Gene-environment interaction research and transgenic mouse models of Alzheimer's disease.

Abstract

The etiology of the sporadic form of Alzheimer's disease (AD) remains largely unknown. Recent evidence has suggested that gene-environment interactions (GxE) may play a crucial role in its development and progression. Whereas various susceptibility loci have been identified, like the apolipoprotein E4 allele, these cannot fully explain the increasing prevalence of AD observed with aging. In addition to such genetic risk factors, various environmental factors have been proposed to alter the risk of developing AD as well as to affect the rate of cognitive decline in AD patients. Nevertheless, aside from the independent effects of genetic and environmental risk factors, their synergistic participation in increasing the risk of developing AD has been sparsely investigated, even though evidence points towards such a direction. Advances in the genetic manipulation of mice, modeling various aspects of the AD pathology, have provided an excellent tool to dissect the effects of genes, environment, and their interactions. In this paper we present several environmental factors implicated in the etiology of AD that have been tested in transgenic animal models of the disease. The focus lies on the concept of GxE and its importance in a multifactorial disease like AD. Additionally, possible mediating mechanisms and future challenges are discussed.

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SAGE-Hindawi Access to Research
International Journal of Alzheimer’s Disease
Volume 2010, Article ID 859101, 27 pages
doi:10.4061/2010/859101
Review Article
Gene-EnvironmentInteraction ResearchandTransgenicMouse
Modelsof Alzheimer’sDisease
L.Chouliaras,1A.S.R.Sierksma,1G.Kenis,1J.Prickaerts,1M.A.M.Lemmens,1I.Brasnjevic,1
E.L.vanDonkelaar,1P. Martinez-Martinez,1M. Losen,1M. H.De Baets,1N.Kholod,1
F.vanLeeuwen,1P.R.Hof,2J. vanOs,1,3H.W.M. Steinbusch,1D.L.A.vanden Hove,1,4
and B. P. F. Rutten1
1School for Mental Health and Neuroscience (MHeNS), Faculty of Health, Medicine and Life Sciences, European Graduate School of
Neuroscience (EURON), Maastricht University Medical Centre, P.O. Box 616, 6200 MD Maastricht, The Netherlands
2Department of Neuroscience, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
3Division of Psychological Medicine, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK
4Department of Psychiatry, Psychosomatics and Psychotherapy, University of W¨ urzburg, 97080 W¨ urzburg, Germany
Correspondence should be addressed to B. P. F. Rutten, b.rutten@np.unimaas.nl
Received 29 May 2010; Accepted 31 July 2010
Academic Editor: A. I. Bush
Copyright © 2010 L. Chouliaras et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The etiology of the sporadic form of Alzheimer’s disease (AD) remains largely unknown. Recent evidence has suggested that gene-
environment interactions (GxE) may play a crucial role in its development and progression. Whereas various susceptibility loci
have been identified, like the apolipoprotein E4 allele, these cannot fully explain the increasing prevalence of AD observed with
aging. In addition to such genetic risk factors, various environmental factors have been proposed to alter the risk of developing
AD as well as to affect the rate of cognitive decline in AD patients. Nevertheless, aside from the independent effects of genetic and
environmental risk factors, their synergistic participation in increasing the risk of developing AD has been sparsely investigated,
even though evidence points towards such a direction. Advances in the genetic manipulation of mice, modeling various aspects
of the AD pathology, have provided an excellent tool to dissect the effects of genes, environment, and their interactions. In this
paper we present several environmental factors implicated in the etiology of AD that have been tested in transgenic animal models
of the disease. The focus lies on the concept of GxE and its importance in a multifactorial disease like AD. Additionally, possible
mediating mechanisms and future challenges are discussed.
1.Introduction
Alzheimer’s disease (AD) is the most common form of
dementia, characterized by an initial loss of short-term
memory, followed by a progressive impairment in multiple
cognitivedomains.Theestimatedlifetimeriskfordeveloping
AD is about 20% for women and 10% for men aged
above 65 [1]. The pathology of AD is characterized by an
accumulation of misfolded proteins, oxidative damage, and
inflammatory changes ultimately resulting in region-specific
loss of synaptic contacts and neuronal cell death [2]. Current
biologicaltheoriesontheetiologyandpathologyofADposit
central roles for age-related molecular and cellular aberra-
tions that induce an imbalance in the production, cleavage,
and clearance of amyloid-β (Aβ), hyperphosphorylation of
the tau protein, and aberrant apolipoprotein E (APOE)
function in the aging brain [1]. Several genetic risk factors
have been linked with an increased risk of developing AD,
such as mutations in the amyloid precursor protein (APP)
and presenilin (PS) 1 and 2 for the familial cases of AD
(FAD), as well as the APOE4 allele for the sporadic late-onset
form of AD (LOAD). Several new genetic findings derived
from powerful genome-wide association studies (GWAS; see
below) have confirmed that AD is a polygenic disorder. The
genes identified in these studies may enlighten unknown
biological pathways involved in AD [3].
Furthermore, various environmental exposures have
been found to modify the risk of AD, such as diet and

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2International Journal of Alzheimer’s Disease
nutrition, physical exercise, exposure to metals, and brain
trauma. Comorbidities, such as vascular disorders or depres-
sion, could also be of considerable importance, since these
have also been suggested to contribute to the risk of AD.
Recent evidence indicates that more attention should be
paid to the role of the environment and its interactions
with underlying genetic susceptibility in triggering disease-
related phenotypes [4]. The gene-environment interaction
(GxE) approach differs from the linear approach of either
genetic or environmental effects by positing a causal role
not only for either genes or environmental exposures in
isolation, but for their synergistic participation in leading
to a certain phenotype (here AD), where the effect of one
is conditional for the other [5–7]. Where epidemiological
studies on AD may reveal statistical evidence for GxE in the
onset and course of AD, animal research can be instrumental
in studying the underlying biological mechanisms.
1.1. Objective. The objective of this review is to give an
overview of the available transgenic mouse studies on AD,
specifically addressing the concept of GxE. We start with a
brief description of the various genetic and environmental
risk factors of AD, and the different available transgenic
mouse models of AD. The main part of the paper describes
the effects of several environmental exposures on AD-related
phenotypes. These sections begin with a brief description of
the epidemiological evidence in AD (when available from
meta-analyses) and continue with describing the findings
from experimental animal studies in which the environmen-
tal factor was manipulated in AD transgenic mice and, when
performed, in wild-type (WT) mice. Thereafter, we discuss
the strengths and limitations of these studies, and we end
with identifying future challenges and prospects.
2.Alzheimer’s Disease
2.1. Genetics of AD. Twin studies on AD have shown a heri-
tability of 60%–80% and a concordance of 18% up to 83%,
depending on for example, the population and age of the
subjects investigated. Thus, both heritable and nonheritable
factors play an important role in AD’s age of onset, risk
and etiology [8–11]. Several genetic risk factors have been
linked to AD. Mutations in APP, PS1, and PS2 genes have
consistently been associated with early-onset FAD. Also for
LOAD several susceptibility loci have been linked with risk
for AD, such as the gene encoding for the APOE4 allele
or loci in the clusterin (CLU), phosphatidylinositol binding
clathrin assembly protein (PICALM), complement receptor
1 (CR1), BIN1 (bridging integrator, amphiphysin) genes, a
locus near the BLOC1S3 (biogenesis of lysosomal organelles
complex1, subunit 3), and MARK4 (microtubule affinity-
regulating kinase 4) genes [3, 12–14]. Other susceptibility
loci have also been associated with AD (see [12, 15],
http://www.alzgene.org/).
2.2. Environment and AD. Although a range of environ-
mental exposures have been linked to AD, well-replicated
and meta-analyses’ evidence for the involvement of clear
environmental factors in AD is sparse. Recent studies,
however,haveshownthatdietaryfactors,suchasexposureto
a Mediterranean diet, fish and high omega-3 diets, cigarette
smoking, head trauma, infections, systemic inflammation,
and metal and pesticide exposure can significantly alter an
individual’s risk of developing AD. In addition, psychosocial
factors such as education, social network, leisure activities
and physical activity, chronic stress, and depression also
seem to be connected to the risk of developing AD [16–
18]. Somatic factors that are under the direct influence of
environmental exposures, such as blood pressure, obesity,
diabetes mellitus, cardio- and cerebrovascular diseases, and
hyperlipidemia, have additionally been implicated in AD
etiology [16, 18].
2.3. Gene-Environment Interactions and AD. The field of
GxE research appears very promising for psychiatry and
neuroscience, albeit still little investigated in AD [19]. The
notion of potential existence of GxE in AD has substantial
impact on the interpretation of reports on genetic and non-
genetic contribution to this disorder. Reported contributions
of environmental and genetic factors to disease risk can be
misleading, since they represent the environmental exposure
in relationship with the genetic susceptibility or resilience to
it [6]. Thus, the advantage of the concept of GxE is that it
includesthegeneticcontrolofsensitivitytotheenvironment.
Additionally, the genome-wide genetic findings identify
associations that also include underlying GxE [6]. In fact,
evidence for GxE in AD has recently started to accumulate.
For example, an interaction between the APOE4 allele and
cholesterol levels has been shown to increase the risk of AD
[20, 21]. Significant statistical interactions were also found
between moderate consumption of alcohol and the APOE4
genotype, as well as for smoking and the APOE4 genotype
[22, 23]. Furthermore, an interaction with this risk genotype
and social factors, such as cohabiting with a partner has been
found; APOE4 carriers who lost their partner before midlife
showed an increased risk of developing AD, compared to
married or cohabiting people [24].
These epidemiological studies indicate that it makes
sense to focus future clinical AD studies on measuring both
genes and environment and analyzing possible interactions,
given that certain environmental factors may only affect a
phenotype when the person is genetically endowed. A major
drawback of epidemiological clinical studies is that they
may indicate merely statistical interactions and thus cannot
easily decipher the biological mechanisms that underlie the
observed statistical interactions. Other major obstacles in
clinical studies are the heterogeneity of the study population
and co-occurrence of various environmental exposures in
the same individuals. Experimental animal research has the
advantage of enabling strict control of genetic and envi-
ronmental variables. Recent advances in transgenesis allow
altering specific genesin isolation, and in a time- and region-
specific manner. As such, transgenic mice form a useful tool
to study the effects of genetic and environmental variations
and to identify the biological mechanisms that underlie
the statistical GxE interactions observed in epidemiological
studies (see Figure 1).

