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International Journal of Alzheimer's Disease
Volume 2011 (2011), Article ID 810981, 9 pages
http://dx.doi.org/10.4061/2011/810981
Review Article

Introducing Human APOE into Aβ Transgenic Mouse Models

1Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL 60612, USA
2Translational Neurobiology, Medtronic Incorporated, Minneapolis, MN 55432, USA

Received 21 July 2011; Accepted 15 August 2011

Academic Editor: Patrizia Mecocci

Copyright © 2011 Leon M. Tai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Apolipoprotein E (apoE) and apoE/amyloid- (A ) transgenic (Tg) mouse models are critical to understanding apoE-isoform effects on Alzheimer's disease risk. Compared to wild type, mice exhibit neuronal deficits, similar to apoE4-Tg compared to apoE3-Tg mice, providing a model for A -independent apoE effects on neurodegeneration. To determine the effects of apoE on A -induced neuropathology, mice were crossed with A -Tg mice, resulting in a significant delay in plaque deposition. Surprisingly, crossing human-apoE-Tg mice with /A -Tg mice further delayed plaque deposition, which eventually developed in apoE4/A -Tg mice prior to apoE3/A -Tg. One approach to address hAPOE-induced temporal delay in A pathology is an additional insult, like head injury. Another is crossing human-apoE-Tg mice with A -Tg mice that have rapid-onset A pathology. For example, because 5xFAD mice develop plaques by 2 months, the prediction is that human-apoE/5xFAD-Tg mice develop plaques around 6 months and 12 months before other human-apoE/A -Tg mice. Thus, tractable models for human-apoE/A -Tg mice continue to evolve.

1. Introduction

Alzheimer’s disease (AD) is the most common form of dementia and represents a serious economic and social burden worldwide. The familial form of AD (FAD) is caused by autosomal dominant mutations that increase levels of the 42 amino acid isoform of the amyloid-beta 42 (A 42) peptide [1, 2]. The primary genetic risk factor for AD is inheritance of the APOE4 gene for apolipoprotein E (apoE), compared to APOE3, with APOE2 reducing risk [35]. The mechanism(s) by which apoE and A affect pathogenesis of the disease is unclear (reviewed [35]). However, evidence suggests that apoE may isoform-specifically modulate A -induced neurotoxicity [4, 5]. To address potential mechanisms in vivo, several transgenic (Tg) mouse models have been developed to assess the structural and functional interactions between apoE and A . However, each of these models has potential drawbacks that affect the interpretation and physiological relevance of the results. This paper will summarize the current models resulting from the introduction of human-APOE into A -Tg mice. The continued development and characterization of both apoE and apoE/A -Tg mouse models is critical to understanding the apoE-isoform effects on AD pathology.

1.1. Amyloid-

Traditional diagnosis of AD is based on pathology that includes extracellular amyloid plaques, composed primarily of A 42, intraneuronal neurofibrillary tangles, hyperphosphorylated tau, neuroinflammation, and neuronal cell loss. A is a 39–43 amino acid (4 kDa) peptide produced via sequential proteolysis of amyloid precursor protein (APP) by -secretase/BACE followed by -secretase (composed of presenilins [PS] 1 and 2), to produce A peptides primarily 40 and 42 amino acids in length. Genetic and experimental evidence indicates that A 42 is the cause of AD pathogenesis [1, 2]: (1) FAD, although only 3–5% of all AD cases is caused by autosomal dominant mutations in APP, PS1, or PS2 that increase levels of A 42 or the A 42 : 40 ratio; (2) Down syndrome is caused by trisomy of chromosome 21 (the location of the APP gene), and is characterized by plaque deposition and dementia by the age 40; (3) A 42 is neurotoxic in vitro and in vivo. A , particularly the more toxic A 42, aggregates to form a variety of higher-order assemblies including oligomers, protofibrils, fibrils, and amyloid plaques [6]. Amyloid plaques themselves exist in different conformations including compact plaques (composed of a dense Thioflavin-S- (ThioS-) positive core), neuritic plaques (identified as ThioS-positive plaques surrounded by a ring of dystrophic neurites), and diffuse plaques (characterized by amorphous wisps of amyloid that lack a central core and are not neurotoxic) [710]. While total plaque burden does not directly correlate with dementia [11], it is an indication of increased A 42 levels, and compact or dense core plaques may be disease relevant [7, 12].

