REVIEW ARTICLE Animal models of amyloid-b-related pathologies in Alzheimer’s disease Ola Philipson1, Anna Lord2, Astrid Gumucio1, Paul O’Callaghan1, Lars Lannfelt1 and Lars N.G Nilsson1 Department of Public Health and Caring Sciences ⁄ Molecular Geriatrics, Uppsala University, Sweden BioArctic Neuroscience AB, Stockholm, Sweden 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 Hammarskjolds vag 20, SE-751 85 ă ă Uppsala, Sweden Fax: +46 18 471 4808 Tel: +46 18 471 5039 E-mail: Lars.Nilsson@pubcare.uu.se In the early 1990s, breakthrough discoveries on the genetics of Alzheimer’s disease led to the identification of missense mutations in the amyloid-b precursor protein gene Research findings quickly followed, giving insights into molecular pathogenesis and possibilities for the development of new types of animal models The complete toolbox of transgenic techniques, including pronuclear oocyte injection and homologous recombination, has been applied in the Alzheimer’s disease field, to produce overexpressors, knockouts, knockins and regulatable transgenics Transgenic models have dramatically advanced our understanding of pathogenic mechanisms and allowed therapeutic approaches to be tested Following a brief introduction to Alzheimer’s disease, various nontransgenic and transgenic animal models are described in terms of their values and limitations with respect to pathogenic, therapeutic and functional understandings of the human disease (Received October 2009, revised 29 November 2009, accepted 30 December 2009) doi:10.1111/j.1742-4658.2010.07564.x 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 diagnosis is based on a patient’s medical history, neurological 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 account the regional distribution and frequency of histopathological hallmarks; specifically, extracellular neuritic plaques and intracellular neurofibrillary tangles (NFTs) in postmortem brain Neuritic plaques mainly consist of b-sheet-containing fibrils of amyloid-b (Ab) that are surrounded by dystrophic neurites and reactive glial cells Diffuse Ab deposits are also present, but these 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 FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS 1389 Animal models of Alzheimer’s disease O Philipson et al lack b-sheet structure and are therefore by definition not amyloid Cerebral amyloid angiopathy (CAA) results in the degeneration of vessel walls and hemorrhages CAA is found in  80% of AD brains, but is not a diagnostic criterion NFTs are intracellular filamentous 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 purification and partial sequencing of Ab from amyloid deposits 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 [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 pyroglutamyl 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 increased APP gene dosage caused by APP gene duplication [8] and Down’s syndrome with trisomy 21 Virtually all Down’s syndrome patients aged 35–40 years develop AD neuropathology, and most experience dementia by 60–70 years of age [9] The major genetic risk factor for developing lateonset AD is the apolipoprotein E (ApoE) e4 allele [10,11] One ApoE e4 allele increases the risk of AD by two- to threefold, and two e4 alleles confer a 12-fold increase in risk In the brain, ApoE is primarily synthesized by astrocytes and serves to regulate the transport of cholesterol-containing lipoprotein particles ApoE binds to Ab and becomes a component of amyloid in AD senile plaques The pathogenic mechanism of ApoE likely relates to altered deposition and ⁄ or clearance of Ab in the brain, although the details are still not fully understood [12] A large number of other disease-related loci and candidate genes have been proposed, but not generally verified, indicating that these genes have a modest impact on the pathogenesis The major risk factors for AD are age and a family history of the disease Low education or cognitive reserve capacity, female gender, head trauma, hypertension, cardiovascular disease and a high-cholesterol diet are proposed risk factors for AD [13] (Fig 2) Nontransgenic animal models Based on the cholinergic hypothesis, scopolamineinduced amnesia, excitotoxic lesions of the basal forebrain and aged primates have been used to assess cognitive deficits Current symptomatic drugs for AD were successfully evaluated in these models, but their etiological relevance is low [14] Nontransgenic rodents Fig 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 1390 FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS O Philipson et al Animal models of Alzheimer’s disease Fig 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 inflammation and oxidative stress, by rather unclear mechanisms These downstream processes give rise to progressive neurodegeneration, 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 experimental support for the hypothesis comes mainly from studies of families in which AD is inherited as a dominant trait due to mutations 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 are poor natural animal models of AD, but intracerebroventricular infusion of Ab [15] or lipopolysaccharide in such animals has been used The latter leads to neuroinflammation with hippocampal neurodegeneration 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 conditioning and show diffuse Ab deposits and vascular inflammation Aged dogs (> 10 