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International Journal of Alzheimer’s Disease3
Genes Environment
G×E
CLU
PICALM
CRI
PS2
APP
PS1
APOE4
Tau
Chronic stress
Environmental
enrichment
Metals
Head trauma
EMF
Diet
Behavioral endophenotypes and AD-like pathology
Figure 1: Research in Alzheimer’s disease (AD) uses both clinical
(human) and preclinical (mouse) methods to elucidate the under-
lying mechanisms of AD etiology. Epidemiological findings such as
genetic and environmental risk factors can provide tools for inves-
tigating their effects on AD etiology separately in mouse models of
AD. In this paper it is, however, postulated that AD research should
move towards a gene-environment (GxE) interaction approach, so
that the synergistic participation of genes and environment can be
scrutinized. Genes in the dashed box represent those genes found
to be implicated with Alzheimer’s disease etiology in humans, while
genes in the solid box resemble the genes that are currently used
in mouse models of Alzheimer’s disease. APOE4: Apolipoprotein
ε4; APP: amyloid precursor protein; CLU: clusterin; EMF: elec-
tromagnetic field; PICALM: phosphatidylinositol-binding clathrin
assembly protein; PS1: Presenilin 1; PS2: Presenilin 2.
2.4. Transgenic Mouse Models of AD. Without the intention
of giving a full overview of the available AD mouse models,
some details on the types of transgenic mice that are
discussed in the present paper can be found in Table 1.
Information on the promoters used for the transgenic
construct, and further details on genetic background are not
further discussed here as these aspects lie outside the scope
of this paper.
ItisnoteworthythattheAβsequenceofWTrodentshasa
three amino acid difference compared to humans, making it
lesslikelytoaggregateanddepositintoamyloidplaques[25].
Therefore, to study Aβ aggregation and plaque formation
in rodents it is necessary to manipulate them genetically
[25].Mosttransgenicmousemodelsfocusonoverexpressing
human APP, PS1, tau, or APOE variants.
3.ChronicStress
3.1.HumanStudiesofChronicStress. Chronicstresshasbeen
implicated in the etiology of AD. The likelihood of develop-
ing AD has been shown to increase by a factor 2.7 with the
personality trait distress proneness [25, 48, 49]. Moreover,
AD patients show elevated plasma cortisol levels [50, 51]
with higher levels of plasma cortisol being associated with
a more rapid disease progression and cognitive deterioration
[51, 52].
Sustained elevated levels of glucocorticoids can cause
volumetric and dendritic changes in the hippocampus of
rats, mice, and tree shrews [53–56], decrease neurogenesis,
and impair long-term potentiation [53, 57, 58]. It has, there-
fore, been proposed that alterations in HPA-axis functioning
might also contribute to the etiology of AD [59–61].
Evidence from studies over the last 20 years indicates that
major depression may serve as a risk factor for developing
AD[62–69].Alifetimehistoryofdepressiveepisodesdoubles
thechanceofdevelopingAD[70].Interestingly,patientswith
major depression show a cerebrospinal fluid (CSF) profile
of Aβ-species that resembles the profile seen in AD. They
display decreased levels of Aβ42 and a decreased Aβ40:Aβ42
ratio [71], which are considered putative biomarkers for AD
[72]. In addition, the severity of depression correlated with
bindingof2-(1-{6-[(2-{18F}Fluoroethyl)(methyl)amino]-2-
naphthyl}ethylidine)malononitrile, also known as FDDNP,
a tracer that binds to plaques and tangles, in the temporal
lobe [73]. Moreover, more plaques and tangles in the
hippocampus as well as a more rapid cognitive decline have
beenobservedinADpatientswithalifetimehistoryofmajor
depression compared to patients without such history [74].
Incontrast,othershavesuggestedthatmajordepressiondoes
notfunctionasanindependentriskfactorforAD,butshould
merely be viewed as an AD prodrome [63, 75, 76].
3.2. Animal Studies of Chronic Stress. Several paradigms have
been used to model the effects of chronic stress in mouse
models of AD. The paradigms that have been applied most
frequently are chronic isolation stress and chronic restraint
or immobilization stress. Table 2 summarizes the current
evidence for effects of stress exposure in transgenic mouse
models of AD.
Chronic isolation stress by subjecting mice to either 3, 5,
or 6 months of social isolation from weaning, has thus far
only been used in the Tg2576 mouse model of AD [77–79].
This resulted in elevated levels of soluble Aβ40 and Aβ42 up
to 59% and increased plaque deposition in the hippocampus
and the neocortex [77, 78]. Moreover, this stress exposure
paradigm caused a rise in basal plasma corticosterone levels,
paralleled with an increased expression of the glucocorticoid
receptor (GR) and corticotropin-releasing factor (CRF)
receptor 1 [78]. In addition, impaired contextual memory
and decreased cell proliferation in the hippocampal dentate
gyrus was observed. Interestingly, the effects of isolation
stressonmemorydeficitsandcellproliferationinthedentate
gyrus could be prevented by a 14-day treatment of fluoxetine
[77].
Another widely used stress paradigm is restraint stress.
Acute short-term restraint stress elevated intracerebral inter-
stitial Aβ levels in Tg2576 mice [79] and stress-induced
corticosterone release in APPswe mice [81]. Administering
CRF or a CRF-antagonist indicated that the interstitial
rise in Aβ depended on CRF levels [79]. Acute restraint
stress furthermore resulted in a 175% increase in blood
glucose levels in APPswe mice, suggesting a wide impact on
metabolism [81].

