Samples

were run in triplicate and data were analyzed

using StepOne Software v2.2 (Applied Biosystems).

Data were normalized to gene expression levels of

control groups.

Immunoblotting

Dissected brain regions were homogenized by sonication

on ice in lysis buffer [50 mM Tris HCl pH 7.5, 0.4% NP-40,

10% glycerol, 150 mM NaCl, 10 mM EDTA, 1 mM sodium

orthovanadate, 100 mM NaF (reagents from Sigma–

Aldrich, St. Louis, MO, USA), Complete mini EDTA-free

protease inhibitor cocktail (from Roche Applied Science,

Monza, Italy)]. Homogenates were centrifuged (13,600g,30 min, 4 �C) and protein levels were quantified in

supernatants using the bicinchoninic acid protein assay

kit (Pierce, Rockford, IL, USA). 40–50 lg proteins were

separated in 8–16% gradient polyacrilamide precast

gels (Pierce Rockford, IL, USA) in a Miniprotean

tetracell apparatus (Bio-Rad, Hercules, CA, USA),

transferred on nitrocellulose membranes, blocked in 5%

skimmed milk in 0.1% Tween20 Tris-buffered saline,

and incubated with primary antibodies, peroxidase-

conjugated secondary antibodies and revealed and

quantified by enhanced chemiluminescence (Pierce) in a

ChemiDoc instrument (Bio-Rad). Antibodies were

purchased from Sigma–Aldrich (monoclonal anti-BDNF,

1:500, Jin et al., 2005), Millipore (Billerica, MA, USA,

polyclonal anti-Sirt1, 1:1000, Sasaki et al., 2008;

polyclonal anti-Pin1 1:1000, Stanya et al., 2008;

monoclonal anti-PS1, 1:1000, De Gasperi et al., 2010;

monoclonal anti-GAPDH 1:2000), Amersham

Biosciences (Little Chalfont, UK, anti-rabbit

Immunoglobulin G 1:2000; anti-mouse Immunoglobulin

G 1:2000). In some instances, membranes were

stripped using RestoreTM Western Blot Stripping buffer

(Pierce) at room temperature for 15 min.

Data analysis

Results were expressed as mean ± S.E.M. (standard

error of the mean). The data were analyzed using a

2-way analysis of variance (ANOVA) approach, with

genotype and brain region as treatment factors. This

was followed by Planned Comparisons on the predicted

means to compare the levels of the selected effect.

Differences were considered statistically significant at

p< 0.05. Statistical analyses were performed with

InVivoStat software (Clark et al., 2012).

RESULTS

Pin1 expression levels

The level of Pin1 mRNA was significantly higher in the

hippocampus of apoE4 mice (1.28 ± 0.09 versus

control group apoE3 = 1.00 ± 0.05, p= 0.020,

Fig. 1A). In contrast, apoE4 genotype was associated

with significantly lower Pin1 expression in the entorhinal

cortex (0.75 ± 0.05 vs. apoE3 = 1.00 ± 0.05,

p= 0.036, Fig. 1A) and in the parietal cortex

(0.68 ± 0.10 vs. apoE3 = 1.00 ± 0.11, p= 0.010,

Fig. 1A). No significant difference between genotypes

was observed in the frontal cortex. Protein levels

measured with immunoblottings did not show significant

changes between groups (Fig. 1B, C).

Sirt1 expression levels

In apoE4 mice, the levels of Sirt1 mRNA in the frontal

cortex were significantly lower in comparison with

respective apoE3 controls (0.80 ± 0.04 vs.apoE3 = 1.00 ± 0.04, p= 0.013, Fig. 2A). No

differences were revealed in the other brain areas

examined. Protein levels did not display statistically

significant variations (Fig. 2B).

PS1 expression levels

Similar to Sirt1, PS1 mRNA levels were significantly lower

in frontal cortex of apoE4 mice (0.77 ± 0.05 vs.

apoE3 = 1.00 ± 0.07, p= 0.005, Fig. 3A). No

differences between genotypes were detected in the

other brain regions. Protein levels did not change

significantly (Fig. 3B).

pro-BDNF expression levels

No statistically significant differences between the levels

of pro-BDNF mRNA were detected in any brain region

of apoE4 versus apoE3 mice (Fig. 4A). No statistically

significant differences were detected in protein levels

(Fig. 4B).

