ILAR Journal Online, Volume 43(2) 2002: Mouse Models of Human Disease

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ILAR Journal V43(2) 2002
Mouse Models of Human Disease

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Plaques

Mouse Models of Alzheimer's Disease: A Quest for Plaques
James A. Richardson and Dennis K. Burns

James A. Richardson, D.V.M., Ph.D., is Professor in the Departments of Pathology and Molecular Biology, and Dennis K. Burns, M.D., is Professor in the Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas.

Abstract

Many genetically altered mice have been designed to help understand the role of specific gene mutations in the pathogenesis of Alzheimer's disease (AD) based on the realization that specific mutations in the genes for amyloid precursor protein--the presenilins and tau--are associated with early-onset familial AD or, in the case of tau mutations, other neurodegenerative diseases with neurofibrillary tangles. However, attempts to reproduce the neuropathology of AD in the mouse have been frustrating. Transgenic designs emphasizing amyloid precursor protein produced mice that develop amyloid plaques, but neurodegeneration and neurofibrillary tangles failed to form. Strategies emphasizing tau resulted in increased phosphorylation of tau and tangle formation, although amyloid plaques were absent. Nevertheless, crossing transgenic animals expressing mutated tau and amyloid precursor protein has produced a mouse that closely recapitulates the neuropathology of AD. A review of the various murine models, their role in understanding the pathogenesis of AD and their use in testing therapeutic regimens, is provided.

Key Words: Alzheimer's disease; amyloid plaque; neurofibrillary tangle; tau; transgenic mice

Introduction

Alzheimer's disease (AD¹) is the most common cause of dementia in the elderly and affects an estimated 4 million people in the United States alone. The majority of cases of AD are sporadic, although in roughly 10% of cases, there is a family history of dementia. These cases of familial AD (FAD¹) have provided important insights into the pathogenesis of AD in recent years. In several pedigrees of early-onset FAD, point mutations in several genes have been identified. Mutations in the genes encoding amyloid-β precursor protein (APP¹), presenilin 1 (PS1¹), and presenilin 2 (PS2¹) are associated with early-onset autosomal dominant AD. In addition, AD is associated with the accumulation of hyperphosphorylated tau within degenerating neurons. The rationale for creating the various transgenic mouse models of AD has been to explore the function of these molecules in vivo and to establish a mouse model with histopathological and clinical features that parallel AD in humans.

Morphological Changes in AD

The brain is typically atrophic in AD, although it may be grossly normal, in the early stages of the disease particularly. When present, atrophy is typically most pronounced in the frontal, temporal, and parietal lobes. Examination of the cut surface reveals symmetrical dilation of the ventricular system in most cases, reflecting a generalized loss of brain parenchyma.

Microscopically, AD is characterized by the presence of filamentous protein aggregates (termed neurofibrillary tangles) within the cytoplasm of neurons in the neocortex, hippocampus, basal forebrain, and some areas of the brainstem (Figure 1A). Specifics regarding the postmortem diagnosis of AD are outlined in a consensus recommendation from the National Institute on Aging (Hyman and Trojanowski 1997). The neurofibrillary tangles are composed of course neuritic processes containing insoluble protein-rich helical filaments, the major component of which is hyperphosphorylated tau protein. Filamentous processes (termed neuropil threads), likely representing altered dendritic processes, also accumulate in the neuropil in AD in a distribution similar to that of the neurofibrillary tangles; and they contain tau and other abnormal cytoskeletal proteins similar to those present in neurofibrillary tangles. Additional accumulations of tau-rich paired helical filaments occur within distal neuronal cell processes (neurites) to form so-called senile plaques (Figure 1B), which appear as aggregates of coarse tortuous neurites in the neuropil of the cerebral cortex and hippocampus. The senile plaques also contain a central core composed of amyloid β protein (Aβ¹). Aβ deposits are usually present within the walls of leptomeningeal as well as smaller parenchymal vessels, a change referred to as amyloid angiopathy.

Figure 1 (A) Section of hippocampus, from an elderly patient with Alzheimer's disease, stained with antibody to hyperphosphorylated tau protein, demonstrates densely staining neurofibrillary tangles within neuronal cell bodies. (B) Immunohistochemical stain from a case similar to (A) demonstrates a typical neuritic plaque, composed of clusters of swollen neuritic cell processes laden with hyperphosphorylated tau protein. Diaminobenzidine-labeled section ×400.

The simple presence of neurofibrillary tangles and/or senile plaques is not, by itself, entirely specific for AD inasmuch as identical structures are also frequently present in the brains of cognitively normal elderly individuals. It is, rather, the density and widespread distribution of these changes in the cerebral neocortex that leads to the diagnosis of AD.