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4International Journal of Alzheimer’s Disease
Table 1: Transgenic mouse models of Alzheimer’s disease with reported environmental effects.
NameMutation BackgroundEffect
Intraneuronal Aβ at 3
months, extracellular at 6,
hippocampal
hyperphosphorylated tau
pathology at 12 months,
synaptic dysfunction
Ref.
3xTg
Injection of APPswe and tauP301L
transgenes in PS1M146V knock-in mice
129/C57BL6
[26]
Aβ PPswe
Carrying the mutant AβPPK670N,
M671L gene
Mixed background of
56.25% C57, 12.5% B6,
18.75% SJL, and 12.5%
Swiss-Webster
Amyloid deposition and
cognitive decline starting at
the age of 8 months
[27]
APOE3, APOE4
APOE knockout mice carry an
inactivated APOE endogenous gene
disrupted by gene targeting in
embryonic stem cells. Human APOE
genomic DNA fragments injected in
single cell emryos fertilized by APOE
knockout mice
cDNA of human APP with the Swedish
double mutation at positions 670/671
combined with the V717I mutation,
inserted to the blunt ended XhoI site of
the expression cassette containing the
murine Thy 1.2 gene
Swedish (KM670/671NL) and London
(V717I) mutation under control of
Thy1 promotor
Human Aβ coding sequence knocked-in
to the endogenous APP gene, combined
with the Swedish (K670N/M671L)
mutation
Double knock-in mouse: APPNLh/NLh
crossed with PS1 P264L knock-in, using
Cre-lox knock-in technology and
endogenous promoters
APOE knockout and
C57BL6
Expression of human APOE
in the brain, high cholesterol
levels
[28]
APP23
C57BL6
Aβ deposition in the
neocortex and hippocampus
at the age of 6 months
[29]
APP715SLCBA/C57BL6
Amyloid plaque deposits at 6
months of age
[30]
APPNLh/NLh
129/Sv
No Aβ depositions, but a
9-fold increase in human Aβ
production compared to
normal human Aβ levels
Increase of Aβ42 levels,
amyloid deposition and
reactive gliosis by 4 months
of age.
Increased Aβ production
and Aβ deposition at 5–7
months of age, decrease in
synaptophysin
immunoreactivity at 2–4
months of age
Amyloid plaque deposition,
cholinergic marker decrease,
memory deficits at 6 months
of age
[31]
APP/PS1 KI
CD-1/129
[32, 33]
APPswe/ind
Expressing human APP with Swedish
mutation (K670N/M671L) and the
V717I Indiana mutation under the
PDGF promoter (J20 line)
C57BL6 × DBA/2
[34]
APPswe/PS1ΔE9
Cross of APPswe and PS1ΔE9
(expressing human PS1 carrying the
exon 9 deleted variant)
C57BL6J
[35]
APPswe/PS1Leu235Pro
APP swedish mutation crossed with
mutant human PS1 Leu235Pro
C3H/HeJ/C57BL/A2G—[36, 37]
APPswe/PS1M146L
Tg2576 combined with PS1 (M146L)
mutation (under PDGF promoter)
C57/B6/SJL/Swiss
Webster
Compared to Tg2576, 41%
increase in Aβ42 which
precedes fibrillar Aβ deposits
in cerebral cortex and
hippocampus. Reduced
spontaneous alternation
performance in the Y-maze.
[38]
APPV717I-C100
Expressing the C-terminal 100 amino
acid of human APP with 717 London
mutation
C57BL6
Intracellular accumulation of
soluble Aβ
[39]

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International Journal of Alzheimer’s Disease5
Table 1: Continued.
NameMutation
The entire human APP gene inserted to
the yeast artificial chromosome (YAC)
B142F9, introduced to embryonic stem
cell by lipofection
BackgroundEffect Ref.
APP-YAC
C57BL6
Significant human APP
expression in the cerebral cortex
[40]
PDAPP
Indiana mutation (V717F) with portions
of APP introns 6–8, driven by the PDGF
promoter
Extracellular Aβ deposits in the
hippocampus from the age of 6
months and neocortex from 8
months of age
[41]
PS1-L286V
Overexpressing human PS1 with L286
mutation under the control of human
PDGF-β promoter
FVB/N
Aβ42 intracellular deposits at 13
months of age
[42]
TASTPM
Carrying human APPswe and PS1 M146V
mutations
C57BL63H
Cerebral Aβ deposition and
cognitive deficits at 6 months of
age
[43]
Tg19959
TgCRND8 mice plus M146L + L286V PS1
transgene in the hamster PrP gene
promoter
Human APPswe (double K670N, M671L)
inserted to hamster prion protein
promoter (PrP) (is also known and
referred to in the text as APPswe)
Expressing the C-terminal 100 amino acid
of human APP (with or without 717
London mutation)
Swedish and Indiana (V717F) APP
mutations
C57/C3H/129SvEv/
Tac/FVB
Amyloid deposits at 1 month of
age
[44]
Tg2576
C57BL6
5-fold increase in Aβ40 and 14
fold increase in Aβ42, behavioral
deficits, amyloid plaques at 9
months of age
[45]
TgC100C57BL6
Intracellular accumulation of
soluble Aβ
[39]
TgCRND8
C57/C3H/129SvEv/
Tac/FVB
Plaques at 3 months of age,
increased Aβ42/40 ratio
Hyperphosphorylated tau
aggregates in the hippocampus,
neurodegeneration, reduced
hippocampal neural activity and
behavioral abnormality
[46]
TgV337M
V337M longest tau, cDNA inserted to the
PDGFβ-chain expression vector
B6SJL
[47]
Chronic restraint stress has so far been performed in 3
different mouse models of AD: APPV717I-C100, Tg2576,
and PS1-L286V mice. Applying chronic restraint stress to
APPV717I-C100 and Tg2576 mice generally resulted in an
increased Aβ plaque load, increased Aβ40 and Aβ42 levels,
increased tau phosphorylation and increased basal plasma
corticosteronelevels[82–84].Chronicrestraintstressapplied
to APPV717I-CT100 mice additionally induced cognitive
impairment as measured for example by using cued food,
that is, powdered chow mixed with a certain aroma, in the
social transfer of food preference task [82]. Chronic restraint
stress has also been associated with neuropathological alter-
ations in AD mouse models. PS1-L286V mice exposed to
chronic restraint stress displayed elevated numbers of degen-
eratingneuronsandadecreasednumberofproliferatingcells
in the hippocampus as compared to nonexposed mice [84].
Chronic restraint stress in APPV717I-CT100 mice caused
elevated numbers of pyknotic cells in the hippocampus [82]
and reduced dendritic arborization of cortical neurons in
Tg2576 mice [83].
Another method to assess the effects of stress is by
mimickingthephysiologicalstressresponsebyadministering
synthetic glucocorticoids, such as dexamethasone, for 7
days. Application of this approach in 3xTg mice resulted in
elevatedAβ-andtau-immunoreactivityinthehippocampus,
amygdala and neocortex and increased levels of insoluble
Aβ40 and Aβ42 and total APP, β-site of APP cleaving enzyme
(BACE1)andAPPfragmentC99levelsinbrainhomogenates
[85].
4.EnvironmentalEnrichment
4.1. Human Studies of Environmental Enrichment. A reduced
riskfordevelopingandaslowerrateofcognitivedeclinehave
been observed in people having a greater purpose in life and
higher levels of physical activity [86, 87].
4.2. Animal Studies of Environmental Enrichment. In the
field of animal research, the environmental enrichment
(EE) paradigm is frequently used to manipulate physical
activity and social interactions. By introducing mates (social
interaction) and/or toys (physical activity) into the cage
of the rodent [88], this paradigm stimulates cognition
as well as sensory and motor behavior with concomitant
intracerebral cellular and molecular changes [89, 90]. To
examine the effect of EE in AD several different paradigms
have been imposed on various mouse models of AD.