years) can show impaired attention, spatial disorientation and disturbed diurnal rhythm Cognitive dysfunction in old dogs is associated with diffuse Ab deposits [20], neuritic dystrophy and gliosis, but few amyloid plaques and no NFTs Ventricular dilation, cortical and hippocampal atrophy, CAA with degeneration of smooth muscle cells (SMC) and hemorrhages can all be found in aged canine brain Interest in nonhuman primate models has grown following the failure to predict meningoencephalitis as a side-effect of the AN1792 vaccination trial from transgenic studies The efficacy and safety of an Ab vaccine has been tested in the Carribean vervet monkey [21] Alternatives are aged lemurs [22], cotton-top tamarins [23], rhesus monkeys [24] or squirrel monkeys [25] An aged chimpanzee with complete AD neuropathology, including neuritic plaques and paired helical filament-containing NFTs, was recently reported [26] FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS 1391 Animal models of Alzheimer’s disease O Philipson et al 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 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 pancreas [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 chromosome, 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 neuropathology and the levels of Ab were low [31] However, 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 mutation is not a prerequisite for amyloid deposition Instead it seems to depend upon producing sufficient Ab levels in the brain to ensure fibrillization To explore the pathogenic impact of individual Ab species, 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 overexpression Transgenic mice expressing BRI–wt-Ab42 developed extensive vascular and parenchymal amyloid pathology, accompanied by dystrophic neurites and astrogliosis In contrast to many APP transgenic models, amyloid deposition in BRI–wt-Ab42 mice began in the cerebellum, where both furin and the transgene were highly expressed This illustrates how the anatom1392 ical location of AD neuropathology can be manipulated simply by enhancing the regional dosage of amyloidogenic proteins and enzymes regulating their metabolism In contrast to BRI–wt-Ab42, no neuropathology was found in aged BRI–wt-Ab40 transgenic mice although they had higher Ab levels when they were young Thus the identity of Ab determines if neuropathology will develop [33] Models with the London-type APP mutation The London mutation (V717I) was the first genetic lesion to be discovered in a family with AD [3], and shortly thereafter the Indiana mutation (V717F) was found in an American pedigree [34] Patients with the Indiana mutation develop short-term learning impairments in the fifth decade, followed by progressive cognitive impairment and dementia with typical AD neuropathology An abundance of NFTs and senile plaques was observed at autopsy, as well as mild CAA [35] Games et al described the neuropathology of PDAPP mice, the first transgenic AD model [36] A mini-gene encompassing a human APP cDNA with the Indiana mutation interposed with introns had been designed Alternative splicing and synthesis of all three isoforms APP695, APP751 and APP770, with strong and selective neuronal expression was enabled by the platelet derived growth factor (PDGF)b promoter Importantly, young PDAPP mice produced high Ab42 levels in the brain, particularly in the hippocampus The animals preferentially accumulated Ab42 peptides and developed senile plaques, but also a substantial number of diffuse Ab deposits at 9–10 months of age [37] Plaque formation began in the cingulate cortex and was accompanied by phospho-tau immunoreactive dystrophic neurites, synaptic loss and gliosis in the adjacent tissue, but not by overt neuronal loss [38,39] Ultrastructural analyses revealed neurons in close proximity to senile plaques and amyloid fibrils The latter had a diameter of 9–11 nm and were surrounded by neuronal membranes and vesicles [40] Young PDAPP mice showed deficits in spatial learning and memory, which worsened with increasing age and Ab burden, although their performance in a novel object-recognition task was unimpaired [41] By contrast, others found age-dependent deficits in object recognition and place learning impairments that were independent of age [42] These discrepancies could be because of differences in experimental procedures or unintentional genetic drift of mouse colonies PDAPP mice are typically bred on a mixed genetic background (Swiss Webster, DBA ⁄ and C57Bl ⁄ 6) Hippocampal volume and corpus callosum length is reduced in FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS O Philipson et al 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 mechanism and its relevance to macroscopic atrophy in AD (if any) is still unclear Van Leuven et al generated several transgenic models, 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 Furthermore, 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 pathology, 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, Animal models of Alzheimer’s disease harbor the Swedish mutation and display both AD-like Ab neuropathology and cognitive deficits [51] The Swedish mutation redirects APP processing to secretory vesicles en route to the cell surface in cell culture [52], whereas APPwt is largely processed in recycling endosomes [53] This difference may be largely irrelevant in the brain, because Ab synthesis along the endolysosomal