Molecular Components in the Pathogenesis of AD

Amyloid precursor protein, PS1, PS2, and tau, are believed to be strongly associated with the incidence of AD in humans. Consequently, the mouse models of AD were created by manipulating these molecules. A review of the pertinent molecular components follows.

One of the principal components of senile plaques in brains affected with AD is amyloid β protein. Aβ is secreted constitutively by normal cells in culture and is detected as circulating peptide in the plasma and cerebrospinal fluid of healthy humans and other mammals. Aβ is derived by endoproteolytic cleavage of APP, which has a large extracellular domain, a single small transmembrane region, and a small cytoplasmic tail. Mutations in the APP gene encoded on chromosome 21 account for a small fraction of the cases of FAD.

APP occurs in several different isoforms, which arise from alternative splicing of a single gene. The shortest of the major isoforms (695 amino acids) is expressed almost exclusively in neurons, whereas the other two common forms (751 and 770 amino acids, respectively) are expressed both in neuronal and non-neuronal cells. Mice null for APP are viable, but they exhibit reactive gliosis and have a decreased locomotor activity and forelimb grip strength (Zheng et al. 1995).

APP has a short half-life and is metabolized by proteases (termed secretases) along two pathways, one nonamyloidogenic and one amyloidogenic (Figure 2). α-Secretase initiates the nonamyloidogenic pathway by cleaving APP between extracellular residues 687 and 688 within the Aβ domain, releasing the large soluble ectodomain, βAPP_S. This initial cleavage is followed by endoproteolytic cleavage of the C-terminal fragment within its transmembrane domain by an enzyme activity (termed γ secretase) to produce a short fragment (termed p3), which is released constitutively by APP-expressing cells during normal metabolism. β-Secretase initiates the amyloidogenic pathway by cleavage of APP after amino acid 671, creating a 99-residue membrane-retained C-terminal fragment having residue 1 of Aβ as its N terminus. The truncated N-terminal fragment of APP, βAPP_S, is released. The C-terminal fragment is then cleaved by γ-secretase to produce Aβ.

Figure 2 Schematic of amyloid precursor protein (APP) processing. The Aβ portion of the molecule is shown in black. When APP is processed by α secretase, it yields a secreted fragment αAPP and the membrane-bound C83. Further cleavage by γ secretase produces p3. Alternatively, cleavage of APP by β secretase produces the membrane-bound fragment C99, which is cleaved by γ secretase to form αβ_40,42.

Of key importance in the pathway described above is the site of the β-secretase cleavage. If the cleavage is between amino acids 712 and 713, Aβ is produced. However, if it is cut after amino acid 714, the larger Aβ₄₀ is formed. The majority of the secreted Aβ peptides are the short soluble Aβ₄₀ variety. However, approximately 10% of the secreted Aβ peptides are the more insoluble Aβ₄₂, which readily aggregate to form extracellular fibrils. This heterogeneity of the Aβ fragment is important inasmuch as Aβ₄₂ is first to appear in the diffuse plaques of Down syndrome patients whereas Aβ₄₀ is not detected until decades later (Selkoe 1998).

Not all of the C-terminal fragments produced by α- and β-secretases are cleaved by γ-secretase to Aβ and p3. Alternatively, proteolytic pathways can fully degrade the C-terminal fragments in endosomes and lysosomes (Selkoe 1998). The identity of the various secretases involved in the cellular processing of APP has been elusive. Recently two β-secretases have been identified: β-site APP-cleaving enzyme (BACE¹) 1 and BACE2 (Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999). BACE1 is highly expressed in the brain and is the major β-secretase for the generation of Aβ peptides by neurons (Cai et al. 2001). In mice, BACE1 cleaves APP at +1 and at +11 to yield Aβ11-40/42 and Aβ1-40-42. Aβ beginning at the +11 site is the major species in rodent brains (Buxbaum et al. 1998).

PS1 and PS2 are ubiquitously expressed transmembrane proteins with six to nine transmembrane domains principally localized in the endoplasmic reticulum and Golgi (Selkoe 1998). Their function is unknown, although we know that deletions result in alterations in processing of APP such that Aβ₄₂ is increased (Scheuner et al. 1996). Although the role played by presenilins in the pathogenesis of sporadic AD remains unknown, mutations in the presenilin genes, especially PS-1, account for a significant proportion of cases of FAD.

Tau is one of the microtubule-associated proteins that stabilizes neuronal microtubules. The single gene that encodes tau generates six isoforms through alternate splicing. Each isoform contains either three (3R tau) or four (4R tau) consecutive imperfect repeat motifs of 31 or 32 amino acids in the carboxy terminal half of the protein. Tau is hydrophilic and soluble; however, when it is hyperphosphorylated on a number of serine and threonine residues, it is prone to aggregate into paired helical filaments that in turn form the neurofibrillary tangles and senile plaques typical of AD. After hyperphosphorylation, tau loses its ability to bind microtubules and is redistributed from the axon to a somatodendritic pattern (Goedert and Hasegawa 1999; Mandelkow and Mandelkow 1998).

