Tài liệu Báo cáo khoa học: Animal models of amyloid-b-related pathologies in Alzheimer’s disease docx

Diffuse Ab deposits are also present, but these Keywords Alzheimer’s disease; amyloid beta-protein; amyloid beta-precursor protein; animal model; apolipoprotein E; neuropathology; presen

Animal models of amyloid-b-related pathologies in

Alzheimer’s disease

Ola Philipson1, Anna Lord2, Astrid Gumucio1, Paul O’Callaghan1, Lars Lannfelt1and Lars

N.G Nilsson1

1 Department of Public Health and Caring Sciences ⁄ Molecular Geriatrics, Uppsala University, Sweden

2 BioArctic Neuroscience AB, Stockholm, Sweden

Introduction

Alzheimer’s disease (AD) accounts for  60–70% of

all dementia cases Prevalence increases with age from

 1% in the 60 to 64-year age group, to 24–33% in

those aged > 85 years There is an insidious onset

with an initial loss of short-term memory, followed by

progressive impairment of multiple cognitive functions

that affect the activities of daily living The AD

diag-nosis is based on a patient’s medical history,

neurolog-ical assessment and neuropsychiatric testing of

cognitive functions Neuroimaging techniques and

biomarkers in cerebrospinal fluid (CSF) are invaluable

in differential diagnosis

The neuropathological diagnosis takes into accountthe regional distribution and frequency of histopatho-logical hallmarks; specifically, extracellular neuritic pla-ques and intracellular neurofibrillary tangles (NFTs) inpostmortem brain Neuritic plaques mainly consist ofb-sheet-containing fibrils of amyloid-b (Ab) that aresurrounded by dystrophic neurites and reactive glialcells Diffuse Ab deposits are also present, but these

Keywords

Alzheimer’s disease; amyloid beta-protein;

amyloid beta-precursor protein; animal

model; apolipoprotein E; neuropathology;

presenilin-1; presenilin-2; tau proteins;

transgenic mice

Correspondence

L Nilsson, Department of Public Health and

Caring Sciences, Molecular Geriatrics,

Uppsala University, Rudbeck Laboratory,

Dag Hammarskjo¨lds va¨g 20, SE-751 85

Uppsala, Sweden

Fax: +46 18 471 4808

Tel: +46 18 471 5039

E-mail: Lars.Nilsson@pubcare.uu.se

(Received 5 October 2009, revised 29

November 2009, accepted 30 December

2009)

doi:10.1111/j.1742-4658.2010.07564.x

In the early 1990s, breakthrough discoveries on the genetics of Alzheimer’sdisease led to the identification of missense mutations in the amyloid-bprecursor protein gene Research findings quickly followed, giving insightsinto molecular pathogenesis and possibilities for the development of newtypes of animal models The complete toolbox of transgenic techniques,including pronuclear oocyte injection and homologous recombination, hasbeen applied in the Alzheimer’s disease field, to produce overexpressors,knockouts, knockins and regulatable transgenics Transgenic models havedramatically advanced our understanding of pathogenic mechanisms andallowed therapeutic approaches to be tested Following a brief introduction

to Alzheimer’s disease, various nontransgenic and transgenic animal modelsare described in terms of their values and limitations with respect to patho-genic, therapeutic and functional understandings of the human disease

Abbreviations

AD, Alzheimer’s disease; ApoE, apolipoprotein E; APP, amyloid-b precursor protein; Ab, Amyloid-b; BACE-1, b-site APP cleaving enzyme-1; CAA, cerebral amyloid angiopathy; CCR2, chemokine (C-C motif) receptor 2; CSF, cerebrospinal fluid; MWM, Morris water maze; NFTs, neurofibrillary tangles; PDGF, platelet-derived growth factor; PS, presenilin; SMC, smooth muscle cells; wt, wild-type.

lack b-sheet structure and are therefore by definition

not amyloid Cerebral amyloid angiopathy (CAA)

results in the degeneration of vessel walls and

hemor-rhages CAA is found in  80% of AD brains, but is

not a diagnostic criterion NFTs are intracellular

fila-mentous lesions with amyloid properties They contain

hyperphosphorylated and aggregated forms of tau, a

microtubule-associated protein that normally serves to

assemble and stabilize microtubules

Genetics and risk factors implicated in

Alzheimer’s disease pathogenesis

Familial forms of AD, with an autosomal dominant

mode of inheritance, account for < 2% of all AD

cases Onset is most often before 65 years of age, and

the penetrance is nearly always complete The

purifica-tion and partial sequencing of Ab from amyloid

depos-its of AD brain in the 1980s [1], led to the cloning and

localization of the amyloid-b precursor protein (APP)

gene on chromosome 21 [2] The first identified AD

mutation was located in the APP gene [3], although

the majority of mutations were caused by genetic

lesions in the presenilin (PS) genes, PS1 and PS2 The

mutations either enhance the steady-state level of Ab,

like the Swedish APP mutation (K670N⁄ M671L) [4],

or selectively increase the level of Ab42 and⁄ or alter

the Ab42⁄ Ab40-ratio, like the PS and London-type

APP mutations do [5] Ab is liberated following

cleavage of APP by b-site APP-cleaving enzyme-1

(BACE-1) and the c-secretase complex, in which

presenilin contributes to the catalytic activity (Fig 1)