1.2. ApoE

ApoE is the only apolipoprotein synthesized within the blood-brain barrier (BBB) and is the primary apolipoprotein associated with lipoprotein particles in the central nervous system (CNS), as peripheral apoE is unable to cross the BBB or blood-cerebrospinal fluid barrier. While the majority of apoE in the CNS is secreted by glial cells, particularly astrocytes, neuronal production of apoE has been observed under specific pathological conditions [13]. CNS lipoproteins are critical for lipid homeostasis, particularly as cholesterol and phospholipids are required for neuronal growth, repair, and synaptogenesis (reviewed [35]). In humans, three apoE polymorphic alleles exist (APOE2, APOE3, and APOE4) which encode three protein isoforms (apoE2, apoE3, and apoE4). Although human-apoE (h-apoE) is a 299 amino acid protein, the three apoE-isoforms differ by a single amino acid substitution at residues 112 or 158: apoE2 contains Cys112,158, apoE3 contains Cys112Arg118, and apoE4 contains Arg112,158 [5]. In the general population, APOE3 is the most common allele (77%), followed by APOE4 (15%) and APOE2 (8%). In contrast, 40% of AD patients express at least one APOE4 allele. Compared to APOE3, inheritance of one or two copies of the APOE4 allele increases the risk for developing AD by 4- and 12-fold, whereas APOE2 decreases risk by 2- and 4-fold [5, 14]. Amyloid plaque deposition is greatest in AD patients with an APOE4 allele(s) [14]. In addition to AD, APOE4 is a risk factor for cerebral amyloid angiopathy (CAA; amyloid deposition in blood vessels) and impairs recovery from cerebral insults such as stroke, cerebral hemorrhage, and brain injury [4]. While apoE knock-out mice (apo ) and h-apoE-Tg models demonstrate that apoE affects neuronal viability independent of A -induced neurotoxicity, the focus of this paper is on the synergism between the h-apoE isoforms and A on neuropathology. To address the latter in vivo, apoE/A -Tg models were subsequently developed to specifically address the isoform-specific effects of h-apoE on A deposition.

2. Transgenic Mice Expressing Human ApoE (Table 1)

tab1
Table 1: ApoE transgenic models. Summary of the effects induced by deletion of mouse apoE (apo ) or the introduction of h-apoE isoforms on markers of neuronal degeneration and behavior. Key: HC: hippocampus; CX: cortex; DG: dentate gyrus; ChAT: choline acetyl transferase; ICH: intracerebral hemorrhage; LTP: long-term potentiation; TR: targeted replacement (knock-in); F: female; M: male; m: month.

Several approaches have been used to make Tg mouse models to assess the function of apoE. Apo Tg-mice were initially used to help understand the role of apoE in the brain [15, 16] although the homology between mouse (m-) and h-apoE is 70%, and mice express only a single isoform, comparable to apoE4 at residues 112 and 158 [54]. Apo mice have been used as the background for several h-APOE-Tg mouse lines. Heterologous promoters have been used to drive the expression of h-apoE in glia or neurons. Examples include glial fibrillar acidic protein (GFAP; glial) [22, 5557], transferrin (neuronal) [58], platelet-derived growth factor (PDGF; neuronal) [59], neuron-specific enolase (NSE; neuronal) [15], and thymocyte differentiation antigen 1 (Thy1; neuronal) [59]. However, these models have limitations inherent in the use of a heterologous promoter and specific to apoE: (1) the expression of protein by a heterologous promoter is not regulated as it would be by the endogenous promoter; (2) the inserted copy number of the transgene cannot be regulated by a traditional Tg mouse approach; (3) while the cell-specific expression of apoE in the brain is controversial [6063], the majority of evidence suggests that glia, not neurons, are the primary cell type to express apoE [13, 6467]; (4) by using the m-apo background and inducing apoE expression via CNS-specific promoters, lack of peripheral apoE becomes a variable of potential importance when interpreting results from these mice; (5) evidence suggests that in both humans and apoE-TR mice, apoE4 levels are significantly lower than apoE2 or apoE3 [6873]. Knock-in or targeted-replacement (TR) mice were developed that express h-apoE under the control of the endogenous mouse promoter and provide an alternative to heterologous expression of h-apoE. In apoE-TR mice, the coding domain for each of the h-APOE isoforms replaces the coding domain for m-APOE. Thus, in apoE-TR mice, glial cells express h-apoE in a native conformation at physiologically regulated levels, and in the same temporal and spatial pattern as endogenous m-apoE. Thus, the interpretation of apoE isoform-specific results is determined by the nature of the apoE-Tg mouse model.