In some cases of dementia, the clinical features of AD may overlap with those of other neurodegenerative diseases. Among the best characterized are cases that share clinical features of both AD and Parkinson's disease. In a significant number of such cases, the protein abnormalities and morphological changes characteristic of AD coexist with intraneuronal inclusions known as Lewy bodies, structures rich in β-synuclein, a protein normally expressed in synapses. Although a comprehensive review of the disorders associated with abnormal β-synuclein accumulation is beyond the scope of this article, recent observations suggest that the β-amyloid accumulations associated with AD may contribute to the development of β-synuclein-rich Lewy bodies in experimental animals. Whether β-synuclein, or other proteins, in turn influence the progression of AD-related changes remains unclear (Masliah et al. 2001).

β Amyloid Precursor Protein Transgenic Mice

Early efforts to create a transgenic mouse model of AD emphasized APP as the first protein found to have a genetic link to AD (Goate et al. 1991). Investigators hoped first to model AD and second to study the biological activity of APP overexpression in vivo. Factors that can affect the phenotype of transgenic mice expressing APP include the host strain, the primary structure of the APP, and the distribution and level of APP expression. Early paradigms used a number of neuron-specific promoters driving the expression of wild-type and AD APP in various murine backgrounds with limited success. The animals often died prematurely or failed to develop AD-like lesions in the brain (Hsiao et al. 1995; Quon et al. 1991). The first reported transgenic mouse was made using human APP under the control of neuron specific enolase. Although these animals had impaired memory and spatial alteration, they developed only rare Aβ deposits in the brain (Moran et al. 1995). LaFerla used the FVB/N strain in which he expressed mouse Aβ, but the model was unsuccessful because >50% died within 1 yr. The mice developed corticolimbic gliosis, apoptosis, and extracellular Aβ deposits (LaFerla et al. 1995).

Rather than emphasizing the extracellular fragment of APP, other investigators made transgenic mice that expressed the intracellular carboxy terminal 100 amino acid fragment of APP. In this model, the transgene is controlled by the brain dystrophin promoter, which directs expression to the hippocampus and neocortex (Neve et al. 1996; Oster-Granite et al. 1996). AP-C100 transgenic mice at 18 to 28 mo exhibited profound loss of neurons in Ammon's horn and the dentate gyrus, but none of the other classic features of Alzheimer's neuropathology developed (Oster-Granite et al. 1996). The finding that overexpression of the AP-C100 was neurotoxic is not surprising considering the recent findings that the C-terminal portion of APP is a component of a DNA transcription complex (Cao and Sudhof 2001) and its overproduction could perturbate the expression of numerous genes.

PDAPP Mouse Model

Games and colleagues (1995) produced the first successful model known as the PDAPP mouse, which is now used extensively (Table 1). The PDAPP mouse expresses a human APP770 mini gene containing the V717F familial AD mutation (hAPP_V717F) under the control of the human platelet-derived growth factor (PDGF¹)-β chain neuronal promoter on a mixed C57BL/6, DBA, and Swiss-Webster strain background. The success of this model was due to the construct used and the high level of APP expression achieved. The transgene contains a splicing cassette that permits expression of all three major APP isoforms. The PDGF-β promoter targets expression preferentially to neurons in the cortex, hippocampus, hypothalamus and cerebellum of the transgenic animals.

The familial AD mutation at residue 717 may be important because it partially shifts production of Aβ from the soluble 40-amino acid form to the more insoluble amyloidogenic 42-residue peptide known to predominate in AD plaques. A greater than 10-fold overexpression of human APP developed in this mouse. Cortical and limbic amyloid deposition began at 3 mo of age in homozygotes and at 6 to 9 mo in heterozygotes. The deposits were associated with reactive neuritic and inflammatory changes (Masliah et al. 1996). Although this model developed amyloid deposits, it failed to meet all criteria of the neuropathology of human AD in the absence of cortical or hippocampal neuronal loss or neurofibrillary tangles in aged transgenic animals (Irizarry et al. 1997b).