However, only a fraction of Ab in postmortem

AD brain is full-length Ab1-42 or Ab1-40 N- and

C-terminally truncated variants are prevalent, and Ab

can undergo racemization, isomerization [6] and

pyro-glutamyl modification [7] The biochemical processes

generating all these Ab species and their significance

to AD pathogenesis are only partially understood.Early-onset AD can also arise as a result of increasedAPP gene dosage caused by APP gene duplication [8]and Down’s syndrome with trisomy 21 Virtually allDown’s syndrome patients aged 35–40 years develop

AD neuropathology, and most experience dementia by60–70 years of age [9]

The major genetic risk factor for developing onset AD is the apolipoprotein E (ApoE) e4 allele[10,11] One ApoE e4 allele increases the risk of AD bytwo- to threefold, and two e4 alleles confer a 12-foldincrease in risk In the brain, ApoE is primarily synthe-sized by astrocytes and serves to regulate the transport

late-of cholesterol-containing lipoprotein particles ApoEbinds to Ab and becomes a component of amyloid in

AD senile plaques The pathogenic mechanism ofApoE likely relates to altered deposition and⁄ or clear-ance of Ab in the brain, although the details are stillnot fully understood [12] A large number of other dis-ease-related loci and candidate genes have been pro-posed, but not generally verified, indicating that thesegenes have a modest impact on the pathogenesis Themajor risk factors for AD are age and a family history

of the disease Low education or cognitive reservecapacity, female gender, head trauma, hypertension,cardiovascular disease and a high-cholesterol diet areproposed risk factors for AD [13] (Fig 2)

Nontransgenic animal models

Based on the cholinergic hypothesis, induced amnesia, excitotoxic lesions of the basalforebrain and aged primates have been used to assesscognitive deficits Current symptomatic drugs for ADwere successfully evaluated in these models, but theiretiological relevance is low [14] Nontransgenic rodents

scopolamine-Fig 1 Disease-causing APP mutations used in transgenic models The Swedish mutation (1) favors b-secretase (b) cleavage, while the Flemish mutation (2) partly disfavors cleavage of APP at the a-secretase (a) site The Arctic, Dutch and Iowa mutations (3), which are located

in the Ab-domain, mainly increase aggregation The London-type APP mutations (4) alter c-secretase (c) cleavage to increase Ab42 or the Ab42 ⁄ Ab40 ratio.

are poor natural animal models of AD, but

intracere-broventricular infusion of Ab [15] or lipopolysaccharide

in such animals has been used The latter leads to

neuroinflammation with hippocampal

neurodegenera-tion and spatial memory deficits [16] The models

require attention to methodological detail and are

difficult to standardize Senescence-accelerated mice

were selectively bred from AKR⁄ J mice In short-lived

SAM-P8, there is an age-related increase in diffuse Ab

deposits and cyclin-dependent kinase 5, cholinergic

deficits and increased blood–brain barrier permeability

The phenotypes likely relate to oxidative stress and

mitochondrial dysfunctions [17] Mice with segmental

trisomy of chromosome 16 have primarily been used to

dissect the genetic mechanisms of Down’s syndrome

phenotypes Ts65Dn mice [18], the most frequently

used model, have interesting synaptic and cognitive

phenotypes with degeneration of cholinergic neurons

that depend on APP gene dosage

Following observations in postmortem brain from

patients with coronary artery disease, rabbits fed a

cholesterol-enriched diet were used as an animal model[19] They are impared in classical eyelid conditioningand show diffuse Ab deposits and vascular inflamma-tion Aged dogs (> 10 years) can show impaired atten-tion, spatial disorientation and disturbed diurnalrhythm Cognitive dysfunction in old dogs is associatedwith diffuse Ab deposits [20], neuritic dystrophy and gli-osis, but few amyloid plaques and no NFTs Ventriculardilation, cortical and hippocampal atrophy, CAA withdegeneration of smooth muscle cells (SMC) and hemor-rhages can all be found in aged canine brain Interest innonhuman primate models has grown following the fail-ure to predict meningoencephalitis as a side-effect of theAN1792 vaccination trial from transgenic studies Theefficacy and safety of an Ab vaccine has been tested inthe Carribean vervet monkey [21] Alternatives are agedlemurs [22], cotton-top tamarins [23], rhesus monkeys[24] or squirrel monkeys [25] An aged chimpanzee withcomplete AD neuropathology, including neuriticplaques and paired helical filament-containing NFTs,was recently reported [26]

Fig 2 AD pathogenesis according to the

amyloid cascade hypothesis This theory

suggests that altered metabolism of Ab, in

particular aggregation-prone Ab species like

Ab42, initiates AD pathogenesis Oligomeric

assemblies of Ab trigger aggregation of tau

and the formation of NFTs, but also

inflam-mation and oxidative stress, by rather

unclear mechanisms These downstream

processes give rise to progressive

neurode-generation, which ultimately results in

dementia The main pathogenic pathway of

AD is illustrated with red arrows, whereas

minor contributory pathways are shown

with thinner brown arrows The

experimen-tal support for the hypothesis comes mainly

from studies of families in which AD is

inherited as a dominant trait due to

muta-tions in APP, PS1 or PS2 The evidence that

the theory applies to sporadic AD is less

solid, although risk factors such as age and

ApoE genotype both strongly impact on Ab

aggregation in transgenic models and post

mortem AD brain.