As discussed, a number of apoE-Tg mouse models have been developed with apoE expression under the control of different promoters [74]. The general phenotypes of apo mice and three examples of the most widely studied apoE-Tg mice (GFAP-apoE, NSE-apoE, and apoE-TR) are briefly discussed (Table 1).

2.1. Apo

Compared to wild type, apo mice have decreased excitatory transmission, spine density, and dendritic length [1618]. These changes may underlie behavioral deficits, as apo mice are cognitively impaired [15, 19, 20]. However, lack of peripheral apoE can have profound effects on plasma lipid homeostasis, potentially leading to a number of confounding variables, including metabolic disease and increased risk for cardiovascular disease, compromising the ability to compensate for oxidative stress or inflammation, deficits that can effect the vasculature of the brain. In addition, the relevance of these mice is unclear as there are no apo humans. Apo provides the background for a number of the h-apoE-Tg mice.

2.2. GFAP-apoE Mice

GFAP is an intermediate filament protein, expressed at high levels by glia in the CNS. To target apoE expression to glia, mice expressing h-apoE under the control of the GFAP promoter were crossed with apo mice [17, 22, 23]. At the cellular level, GFAP-apoE4 mice show increased CA1 cellular atrophy and decreased spine density compared to GFAP-apoE3 mice [75]. In addition, compared to GFAP-apoE3 mice, GFAP-apoE4 mice demonstrate impaired cognition and increased anxiety; common symptoms in AD patients [17, 23, 24]. Interestingly, cognitive impairment is evident at an earlier age in female compared to male GFAP-apoE4 mice [23, 24]. A limitation of this model is that the effect of apoE isoform on neuroinflammatory responses cannot be interpreted, as apoE is expressed under the control of a promoter that is induced by neuroinflammation.

2.3. Neuronal apoE Expression

Neuronal apoE expression remains controversial, although expression has been identified under conditions of stress [25]. To investigate the role of neuronal expression, apoE-Tg mice expressing h-apoE under the control of the NSE promoter were crossed with apo mice [2527]. Kainic acid was used to induce apoE expression in these mice and resulted in the protection of NSE-apoE3 mice from age- and kainic acid-induced presynaptic and dendritic degeneration compared to NSE-apoE4 mice [25]. In addition, female NSE-apoE4 mice demonstrate impaired cognition compared to female NSE-apoE3 mice [25]. In contrast, male NSE-apoE4 mice do not exhibit cognitive deficits [15]. Thus, the effect of apoE4 on cognition may be modulated by gender. This is similar to the early development of cognitive deficits in GFAP-apoE4.

2.4. ApoE-TR

To investigate the function(s) of apoE in a more physiologically relevant apoE-Tg model, apoE-TR mice were developed to express each h-apoE isoform under the control of the endogenous m-APOE promoter [2835]. Although apoE protein levels in brain homogenates of these mice were initially described as being similar for each h-apoE isoform [32], subsequent analysis demonstrated that apoE4 levels are significantly lower in plasma, CSF, and brain homogenates than apoE2 and apoE3 [28, 30], similar to the levels of apoE isoforms in humans [6873]. Compared to apoE3-TR mice, apoE4-TR mice demonstrate decreased spine density and dendrite length, reduced excitatory transmission, and long-term potentiation (Table 1). Again, similar to the cognitive deficits observed in female GFAP-apoE4 mice and NSE-apoE4 mice, female apoE4-TR mice are cognitively impaired compared to female apoE3-TR mice [31, 32]. However, male apoE3-TR and apoE4-TR mice are not significantly different. Interestingly, both apoE3- and apoE4-TR females are cognitively impaired when compared to apoE-isoform-matched males.