pathway is clearly important in Tg2576 [54] A substantial amount of CAA is often found in transgenic mice with the Swedish mutation, which is likely to be because of the high rate of synthesis and accumulation of Ab1-40 In Tg2576, more than fivefold overexpression of the human APP695 isoform with the Swedish mutation is generated by the prion promoter APP cDNA was cloned into a  40 kb genomic fragment (cosSHaPrP) [55] from the hamster prion protein gene A significant proportion of Tg2576 mice die at a young age, and the severity of this phenotype depends upon the genetic background It has been found that colonies are best maintained by mating heterozygous C57BL ⁄ males with B6SJLF1 females At around  11 months of age, Tg2576 mice show extracellular Ab deposits which are largely soluble in SDS and mainly contain Ab40 ( 75%) CSF levels of Ab42, but not Ab40, decrease with age and amyloid deposition [56], and pathogenesis is accelerated in female Tg2576 mice [57] Both these observations fit well with biochemical and epidemiological findings in AD [13] Borchelt et al used the prion promoter to generate line C3-3 [58] A chimeric cDNA clone encoding murine APP695 was used, in which the region in and around the murine Ab domain was replaced with the human Ab sequence and the Swedish mutation The MoPrP.Xho vector was much smaller than the cosSHaPrP, and selectively directed twofold overexpression of APP to the brain [58,59] In an even more refined strategy, the murine Ab sequence was humanized and the Swedish mutation introduced with gene targeting In this knockin model, APPNLh ⁄ NLh, only five single amino acids were altered in the entire murine genome Consequently, APP protein synthesis remained unchanged in terms of its spatial and temporal expression pattern and mRNA localization The Swedish mutation led to markedly enhanced b-secretase 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 developed Ab neuropathology [61], but young mice were elegantly used to estimate the turnover of Ab, APP and APP fragments in vivo [62] In another genomic approach, a 650 kb yeast artificial chromosome vector harboring the whole human APP gene locus with the FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS 1393 Animal models of Alzheimer’s disease O Philipson et al 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 cDNAbased 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  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 surface 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 months of age in APP23 mice By 25 months, the mice underperformed 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 they overexpress chimeric APP mRNA under a heterologous 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 interactions 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 1394 mutations were combined in a cDNA-strategy to create twofold overexpression under the human Thy1 promoter [65] The Tg-CRND8 model was designed by inserting human APP695 with Swedish and Indiana mutations in the cosSHaPrP vector [55] It resulted in an aggressive neuropathology with onset of amyloid deposition and place learning impairment as early as months of age There was postnatal lethality, like many other APP transgenic models, which could be mitigated by maintaining colonies on a favorable genetic background [69] Lines J9 and J20, developed by Mucke et al., also combined these two mutations, but the expression was regulated by the PDGFb promoter There was a loss of presynaptic synaptophysin immunoreactivity which was unrelated to plaques, and it could be shown that not only the level of Ab42, but also the ratio of Ab42 ⁄ Ab40, determined the onset of plaque formation [70] Consistent with this idea, Ab40 inhibited amyloid formation when uncoupled from APP processing in transgenic mice expressing BRI–Ab fusion proteins [71] Tetracycline-regulated systems have not often been used in the Ab-based transgenic models, perhaps because of their complicated nature with technical caveats regarding ‘leakage’ They offer a way to tightly control the induction or repression of a transgene In TetO-APP-Swe ⁄ Ind (line 107), a tetracycline-responsive promoter was linked to a chimeric mouse ⁄ human APP695 isoform, designed like the C3-3 line, but with both the Swedish and Indiana mutations The mice were crossed with animals expressing the tetracycline transactivator under the control of the calciumcalmodulin kinase IIa promoter Repression of the transgene was thereby restricted to the forebrain Amyloid deposition was found in crossed rTA ⁄ APP mice from  weeks of age, a consequence of 10 to 30-fold increased APP expression and two pathogenic APP mutations APP transgene expression was suppressed > 95% when 4-week-old mice were given doxycycline for weeks Relative to doxycycline-free mice, Ab in PBS- and SDS-soluble pools were efficiently cleared, although Ab42 partially remained in the formic acid soluble pool By contrast, brains of animals reared on doxycycline from birth to weeks of age contained essentially no human Ab, suggesting a very early onset of Ab aggregation and a tightly controlled transgene expression with little leakage Interestingly, suppression of the transgene in 6-month-old mice arrested amyloid deposition, 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 FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS O Philipson et al eliminate existing Ab aggregates and secondary