Transgenic animal models

Models devoid of any disease-causing APP

mutations

Animal models expressing wild-type (wt) human APP

are of interest because the great majority of sporadic

AD patients do not carry any disease causing APP

mutation In early transgenic attempts, APP processing

was bypassed altogether and human Ab was directly

expressed under a promoter Natural inclusions in the

brain were mistakenly identified as amyloid-like fibrils

in these mice [27] Fusion proteins, in which the signal

peptide and C-terminal fragment (C99) of wild-type

APP were joined and expressed under the control of

the cytomegalus enhancer⁄ chick b-actin promoter,

were also generated Ab levels in plasma from these

transgenic mice were in the nm range, but Ab deposits

did not form in the brain Instead, intracellular Ab

aggregates or amyloid deposists were found in the

pan-creas [28], intestine [29] or skeletal muscles [30] The

level of plasma Ab in C99-based models was similar to

Tg2576, an APP transgenic model with high peripheral

promoter activity

In an alternative strategy, a yeast artificial

chromo-some, harboring the whole APPwt gene, was used to

maintain transcriptional regulation, alternative splicing

and normal APP processing In these Py8.9 mice,

proper APP protein synthesis and alternative splicing

was demonstrated, but the brain was devoid of

neuro-pathology and the levels of Ab were low [31]

How-ever, when wild-type human APP was expressed at

very high levels, under the Thy1 promoter, sparse

parenchymal and vascular amyloid deposits were

found in aged mice [32] Thus a pathogenic APP

muta-tion is not a prerequisite for amyloid deposimuta-tion

Instead it seems to depend upon producing sufficient

Ab levels in the brain to ensure fibrillization To

explore the pathogenic impact of individual Ab

spe-cies, a fusion protein, BRI–wt-Ab42, was designed

from which Ab was released by furin-like enzymes on

the cell surface BRI is a transmembrane protein that

is involved in amyloid deposition in British familial

dementia The fusion design permitted the synthesis of

high Ab levels in the brain in a manner similar to APP

transgenic mice, but in the absence of APP

overexpres-sion Transgenic mice expressing BRI–wt-Ab42

devel-oped extensive vascular and parenchymal amyloid

pathology, accompanied by dystrophic neurites and

astrogliosis In contrast to many APP transgenic

mod-els, amyloid deposition in BRI–wt-Ab42 mice began in

the cerebellum, where both furin and the transgene

were highly expressed This illustrates how the

anatom-ical location of AD neuropathology can be lated simply by enhancing the regional dosage ofamyloidogenic proteins and enzymes regulating theirmetabolism In contrast to BRI–wt-Ab42, no neuropa-thology was found in aged BRI–wt-Ab40 transgenicmice although they had higher Ab levels when theywere young Thus the identity of Ab determines ifneuropathology will develop [33]

manipu-Models with the London-type APP mutationThe London mutation (V717I) was the first geneticlesion to be discovered in a family with AD [3], andshortly thereafter the Indiana mutation (V717F) wasfound in an American pedigree [34] Patients with theIndiana mutation develop short-term learning impair-ments in the fifth decade, followed by progressivecognitive impairment and dementia with typical ADneuropathology An abundance of NFTs and senileplaques was observed at autopsy, as well as mild CAA[35] Games et al described the neuropathology ofPDAPP mice, the first transgenic AD model [36] Amini-gene encompassing a human APP cDNA with theIndiana mutation interposed with introns had beendesigned Alternative splicing and synthesis of all threeisoforms APP695, APP751and APP770, with strong andselective neuronal expression was enabled by the plate-let derived growth factor (PDGF)b promoter Impor-tantly, young PDAPP mice produced high Ab42 levels

in the brain, particularly in the hippocampus The mals preferentially accumulated Ab42 peptides anddeveloped senile plaques, but also a substantial num-ber of diffuse Ab deposits at 9–10 months of age [37].Plaque formation began in the cingulate cortex andwas accompanied by phospho-tau immunoreactivedystrophic neurites, synaptic loss and gliosis in theadjacent tissue, but not by overt neuronal loss [38,39].Ultrastructural analyses revealed neurons in closeproximity to senile plaques and amyloid fibrils Thelatter had a diameter of 9–11 nm and were surrounded

ani-by neuronal membranes and vesicles [40] YoungPDAPP mice showed deficits in spatial learning andmemory, which worsened with increasing age and Abburden, although their performance in a novelobject-recognition task was unimpaired [41] Bycontrast, others found age-dependent deficits in objectrecognition and place learning impairments that wereindependent of age [42] These discrepancies could bebecause of differences in experimental procedures orunintentional genetic drift of mouse colonies PDAPPmice are typically bred on a mixed genetic background(Swiss Webster, DBA⁄ 2 and C57Bl ⁄ 6) Hippocampalvolume and corpus callosum length is reduced in