Tg2576 Mouse Model

Hsiao and colleagues (1996) produced the second popular transgenic model of AD known as the Tg2576 mouse. This mouse expresses human APP695 and contains the Swedish familial AD double mutation K670N, M671L (hAPP_Sw) controlled by the hamster prion protein (PrP) on a C57B6/SJL background. The mouse expresses human APP at a level more than sixfold higher than endogenous murine APP. The mice have a fivefold increase in the concentration of Aβ₄₀ and a 14-fold increase in that of Aβ₄₂. Both diffuse and dense core amyloid deposits developed in the same distribution as those found in the Games et al.(1995) mouse beginning in cortical and limbic regions by 9 mo of age. The amyloid deposits were associated with dystrophic neurites, punctate immunoreactivity to hyperphosphorylated tau, astrocytosis, microgliosis, and vascular amyloidosis, without significant CA1 neuronal loss or formation of neurofibrillary tangles (Irizarry et al. 1997a). The elevation of Aβ correlated with the appearance of memory and learning deficits in the oldest group of transgenic mice.

APP23 Mouse Model

The APP23 mouse (Bornemann and Staufenbiel 2000; Sturchler-Pierrat and Staufenbiel 2000; Sturchler-Pierrat et al. 1997) carries the same human Swedish double mutation at positions 670/671 as the Tg2576 mouse. Unlike the Tg2576 mouse, the APP23 mouse carries the APP751 isoform and the transgene is under the control of the murine Thy1 promoter. These mice express human APP at a level sevenfold higher than murine endogenous APP. They developed amyloid plaques in the neocortex and hippocampus at 6 mo of age. The vast majority of the amyloid deposits were fibrillar. The plaques were almost exclusively congophilic at their first appearance and were associated with a pronounced glial reaction. A considerable amount of vascular amyloid was detected. Biochemical analysis revealed that the Aβ₄₀ isoform was more prominent than the Aβ₄₂ isoform. Both substantial neurodegeneration and a reduction of neuron numbers were apparent. Dystrophic neurites surrounded the plaques. Most importantly, hyperphosphorylated tau was detected in distorted neurites associated with congophilic plaques. However, no neurofibrillary tangles developed.

General Considerations

Each of the models described above is remarkable in that the anatomical pattern of plaque formation parallels that seen in human AD. Furthermore, the morphology of amyloid plaques in aged APP transgenic mice recapitulates amyloid pathology in human AD inasmuch as the plaques span the continuum from diffuse Aβ deposits to compact core plaques with inflammation and neuritic dystrophy. However, none of these models reflects a complete picture of the neuropathology of AD because neurofibrillary tangles are not identified and prominent neurodegeneration and cerebral atrophy do not occur.

Further underscoring the difficulty of establishing a murine model of AD is a recent publication in which the authors report that the amyloid fibrils deposited in the brains of APP23 transgenic mice are chemically and morphologically distinct from those that develop in human brains with AD (Kuo et al. 2001). Mouse fibrils are completely soluble in buffers containing sodium dodecyl sulphate whereas human fibrils are insoluble. This difference occurs either because insufficient time is available in murine models for Aβ structural modifications to occur or because the complex species-specific environment of the human disease is not precisely replicated in the transgenic mouse. The authors caution that the evaluation of therapeutic agents or protocols must be considered in the context of the difference in plaques between the transgenic mouse and humans.

Recently the enzymes responsible for cleaving APP at the β-secretase site, BACE1 and BACE2, have been identified. Cai and colleagues (001) established in neuronal cell cultures that BACE1, which is expressed at high levels in the brain, is the major β-secretase for the generation of Aβ peptides in neurons. In contrast, BACE2, which is expressed at very low levels in the brain, cleaves APP within the Aβ domain and precludes the formation of Aβ (Farzan et al. 2000). These in vitro studies suggest that BACE1 could be an exciting therapeutic target for protease inhibitors in AD. However, the following questions remain: (1) Would inhibition of BACE1 reduce the accumulation of Aβ peptides in vivo? (2) Would perturbation of BACE1 be neurotoxic? Knocking out BACE1 in mice proved that interfering with BACE1 did not have untoward effects. Mice deficient in BACE1 were healthy and fertile and appeared normal in gross anatomy, tissue histology, and clinical chemistry (Luo et al. 2001).

It is interesting that the BACE1 -/- mouse is viable inasmuch as BACE1 is expressed in many tissues. The finding that there are no apparent adverse effects associated with BACE1 deficiency in mice suggests that inhibitors of BACE1 in humans may not be toxic. Although it was documented in neuronal cell cultures that BACE1-deficient cells produced less Aβ in vitro, transgenic animal experiments would confirm whether or not similar changes would occur in vivo. When BACE1 -/- mice were bred to Tg 2576 APP-overexpressing mice, which readily develop increased levels of Aβ in the brain by 3 mo of age (Hsiao et al. 1996), Aβ-40 levels in BACE1 -/- APP+ brain extracts were only 5 to 7% of those in BACE1 +/+ APP+ mice.