pathology, 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 leukoencephalopathy, 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 mutations 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  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 Animal models of Alzheimer’s disease spongiosis and gliosis in APP ⁄ Fl mice, but no Ab or tau pathology Male APP ⁄ Fl mice, which were bred on the FVB background, were aggressive and suffered premature death and seizures APP expression was likely insufficient to generate neuropathology and it is unclear if the findings in APP ⁄ Fl were specifically caused by the Flemish mutation An APP ⁄ Du model, developed in parallel, displayed similar phenotypes [81] The Arctic APP mutation (E693G) [79] is associated with clinical features of early-onset AD commencing at 52–62 years There are NFTs, severe CAA in the absence of hemorrhage and an abundance of parenchymal Ab deposits lacking amyloid cores in postmortem brain [82] The Arctic mutation promotes Ab protofibril and fibril formation, but also favors intracellular b-secretase processing of APP [79,83,84] Tg-ArcSwe models with both the Swedish and Arctic mutation were developed by two independent groups In young mice, the Arctic mutation increased intraneuronal Ab accumulation in an age-dependent manner [85,86] Tg-ArcSwe mice without Ab deposition showed cognitive deficits in MWM and two-way active avoidance [85,87], and their performance correlated inversely with soluble Ab protofibril levels [87] Building on the combinatorial principle, lines Arc6 and Arc48 with Swedish, Arctic and Indiana mutations were generated Again, the Arctic mutation accelerated amyloid formation despite a reduced proportion of Ab42 in young mice [88] Recently, a mouse model expressing human APP with only the Arctic mutation (APParc) under the control of the neuron-specific Thy1 promoter was reported [89] In old APParc mice, both parenchymal and vascular congophilic Ab deposits were found in the subiculum and thalamus In contrast to transgenic models with both the Arctic and Swedish mutation [85,86], APParc mice did not show any punctate intraneuronal immunoreactivity Locomotor activity and exploratory behavior of APParc mice was normal, although aged female mice displayed spatial learning and memory deficits Accelerated amyloid pathology in female APParc mice is consistent with findings in Tg2576 mice [57] 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) and presenilin-2 (PS-2) AD mutations Metabolic Ab42 levels were selectively increased in PS-1 transgenic mice [58,90] and, in comparison with APP transgenic mice, amyloid deposition was markedly accelerated in FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS 1395 Animal models of Alzheimer’s disease O Philipson et al 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 doubletransgenic mice were created by cross-breeding with Tg2576 a more complete AD neuropathology was generated Moreover tau pathology, but not Ab pathology, 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 expression 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 accumulated Ab in brain was similar in single- and doubletransgenic mice [96] The findings are relevant to the Lewy body variant of AD with a-synuclein inclusions Instead of cross-breeding two single-transgenic models, several vector constructs can be coinjected into fertilized oocytes The transgenes will typically cointegrate at one location in the genome, and thus be inherited 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 transgenes were subcloned into the Thy1.2 expression cassette Intraneuronal Ab was visible in 3-month-old 1396 mice and extracellular plaques in to 12-month-old animals Phospho-tau immunoreactivity was detected in 12 to 15-month-old mice, whereas paired helical filament-1 immunoreactivity and Gallyas staining, which indicate NFT formation, were not seen until 18 months of age [98] In more recent studies of this model, amyloid deposition commenced at  15 months in the hippocampus and was widespread > 18 months [99] Triple-transgenic mice have been elegantly used to study interactions between Ab and tau pathologies and their impact on phenotypes of synaptic and cognitive dysfunction [100,101] Advanced animal models have recently been generated in which neuronal degeneration is clearly evident In the 5xFAD transgenic model, Thy1 promoter-driven transgenes of APP (with the Swedish, Florida and London AD mutations) and PS-1 (with the AD mutations M146L and L286V) were coinjected into pronuclei of C57BL ⁄ 6xSJL mice The model was made in an effort to alter the Ab42 ⁄ Ab40 ratio in favor of Ab42 synthesis ([102] and references therein) Indeed the strategy resulted in a high level of Ab42 and an Ab42 ⁄ Ab40 synthesis ratio of 25 : in young mice, in comparison with 0.1–0.2 : in Tg2576 mice with only the Swedish APP mutation Amyloid deposits formed within months, and the mice also developed intraneuronal Ab aggregates The intraneuronal deposits were in a b-pleated sheet conformation, and located to large pyramidal neurons of cerebral cortex layer V Interestingly, in 9-month-old 5xFAD-mice there was a selective loss of these neurons and a decrease of several synaptic markers Importantly, these phenotypes and age-dependent cognitive deficits seemed to depend upon 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 APP transgene harboring the Swedish and London