PDAPP mice and this depends on APP gene dosage,

but it is unrelated to age-dependent Ab accumulation

[43] Certainly this abnormality could impact on the

behavior of PDAPP mice, but the molecular

mecha-nism and its relevance to macroscopic atrophy in AD

(if any) is still unclear

Van Leuven et al generated several transgenic

mod-els, including mice with only the London mutation,

APP-London As expected, a markedly increased level

of Ab42 was found in young mice and predominantly

Ab42-immunoreactive diffuse and neuritic plaques in

aged animals, compared with models harboring the

Swedish mutation Impaired long-term potentiation in

hippocampal slices and deficits in spatial learning and

memory in the Morris water maze (MWM) were

reported The mice were on a FVB⁄ N background and

displayed neophobic behavior They were sensitive to

glutamate antagonists and died prematurely These

phenotypes were noted in young mice prior to the

onset of plaque formation, and could possibly be

caused by the combination of APP overexpression and

FVB⁄ N genetic background [44] In older mice

(> 15 months), CAA was found in the arteries and

pial arterioles in association with disruption of external

elastic lamina and the formation of aneurysm

Further-more, the ratio of Ab42⁄ Ab40 levels in leptomeninges

was eight times lower than in neocortical tissue

extracts Ab42 could still have initiated deposition of

Ab in vessels, because some focal lesions were only

Ab42-immunoreactive [45] By contrast, brains from

patients with the London mutation contained mainly

Ab-immunoreactive plaques and cytoskeletal

pathol-ogy, but only modest or little CAA [46] Thus, the

CAA phenotype in the transgenic mice might not have

been caused by the London mutation Instead it may

be the result of strong APP expression, advanced age,

strain background (FVB⁄ N) and ⁄ or differences in APP

processing between species

Models with the Swedish APP mutation

The Swedish mutation (KM670⁄ 671NL) is located just

outside the N-terminus of the Ab domain in APP It

was identified in 1992 [47] and shown to increase Ab

levels by six- to eightfold [4] These discoveries created

intense interest in APP processing and paved the way

for the development of more sophisticated ELISAs to

selectively measure Ab40 and Ab42 [48] Later, the

Swedish mutation became essential in the identification

and characterization of BACE-1 [49] The clinical and

neuropathological features associated with the Swedish

mutation are those of typical AD [47,50] Tg2576

mice, the most frequently used APP transgenic model,

harbor the Swedish mutation and display both AD-like

Ab neuropathology and cognitive deficits [51] TheSwedish mutation redirects APP processing to secre-tory vesicles en route to the cell surface in cell culture[52], whereas APPwt is largely processed in recyclingendosomes [53] This difference may be largely irrele-vant in the brain, because Ab synthesis along theendolysosomal pathway is clearly important in Tg2576[54] A substantial amount of CAA is often found intransgenic mice with the Swedish mutation, which islikely to be because of the high rate of synthesis andaccumulation of Ab1-40

In Tg2576, more than fivefold overexpression of thehuman APP695 isoform with the Swedish mutation isgenerated by the prion promoter APP cDNA wascloned into a  40 kb genomic fragment (cosSHaPrP)[55] from the hamster prion protein gene A significantproportion of Tg2576 mice die at a young age, and theseverity of this phenotype depends upon the geneticbackground It has been found that colonies are bestmaintained by mating heterozygous C57BL⁄ 6 maleswith B6SJLF1 females At around  11 months ofage, Tg2576 mice show extracellular Ab deposits whichare largely soluble in SDS and mainly contain Ab40( 75%) CSF levels of Ab42, but not Ab40, decreasewith age and amyloid deposition [56], and pathogenesis

is accelerated in female Tg2576 mice [57] Both theseobservations fit well with biochemical and epidemio-logical findings in AD [13]

Borchelt et al used the prion promoter to generateline C3-3 [58] A chimeric cDNA clone encodingmurine APP695 was used, in which the region in andaround the murine Ab domain was replaced with thehuman Ab sequence and the Swedish mutation TheMoPrP.Xho vector was much smaller than the cos-SHaPrP, and selectively directed twofold overexpres-sion of APP to the brain [58,59] In an even morerefined strategy, the murine Ab sequence was human-ized and the Swedish mutation introduced with genetargeting In this knockin model, APPNLh⁄ NLh, onlyfive single amino acids were altered in the entire mur-ine genome Consequently, APP protein synthesisremained unchanged in terms of its spatial and tempo-ral expression pattern and mRNA localization TheSwedish mutation led to markedly enhanced b-secre-tase activity and a ninefold increase in Ab level,compared with normal aged human brain [60] By

22 months of age, APPNLh⁄ NLh mice had not oped Ab neuropathology [61], but young mice wereelegantly used to estimate the turnover of Ab, APPand APP fragments in vivo [62] In another genomicapproach, a 650 kb yeast artificial chromosome vectorharboring the whole human APP gene locus with the

devel-Swedish mutation was used In the homozygous mice

(R.1.40), in which Ab42 levels were 15 to 20-fold

higher than in mice expressing wild-type human APP,

fibrillar Ab deposition began at 14–15 months of age

There were region-dependent differences with more

cortical Ab deposits in old R.1.40 mice than

cDNA-based Tg2576 mice, despite lower Ab40 levels in young

mice [63] Heterozygous aged R.1.40 mice had only

diffuse Ab deposits, illustrating how a small change in

Ab levels in adolescence influences the speed and type

of AD-like pathology [64]

The APP23 model was generated by inserting human

APP751 with the Swedish mutation into the murine

Thy1 cassette It led to strong and highly specific

expression in postmitotic neurons and at  7 months

of age the animals developed a progressively increasing

burden of Congo Red-positive plaques The plaques

were surrounded by gliosis and distorted neurites that

were immunopositive for hyperphosphorylated tau

[65] APP23 mice have often been used to study CAA

pathogenesis It is most frequent at the adventitial

sur-face of SMC in arteries⁄ arterioles and is accompanied

by degeneration of vascular SMCs, disruption of the

blood–brain barrier and microhemorrhages in severely

amyloid-laden vessels [66,67] Behavioral and cognitive

effects with changes in activity levels that depended on

circadian rhythm were apparent from 3 months of age

in APP23 mice By 25 months, the mice

underper-formed in passive avoidance and small MWM tasks

[68]