Presenilin Transgenic Mice

Point mutations in the PS1 gene are a major cause of familial AD. Knocking out the gene offered no clues to its role in AD because mice null for PS1 die late in gestation (Shen et al. 1997). It has been proposed that the phenotype is a result of disturbed notch signaling. Because embryonic lethality precludes further analysis of the possible effects of PS1 on APP metabolism in living animals, brain cultures were generated from PS1 null embryos. In vitro, cleavage of the extracellular domain of APP by α- and β-secretase was not affected by the absence of PS1, but the activity of γ-secretase on the transmembrane domain of APP was prevented. This inhibition caused carboxyl-terminal fragments of APP to accumulate and resulted in a concurrent fivefold drop in the production of amyloid peptide (De Strooper et al. 1998).

PS1 Null Model

These in vitro findings support the idea that PS1 facilitates γ-secretase activity, which cleaves the integral membrane domain of APP, and that clinical mutations in PS1 result in a gain of the function of PS1. Further support for the gain of function theory is offered by knockin experiments using the PS1 null mouse (Qian et al. 1998). Transgenes expressing either the wild-type human PS1 or PS1 containing the FAD-associated mutation, A246E, under the transcriptional control of the human Thy-1 promoter, rescued the PS1 knockout mouse from embryonic lethality. Brain Aβ measurements revealed that mice expressing the mutant PS1 protein on the murine PS1 null background had a highly significant increase in the level of Aβ1-42/43, whereas reduction of PS1 activity in heterozygous PS1 knockout mice did not lead to an increase in Aβ1-42/43.

PDF Promoter Model

A second PS1 transgenic model expressing human mutant (M146L or M146V) and wild-type PS1 under the control of the PDGF promoter offered similar results (Duff et al. 1996). Expression of mutant or wild-type PS1 had no significant effect on brain Aβ40, whereas expression of mutant but not wild-type PS1 increased endogenous mouse Aβ1-42/43 in brain homogenates. Expression of wild-type PS1 did not significantly increase the levels of Aβ1-42/43 even though expression of wild-type PS1 was substantially increased to levels comparable with those for mutant PS1. Histopathological analysis of the mice at ages 3 to 4 wk revealed no Aβ deposition or other pathology. This finding is not surprising inasmuch as APP transgenic mice do not develop significant pathology until they are 12 mo old.

General Considerations

Because expression of mutant PS1 in mouse brain results in the accumulation of Aβ42/43 from endogenous murine APP, the next logical step was to determine whether mutated PS1 would have a similar effect in mice transgenic for human APP. This question was studied by crossing mice expressing FAD-linked human PS1 variant (A246E) with a chimeric mouse/human APP harboring mutations (K595N, M596L) linked to Swedish FAD kindred (APP swe). The young double transgenic progeny had an elevated Aβ1-42/Aβ1-40 ratio in brain homogenates. The brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP revealed no alteration.

These studies imply that mutant PS1 causes AD by increasing the extracellular concentration of Aβ1-42/43 (Borchelt et al. 1996). Extension of these studies (Borchelt et al. 1997) to include neuropathological examination of the brain revealed that the mice transgenic for both genes developed numerous amyloid deposits much earlier than age-matched mice expressing APP swe and wild-type Hu PS1 and APP swe alone. Interestingly, the majority of Aβ deposits in the double transgenic mice were not immunoreactive with antisera against Aβ1-42 but instead were stained with antisera to Aβ1-40.

Crossing the Tg2576 transgenic mice, which express mutant APP_{K670N, M671L}, with mice transgenic for PS1_M146L, Holcomb and colleagues (1998) found that doubly transgenic mice revealed a selective 41% increase in Aβ1-42/43 in homogenates of their brains. The AD-like pathology was substantially enhanced, and the mice revealed deterioration of brain function in the "y" maze.

Frequently double-transgenic mice have a complex genetic background in that the transgenic lines are on mixed backgrounds. To overcome this problem, Citron and colleagues (1997) bred a transgenic mouse bearing a human wild-type APP695 gene with mice bearing different human PS1 transgenes containing FAD-linked mutations to produce offspring expressing wild-type human APP695 alone or both wild-type APP95 and either mutant or wild-type human PS1. In this case, both genes were under the control of the cytomegalovirus promoter. Use of the same cytomegalovirus promoter element for both transgenes offered the advantage that both transgenes were likely to be expressed in adequate quantities in the same cells. In addition, both transgenic lines were on the same inbred FVB/N strain. With this strategy, the only difference between double and single transgenic animals was the presence or absence of the human PS1 transgene and any associated insertional mutation. Mutant but not wild-type PS1 transgenic mice revealed significant overproduction of Aβ42 in the brain, and this effect was detectable as early as 2 to 4 mo of age. These findings confirm that mutations in the presenilin gene cause a dominant gain of function and may induce AD by enhancing Aβ42 production, thus promoting cerebral β-amyloidosis.