mutation In young mice intraneuronal Ab aggregates, positive for thioflavine S, were found in neurons that degenerated with aging, as in the 5xFAD model Their brains contained a substantial amount of N-truncated and 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 the understanding of molecular pathogenesis Steady-state levels of Ab in brain and CSF, and the ratio between them in young transgenic mice are quite similar between the different models and conditions in healthy FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS 1.6 nM 220 90 pM 20 Diffuse and neuritic plaques and CAA NFTs, atrophy Murine Thy1 Healthy [164,165] human APP695 cDNA KM670 ⁄ 671NL (Swedish) + E693G (Arctic) TgArcSwe [86] Murine Thy1 APP23 [65] Tg2576 [51,56] APP minigene V717F (Indiana) APP695 cDNA KM670 ⁄ 671NL (Swedish) APP751 cDNA KM670 ⁄ 671NL (Swedish) PDAPP [36,37,105] Hamster PrP 12 BioArctic 1.5 nM 300 10 pM 5–6 Novartis 11 nM 500 80 pM 40 Taconic 13 nM 4.5 nM 40 9–10 Elan ⁄ Eli Lilly nM 500 40 pM 20 6–9 Diffuse and neuritic plaques, little CAA Neuritic plaques substantial CAA Neuritic plaques profound CAA some neuron loss Neuritic plaques and CAA Neuropathology Promoter Transgene Model PDGF Contact Ratio brain Ab ⁄ CSF Ab CSF Ab Ratio brain Ab ⁄ plasma Ab Plasma Ab Brain Ab (pmolỈg)1) Age-of -onset (months) 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 transgenic 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 neuropathology With live imaging, the arrangement of vascular SMC is found to be disrupted by CAA in transgenic mice It leads to impaired vasodilator reactivity, distinct loss of SMC and hemorrhages [106], which is similar to the pathogenesis in human brain [107] Enthorhinal cortex lesion or transection of the perforant 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 microscopy [112] The great majority of amyloid plaques Animal models of Alzheimer’s disease Table Metabolic levels of amyloid-b (Ab) in brain, cerebrospinal fluid (CSF) and plasma of young transgenic mice and healthy human Measures in PDAPP refer to Ab 1–x, in Tg2576, APP23 and humans to the sum of Ab1–40 and Ab1–42, and in tgArcSwe they refer to Ab1–40 A density gỈL)1 for brain tissue has been assumed when the ratio of brain Ab ⁄ plasma Ab has been calculated Thus, for example, 20 pmolỈg)1  20 nM Studies on the initial description and on metabolic levels of brain, CSF and plasma Ab that were used as sources of information are cited APP, amyloid-b precursor protein; CAA, cerebral amyloid angiopathy; NFT, neurofibrillary tangles; PDGF, platelet-derived growth factor O Philipson et al 1397 Animal models of Alzheimer’s disease O Philipson et al formed very rapidly, within 1–2 days, reaching a size that was surprisingly stable Within 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 not change in structure [116] Loss of CA1 pyramidal neurons in the hippocampus 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 analyses of mouse brain in which large amounts of Ab have accumulated provide no or very sparse support for this hypothesis Neurodegenerative mechanisms of proteopathies are still largely unknown Perhaps the neurotoxicity is sparse because APP trafficking and subcellular Ab accumulation in AD brain is poorly mimicked in most models, as chimeric APP mRNAs are overexpressed 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 truncated 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 mechanisms of Ab truncation and modification Most transgenic models produce mainly full-length Ab, but by manipulating the regulatory mechanisms or by uncoupling 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 1398 expressed under the glial fibrillary acidic protein promoter in PDAPP mice lacking murine ApoE Ab burden was then accelerated by the risk allele ApoE e4 and decelerated by the protective allele ApoE e2, relative to the ApoE e3 allele [120] These findings fit well with observations in postmortem AD brain [121] However, although murine ApoE facilitates Ab deposition in a gene-dose-dependent manner, human ApoE decelerates Ab deposition compared with murine ApoE [122] This may be because a human transgene was introduced into a complex feedback network involving murine lipoprotein receptors Alternatively, ApoE may affect both Ab clearance and deposition It illustrates the complexity of detailed mechanistic studies Deletion of apolipoprotein J, which also binds to Ab, decelerated amyloid formation [123], whereas ablation of both ApoE and apolipoprotein J strongly increased Ab deposition [124] Possibly the lipoprotein metabolism in the brain is altered when two abundantly expressed apolipoproteins, E and J, are both absent Lipidation of ApoE-containing lipoparticles via the ATP-binding cassette family of active transporters regulates Ab deposition PDAPP mice overexpressing murine ATP-binding cassette family of active transporters are phenotypically similar to those devoid of murine ApoE In contrast, Ab and amyloid deposition is accelerated in APP transgenic mice devoid of ATPbinding cassette family of active transporters ([12] and references therein) Neuroinflammation has been suggested to influence AD pathogenesis Astroglial expression of a1-antichymotrypsin, a constituent of senile plaques in AD brain [125], accelerated both diffuse and senile plaque formation [126–128] Transforming growth factor b1, a multifunctional cytokine, increased the level of extracellular matrix