3. ApoE in A Transgenic Models (Table 2)

tab2
Table 2: APOE in A transgenic models. Genetic deletion of mouse apoE (apo ) from A -Tg mice delays amyloid deposition, which is further delayed by the introduction of h-APOE into A -Tg mice. *LaDu Lab unpublished observations.
3.1. A -Tg

Although amyloid plaques are a hallmark of AD, the mechanisms underlying A -induced toxicity remain unknown. To help determine the effects of A and amyloid deposition on neurotoxicity in vivo, A -Tg mouse models have been produced that express human FAD mutations. These models include but are not limited to: PDAPP (APPV717F), Tg2576 (APPK670N, M671L), J9 (PS1M146V,L286V, APPV717F), as well as 5xFAD (APPK670N, M671L,I716V,V717I, PS1M146V,L286V) (reviewed in [2]). These A -Tg mice express either APP mutations alone (PDAPP, Tg2576) or in combination with PS mutations (J9, 5xFAD). Plaque development generally begins at 6 months of age in these mice (with the exception of 5xFAD), and the onset of cognitive deficits is dependent on the model (e.g., PDAPP at 6 months, Tg2576 at 9 months). Because the APPK670N, M671L mutation is linked primarily to CAA, the Tg2576 mice also develop A deposition around blood vessels, particularly leptomeningeal and cortical vessels. However, one limitation of A -Tg models is the relative lack of neuronal loss as observed in AD (reviewed [76]) although there are some exceptions (e.g., 5xFAD [49], APPSLPS1KI, and TBA2 mice [76]).

3.2. Apo /A -Tg

Initially, the effect of apoE on A deposition was investigated using apo crossed with A -Tg mice (Table 2). Specifically, apo mice were crossed with PDAPP [7779] or Tg2576 mice [80]. In both models, the absence of apoE significantly delayed ThioS-positive plaque deposition by 6 months and decreased A levels in the hippocampus and cortex, as measured biochemically from brain homogenates and by A immunoreactivity [42, 77] (Table 2). APOE affects plaque deposition in a gene-dose-dependent manner, as plaque levels were intermediate in apo /PDAPP compared with apo /PDAPP and apo /PDAPP mice [78]. In addition, the A 40 CAA present in the Tg2576 mice was absent in the apo /Tg2576 mice [80].

3.3. GFAP-apoE/A -Tg

The initial studies demonstrating that the lack of m-APOE delayed plaque deposition in A -Tg mice led to the question of what would be the effect of introducing h-APOE into apo /A -Tg mice (Table 2). To initially address this issue, GFAP-apoE mice was crossed with apo /PDAPP mice [41, 42]. Surprisingly, the introduction of h-APOE did not result in the expected reduction in the age of onset of A pathology, rather the presence of h-APOE further delayed A deposition. Amyloid accumulation is delayed from 6 to 12 months in apo /PDAPP mice, and to 15 months in GFAP-apo /PDAPP mice (Table 2). Once plaque pathology returned, the greatest plaque burden was found in GFAP-apoE4/PDAPP mice, compared with GFAP-apoE2/PDAPP and GFAP-apoE3/PDAPP mice. One potential confounding factor in these mice, as well as the NSE-apoE mice described below, is the equal expression of the h-APOE isoforms, in contrast to human data where inheritance of an APOE4 allele results in lower apoE levels [68, 69, 73].

3.4. Neuronal apoE Expression/A -Tg

To determine the effect of neuronal h-APOE expression on A pathology, NSE-apoE were crossed with J9 mice (PS1M146V,L286V, APPV717F) [81]. Similar to GFAP-apoE/PDAPP-Tg mice, the introduction of h-APOE to J9 mice delayed plaque pathology from 8 to 19 months, with deposition greater in the NSE-apoE4/J9 than NSE-apoE3/J9 [26].