Tau Transgenic Mice

In addition to their presence in AD, filamentous tau inclusions accompanied by extensive gliosis and loss of neurons are the neuropathological hallmarks of an expanding family of other neurodegenerative diseases that are sometimes designated as tauopathies. The discovery of autosomal dominant pathogenic tau gene mutations in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17¹) has led to the rapid emergence of new insights into mechanisms underlying FTDP-17, AD, and other tauopathies (Hutton et al. 1998).

Early efforts to produce animal models with tau pathology were based on the expectation that transgenic mouse models of AD that developed amyloid plaques would develop the other classic lesions of AD. However, tau-positive neurofibrillary tangles were never observed. Even the more complex transgenic mouse models (e.g., models that overexpressed FAD-linked PS1 mutations alone or FAD-linked PS1 and APP mutations together) did not develop tau pathology. Therefore, attention turned from classic mouse models for AD to mice transgenic for specific mutations of the tau protein.

Mutations in the tau gene in FTDP-17 can alter splicing and produce a shift from the short tau isoform with three repeats to the longer isoform, which contains four repeats. This finding suggests that overproduction of four-repeat tau may be sufficient to cause frontotemporal dementias. Reports from initial studies of mice transgenic for human tau indicated that the mice developed pretangle tau pathology but no filamentous tau inclusions (Brion et al. 1999; Gotz et al. 1995). The lack of tau filament formation in these transgenic models may have been due to production of a low level of human tau in that only 10 to 20% of total mouse brain tau was derived from the transgene.

Model of Shortest Human Tau Expression

Ishihara and colleagues (1999) were successful in creating a transgenic line that expressed high levels of the shortest human tau under the control of the mouse prion promoter. These mice developed insoluble intraneuronal filamentous hyperphosphorylated tau inclusions by 12 mo of age. These argyrophilic inclusions formed by aggregated 10- to 20-nm filaments composed of tau and neurofilament proteins were found in cortical and brainstem neurons but were most abundant in spinal cord neurons where they were associated with axon degeneration and reduced axonal transport in ventral roots as well as spinal cord gliosis and motor weakness. However, the filaments within the inclusions did not exhibit the ultrastructural features of paired-helical filaments typical of classic AD neurofibrillary tangles. The phenotype of these transgenic mice was more similar to that seen in tauopathies such as FTDP-17 than to that seen in AD inasmuch as the tau pathologies were more abundant in the spinal cord and brain stem. In addition, these inclusions differed from authentic neurofibrillary tangles (NFTs¹) in AD and other human tauopathies in that the tau inclusions were not stained by thioflavin-S or Congo red and contained straight 10- to 20-nm-diameter filaments that were admixed with neurofilaments. Even though tau protein isolated from brain and spinal cord of these mice became progressively insoluble and more phosphorylated with age, overexpression of human tau did not result in neurofibrillary tangles. If the ages of the mice described above were beyond 12 mo, however, the character and distribution of the inclusions changed (Ishihara et al. 2001). In contrast to those in younger animals, the inclusions were congophilic, similar to those found in human tauopathies. Ultrastructurally, the lesions contained straight tau filaments composed of both mouse and human tau proteins but not other cytoskeletal proteins. The tau inclusions developed in the hippocampus and associated limbic areas.

JNPL3 Mouse Model

Transgenic mice designated JNPL3 express human four-repeat tau containing the most common FTDP-17 mutation (P301L) under the control of the mouse prion promoter and developed motor and behavioral deficits (Lewis et al. 2000). These deficits were associated with age- and gene-dose-dependent development of congophilic neurofibrillary tangles, which formed in the amygdala, septal nuclei, preoptic nuclei, hypothalamus, midbrain, pons, medulla, deep cerebellar nuclei, and spinal cord. Tau-immunoreactive pre-tangles were found in the cortex, hippocampus, and basal ganglia, but at a lower level than commonly found in human disease. The tangles were associated with neuronal degeneration, especially in the spinal cord where motor neurons were reduced by approximately 48%. Interestingly, this transgenic model had a surprisingly severe phenotype given the relatively low-level expression of the transgene. This severity presumably reflects the inclusion of the P301L mutation that is associated with FTDP-17. Phenotypically, this mouse recapitulates features of the human tauopathies rather than the classic features of AD. However, it will be valuable in breeding experiments to produce model systems that more accurately recapitulate the hallmark pathology of AD by crossing it with transgenic mouse models of AD Aβ amyloidosis.