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6International Journal of Alzheimer’s Disease
Table 2: Effects of stress exposure in transgenic mouse models of Alzheimer’s disease.
Mouse modelExposure
Duration of the
experiment
Age at the start Effects on the brain
Effects on
behavior
Reference
Tg2576
Chronic
isolation stress
3 monthsFrom weaning
↑ soluble Aβ40 (38%) and Aβ42
(59%) in hippocampus, no change
in Aβ40:Aβ42 ratio, no difference
in APP, α- or β-CTF levels, no
changes in IDE, NEP (neprilepsyn)
or APOE levels
Not measured[79]
Tg2576
Chronic
isolation stress
5 monthsFrom weaning
↑ Aβ plaques
↓ proliferation in DG
↓ contextual
memory at 6
months
[77]
Tg2576
Chronic
isolation stress
6 monthsFrom weaning
↑ Aβ40 + Aβ42 levels and plaque
deposition in neocortex and
hippocampus
↑ expression of GR and CRFR1 in
neocortex and hippocampus
↑ basal corticosterone in plasma
No changes in basal corticosterone
levels
↓ soluble Aβ40 levels in the frontal
cortex + hippocampus
↓ insoluble Aβ42 levels in frontal
cortex + hippocampus, no
difference in endocannabinoid
levels in frontal cortex and
hippocampus
Not measured[78]
TASTPM
Repeated novel
cage exposure
(1h/day,
4x/week)
5 weeks4 months
No difference
in locomotion,
nor in anxiety
levels
contextual
memory ↑
[80]
APPswe
Acute restraint
stress (for 4h)
4 hours 19 months
↑ 175% in blood glucose levels,
dropping to below basal values 2
hours after restraint
↑ in stress-induced corticosterone
release
Not measured [81]
Tg2576
Acute restraint
stress (for 3h)
3 hours 3-4 months
↑ interstitial fluid Aβ, no difference
in APP or β-CTF levels
↓ α-CTF levels, no changes in IDE,
NEP (neprilysin) or APOE levels
Not measured[79]
APPV717I-CT100
Chronic
immobilization
stress (6h/day,
4x/week)
8 months3 months
↑ Aβ plaques in hippocampus,
entorhinal + piriform cortex
↑ APP-CTFs
↑ pyknotic cells in hippocampus +
entorhinal cortex
↑ phospho-tau in CA3 + entorhinal
cortex
↑ Corticosterone in plasma
↑ cognitive
impairment
[82]
Tg2576
Chronic
immobilization
stress (6h/day,
4x/week)
6 months 3 monthsNot measured
↓ cued food
preference
[82]
Tg2576
Chronic
restraint stress
(2h/day)
16 days14 months
↑ Aβ plaques in hippocampus, PFC,
cingulate, motor, parietal and
piriform cortex
↑ Aβ40 + Aβ42 in cortical
homogenates
↑ immunoreactive astrocytes near
plaques
↑ phospho-tau
↓ dendritic arborization of cortical
neurons
↓ MMP-2 (Aβ-degrading enzyme)
↑ basal corticosterone levels
Not measured[83]

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International Journal of Alzheimer’s Disease7
Table 2: Continued.
Mouse modelExposure
Duration of the
experiment
Age at the startEffects on the brain
Effects on
behavior
Reference
PS1-L286V
Chronic
restraint stress
(6h/day)
3 or 15 weeks 7 weeks
3weeks exposure
↓ body weight
↑ adrenal gland weight
↑ corticosterone levels in plasma
↑ number of degenerating neurons
in DG, CA3, and retrosplenial
cortex, no effect on number of
granule neuron precursors (Pax6)
or proliferating cells (Ki67) in DG
and/or SGZ
↓ BrdU-positive cells
↑ DCX-positive neuronal
progenitor cells
15weeks exposure
↓ body weight
↑ adrenal gland weight
↑ number of degenerating neurons
in DG, CA3 and retrosplenial
cortex, no effect on # of granule
neuron precursors or proliferating
cells in DG and/or SGZ
↑ Aβ in hippocampus, neocortex,
amygdala
↑ tau in dendrites and axons in
hippocampus, neocortex, amygdala
↑ insoluble Aβ40 and Aβ42
↑ total APP, BACE1, C99 levels
↑ basal corticosterone levels from 9
months on
Not measured[84]
3xTg
Dexamethasone
administration
(1 or 5mg/kg)
i.p.
7 days4 months
Not measured[85]
Table 3 summarizes the effects of EE in transgenic mouse
models of AD.
4.2.1. APP Mice. EE, in terms of housing multiple mice in a
larger cage with platforms, running wheels, toys, and other
novel habitats, for a period of 6 months improved cognition
in a battery of tests such as Morris water maze (MWM),
circular platform, platform recognition and radial arm water
maze, despite signs of stable Aβ deposition in 16-month-
old APPswe mice [91]. EE for 4 months in 5-month-old
TgCRND8micedidnotsignificantlyaltersolublelevelsofAβ
in the brain or the blood, but did enhance mRNA expression
of angiogenic genes [92]. EE in this mouse model attenuated
age-related reductions in cell proliferation, neurogenesis and
synaptic plasticity [93], while the same paradigm in another
laboratory elevated Aβ plaque load without compromising
behavioral phenotypes such as feeding and drinking pattern,
grooming, locomotion or cognition [94, 105]. In an attempt
to disentangle the exact components of EE that influence
phenotypes in APP mutant mice, Wolf et al. [96] exposed
APP23 transgenic mice to either an enriched environment
or unlimited access to a running wheel and compared both
conditions with standard housing. EE had differential effects
upon improving performance in the MWM as compared
to the increased physical activity and standard housing
groups, however, no differential effects on plaque load in the
neocortex or hippocampus were found [95, 96]. Moreover,
miceexposedtoEEexhibitedsignsofincreasedhippocampal
neurogenesis and neurotrophic support [95, 96].
4.2.2. APP/PS1 Mice. When comparing social interaction
andphysicalactivity,differentialeffectsofEEcanbeobserved
on learning and memory processes, Aβ plaque load and
synaptophysin immunoreactivity of 9-month-old APP/PS1
transgenic mice [97]. EE in APPswe/PS1ΔE9 mice reduced
cortical and hippocampal Aβ deposition with mice being
more active in the running wheel showing an even more
marked decrease in Aβ [98]. Furthermore, EE in PS1/PDAPP
mice attenuated cognitive impairments [99].
Possibly in contrast with overt beneficial effects, 2-
month-old female APPswe/PS1ΔE9 mice exposed to EE
for several months displayed increased Aβ levels in the
neocortex, and hippocampus [100]. After further backcross-
ing these mice to a C57Bl6 background strain in order
to attain fewer genetic background differences, the same
group demonstrated that EE in 2-month-old transgenic
APPswe/PS1ΔE9 female mice, attenuated cognitive deficits
[101], but still exhibited a 25% increase in Aβ deposits in
cortical and hippocampal brain regions [101]. One could
arguethatenhancedsecretionanddepositionoftoxicsoluble
Aβ species (scavenging the toxic species into packages away