Models with both Swedish and London-type APP

mutations

Patients never inherit multiple pathogenic mutations in

APP, presenilin, tau or a-synuclein genes, nor do they

overexpress chimeric APP mRNA under a

heterolo-gous promoter Thus none of the transgenic models

fully mimic the genetics of familial AD By combining

genetic lesions one can accelerate Ab aggregation and

lower the cost of research One can also confer certain

characteristics to Ab and dissect molecular

interac-tions The Swedish mutation has often been used

together with other mutations in transgenic models

because it is located outside the Ab domain and serves

to enhance Ab levels

No Ab pathology was evident at 24 months of age

in J.1.96 homozygous transgenic mice when a genomic

vector with both the Swedish and London mutations

was introduced, despite life-long exposure to a four- to

sixfold increased levels of Ab42, compared to human

APPwt [64] In contrast, APP22 mice presented with

diffuse Ab deposits and few amyloid plaques when the

mutations were combined in a cDNA-strategy tocreate twofold overexpression under the human Thy1promoter [65]

The Tg-CRND8 model was designed by insertinghuman APP695with Swedish and Indiana mutations inthe cosSHaPrP vector [55] It resulted in an aggressiveneuropathology with onset of amyloid deposition andplace learning impairment as early as 3 months of age.There was postnatal lethality, like many other APPtransgenic models, which could be mitigated by main-taining colonies on a favorable genetic background[69] Lines J9 and J20, developed by Mucke et al., alsocombined these two mutations, but the expression wasregulated by the PDGFb promoter There was a loss

of presynaptic synaptophysin immunoreactivity whichwas unrelated to plaques, and it could be shown thatnot only the level of Ab42, but also the ratio ofAb42⁄ Ab40, determined the onset of plaque formation[70] Consistent with this idea, Ab40 inhibited amyloidformation when uncoupled from APP processing intransgenic mice expressing BRI–Ab fusion proteins[71]

Tetracycline-regulated systems have not often beenused in the Ab-based transgenic models, perhapsbecause of their complicated nature with technicalcaveats regarding ‘leakage’ They offer a way to tightlycontrol the induction or repression of a transgene InTetO-APP-Swe⁄ Ind (line 107), a tetracycline-responsivepromoter was linked to a chimeric mouse⁄ humanAPP695 isoform, designed like the C3-3 line, but withboth the Swedish and Indiana mutations The micewere crossed with animals expressing the tetracyclinetransactivator under the control of the calcium-calmodulin kinase IIa promoter Repression of thetransgene was thereby restricted to the forebrain Amy-loid deposition was found in crossed rTA⁄ APP micefrom 8 weeks of age, a consequence of 10 to 30-foldincreased APP expression and two pathogenic APPmutations APP transgene expression was suppressed

> 95% when 4-week-old mice were given doxycyclinefor 2 weeks Relative to doxycycline-free mice, Ab inPBS- and SDS-soluble pools were efficiently cleared,although Ab42 partially remained in the formic acidsoluble pool By contrast, brains of animals reared ondoxycycline from birth to 6 weeks of age containedessentially no human Ab, suggesting a very early onset

of Ab aggregation and a tightly controlled transgeneexpression with little leakage Interestingly, suppression

of the transgene in 6-month-old mice arrested amyloiddeposition, but did not promote clearance Moreover,astrogliosis and ubiquitin-positive dystrophic neurites

in the vicinity of senile plaques were unchanged Thus,the endogenous clearance systems were unable to

eliminate existing Ab aggregates and secondary

pathol-ogy, at least in this aggressive transgenic model [72]

Models with the Flemish, Arctic, Dutch or Iowa

APP mutation inside the Ab domain

Mutations at positions 21–23 in the Ab domain of

APP, near the hydrophobic cluster, are a heterogeneous

group of genetic lesions They affect Ab aggregation

and degradation, but also APP processing The Dutch

(E693Q) [73] and Iowa (D694N) [74] mutations are

associated with CAA and diffuse Ab deposits, resulting

in hemorrhagic strokes and⁄ or infarcts and dementia

The neuropathology also consists of

leukoencephalopa-thy, degenerating neurites and NFTs [73,74] Transgene

expression in APPDutch mice, with only the Dutch

mutation, is regulated by the neuron-specific Thy1

promoter [32] The ratio of Ab40⁄ Ab42 was increased

in APPDutch mice, compared with APPwt, with the

Dutch mutation favoring the production and⁄ or

increased resistance of Ab40 to proteolysis In aged

animals there was an extensive accumulation of CAA

in leptomeningeal and cortical vessels with few diffuse

plaques, severe loss of SMCs, weakening of vessel walls

leading to hemorrhage and perivascular microgliosis

and astrocytosis The Dutch and Iowa mutations both

reduce the negative charge of Ab and stimulate the

formation of amyloid fibrils and attachment to the cell

surface of human cerebrovascular SMCs The two

mutations were combined with the Swedish mutation

in the SweDI transgenic model [75], because in vitro

studies had shown that Ab peptides with both

muta-tions induced greater vascular SMC degeneration [76]