DISCUSSION

The present study was aimed at gaining insight in the

molecular mechanism of the robust and consistent

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Hip EntCx ParCx FrCx

Pin1

apoe3

apoe4

*

* *

Pin1

GAPDH

apoE3 apoE4

Hippocampus

28

17

kDa

38

Pin1

GAPDH

apoE3 apoE4

Entorhinal Cortex

28

17

kDa

38

A

B C Fig. 1. (A) Pin1 relative gene expression (mRNA) levels were determined by real-time PCR in the hippocampus, entorhinal cortex, parietal cortex,

and frontal cortex of apoE3 and apoE4 transgenic mice. Results are expressed as the amount of Pin1 mRNA normalized to GAPDH endogenous

expression compared to control values (apoE3). Data represent mean ± S.E.M. calculated by the DDCt method. ⁄p< 0.05 (n= 6). Detection of

Pin1 protein levels by immunoblotting in hippocampus (B) or entorhinal cortex (C).

F. Lattanzio et al. / Neuroscience 256 (2014) 360–369 363

association between the apoE4 isoform and late-onset

AD by investigating alterations of gene and protein

expression in brain regions vulnerable to AD-related

neurodegeneration. Although the association between

apoE4 and AD is well-demonstrated, the presence of the

genotype is not necessary or sufficient for bringing about

the disease; therefore several hypotheses have been

proposed to explain the molecular mechanism of its

facilitating effect (Bu, 2009; Kim et al., 2009; Zhong and

Weisgraber, 2009; Huang, 2010; Hauser et al., 2011; Liu

et al., 2013). In apoE TR mice, alternative human isoforms

replaced mice apoE while maintaining endogenous murine

apoE promoter elements (Sullivan et al., 1997). Therefore,

this model allows examining the specific effect of the

disease-associated isoform when expressed at similar

levels and brain localization as in non-demented humans

(Sullivan et al., 1997; Sullivan et al., 2004). TR mice carrier

of the human apoE4 genotype show moderate cognitive

deficits that mainly entail impairments in spatial memory

performances. Deficits are already apparent in young

animals and worsen at later ages (Grootendorst et al.,

2005; Bour et al., 2008; Reverte et al., 2012; Rodriguez

et al., 2013). Impaired memory performances take place in

the absence of neuropathological markers of AD, since no

signs of gliosis, amyloid deposition or NT are observed in

these mice (Wang et al., 2005; Hunter et al., 2012). ApoE3

and apoE4 mice show similar brain Ab1–42 levels, and

only slightly increased levels of Ab1–40 in hippocampus

and cortex of apoE4 carriers (Hunter et al., 2012).

Therefore, it has been suggested that apoE TR mice may

represent a model of apoE4-dependent susceptibility to

cognitive decline (Wang et al., 2005; Bour et al., 2008;

Klein et al., 2010). In apoE4 mice, memory impairments

are associated with dysfunctions in neural plasticity (Levi

et al., 2003), including reduced magnitude of hippocampal

long-term potentiation (Trommer et al., 2004) and deficient

cholinergic modulation (Yun et al., 2005). Synaptic deficits

were also described in excitatory transmission of the lateral

amygdala and both regions are critical for memory

formation (Wang et al., 2005). Anatomical and molecular

dysregulations that may play a role in apoE4 cognitive

deficits were described in the brain cortex, with apoE4 TR

mice showing reduced spine density and dendritic

arborization in lateral amygdala and brain cortex,

particularly at older ages (Wang et al., 2005; Dumanis

et al., 2009).