Apolipoprotein E Transgenic Mice

Apolipoprotein E (ApoE¹) appears to play an important role in the pathogenesis of AD inasmuch as the relative risk of developing late-onset senile dementia of the AD type is increased in individuals who inherit an APOEε4 allele. A consistent consequence of carrying the APOEε4 allele is an increased number of amyloid plaques in brain and more abundant amyloid deposition in the cerebral vasculature. The mechanism by which ApoE4 contributes to the development of neurodegeneration remains unknown, but it may modify the ability of the brain to respond to environmental stresses or alter the blood-brain barrier (Strittmatter 2000).

Employing a knockout strategy, two distinct AD models, Tg2576 and PDAPP, have been crossed with ApoE null mice to determine whether the lack of ApoE would affect the neuropathological phenotype. The ApoE null animals revealed that the absence of ApoE altered the quantity, character, and distribution of Aβ deposits in the transgenic animals.

Tg2576xApoE Mouse Model

As Aβ accumulates in the brains of Tg2576 transgenic mice, the brain ApoE increases by 60% relative to control mice. The ApoE accumulates in neuritic plaques that are thioflavin-S positive, suggesting that elevation of brain ApoE in Tg2576 mice participates in an age-related abnormal regulation of Aβ clearance (Kuo et al. 2000).

When examined at 1 yr of age, mice heterozygous for APP on an ApoE null background had significantly reduced Aβ deposition, lacked thioflavine-S-positive deposits, had no neurodegeneration, and developed less vascular amyloid compared with heterozygous mice with endogenous mouse ApoE. The pattern of amyloid deposits in the cortex and hippocampus did not change in the ApoE null background.

PDAPPxApoE Mouse Model

In experiments crossing PDAPP homozygous mice (Games et al. 1995) with ApoE null mice, the Aβ burden in the cortex and hippocampus was markedly reduced. Interestingly, the character of the plaques changed in this model. Elimination of ApoE prevented the formation of compact, thioflavine-S-staining plaques.

In animals examined at 12 mo of age, a dramatic redistribution of deposits occurred. PDAPP mice with ApoE had compact deposits scattered throughout the frontal cortex. PDAPP+/+ApoE-/- mice had diffuse deposits only in the deep cortical layers. Within the hippocampal subfields, the pattern of Aβ staining in the ApoE null mice was altered in a very specific anatomic manner. In PDAPP+/+ApoE+/+ mice, amyloid deposited prominently in a band in the outer layer of the dentate gyrus, with focal deposits throughout the other hippocampal subfields as in human AD. In ApoE null mice, the dentate gyrus was remarkably free of Aβ immunoreactivity.

The findings that the levels of APP mRNA by RT-polymerase chain reaction, the levels of APP protein by Western blot, or total Aβ and Aβ1-42 by enzyme-linked immunosorbent assay in the hippocampus or cortex did not change in the ApoE null background implicate ApoE in Aβ fibrillogenesis, stabilization of fibrillar Aβ, and/or maturation of amyloid plaques (Bales et al. 1997; Irizarry et al. 2000). In contrast, transgenic experiments wherein ApoE was overexpressed yield entirely different results from those described in ApoE knockout mice. Overexpression of the human APOE4 allele in neurons resulted in hyperphosphorylation of protein tau (Tesseur et al. 2000b). In three independent transgenic lines using two different promoter constructs, increased phosphorylation of protein tau was correlated with ApoE expression levels. Hyperphosphorylation of tau increased with age. These findings suggest a role for ApoE4 in neuronal cytoskeletal stability and metabolism. No neurofibrillary tangles or other neurofibrillary inclusions were found; however, axonal dilations with accumulation of synaptophysin, neurofilaments, mitochondria, and vesicles were documented, suggesting impairment of axonal transport (Tesseur et al. 2000a). This current transgenic mouse offers the opportunity to investigate the interaction between ApoE 4 and protein tau.

Neprilysin Transgenic Mice

At the time of this writing, most research using transgenic mouse models of AD has focused on the secretases that free Aβ from APP. However, investigators have recently discovered two proteases that actively degrade Aβ: insulin-degrading protein (Vekrellis et al. 2000) and neprilysin (Shirotani et al. 2001; Takaki et al. 2000). If these enzymes were defective, Aβ could theoretically accumulate.

Neprilysin-null mice offer direct evidence that neprilysin could be a natural Aβ-degrading enzyme (Iwata et al. 2001). When Aβ is given by injection into the brains of normal mice, it is degraded in about 30 min; however, in neprilysin knockout mice, almost all of the peptide persists. In mice heterozygous for neprilysin, more of the injected Aβ persisted than in the wild-type, but less than in the null. The Aβ levels in knockout mice were highest in the hippocampus and cortex, where Alzheimer's plaques are most prominent. Relevance of neprilysin to AD in humans was suggested when it was discovered that there are low levels of neprilysin in plaque regions in patients who died of AD (Yasojima et al. 2001). With the availability of transgenic mice that accumulate Aβ in the brain, it will be of interest to determine whether the accumulation of Aβ can be accelerated by breeding them to neprilysin null mice.