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8International Journal of Alzheimer’s Disease
Table 3: Environmental enrichment in transgenic transgenic mouse models of Alzheimer’s disease.
Mouse modelExposure
Duration of
the exposure
Age at the
start
Effect on brainEffect on behavior Reference
APPswe
Enriched housing
(multiple mice in a large
bin containing an inner
cage with platforms,
passageways, running
wheels, toys, and novel
habitats) + novel
complex environment 3x
weekly for several hours
Enriched housing
(equipped with diverse
physically and
cognitively stimulating
objects, for example,
gnawing wood, tunnels,
balls, running wheels,
and ladders)
Enriched housing
(equipped with diverse
physically and
cognitively stimulating
objects, for example,
gnawing wood, tunnels,
balls, running wheels,
and ladders)
4 months16 months
No differences in total
Aβ load
Improved MWM
performance
[91]
TgCRND8
4 months1 month
↑ angiogenesis
↑ ApoE, LRP1, A2M
↓ RAGE
Not measured[92]
TgCRND8
4 months1 month
↑ BrdU-positive cells
↑ synaptophysin
immunoreactivity in
the hippocampus
Not measured[93]
TgCRND8
Enriched housing
(plastic inset, wooden
climbing frame, and a
nesting material)
4 months1 month Not measured
↑ exploratory behavior
↓ anxiety-related
behavior
No effects in learning
and memory,
as assessed with MWM,
barrier test, open-field,
elevated plus maze,
object recognition task,
and Barnes maze
[94]
APP23
Enriched environment
(multiple mice housed
in large cages with a
rearrangeable system of
plastic tubes and
cardboard boxes)
1 months6, 18 months
No differences in plaque
load
↑ DCX/CR ratio
↑ DCX- and
calretinin-positive
neurons in the
hippocampus
No differences in plaque
load in neocortex or
hippocampus
↑ hippocampal
neurogenesis (DCX,
calretinin)
↑ BDNF, NT-3 in the
hippocampus
↓ total Aβ deposition,
28% in the
hippocampus and 36%
in entorhinal cortex
↑ synaptophysin in CA1
and CA3
[95]
APP23
Enriched housing
(spacious cage equipped
with a rearrangeable
system of tubes, a
cardboard box house,
wire mesh ladders, and a
crawling ball)
9 months2 months
Improved learning and
memory in MWM
[96]
APPswe/PS1ΔE9
Enriched housing
(multiple mice in a large
cage with crawl-tubes,
platforms, running
wheels and toys,
changed weekly)
6 months 1.5 month
Improved performance
in MWM, RAWM
[97]

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International Journal of Alzheimer’s Disease9
Table 3: Continued.
Mouse model Exposure
Duration of
the exposure
1 month, 3
hours daily,
next 4 months
three times per
week
Age at the
start
Effect on brainEffect on behaviorReference
APPswe/PS1ΔE9
Enriched environment
(large cages, running
wheels, colored
tunnels, toys, and
chewable material)
Enriched housing
(multiple mice in a
large bin containing an
inner cage with
platforms,
passageways, running
wheels, toys, and novel
habitats) + novel
complex environment
3/weekly for several
hours
Enriched housing
(larger cages with
running wheels, plastic
play tubes, cardboard
boxes, and nesting
material that were
changes or rearranged
weekly)
Enriched housing
(larger cages with
running wheels, plastic
play tubes, cardboard
boxes, and nesting
material that were
changes or rearranged
weekly)
1 month
↓ neocortical and
hippocampal Aβ
deposits
↑ increased neprilysin
expression
Not measured[98]
PS1/PDAPP
5 monthsweaning
↑ gene expression of
TTR, NF-κB inhibitors,
Improved performance
in MWM, RAWM and
platform recognition
tasks
[99]
APPswe/PS1ΔE9
6 months2 months
↑ 68% of plaque area in
the hippocampus
↑ 52% of total Aβ in
hippocampus
Not measured[100]
APPswe/PS1ΔE9
6 months 2 months
↑ 50% Aβ42 in the
hippocampus
↑ 25% in hippocampal
plaque load
Improved performance
in MWM, RAWM
[101]
APOE3, APOE4
Cages with exploratory
objects (toys, tunnels,
and running wheels)
5 months3 weeks
improvement in
T-maze performance in
APOE3 only
↑ expression of NGF
↑ Synaptophysin in the
hippocampus of
APOE3 only
[102]
APOE3, APOE4
Enriched housing (
cage with running
wheel, labyrinth,
bedding, house, chains,
and wooden blocks)
5 months3 weeks
↑ hippocampal Aβ
deposits in the APOE4
Not measured[103]
APOE3, APOE4Wheel running 6 weeks 10 months
↑ BDNF in both
↑ TrkB, PAK and
synaptophysin only in
APOE4
Improved performance
in place recognition in
both genotypes
Improved RAWM in
APOE4 only
[104]
from intracellular and synaptic compartments) may be a
mechanistic explanation for these findings.
4.2.3. APOE Mice. EE in mice carrying the APOE3 allele
improved learning and memory, as assessed with the T-
maze test, while it had no effect in the ones carrying the E4
allele. The improved cognitive performance in APOE3 mice
was associated with increased neocortical and hippocampal
synaptophysin- and nerve growth factor-immunoreactivity,
which was not observed in the APOE4 mice [102].
In conclusion, the majority of studies indicate that EE
affects AD-related phenotypes in transgenic mouse models
of AD pathology, mostly in a beneficial manner, particularly
withregardstobehavior.However,contradictoryresultshave
been reported which can possibly be explained by different
experimental paradigms, age,sex, and genetic background of
the mice used.

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10International Journal of Alzheimer’s Disease
5.Metal Exposure
5.1. Lead. Lead exposure has been proposed as a risk factor
for AD by some authors [106, 107] while others have argued
against it [108]. No studies to date have performed lead
exposure experiments in mouse models of AD although
other animal work has indicated that lead exposure early in
lifemaycontributetotheonsetofAD-relatedpathologylater
in life [109, 110].
5.2. Aluminum. While it has been proposed that occupa-
tional aluminum exposure is not a significant risk factor
for AD [108], prolonged exposure to aluminum in drinking
water is significantly associated with an increased risk of
developing AD in a dose-dependent manner withthe relative
risks varying from 1.00 to 2.14 (for review see [111]). These
findings should be regarded with some caution as aluminum
concentrations varied highly between the different studies
and many variables (such as interaction with other chemical
constituents in the drinking water as well as alternative
sources of aluminum, for example through antacid use or
dietary intake) have often been overlooked in these studies
[111].
Products made of baking-powder often contain high
levels of aluminum, and it has been observed that AD
patients were more frequently exposed to ingestion of
foods containing baking-powder (retrospectively investi-
gated) than age-matched controls [112]. Other studies
suggested that AD patients have significantly enhanced
gastrointestinal absorption of aluminum (up to 1.64 times
higher) compared to age-matched controls, and indicate that
differential gastrointestinal function may lead to a systemic
rise of aluminum [113, 114].
Pratic` o and colleagues [115] reported that Tg2576 mice
exposedchronicallytodietaryaluminumdisplayedincreased
Aβ40 and Aβ42 levels, plaque deposition, and markers of
oxidativestressinthehippocampusandneocortexcompared
to non-exposed Tg2576 mice. Others authors, however, were
not able to replicate these findings. They reported that
chronic aluminum treatment in Tg2576 mice did not affect
Aβ load in the cerebral cortex or oxidative stress reactions in
the hippocampus, nor impair spatial cognition, as measured
by the MWM [115–117]. Aluminum treatment did raise the
levels of aluminum and other metals in the hippocampus,
neocortex and cerebellum, but no major differential effects
could be found between Tg2576 and WT mice [116, 118].
The differential effect of aluminum exposure in these mice
could possibly be explained by higher concentrations of
aluminum in the chow, differential ages at the start of the
experiment and a shorter duration of exposure.
5.3. Iron, Zinc, and Copper. The endogenous biometals iron,
zincandcopperhaveoftenbeenimplicatedinAD,astheyare
present in and around amyloid deposits in the AD brain and
their presence can promote aggregation of Aβ [119–121].
Although no report has confirmed a direct link between
iron exposure and the risk of developing AD, Dwyer and
colleagues do propose a ferrocentric model of AD [122].
Higher levels of ferritin iron in the basal ganglia have been
considered a risk factor for AD [123, 124]. AD patients show
elevated levels of iron in the hippocampus [125], and this
metal seems to concentrate in the core and rims of plaques in
the amygdala [126].
Rodent research has indicated that gestational or early
developmental iron deficiency can alter the expression of the
APP and CLU genes implicated in synaptic plasticity, den-
dritic outgrowth, and AD pathogenesis [127–129]. Neonatal
administration of iron for 3 days to APPswe/PS1ΔE9 mice
was found not to alter Aβ deposition in the hippocampus
and temporal cortex at 6 months of age but did cause
changes in lipid composition, decreased steady-state levels of
oxidative damage markers, and increased astrocyte levels in
the temporal cortex [130].
Zinc seems to play a double role in AD etiology. Low
levels of zinc have been reported to be protective against
Aβ formation [131] and metalloproteases, such as neprilysin
and insulin-degrading enzyme (IDE), that degrade Aβ are
zinc dependent [132]. It has also been found that high
levels of zinc elevate Aβ toxicity [131, 133] and promote
total Aβ aggregation [121]. AD patients displaying higher
levels of zinc in hippocampus and amygdala [125, 126]
exhibited normal zinc serum levels, but significantly lower
zinc levels in CSF compared to matched controls [134].
This may be explained by the binding of zinc to Aβ in
the brain parenchyma [135]. However, to our knowledge,
no reports have been published on putative associations
between zinc exposure and risk of AD in the human
population.Nonetheless,severalstudieshaveinvestigatedthe
effects of altered zinc intake in AD mouse models.
Stoltenberg et al. [136] reported that lowering zinc by
a 3-month dietary deficiency increased the plaque load in
APPswe/PS1ΔE9 mice by 25%, without changing zinc ion
distribution, zinc transporter mRNA expression levels nor
inducing oxidative stress [136]. Alternatively, administrating
zinc to TgCRND8 and Tg2576 mice through the drinking
water for a period of 5 and 9 months, respectively, lowered
the amyloid plaque burden in the hilar and molecular region
of the dentate gyrus, while impairing spatial memory in
MWM [137]. Concurrently, long-term administration of
high zinc concentrations in TgC100 mice did not signif-
icantly affect soluble Aβ levels or levels of glial fibrillary
acidic protein (GFAP), superoxidase dismutase 1, APP, β-
secretase-cleaved carboxyl-terminal fragment, or neurofila-
ment200,amarkerforneuronaldamage[138].Interestingly,
a genetic reduction of zinc in the brain of Tg2576 mice,
by crossing these mice with a zinc transporter 3 deficient
mouse (ZnT3−/−), significantly reduced the plaque load in
the hippocampus and neocortex while increasing the ratio
between soluble versus insoluble Aβ [139].
AD patients have been shown to display reduced copper
levels in the amygdala and hippocampus, while copper
levels are specifically elevated in amyloid plaques [125, 126].
Copper intake in AD patients decreases the reduction of
Aβ42inCSFmosttypicallyseenasthediseaseprogresses,but
does not ameliorate cognitive performance [140]. Adding
copper to drinking water of cholesterol-fed rabbits causes
accumulation of Aβ and the formation of plaque-like
structures [141].