The SweDI mice, with a low transgene expression

regulated by the Thy1.2 promoter, accumulated large

amounts of Ab in microvessels Severe CAA was

observed at  3 months of age with predominantly

diffuse parenchymal Ab deposits [75], and the mice

were cognitively impaired from a young age [77] The

vessel density in the hippocampus and thalamus was

reduced in parallel with increasing CAA, but was

not reduced in the frontotemporal cortex where

mainly diffuse parenchymal Ab deposits accumulated

The microvascular pathology was accompanied by

astro- and microgliosis as well as increased levels of

proinflammatory cytokines

The Flemish (A692G) [78] mutation can start either

with presenile dementia or CAA In contrast to the

other intra-Ab mutations, it makes APP an inferior

substrate for a-secretase and increases Ab levels

[79,80] APP cleavage by b-secretase was favored in

transgenic mice with the Flemish mutation (APP⁄ Fl),

with modestly increased Ab1-40 levels There was

spongiosis and gliosis in APP⁄ Fl mice, but no Ab ortau pathology Male APP⁄ Fl mice, which were bred onthe FVB background, were aggressive and sufferedpremature death and seizures APP expression waslikely insufficient to generate neuropathology and it isunclear if the findings in APP⁄ Fl were specificallycaused by the Flemish mutation An APP⁄ Du model,developed in parallel, displayed similar phenotypes[81]

The Arctic APP mutation (E693G) [79] is associatedwith clinical features of early-onset AD commencing at52–62 years There are NFTs, severe CAA in theabsence of hemorrhage and an abundance of paren-chymal Ab deposits lacking amyloid cores in postmor-tem brain [82] The Arctic mutation promotes Abprotofibril and fibril formation, but also favors intra-cellular b-secretase processing of APP [79,83,84].Tg-ArcSwe models with both the Swedish and Arcticmutation were developed by two independent groups

In young mice, the Arctic mutation increased ronal Ab accumulation in an age-dependent manner[85,86] Tg-ArcSwe mice without Ab depositionshowed cognitive deficits in MWM and two-way activeavoidance [85,87], and their performance correlatedinversely with soluble Ab protofibril levels [87] Build-ing on the combinatorial principle, lines Arc6 andArc48 with Swedish, Arctic and Indiana mutationswere generated Again, the Arctic mutation acceleratedamyloid formation despite a reduced proportion ofAb42 in young mice [88] Recently, a mouse modelexpressing human APP with only the Arctic mutation(APParc) under the control of the neuron-specificThy1 promoter was reported [89] In old APParc mice,both parenchymal and vascular congophilic Ab depos-its were found in the subiculum and thalamus Incontrast to transgenic models with both the Arctic andSwedish mutation [85,86], APParc mice did not showany punctate intraneuronal immunoreactivity Loco-motor activity and exploratory behavior of APParcmice was normal, although aged female mice displayedspatial learning and memory deficits Acceleratedamyloid pathology in female APParc mice is consistentwith findings in Tg2576 mice [57]

intraneu-APP transgenic models harboring a presenilin,tau or a-synuclein transgene

Presenilin transgenic mice were generated in response

to the identification of the presenilin-1 (PS-1) andpresenilin-2 (PS-2) AD mutations Metabolic Ab42levels were selectively increased in PS-1 transgenicmice [58,90] and, in comparison with APP transgenicmice, amyloid deposition was markedly accelerated in

bigenic PS-1· APP transgenic mice [91,92] Borchelt

et al generated PS-1 transgenic models expressing

mutant protein (M146L, A246E or PS1DE9), which

were cross-bred with APP transgenic mice with the

Swedish mutation, line C3-3 [58,91] Other researchers

cross-bred Tg2576 with PS-1 transgenic mice, in which

PS-1 cDNA (M146L or M146V) had been linked to

the PDGFb2 promoter [90], resulting in the PSAPP

model [92] PS2APP mice, generated by crossing PS2

(N141I) and APP-Swe, also displayed an aggressive

Ab pathology with age-dependent spatial learning and

memory deficits [93]

Autosomal dominant mutations in tau causing

frontotemporal lobe dementia were quickly utilized for

transgenic experiments The JNPL3 model, expressing

4R0N tau isoform with the P301L mutation, was the

first model with Gallyas-positive NFTs When

double-transgenic mice were created by cross-breeding with

Tg2576 a more complete AD neuropathology was

generated Moreover tau pathology, but not Ab

patho-logy, was enhanced in these mice, suggesting that effects

on tau are downstream of Ab in AD pathogenesis [94]

(Fig 2) However triple-transgenic mice, which were

generated by crossing mice producing the wild-type tau

isoform (3R0N) with mice carrying the Swedish and

London APP mutations and a PS-1 mutation (M146L),

only resulted in somadendritic accumulation of tau and

cytoskeletal changes Thus Ab-driven transgene

expres-sion failed to facilitate NFT formation in the presence

of human wild-type tau [95] In a similar strategy, the

pathogenic interaction between Ab and a-synuclein

was investigated by crossing PDGF–a-synuclein

and APP-SweInd, which led to a 1.6-fold increase in

a-synuclein inclusions in comparison with transgenic

mice that expressed only wild-type human a-synuclein

Similar to crossed APP· Tau mice, the level of

accumu-lated Ab in brain was similar in single- and

double-transgenic mice [96] The findings are relevant to the

Lewy body variant of AD with a-synuclein inclusions

Instead of cross-breeding two single-transgenic

mod-els, several vector constructs can be coinjected into

fertilized oocytes The transgenes will typically

cointe-grate at one location in the genome, and thus be

inher-ited as a single transgene ([97] and references therein)