In brain, apoE is mainly synthesized and released by

astrocytes (Vance and Hayashi, 2010). Its functions are

accomplished by binding to specific receptors belonging

to the low-density lipoprotein (LDL) receptor

superfamily, including LDL receptors, LDL-receptor-

related protein 1, very low-density lipoprotein receptors,

and apolipoprotein E receptor-2, which are located in

neurons and glia (Vance and Hayashi, 2010; Hauser

et al., 2011). In addition to the crucial role as lipid

carrier, apoE is involved in the modulation of other

important physiological functions. Signal transduction

cascades are activated in consequence to receptor

0

0.2

0.4

0.6

0.8

1

1.2

Hip EntCx ParCx FrCx

Sirt1

apoe3

apoe4

*

Sirt1

GAPDH

apoE3 apoE4

Frontal Cortex

188

98

kDa

38

A

B Fig. 2. (A) Sirt1 relative gene expression (mRNA) levels were determined by real-time PCR in the hippocampus, entorhinal cortex, parietal cortex,

and frontal cortex of apoE3 and apoE4 transgenic mice. Results are expressed as the amount of Sirt1 mRNA normalized to GAPDH endogenous

expression compared to control values (apoE3). Data represent mean ± S.E.M. calculated by the DDCt method. ⁄p< 0.05 (n= 6). (B) Detection

of Sirt1 protein levels by immunoblotting in the frontal cortex.

364 F. Lattanzio et al. / Neuroscience 256 (2014) 360–369

binding, ultimately influencing calcium signaling, long-

term potentiation, neurite growth, brain development,

neuronal survival, and modulation of inflammatory

responses (Vance and Hayashi, 2010; Hauser et al.,

2011; Zhang et al., 2011). It is documented that apoE3

and apoE4 isoforms are able to activate the receptors in

a different manner and that they specifically affect the

modulated cellular functions, possibly because of

diverse structural characteristics (Bu 2009; Hauser

et al., 2011; Verghese et al., 2011; Liu et al., 2013).

Therefore, it is conceivable that the altered gene

expression levels revealed in this study rely on the

distinct impact on signal transduction pathways. In

particular, apoE can reduce inflammatory responses by

blocking the activation of NF-jB (Singh et al., 2008).

Moreover, compelling evidence suggests that signal

transduction systems downstream of apoE-activated

receptors include the Phosphoinositide 3-kinase/Akt/

Forkhead box protein O (FoxO) pathway (Beffert et al.,

2002; Shen et al., 2011). Since putative regulatory

sequences for NF-jB are present in the promoter

region of Pin1 and PS1, and FoxO-recognized

sequences exist in the promoter region of both Sirt1 and

PS1 (http://www.genecards.org/), it is tempting to

speculate that the modulation of these genes rely on the

different influence on transduction pathways exerted by

apoE3 versus apoE4 isoforms.

We demonstrated that in 18-month-old apoE4 mice,

Pin1 levels were increased in the hippocampus and

reduced in entorhinal and parietal cortices as compared

to apoE3 controls. Available evidence sustains an

association between reduced Pin1 levels and AD. In AD

brains, Pin1 binds to phosphorylated tau in NT, resulting

in depletion of soluble Pin1, reducing phosphorylated

tau binding to microtubules to promote microtubule

assembly and ultimately contributing to neuronal loss

(Lu et al., 1999). In normal brains, Pin1 expression

inversely correlates with neuronal vulnerability, and in

AD-affected brains it inversely correlates with

neurofibrillary degeneration. In mice, Pin1 deletion

brings about age-dependent neuropathological changes,

including tau hyperphosphorylation, tau filament

formation and neuronal degeneration (Liou et al., 2003).

In addition, it has been demonstrated that Pin1

deregulation can also contribute to plaque pathology,

since reduced Pin1 levels increase amyloidogenic APP

processing and elevate insoluble Ab42 (Pastorino et al.,

2006). Nevertheless, Pin1 function may vary during the

disease, since it has been reported that in the frontal

cortex of mild-cognitive impairment and AD patients,

Pin1 protein levels and activity increased with respect to

controls, suggesting that compensatory mechanisms

may also take place (Wang et al., 2007). In analogy, it

is possible that the increased Pin1 levels in the

0

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1

1.2

Hip EntCx ParCx FrCx

Ps1

apoe3

apoe4

**

Ps1

GAPDH

apoE3 apoE4

Frontal Cortex

28

17

kDa

38

A

B Fig. 3. (A) PS1 relative gene expression (mRNA) levels were determined by real-time PCR in the hippocampus, entorhinal cortex, parietal cortex,

and frontal cortex of apoE3 and apoE4 transgenic mice. Results are expressed as the amount of PS1 mRNA normalized to GAPDH endogenous

expression compared to control values (apoE3). Data represent mean ± S.E.M. calculated by DDCt method. ⁄⁄p< 0.01 (n= 6). (B) Detection of

PS1 protein levels by immunoblotting in the frontal cortex.