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International Journal of Alzheimer’s Disease11
Exposing AD mouse models to chronic upregulation
of copper has yielded conflicting results. Chronic copper
administration to APP715SL mice did not alter copper, zinc,
iron, Aβ nor APP levels in the brain [142]. Long-term
administration of high levels of copper resulted in a 18%
decrease in soluble Aβ40 and increased zinc levels in the
brain without changing GFAP, SOD1, APP, C100, or NF200
levels to TgC100 mice. Yet, copper exposure in 3xTg mice
led to elevated steady-state levels of APP, and C99 as well
as to increased Aβ production and tau phosphorylation in
the brain [143]. Interestingly, copper, APP and Aβ seem
to be closely connected; Tg2576 mice displayed an overall
reduction of copper in the brain whereas the ablation of
APP and amyloid precursor-like protein 2 increased overall
central copper levels [144, 145].
In summary, both human and rodent research on the
exact contributing roles of metal exposures in interaction
with AD risk genes APP and PS1 remain largely inconclusive.
For an overview of metal exposure in the transgenic animal
models of AD listed above, see Table 4.
6.TraumaticBrainInjury
6.1. Human Studies of Traumatic Brain Injury. Traumatic
brain injury (TBI) has repeatedly been identified as a risk
factor for AD. It has been suggested that TBI accelerates the
onset of AD and that the severity of the injury increases
the risk of AD [147]. AD-like pathology has been observed
afteracutebraintrauma,eveninbrainsofyoungindividuals.
A polymorphism in the promoter of the gene that encodes
neprilysin, causing a greater length in GT repeats, has been
associated with the acute development of plaques following
TBI [148]. In addition, carriers of the APOE4 genotype have
been associated with poorer outcome after TBI [147].
6.2. Animal Studies of Traumatic Brain Injury. For an
overview of TBI in mouse models of AD, see Table 5.
After corticol contusion, 10- to 16-month-old PDAPP mice
did not show significant differences in behavior or Aβ
neuropathology following TBI, as compared to WT controls
that underwent the same procedure of experimental brain
injury [149]. Inducing TBI in the PDAPP mouse model
at 4 months of age, accelerated memory loss as assessed
with the MWM test. TBI also resulted in hippocampal
neuronal loss one week after injury, which was associated
with an increase in hippocampal Aβ40 and Aβ42 [150].
Furthermore, TBI resulted in long-term effects at 2, 5, and
8 months after TBI: a significant reduction in Aβ plaque load
was found which was accompanied with more pronounced
hippocampal atrophy. TBI induction in APPNLh/NLhmice
causedatwofoldincreaseinsolublehippocampalAβ levelsat
3and7daysafterTBI.Additionally,post-TBIadministration
of caspase-3 inhibitors and the hypolipemic simvastatin were
able to attenuate impaired hippocampal synaptic function,
microglial activation and MWM performance after TBI
induction in APPNLh/NLhmice [151, 152]. TBI induced by
cortical impact provoked gene expression changes in 22-
month-old APPswe mice compared to WT mice. Expression
changes were detected in genes involved in various biological
pathways such as immune response, cell cycle and cell death,
cellular development, tissue development and connective
tissue function and development, cellular movement, and
hematological systems [153].
Single and repetitive mild TBI, using a cortical impact
device, in 9-month-old Tg2576 mice as compared to sham
treated transgenic and WT, resulted in significant cognitive
dysfunction(measuredwithMWM)withoutaffectingmotor
performance 16 weeks after TBI. However, only repetitive
TBI caused increased Aβ burden in the hippocampus and
neocortexwithaparallelincreaseinisoprostane,anindicator
for increased oxidative stress [154]. TBI in 10-month-old
transgenic mice overexpressing either human APOE3 or 4,
was associated with differential gene expression, particularly
in genes related to oxidative stress, with an increased
expression of antioxidant genes in the APOE3 mice as
compared to the APOE4 [155].
Thus, accumulating evidence indicates that TBI interacts
with AD-related genes.
7.ElectromagneticField Exposure
Occupational exposure to electromagnetic field (EMF) has
been proposed as a risk factor of AD. In particular, extremely
low-frequency exposure has been implicated to increase the
odds to develop AD up to 2.03 (as reviewed in [155]).
Strikingly, Arendash et al. [27] demonstrated that long-
term high-frequency exposure to EMF (i.e., similar to that
generated by cell-phone use) was beneficial to AβPPswe
mice (see Table 4). EMF exposure in young adult AβPPsw
mice prevented the age-related genotype-specific cognitive
impairment, while EMF in aged AβPPsw mice was also able
to reverse cognitive impairment in these animals. Chronic
EMF exposure furthermore influenced Aβ aggregation in the
brain, with higher levels of soluble Aβ and less Aβ plaques
in the hippocampus and entorhinal cortex of AβPPsw
mice. EMF exposure has been proposed to contribute to a
decreaseinAβaggregation,viaalteringlevelsoftransthyretin
[158]. Transthyretin is known to sequester Aβ in CSF,
therebyhinderingitsaggregationintoamyloidplaques[158].
Interestingly, AD patients show a significant decrease in
CSF transthyretin levels [159] while decreased transthyretin
levels have also been found in blood serum of long-term
wireless phone users [158]. Thus, effects of EMF are quite
puzzling while the association with AD remains to be firmly
established.
8.Effects of Diet andNutritionalFactors
8.1. Mediterranean Diet. Various dietary and nutritional
factors seem to be protective or detrimental in the develop-
ment and course of AD. One of the most prominent is the
Mediterranean type of diet which has been linked to reduced
risk of developing AD and showing a dose-response effect
(high adherence to Mediterranean diet, OR: 0.76; moderate
adherence, OR: 0.47) [160–162].
A typical Mediterranean diet is characterized by higher
consumption of vegetables, fruits, cereals, fish, and olive