The approach saves time and generates transgenic mice

on a homogenous genetic background, which reduces

variability and the number of animals needed for

experiments LaFerla et al developed 3xTg-AD by

coinjecting transgenes encoding both APP695-Swedish

and Tau isoform 4R0N with the P301L mutation into

pronuclei of PS-1 (M146V) knockin mice Both

transg-enes were subcloned into the Thy1.2 expression

cassette Intraneuronal Ab was visible in 3-month-old

mice and extracellular plaques in 6 to 12-month-oldanimals Phospho-tau immunoreactivity was detected

in 12 to 15-month-old mice, whereas paired helicalfilament-1 immunoreactivity and Gallyas staining,which indicate NFT formation, were not seen until

18 months of age [98] In more recent studies of thismodel, amyloid deposition commenced at 15 months

in the hippocampus and was widespread > 18 months[99] Triple-transgenic mice have been elegantly used tostudy interactions between Ab and tau pathologies andtheir impact on phenotypes of synaptic and cognitivedysfunction [100,101]

Advanced animal models have recently been ated in which neuronal degeneration is clearly evident

gener-In the 5xFAD transgenic model, Thy1 promoter-driventransgenes of APP (with the Swedish, Florida andLondon AD mutations) and PS-1 (with the AD muta-tions M146L and L286V) were coinjected into pronu-clei of C57BL⁄ 6xSJL mice The model was made in aneffort to alter the Ab42⁄ Ab40 ratio in favor of Ab42synthesis ([102] and references therein) Indeed thestrategy resulted in a high level of Ab42 and anAb42⁄ Ab40 synthesis ratio of 25 : 1 in young mice, incomparison with 0.1–0.2 : 1 in Tg2576 mice with onlythe Swedish APP mutation Amyloid deposits formedwithin 2 months, and the mice also developed intran-euronal Ab aggregates The intraneuronal depositswere in a b-pleated sheet conformation, and located tolarge pyramidal neurons of cerebral cortex layer V.Interestingly, in 9-month-old 5xFAD-mice there was aselective loss of these neurons and a decrease of severalsynaptic markers Importantly, these phenotypes andage-dependent cognitive deficits seemed to dependupon Ab because they did not occur in aged 5xFAD⁄BACE-KO mice [103] Neuronal loss was also found

in APP⁄ PS1 KI, homozygous PS-1 knockin mice(M233T⁄ L235P) with a Thy1 promoter-driven APPtransgene harboring the Swedish and Londonmutation In young mice intraneuronal Ab aggregates,positive for thioflavine S, were found in neurons thatdegenerated with aging, as in the 5xFAD model Theirbrains contained a substantial amount of N-truncatedand modified Ab peptides [104]

Insight into AD pathogenesis from experiments with transgenic models

Although far from perfect animal models of AD,transgenic mice have contributed significantly to theunderstanding of molecular pathogenesis Steady-statelevels of Ab in brain and CSF, and the ratio betweenthem in young transgenic mice are quite similarbetween the different models and conditions in healthy

humans (Table 1) It has been proposed that Ab

exists in a state of dynamic equilibrium between the

plasma and central nervous system (brain and CSF)

[105] If so, the ratio of [Ab]brain⁄ [Ab]plasma or

[Ab]CSF⁄ [Ab]plasma should be the same in all

trans-genic models A much higher plasma Ab level in

Tg2576 mice than in APP23, but a comparable

central nervous system Ab level, is inconsistent with

the idea of a dynamic equilibrium The higher plasma

Ab levels in Tg2576 is most likely explained by the

stronger peripheral activity of the hamster PrP

promoter, compared with the neuron-specific Thy1

promoter in APP23 This emphasises the influence of

promoter selection on differential expression patterns

of APP and steady-state Ab levels in the central

nervous system and in peripheral tissue; consequently,

interpretations from transgenic models regarding Ab

dynamics should be made with caution

In the 1980s it was debated whether Ab amyloid

deposits, and in particular CAA at the cerebral vessel

wall, had a central nervous system or a peripheral

source Models driven by the Thy1 promoter, like

APPDutch transgenic mice, with almost exclusive

neuronal central nervous system expression of APP

develop almost only CAA, but by introducing a

presenilin transgene and raising the ratio of

Ab42⁄ Ab40 synthesis instead mainly parenchymal

senile plaques develop [32] By contrast, models with

peripheral APP synthesis and high plasma Ab levels

present with amyloid deposits in peripheral organs

and neither CAA nor Ab plaques are found in the

brain of such mice [28–30] Thus, studies in transgenic

mice strongly suggest that neuronal Ab produced in

the brain generates cerebrovascular Ab

neuropathol-ogy With live imaging, the arrangement of vascular

SMC is found to be disrupted by CAA in transgenic

mice It leads to impaired vasodilator reactivity,

dis-tinct loss of SMC and hemorrhages [106], which is

similar to the pathogenesis in human brain [107]