Aβ Immunization and AD

Clearly, transgenic mice have played a pivotal role in understanding the various molecular elements in the pathogenesis of AD. A significant recent development has been the use of these models to evaluate treatment modalities. Given the paucity of inflammatory changes in AD, it is both surprising and exciting to find that vaccination with Aβ₄₂ can alter the course of plaque formation in murine models of AD. Immunization with Aβ₄₂ offered promising results either at 6 wk of age, before the onset of AD-type neuropathology, or at 11 mo, during amyloid-β deposition (Schenk et al. 1999). The immunization of young animals essentially prevented the development of β-amyloid plaque formation, neuritic dystrophy, and astrogliosis. Treatment of older animals also markedly reduced the extent and progression of the AD-like neuropathology.

There are two possible mechanisms to account for the success of the vaccination protocol. The anti-Aβ antibodies could reduce the plaques either by facilitating clearance of amyloid-β before deposition or by triggering monocytic/microglial cells to clear established plaques through signals mediated by Fc receptors. The latter mechanism dominates. Studies using the same model revealed that antibodies to Aβ cross the blood-brain barrier and decorate the plaques triggering microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation (Bard et al. 2000).

Vaccination has an obvious effect not only on the morphology of β-amyloid plaques but also on the amelioration of cognitive dysfunction. Using Tg 2576 APP transgenic mice (Hsiao et al. 1996) crossed to PS1 transgenic mice (Duff et al. 1996) that have age-related cognitive impairment (Arendash et al. 2001), investigators found that vaccinated mice performed in cognitive testing as well as nontransgenic mice (Morgan et al. 2000). Parallel cognitive improvement was also seen in the TgCRND8 murine model. This mouse is a modified Tg2576 mouse that carries a double mutated human APP-β (K670N, M671L and V717F) on a C3H/B6 background (Janus et al. 2000).

Conclusions

It is clear that none of the mouse models to date recapitulate the complete neuropathology of AD. Some models develop plaques and others, NFTs and neurodegeneration. Nonetheless, the genetically altered mouse has offered tremendous insight into the function of the various molecular elements in the pathogenesis of AD, although questions remain. It is still unclear as to how Aβ and tau are related. Two camps exist, each purporting the primacy of either tau or Aβ. The existing models offer a solid platform from which to explore this debate, and two recently published papers help bring the two camps together.

Gotz and colleagues (2001) describe a model wherein P301L tau transgenic mice develop neurofibrillary tangles after the intracerebral injection of Aβ42. The Aβ was given by injection into the CA1 region of the hippocampus, and the neurofibrillary tangles developed in the respective cell bodies of projection neurons in the amygdala as soon as 18 days after Aβ injection. These experiments indicate that the interaction of β-amyloid with the P301L mutation is required for NFT formation. Neither β-amyloid nor the mutation in tau alone is sufficient to generate high numbers of NFTs. It will be interesting to learn whether vaccination with Aβ will be effective in preventing NFT formation in this model.

Double mutants produced from crossing JNPL3 transgenic mice expressing mutant P301L tau (Lewis et al. 2000) with Tg 2576 mice (Hsiao et al. 1996) expressing the APP Swedish mutation also offered proof that Aβ influences the development of NFTs (Lewis et al. 2001). The double mutants exhibited neurofibrillary tangle pathology that was substantially enhanced in the limbic system and the olfactory cortex. These results suggest that APP or Aβ augments the formation of neurofibrillary tangles in the regions of the brain vulnerable to the formation of these lesions. This model most closely recapitulates the lesions of AD in humans inasmuch as the mice develop not only amyloid deposition and NFTs but also neuronal loss. This model is not likely to be the final murine model of AD. It and other models to come will be invaluable in determining the pathogenesis of AD and in evaluating therapeutic protocols designed to prevent the disease.

¹Abbreviations used in this article: Aβ, amyloid β protein; AD, Alzheimer's disease; ApoE, apolipoprotein E; APP, amyloid precursor protein; BACE1, β-site APP-cleaving enzyme 1; BACE2, β-site APP-cleaving enzyme 2; FAD, familial Alzheimer's disease; FTDP-17, frontotemporal dementia with Parkinsonism linked to chromosome 17; NFT, neurofibrillary tangles; PDGF-β, platelet-derived growth factor β PS1, presenilin 1; PS2, presenilin 2.

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