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12 International Journal of Alzheimer’s Disease
Table 4: Environmental exposure to metals and electromagnetic fields in transgenic mouse models of Alzheimer’s disease.
Mouse model Exposure
Duration of
the exposure
Age at the
start
Effect on brain
Effect on
behavior
Reference
TgV337M
Aluminum-mltolate i.p.
injection at various
concentrations
(50–100–200μM)
Max 14 days3 months
Al levels were too low to induce
changes in tau phosphorylation
in brain homogenates, but Al
concentration was lethal
↑ soluble and insoluble Aβ40 and
Aβ42 in neocortical and
hippocampal homogenates
↑ plaque load in hippocampus
and neocortex
↑ oxidative stress markers
No significant differences in
Aβ40 and Aβ42 in cortical
homogenates, no alterations in
proliferation, survival or
differentiation of BrdU-positive
neurons in DG
↑ Al concentration in
hippocampus and cerebellum
↑ Cu in hippocampus
↓ Fe in cerebellum
↑ Mn and Zn in neocortex,
hippocampus and cerebellum
↑ Al concentrations in the
hippocampus, but no difference
between WT and Tg animals, no
difference in oxidative stress
reaction in the hippocampus
between WT and Tg
No difference in Aβ plaque load
in hippocampus and temporal
cortex, no difference in
microglial activity
↑ GFAP levels in temporal cortex
↑ saturated fatty acids
↓ unsaturated fatty acids
↓ oxidative damage markers
No significant difference in
serum zinc levels, no difference
cortical volume
↑ 25% in total plaque volume, no
difference in number of plaques
or laminar distribution, no
difference in oxidative stress
markers
Not measured[146]
Tg2576
Dietary aluminum
(2mg/kg diet)
9 months3 months
Not measured[115]
Tg2576
Dietary aluminum
lactate (1mg/g diet)
120 days5 months
No
improvement
MWM
[117]
Tg2576
Dietary aluminum
lactate (1mg/g diet)
6 months5 months
Not measured[118]
Tg2576
Dietary aluminum
lactate (1mg/g diet)
6 months5 months
Not measured[116]
APPswe/PS1ΔE9Iron carbonyl (1mg/ml) 3 daysP12
Not measured [130]
APPswe/PS1ΔE9
Zinc-deficient (<10 parts
Zn per million (ppm))
3 months 9 months
Not measured [136]
Tg2576
Zinc in drinking water
(10 ppm/0.153mM Zn)
±12 months
From
conception
↓ Aβ deposits in hilar and
molecular region of the DG
↓ spatial
memory in
MWM both in
Tg and WT,
buth most
pronounced in
Tg
↓ spatial
memory in
MWM both in
Tg and WT,
buth most
pronounced in
Tg
[137]
TgCRND8
Zinc in drinking water
(10ppm/0.153mM Zn)
5 months
From
weaning
No significant differences
[137]

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International Journal of Alzheimer’s Disease13
Table 4: Continued.
Mouse modelExposure
Duration of
the exposure
Age at the
start
Effect on brainEffect on behaviorReference
TgC100
Zinc in diet
(ZnSO4, 1000, 500
or 300ppm)
15 months7 weeks
↑ Brain Zn levels in brain
homogenates
↓ Cu levels (n.s.)
↓ Cu/Zn ratio
↓ 13% soluble Aβ 40 (trend)
No changes in GFAP, SOD1,
APP, C100, nor NF200
(neuron loss), no difference in
intensity or distribution of Aβ
or GFAP staining
No significant differences in
Cu levels in brain homogenates
↑ Zn levels
↓ 18% soluble Aβ 40, no
changes in GFAP, SOD1, APP,
C100, nor NF200 (neuron
loss), no difference in intensity
or distribution of Aβ or GFAP
staining
3 months exposure:
↑ steady-state levels APP, C99
and BACE1
↑ Aβ 40 in total plaque load in
hippocampus, no alterations
in total tau, phospho-tau nor
Thy1.2 transcription activity
↑ AT8-positive neurons in
CA1, no changes in
steady-state levels of cdk5,
p35/p25, GSK-3β or
phospho-GSK-3β
↓ SOD1 activity in brain
homogenates
9 months exposure:
↑ steady-state levels APP, C99,
C83, BACE1, ADAM10
↑ soluble Aβ40, phospho-tau,
no alterations total tau levels
↑ p25 formation
↓ SOD1 activity
Not measured[138]
TgC100
Copper in diet
(CuSO4150 or
100ppm)
7 weeks9 months
Not measured[138]
3xTg
Copper sulfate
(250ppm) in 5%
sucrose drinking
water
3 or 9 months2 months
Not measured[143]
AβPPsw
Electromagnetic
field exposure
(918MHz,
0.25W/kg ± 2dB)
2 ×1hp/d
7-8 month
exposure
2 months
5 months
Young adult 7 months exposure,
no significant differences in
soluble Aβ in hippocampus +
neocortex, no effect op
hippocampal DNA repair
enzymes, antioxidant enzyme
markers, protein oxidative
damage, nor striatal DNA
oxidation
Aged adult 8 months exposure
↓ Aβ plaque load in
hippocampus (−35%) and
entorhinal cortex (−32%)
↑ soluble Aβ in hippocampus
+ neocortex
Young adult 7
months exposure
Prevention of
cognitive deficits
in retroactive
interference
↑ Y-maze
spontaneous
alternation level
No differences in
open field activity,
balance beam,
string agility, and
elevated plus
maze
Aged adult 8
months exposure
Reversal of
cognitive deficits
[27]

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14International Journal of Alzheimer’s Disease
Table 5: Traumatic brain injury in transgenic mouse models of Alzheimer’s disease.
Mouse
model
Exposure
Duration
exposure
Age at start
exposure
Effects on brainEffects on behaviour Reference
APP-YAC
Cortical contusion impact
(3-mm diameter impounder
onto the left parietal cortex,
100ms; velocity (v) =
4.8−5.2m/s; depth = 1mm)
Cortical impact brain injury
(3-mm diameter impounder
onto the left parietal cortex,
100ms; v = 4.8−5.2m/s; depth
= 1mm)
Controlled cortical impact
(3-mm diameter impounder
onto the left parietal cortex,
100ms; v = 4.8−5.2m/s; depth
= 1mm)
Controlled cortical impact
(3-mm diameter impounder
onto the left parietal cortex,
100ms; v = 4.8−5.2m/s; depth
= 1mm)
Controlled cortical impact
(3-mm diameter impounder
onto the left parietal cortex,
47ms; v = 5.82m/s; depth =
1.2mm, driving pressure 73psi)
Controlled cortical impact
(mild to moderate, 2-mm
diameter impounder onto the
right cortex, v = 3.3m/s; depth
= 1mm)
Controlled cortical impact
(3-mm diameter impounder
onto the left parietal cortex,
100ms; v = 4.8−5.2m/s; depth
= 1mm)
—10–16 monthsNo difference
No difference in
MWM
[149]
PDAPP
—4 months
Increased hippocampal
neuronal death
MWM memory
impairment in
transgenic as
compared to
controls
[150]
PDAPP
— 6 months
Hippocampal
atrophyDecrease the
hippocampus and
cingulate cortex 3
months after TBI
Not measured[156]
PDAPP
— 24 months
Increased hippocampal
neuronal loss and gliosis
Regression of Aβ in the
hippocampus
Not measured [157]
APPswe—3 months
2× increase in Aβ40 and
Aβ 42
Reduced CA3
ynaptophysin
immunoreactivity
MWM performance
deficit
[152]
Tg2576
— 22 months
Gene expression
differences in
inflammation, immune
response and cell death
Not measured[153]
Tg2576
Repetitive
(2×)
9 months
Increased hippocampal
amyloid deposition
MWM cognitive
dysfunction
[154]
oil and associated with a general higher consumption of
unsaturated fatty acids and lower consumption of saturated
fattyacids,usuallyaccompaniedbymildormoderatealcohol
intake (preferably red wine taken with meals) [160]. The
exact factors and mechanisms by which the Mediterranean
diet is protective remains to be elucidated, although it has
been speculated that this diet can attenuate the detrimental
effects of oxidative stress and inflammation [161].
8.2. Western Diet and Obesity. Obesity during mid-life is
associated with an increased risk for AD, with an OR of
2.4, additively increasing up to 6.2 when combined with
high total cholesterol levels and high systolic blood pressure
[163]. Higher intake of calories and fat have been associated
withincreasedriskfordevelopingAD,particularlyinAPOE4
carriers, with a hazard ratio of 2.3 [164]. Western, high-
fat and low carbohydrate diet for 4 months in 1-month-
old Tg2576 mice, increased levels of soluble Aβ in brain
homogenates, while the treatment did not have any effect
on plaque load [165]. Further, insulin resistance induced
by 5 months of high-fat diet, in 9-month-old Tg2576
mice, was associated with to a twofold increase of Aβ40
and Aβ42 peptide content in the hippocampus and a
twofold increase in plaque burden in the neocortex, with a
concomitantaccelerationofcognitivedeclineasmeasuredby
the MWM. In addition, γ-secretase activity was increased,
while the expression of IDE was decreased by this diet
[166]. APP/PS1 KI mice exposed to Western, high-fat diet
showed increased oxidative stress markers as measured in
brain homogenates of 2-month-old mice when compared to
nontransgenic controls, but Aβ levels were not altered [167].
In another study, Western diet increased Aβ deposition in
the hippocampus of the APPswe/PS1ΔE9 transgenic mice
at 18 months of age, after a period of 12 months on a
high-fat diet [168]. In the 3xTg mouse model of AD, a
high fat diet starting at the age of 4 months for a total
period of 13 months, induced similar effects in the frontal
cortex [169].