proteins and induced CAA in aged mice When glial fibrillary acidic protein ⁄ transforming growth factor b1 mice were crossed with PDGF–APP transgenic mice (lines H6 or J9) [70] CAA was increased and parenchymal amyloid deposition reduced In postmortem AD brain, the extent of CAA correlated with expression of transforming growth factor b1 [129,130] Bone marrow-derived microglia can reduce both the size and number of senile plaques in transgenic mice [131,132] The chemokine (C-C motif) ligand 2, and its receptor CCR2, is a key system in the recruitment of mononuclear phagocytes into the CNS Astroglial overexpression of chemokine (C-C motif) ligand led to microgliosis and more diffuse Ab deposits [133] When CCR2 was deleted, microglial activation was mitigated and perivascular Ab deposition accelerated in crossed Tg2576 ⁄ CCR2-knockout mice [134] FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS O Philipson et al There is considerable in vitro evidence that metal ions like Zn2+ and Cu2+ play a role in Ab biology, and possibly in AD pathogenesis Both ions bind APP and Ab with high affinity, and stimulate Ab aggregation and oxidizing effects of Ab in vitro By deleting the gene encoding a zinc transporter, ZnT3, the endogenous pool of synaptic Zn2+ was depleted Senile plaque deposition was then markedly decelerated in Tg2576 mice in a dose-dependent manner, whereas soluble Ab was modestly increased Synaptic zinc and ZnT3 changed in response to ovariectomy and estrogen replacement possibly explaining why increased Ab burden is often observed in female APP transgenic mice [57,135,136] As described above, knowledge on pathogenic mechanisms has often been gained by removing or expressing normal or mutant genes and examining APP ⁄ Ab-related phenotypes A major caveat when interpreting the results from a cross-breeding experiment is the influence of the genetic background This problem can be circumvented by expressing a transgene at a specific location in the brain with a lentiviral vector [137] The relevance of cross-breeding experiments is strengthened if expression and deletion of the gene(s) results in the opposite outcome, or if the results are substantiated by in vitro studies or analyses of clinical samples The ability to track pathogenic events in living animals with intravital multiphoton microscopy, small animal PET cameras and microdialysis has considerably enhanced our understanding of AD pathogenesis Therapeutic studies in AD models Access to good animal models is crucial to success in developing disease-modifying therapeutics However, AD neuropathology is incomplete in the Ab-expressing animal models discussed, and their ability to predict the outcome of clinical studies is limited There are publications on a great variety of therapeutic strategies in APP transgenics and most often they have had a positive outcome [13] Does this mean that virtually anything can clear Ab deposits in transgenic mice and that the models lack predictive validity altogether? We would argue that it does not, but that many therapeutic studies have, in fact, been poorly designed and have had insufficient power and that their appearance in the literature is caused by publication bias If experimental groups are not randomized it can result in the drugtreated group of mice having by chance lower Ab burden prior to the therapeutic intervention Wellknown to many researchers, but seldom discussed in the literature, are both gender- [57] and litter-depen- Animal models of Alzheimer’s disease dent differences in the speed of Ab accumulation Moreover, transgene expression can differ across generations and give rise to unstable Ab phenotypes because of loss of transgene copies or more complex genetic mechanisms Transgenic mice are often bred on a mixed background, and complex genetics affects the steady-state level of Ab and Ab accumulation [138] A difference in genetic background between drug- and placebo-treated groups can then lead to a systematic error that is mistakenly interpreted as evidence of therapeutic efficacy Many APP transgenic models also suffer spontaneous death, which drives selection in a cohort of animals by unknown mechanisms Consequently, a drug under investigation might modulate spontaneous death and not AD-pathogenesis Randomizing experimental groups, matching them for gender, and having sufficient power are therefore extremely important in minimizing the influence of confounding factors arising from inherent problems associated with breeding APP transgenic mice and maintaining stable phenotypes We would argue that a single report that has not been replicated, preferably by other researchers, provides weak evidence of therapeutic efficacy Knowledge of the pathway one intends to target and the pharmacological mechanism of the drug are equally important In vitro and in vivo pharmacology of the drug should preferably be carried out before efficacy is tested in APP transgenic mice Unfortunately, many drug candidates or dietary supplements are simply directly tested for in vivo efficacy in the absence of prior pharmacological and pharmacokinetic experiments, and without mechanistic knowledge One also needs to carefully consider which transgenes to express ⁄ suppress and what pathogenic AD mutation to include in the animal model For example, a putative BACE-1 inhibitor can be evaluated in a transgenic model with the Swedish mutation because Ab levels would be high and