F. Lattanzio et al. / Neuroscience 256 (2014) 360–369 365

hippocampus shown in this study may try to counteract

the harmful effects of the apoE4 genotype on spatial

memory, a function largely relying on this brain region.

In summary, although a large body of data supports the

involvement of Pin1 in AD neuropathology, its molecular

mechanisms are not fully understood (Balastik et al.,

2007; Lu and Zhou, 2007). Since a correlation between

apoE4 genotype and regulation of Pin1 levels in brain

has not been demonstrated before, our results show for

the first time that Pin1 dysregulation may occur in an

apoE4 expression model in which no AD-related

neuropathological changes are observed. These

findings suggest that apoE4 genotype may contribute to

AD susceptibility also by modulating Pin1 expression

levels in brain regions relevant for AD-related

neurodegeneration. In keeping with these findings,

previous data showed that in the peripheral blood

mononuclear cells of late-onset AD patients, Pin1

expression was significantly increased, whereas a

reduction in Pin1 phosphorylation and promoter

methylation was observed. Interestingly, the lowest level

of Pin1 gene promoter DNA methylation was associated

to apoE4 carriers (Arosio et al., 2012).

We found reduced Sirt1 levels in apoE4 mice frontal

cortex as compared to apoE3 controls. The concept that

Sirt1 may exert a protective role in AD is based on its

potential role in diminishing the formation of plaques

and NT. APP is alternatively processed through

a- followed by c-secretases to soluble, neuroprotective

segments, or by b- and c-secretases that produce toxic

Ab species. The competition between these two

pathways may be modulated by Sirt1, which helps

switching the balance toward the non-amyloidogenic

direction (Qin et al., 2006; Donmez et al., 2010).

Moreover, Sirt1 may also protect neurons from

neurofibrillary degeneration by promoting proteasome-

mediated degradation of pathogenic forms of tau (Min

et al., 2010). An anti-inflammatory mechanism that

could reduce Ab toxicity due to microglia activation may

play an additional role in Sirt1 neuroprotective action

(Chen et al., 2005). Here, we show for the first time that

reduced expression of this histone deacetylase is

observed in association with the apoE4 genotype.

Therefore, Sirt1 reduction may constitute part of the

mechanism for

apoE4-induced susceptibility to AD, possibly also by

interfering with its activity on epigenetic regulations. It is

conceivable that in this model Sirt1 reduced expression

may bring about (or at least be a factor in) memory

impairments demonstrated in apoE4 TR mice, since

accumulating findings show that Sirt1 modulates

synaptic plasticity and memory formation (Gao et al.,

0

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1

1.2

1.4

Hip EntCx ParCx FrCx

Pro-BDNF

apoe3

apoe4

GAPDH 38

Pro-BDNF 38

apoE3 apoE4

Hippocampus

kDa

B

A

Fig. 4. (A) pro-BDNF relative gene expression (mRNA) levels were determined by real-time PCR in the hippocampus, entorhinal cortex, parietal

cortex, and frontal cortex of apoE3 and apoE4 transgenic mice. Results are expressed as the amount of pro-BDNF mRNA normalized to GAPDH

endogenous expression compared to control values (apoE3). Data represent mean ± S.E.M. calculated by DDCt method (n= 6). (B) Detection of

pro-BDNF protein levels by immunoblotting in the hippocampus.

366 F. Lattanzio et al. / Neuroscience 256 (2014) 360–369

2010; Michan et al., 2010) and that alterations of

Sirt1 mRNA levels correspond to some kind of memory

deficits induced by high-fat diet (Heyward et al., 2012).