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International Journal of Alzheimer’s Disease15
8.3. Cholesterol. As the generation, deposition, and clear-
ance of Aβ is regulated by cholesterol, many studies have
specifically focused on the implication of lipids, cholesterol
metabolism, related vascular disease, APOE genotype, and
their interrelationships on the development of AD [170–
172]. The precise mechanisms underlying cholesterol and
APOE4 need further investigation, as it is not clear whether
cholesterol and the APOE4 genotype act as independent
factors or interact with one another or whether the
effect of APOE4 is partially mediated by high cholesterol
levels [171–174]. Also, hypercholesterolemia in 3-month-
old APPswe/PS1M146L mice has been shown to accelerate
Aβ accumulation while drug-induced hypocholesterolemia
reduced the amyloid pathology [175, 176].
8.4. Docosahexaenoic (DHA). Studies in mouse models of
AD amyloidosis, such as Tg2576, APPswe/PS1ΔE9, and
3xTg, have shown that a diet rich in the omega-3 fatty
acid DHA reduces Aβ accumulation and somatodendritic
tau accumulation, improves cognition, and induces cerebral
hemodynamic changes [168, 177–180]. Such findings are in
line with evidence from epidemiological studies showing a
protective effect of diets rich in omega-3 fatty acids [181–
183]. More specifically, DHA-enriched diet was shown to
increase relative cerebral blood volume with a concomitant
improvement in spatial memory and reduction of Aβ load
in APPswe/ PS1M146Lmice [184]. Exposing APPswe/PS1ΔE9
mice to a diet high in omega-3 fatty acids, however, neither
improved cognition in APPswe/PS1ΔE9 mice nor reduced
hippocampal Aβ, but increased omega-3 fatty acid levels in
their brain [185]. Interestingly, high levels of omega-6 were
linked to cognitive impairment [185].
8.5. Vitamins. Dietary deficiency of B6, B12, and folate
for 7 months increased Aβ levels in the brains of 15-
month-old Tg2576 mice, without altering APP, BACE-1, A
disintegrin and metallopeptidase 10 (ADAM-10), nicastrin,
IDE, APOE, or neprilysin [186]. Additionally, the same
pattern of dietary vitamin B deficiency led to increased
expression of PS1 via DNA demethylation of the promoter
region of the encoding gene in brain homogenates of
TgCRND8 mice [187]. In the brains of mice of the same
animal model, vitamin B deficiency increased the levels
of glycogen synthase kinase 3β (GSK3β) and reduced the
activity of protein phosphatase 2A, which are both involved
inthehyperphosphorylationoftau[188].Furthermore,folic
acid deficiency for 3 months in APPswe mice did not affect
the Aβ plaque load, but induced neuron loss in the CA3
regionofthehippocampusandenhancedhippocampalDNA
damage, as compared to controls [189]. Besides B6 and B12,
deficiencyofB1,alsocalledthiamine,exacerbatedAβpathol-
ogy via an upregulation of BACE1 in brains of Tg19959
mice [190].
Furthermore, dietary supplementation with the coen-
zyme Q10 for 2 months delayed hippocampal atrophy in 22-
month-old APPswe/PS1Leu235Promice as compared to vehicle
treated controls [36, 37], with concurrent reduction in
plaque load [36, 37].
Deficiency of vitamin A has been implicated in Aβ
accumulation, loss of long-term potentiation and memory
impairment, while administration of its active metabolite
retinoic acid for a duration of 2 months was able to rescue
these deficits in the frontal cortex and hippocampus of 7-
month-old APPswe/PS1ΔE9 transgenic mice [191].
8.6. Caffeine and Green Tea. Besides the various nutritional
factors, other lifestyle habits have also been associated with
AD. Longitudinal studies have shown that coffee and tea
drinking are associated with decreased risk for cognitive
decline, dementia and AD in various population samples
[192, 193]. Another study showed a protective effect of
caffeine only in women, with a relative risk of 0.49 [194],
but a meta-analysis estimated an overall protective effect
againstdementia,witharelativeriskof0.84,thoughpointing
out the large heterogeneity in the methods of the various
epidemiological studies [195].
Acute and long-term caffeine consumption was recently
shown to delay cognitive decline and lower Aβ pathol-
ogy in the hippocampus of 15-month-old APPswe and
APPswe/ PS1M146L mice, by suppressing β- and γ-secretase
levels [196, 197]. Furthermore, oral or intraperitoneal
administrationofepigallocatechin-3gallate,whichisderived
fromgreentea,for2or6months,exertedbeneficialeffectsin
APPswe transgenic mice, at the age of 14 months. The bene-
ficial effects consisted of a reduction in Aβ pathology in the
neocortex and hippocampus, with a parallel improvement of
working memory [198–200]. Furthermore, administration
of the citrus-derived flavonoid luteolin and its analogue
diosmin for a total of 30 days, significantly reduced Aβ
pathology in the hippocampus and neocortex of 9-month-
old Tg2576 mice. This effect was mediated via an inhibition
of GSK3β, which increased PS1 phosphorylation [201].
8.7. Wine. Moderate red wine consumption has been shown
to be beneficial. Cabernet sauvignon administration for
7 months in 4-month-old Tg2576 mice attenuated the
cognitive impairment that is observed in these mice, in
terms of spatial memory, when compared to ethanol-
consuming and tap water Tg2576 controls. Cabernet sauvi-
gnon consumption decreased cortical and hippocampal Aβ
plaque load in these mice, by promoting nonamyloidogenic
processing in the direction of α-secretase cleavage [202].
Further, in vitro studies in hippocampal neuron cultures
derived from Tg2576 mice, showed that the polyphenol
extracts from the Cabernet sauvignon grapes increased the
levels of α-secretase, which promotes the nonamyloidogenic
cleavage of APP that reduced the levels of Aβ peptides
[202]. Furthermore, consumption of the muscadine wine
wasproventoattenuateAβpathologyinbrainsof14-month-
oldTg2576mice,followinga10-monthwinetreatment,with
a different mechanism of action. In this case, muscadine
consumption reduced the aggregation of Aβ, with a parallel
improvementinspatialmemory[203].Thedifferentialeffect
of the two types of wine was attributed to their distinct
composition in polyphenolic compounds, which have a
differential effect on APP processing [203].