even a modest reduction by a drug candidate would be easy to detect The conditions would not, however, reflect those of sporadic AD in terms of APP trafficking and enzyme substrate, since BACE-1 cleaves APP with the Swedish mutation much more efficiently than wildtype APP Another example are c-secretase modulators whose efficacy differs between wild-type and mutant PS, and also among different PS mutations [139] The choice of transgene and mutation can also markedly affect the solubility of Ab deposits [140] Thus, testing a drug that stimulates clearance in an animal model carrying the Swedish mutation may generate a positive outcome, but then fail in patients, because AD deposits are far more resilient FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS 1399 Animal models of Alzheimer’s disease O Philipson et al Complementary studies in AD models where plaques are more resistant to degradation, for example, Tg-ArcSwe [140] or PSAPP transgenic mice [72] may then be worthwhile A substance that has been proven to target Ab aggregation ⁄ clearance or APP processing in vitro in convincing dose–response experiments is a good candidate for an efficacy study in APP transgenic mice One can then investigate drug metabolism, determine if relevant concentrations reach its target in the brain and compare dose-response findings in vivo and in vitro It is also important to consider how the pathological Ab-lesions relate to dementia in AD patients Unfortunately, in a clinical trial AD patients (and likely some healthy subjects) will have substantial amounts of Ab and tau pathology at the commencement of treatment Protein aggregation has often only been prevented in APP transgenic mice, but it is far more difficult to clear existing Ab deposits This has also been seen in, for example, superoxide dismutase transgenic mice models of amyotrophic lateral sclerosis [141] Therefore, to avoid overinterpreting therapeutic studies in transgenic mice, it is important to record the age and evaluate the stage of neuropathology when the animals were first given the drug Here we present a few examples of Ab-based drug candidates that are in preclinical or clinical development Ab immunotherapy is perhaps the most promising disease-modifying treatment strategy for AD, and it also illustrates the potential clinical value of transgenic mice Immunization would probably never have been pursued if APP transgenic mice had not been available Human clinical trials of active immunization with fibrillar Ab1–42 (AN1792) were rapidly initiated when biochemical and functional efficacy had been proven in APP transgenic mice [142–144] Studies were halted in phase II, because meningoencephalitis developed in a subgroup of patients [145] Encouragingly, there was evidence of Ab plaque clearance in postmortem brain of vaccinated patients, resembling that of transgenic mice [146] Passive immunization, i.e direct administration of N-terminal Ab antibodies has proven efficacious and possibly safer [147] It permits dosage control and circumvents problems of insufficient humoral immune response among the elderly [148] Reduced Ab pathology has been shown with several types of Ab antibodies in APP transgenic mice [147,149,150] The outcome of ongoing phase III trials will likely decisively influence the future of immunotherapy, and also the procedures whereby immunotherapeutic strategies are evaluated at the preclinical stage Reduction of Ab synthesis with inhibitors of b- and c-secretase was pursued long before the molecular 1400 identities of the drug targets were known or APP transgenic mice were available With robust and sensitive Ab ELISAs, the efficacy of b- and c-secretase can be tested in nontransgenic animals Pharmacological studies with the c-secretase inhibitor semagacestat (LY450139) showed that temporal changes in plasma and CSF Ab in patients, were better reflected by observations in beagle dogs than in PDAPP mice [151– 153] This illustrates that nontransgenic animal models are needed, at least as a complement in the drugdevelopment process Nonetheless, pharmacological evaluation of b- and c-secretase inhibitors in transgenic mice permits investigators to determine if a treatment is tolerable at a dosage which impacts on neuropathology and cognitive dysfunction It should also be remembered that transgenic models have contributed significantly to research on b- and c-secretase inhibitors by identifying potential side effects and providing suggestive biomarkers to be used in clinical trials Examples of pioneering studies are the demonstration of impaired remyelination of sciatic nerves and reduced cleavage of the neuregulin-1 precursor in BACE-1 knockout mice [154], and lethal phenotypes observed in PS-1 knockout mice The latter experiments led to the realization that the c-secretase complex also regulates the Notch cell signaling pathway [155] Functional studies with AD models We regard behavioral studies with APP transgenic mice as being relevant to prove that a certain Ab species or a pathological lesion has a functional effect on neurotransmission Because our knowledge of the mechanisms and neuropathology of AD are both incomplete, it is not possible to predict if a new drug will impact on AD symptomatology based on functional studies with APP transgenic mice Effects of the promoter, transgene overexpression and strain background all need to be considered when interpreting functional studies