Enthorhinal cortex lesion or transection of the

per-forant pathway have been used to demonstrate that

senile plaque formation depends on the synaptic

release of Ab and anterograde axonal transport of

APP [108,109] Senile plaque formation can also be

induced in APP transgenic mice if Ab-containing

brain extracts from plaque-laden mice or AD brain

are brought in direct contact with the central nervous

system The Ab phenotype then depends on both the

seeding agent and the host environment, similar to

prion disorders [110,111] The growth and stability of

dense-cored plaques have been investigated using

open skull window surgery and multiphoton

micros-copy [112] The great majority of amyloid plaques Table

formed very rapidly, within 1–2 days, reaching a size

that was surprisingly stable Within 1 week, early

changes were accompanied by the recruitment of

reactive microglia, and shortly thereafter by neuritic

dystrophy [113] However, in a subsequent study,

plaque growth occurred over a period of weeks when a

thinned-skull cranial window was used instead [114]

There is also a local neurotoxic effect on nerve endings

near amyloid plaques in APP transgenic models and in

AD postmortem brain [115], whereby dendritic spines

decrease in density but do not change in structure

[116] Loss of CA1 pyramidal neurons in the

hippo-campus was reported in aged APP23 mice with a high

plaque load [117], although subtle cell loss is difficult

to distinguish from physical displacement Today,

almost every research article in the AD field contains

an introductory statement in which the neurotoxicity

of Ab is described as a well-established fact, yet

analy-ses of mouse brain in which large amounts of Ab have

accumulated provide no or very sparse support for this

hypothesis Neurodegenerative mechanisms of

proteop-athies are still largely unknown Perhaps the

neurotox-icity is sparse because APP trafficking and subcellular

Ab accumulation in AD brain is poorly mimicked in

most models, as chimeric APP mRNAs are

overexpres-sed under heterologous promoters This hypothesis is,

however, inconsistent with knockin mice, APPNLh⁄ NLh,

showing no neurodegeneration Murine neurons could

be devoid of the downstream pathways necessary for

Ab to induce toxicity, and a prime suspect is of course

the processes leading to tau aggregation and NFTs in

AD brain More than 10 years ago modified and

trun-cated Ab peptides were demonstrated in AD brain

[6,7], but the observations were partially ignored It

could be that only certain species of Ab are neurotoxic

and that by using mutations linked to familial AD we

poorly replicate the processes of Ab production and

aggregation in sporadic AD brain There is now a

renewed interest in studying APP processing in

sporadic AD brain, and in understanding the

mecha-nisms of Ab truncation and modification Most

trans-genic models produce mainly full-length Ab, but by

manipulating the regulatory mechanisms or by

uncou-pling Ab synthesis from APP processing one can

generate transgenic models producing certain Ab

species These can indeed be neurotoxic, for example,

Ab3(pE)-42 in TBA2 mice [118], and thus not only in

a cell culture

The effect of ApoE on Ab neuropathology was first

examined in APP transgenic mice lacking murine

ApoE Ab burden, and more markedly amyloid

burden, was reduced in a gene-dose-dependent manner

[119] The human ApoE isoforms (e2, e3 and e4) were

expressed under the glial fibrillary acidic proteinpromoter in PDAPP mice lacking murine ApoE Abburden was then accelerated by the risk allele ApoE e4and decelerated by the protective allele ApoE e2, rela-tive to the ApoE e3 allele [120] These findings fit wellwith observations in postmortem AD brain [121].However, although murine ApoE facilitates Ab deposi-tion in a gene-dose-dependent manner, human ApoEdecelerates Ab deposition compared with murineApoE [122] This may be because a human transgenewas introduced into a complex feedback networkinvolving murine lipoprotein receptors Alternatively,ApoE may affect both Ab clearance and deposition Itillustrates the complexity of detailed mechanistic stud-ies Deletion of apolipoprotein J, which also binds to

Ab, decelerated amyloid formation [123], whereas tion of both ApoE and apolipoprotein J stronglyincreased Ab deposition [124] Possibly the lipoproteinmetabolism in the brain is altered when two abun-dantly expressed apolipoproteins, E and J, are bothabsent Lipidation of ApoE-containing lipoparticles viathe ATP-binding cassette family of active transportersregulates Ab deposition PDAPP mice overexpressingmurine ATP-binding cassette family of active transport-ers 1 are phenotypically similar to those devoid ofmurine ApoE In contrast, Ab and amyloid deposition

abla-is accelerated in APP transgenic mice devoid of binding cassette family of active transporters 1 ([12] andreferences therein)

ATP-Neuroinflammation has been suggested to influence

AD pathogenesis Astroglial expression of chymotrypsin, a constituent of senile plaques in ADbrain [125], accelerated both diffuse and senile plaqueformation [126–128] Transforming growth factor b1, amultifunctional cytokine, increased the level of extra-cellular matrix proteins and induced CAA in agedmice When glial fibrillary acidic protein⁄ transforminggrowth factor b1 mice were crossed with PDGF–APPtransgenic mice (lines H6 or J9) [70] CAA wasincreased and parenchymal amyloid depositionreduced In postmortem AD brain, the extent of CAAcorrelated with expression of transforming growthfactor b1 [129,130] Bone marrow-derived microgliacan reduce both the size and number of senile plaques

a1-anti-in transgenic mice [131,132] The chemoka1-anti-ine (C-Cmotif) ligand 2, and its receptor CCR2, is a key system

in the recruitment of mononuclear phagocytes into theCNS Astroglial overexpression of chemokine (C-Cmotif) ligand 2led to microgliosis and more diffuse Abdeposits [133] When CCR2 was deleted, microglialactivation was mitigated and perivascular Ab deposi-tion accelerated in crossed Tg2576⁄ CCR2-knockoutmice [134]