In apoE4 mice we detected lower PS1 gene

expression in frontal cortex as compared to apoE3

mice. It is well established that families harboring

mutated versions of the gene encoding for non-

functional forms of PS1 are associated with AD, thus

indicating the important role of the c-secretase pathway

for APP processing (Cacquevel et al., 2012). Our

findings support a further correlation with the other most

important genetic risk factor, since data show that the

human apoE4 isoform induces a reduction of PS1 levels

that may impact on c-secretase function. Although

plaques and NT are not produced in apoE TR mice, the

association between apoE4 genotype and worst

neuropathological outcomes has been demonstrated in

double transgenic mice lines in which apoE TR mice

have been crossed with models that show AD-like

neuropathological degenerations (e.g. Holtzman et al.,

2000; Buttini et al., 2002; Fryer et al., 2005; Bales et al.,

2009; Kim et al., 2011; Fitz et al., 2012; Youmans et al.,

2012). Reduced PS1 mRNA levels may contribute to

the enhanced susceptibility to neurodegeneration

associated with the apoE4 genotype, due to hampered

c-secretase function.

It has been proposed that decreased neurotrophic

function may be associated with AD pathogenesis and

may represent an avenue for future therapeutic

strategies. BDNF could interfere with Ab deposition,

play a neuroprotective role against its neurotoxicity or

could decrease tau phosphorylation, thus ultimately

reducing NT formation (Fumagalli et al., 2006; Tapia-

Arancibia et al., 2008; Zhang et al., 2012). Prompted by

these findings, we examined whether altered BDNF

levels could be revealed in apoE4 mice. We could not

detect significant differences between genotypes, in

keeping with findings obtained in younger animals

(Maioli et al., 2012; Reverte et al., 2012).

The region-specific gene expression modulations

revealed in this study may be associated with previously

reported region-specific changes, in agreement with the

different susceptibility to AD-related neuropathological

degeneration. ApoE4 TR mice show a decrease in apoE

protein levels which is especially massive in the

hippocampus as compared to the brain cortex, although

absolute apoE levels are higher in the former region in

all genotypes (Riddell et al., 2008; Kim et al., 2011;

Sullivan et al., 2011; Hunter et al., 2012; Maioli et al.,

2012). In apoE4 mice, dysregulations of region-specific

functions are also observed, such as reduced LTP

amount and decreased neurogenesis that specifically

F. Lattanzio et al. / Neuroscience 256 (2014) 360–369 367

take place in the hippocampus. Morphological anomalies

show region-specific patterns as well, such as reduced

spine density that appears in the cortex and amygdala,

whereas the hippocampus exhibits normal dendrite

shape and number (Dumanis et al., 2009).

In the present study, although differential mRNA levels

for Pin1, Sirt1, and PS1 were demonstrated in brain

regions primarily affected during AD neuropathology,

parallel modulations of protein expression did not

overlap with gene expression changes. In AD patients,

different regulations for mRNA and protein levels were

reported for Pin1 in peripheral blood cells (Arosio et al.,

2012). Nevertheless, a possible alternative explanation

for these findings may be related to the different

sensitivity of the methodological techniques. Indeed,

whereas RT-qPCR is a quantitative method allowing the

detection of fine modulations, which are endowed with

physiological significance in the regulation of brain

functions, immunoblotting is a semi-quantitative method

that is not most suitable to appreciate small variations

that may go undetected due to type 2 errors.

Nevertheless, it is also possible that different

degradation cycle times do not consent the variation in

mRNA levels to be reflected in different protein

amounts. Whether the observed regulation was

generated at the level of mRNA synthesis, stabilization

or degradation was not addressed in this investigation.

CONCLUSION

Our study demonstrates for the first time that the apoE4

genotype modulates brain mRNA levels of Pin1, Sirt1,

and PS1 differently in the distinct cerebral areas,

suggesting that dysregulated expression of these genes

may play a role in the mechanism of AD vulnerability

induced by this genotype.

Acknowledgement—Financial support for this work was provided

by grants from the University of Bologna (RFO10 to SC and

RFO11 to PR).

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(Accepted 9 October 2013)(Available online 23 October 2013)