These parameters can have a major impact on behavior and also generate variability It is also difficult to distinguish between deficits caused by Ab or APP overexpression A transgenic model should ideally first be investigated in a comprehensive battery of cognitive and sensorimotor tests [156], which is labor intensive but can be rewarding if combined with statistical analyses [157] The MWM task is one of the most widely used cognitive tests It depends on hippocampal formation, which is damaged by pathological lesions early in AD The animal swims in a pool and is required to find and remember where a submerged platform is hidden, by FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS O Philipson et al Animal models of Alzheimer’s disease using distal visual cues [158] This is believed to trigger increased activity of place cells in the hippocampus, whereby a spatial map is engrained [159] By analyzing the swim speed and pattern or by elevating the platform above the surface (visual learning), one can exclude sensorimotor disturbances or motivational shortage More challenging protocols of MWM, in which the platform location alternates [41], or the radial arm water maze [160] will increase the demand on working memory Spatial alternation tasks (e.g Y- and T-maze) are other hippocampal-dependent behavioral tests in which spontaneous or rewarded exploration behaviour and working memory can be assessed Functional study results are often difficult to reproduce between laboratories, and even between cohorts of an animal model in the same laboratory Conventional tests are also time-consuming and greatly influenced by individual handlers IntelliCages are automated learning cages where animals carrying transponders are housed in groups and trained in learning corners Each individual is recognized by a distinct antenna signal [161] Standardized and validated protocols with the Intellicage system, or a similar system [162], could circumvent practical drawbacks and limit variability associated with conventional behavioral tasks These systems should facilitate reproducible functional studies with animal models of AD Concluding remarks and future perspectives Transgenic techniques have revolutionized our ability to develop animal models of AD, and also contributed significantly to the understanding of molecular pathogenesis Today a wide range of animal models are available for mechanistic, therapeutic and functional studies They offer an appealing means to rapidly move from simplistic in vitro experiments to clinical trials It is important to understand the strengths and limitations of the models (Table 2) We foresee that technical advances in RNA interference and gene targeting with, for example, zinc-finger nucleases will be increasingly utilized in the future This could lead to new animal models of AD where proteins are not overexpressed and also to more sophisticated studies of pathogenic mechanisms in APP transgenic mice Moreover, chimeric proteins will frequently be designed to target transgene expression to certain subcellular locations in postmitotic neurons and to then express only a defined Ab peptide The first transgenic nonhuman primate model of AD will be developed, as a result of recent successes in modeling Huntington’s disease [163] This will enable limited therapeutic studies where effects of a new drug on higher cognitive functions can be better evaluated with high-resolution imaging and neuropsychology Animal models will continue to be crucial in translational research, but Table Neuropathological characteristics of some common transgenic mouse models +++, extensive phenotype; +, detectable; ), not detected; nr, not reported Model [Ref] Age of plaque onset (mo) Neuritic plaques Diffuse plaques Bri-wt-Ab42A [33] PDAPP [36] APP-London [44] Tg2576 [51] APPNLh ⁄ NLh [60,61] C3-3 [58] R.1.40 [64] APP23 [65] Tg-CRND8 [69] APPDutch [32] SweDI [75] Tg-ArcSwe [86] APParc [89] PSAPP [92] 3xTg-AD [98] 5xFAD [102] APP ⁄ PS1 KI [104] TBA2 [118] 6–8 > 12 9–11 > 22 18 14 22–25 > 12 6 2–3 +++ +++ +++ +++ nr + +++ +++ +++ ) ) +++ + +++ +++ +++ +++ nr +++ +++ +++ + nr + +++ + + + +++ + + + +++ + + +++ CAA Intraneuronal Ab accumulation CNS specific expression Neurodegeneration N-terminal truncated Ab + + + + nr nr + + + +++ +++ + + + + nr nr ) nr nr nr + nr nr nr nr nr + nr +++ ) nr +++ +++ +++ +++ + +++ +++ + ) + ) +++ + +++ +++ +++ +++ + +++ +++ +++ +++ ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + + +++ nr nr nr + nr nr nr nr nr nr nr nr nr nr nr nr +++ +++ FEBS Journal 277 (2010) 1389–1409 ª 2010 The Authors Journal compilation ª 2010 FEBS 1401 Animal models of Alzheimer’s disease O Philipson et al careful in vitro experiments and advanced early clinical studies will provide important contributions to the development of the first approved disease-modifying drug for AD Acknowledgements Mattias Staufenbiel and Thomas Bayer are greatly acknowledged for providing information on the APP23 and TBA ⁄ models, and the Swedish Research Council (2009-4567, LL; 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confounding factors arising from inherent problems associated with breeding APP transgenic mice and maintaining stable phenotypes We would argue that a single... similar in single- and doubletransgenic mice [96] The findings are relevant to the Lewy body variant of AD with a-synuclein inclusions Instead of cross-breeding two single-transgenic models, several