3.5. ApoE-TR/A -Tg

The physiologic advantages of using apoE-TR mice to study the function(s) of apoE in vivo led to the generation of apoE-TR/PDAPP [30] and apoE-TR/Tg2576 mice [47] (Table 2). The resulting data confirmed that h-APOE delayed plaque deposition. In apoE-TR/PDAPP mice, plaque deposition was delayed from 6 months to 18 months of age. An apoE isoform-specific effect on A pathology was also observed, with ThioS staining, A immunoreactivity, and A levels in brain homogenates highest with apoE4 [30]. In Tg2576 mice, plaque deposition initiates at 9 months of age, while in apoE-TR/Tg2576 mice there is minimal plaque staining at 15 months. Interestingly, at 15 months, the isoform effect in these mice is primarily on CAA ( ), as amyloid deposition in the parenchyma is minimal [47].

3.6. Addressing h-APOE-Induced Delay in A Pathology

The major drawback to the apoE/A -Tg crosses described thus far is the significant h-APOE-induced delay in A pathology. For example, in apoE-TR/PDAPP mice, plaque deposition is not identified until mice are ≥18 months of age (Table 2). This substantial delay precludes timely analyses of apoE isoform-specific effects on early aspects of A pathology. One approach to address this temporal delay is to introduce an additional insult, such as traumatic brain injury (TBI) [82], kainic acid [25], nitric-oxide-synthase-2- (NOS2-) knock-out [83], or by blocking A degradation via neprilysin inhibition [84]. Although no amyloid deposition is present in 12-month-old GFAP-apoE/PDAPP mice, TBI at 9 months leads to amyloid deposition at 12 months [82], which is greater with apoE4 compared to apoE3. Kainic acid decreases synaptophysin and MAP-2 staining in apo and NSE-apoE Tg mice, with the effect more pronounced with apoE4 than apoE3. Nitric oxide, produced by inducible NOS (encoded for by the NOS2 gene), is an important signaling and redox factor that plays a key role in neuroinflammation and neurodegeneration [83]. NOS mice have been crossed with multiple A -Tg mouse models, and results demonstrate increased tau phosphorylation and neuronal loss in NOS /A -Tg mice [83]. In the Tg2576/NOS2-/- mice, A deposition is higher compared to Tg2576 mice. Inhibition of neprilysin, an enzyme that degrades extracellular A , with thiorphan, induces fibrillization and deposition of A and in wild-type mice. Thiorphan treatment of apoE-TR mice leads to aggregation of mouse A 1 week after treatment, with higher A deposition in apoE4-TR compared to apoE3-TR mice [84, 85]. Thus, thiorphan treatment of h-apoE-A -Tg mice represents a potential method of accelerating human A deposition.

An alternative method to address the h-APOE-induced temporal delay in A accumulation is to cross apoE-TR mice with A -Tg mice that have rapid-onset A pathology, such as 5xFAD [49], 3xTR [86, 87], APPSLPS1KI [88], APPPS1 [89], or TgCRND [90]. For example, because 5xFAD mice develop plaques by 2 months, the prediction is that apoE-TR/5xFAD-Tg mice develop plaques around 6 months and 12 months before other human-apoE/A -Tg mice (LaDu lab, unpublished observations and [5053]). The approaches described herein will lead to more tractable apoE/A -Tg-models to assess the apoE isoform-specific effects on A pathology.

4. Concluding Remarks

ApoE is the greatest risk factor for AD. Apo mice exhibit neuronal and cognitive deficits. Human apoE-Tg mouse models demonstrate that, compared to apoE3, apoE4 increases markers of neurodegeneration and cognitive impairment. Initially, to determine the effect of apoE on A pathology, A -Tg mice were crossed with apo mice, resulting in a significant delay in plaque deposition. Surprisingly, the introduction of h-APOE to several apo /A -Tg mouse models further delayed plaque deposition. This temporal delay restricts the usefulness of the current apoE/A -Tg mice for investigating the process of A accumulation and the resulting neurotoxicity. To accelerate A deposition, current apoE/A -Tg models could be treated with an additional insult such as TBI, crossed with other Tg models of neurodegeneration (NOS ), or treated with drugs that decrease A degradation. Alternatively, the development of A pathology could be accelerated by crossing A -Tg models with a rapid onset of A pathology, such as 5xFAD mice with apoE-TR mice. Transgenic models such as these provide tractable models for identifying biomarkers and the efficient initial validation of therapeutic targets.

Acknowledgments

Current LaDu lab funding includes NIH (NIA) P01AG03012801, Alzheimer’s Association ZEN-08-99900, UIC CCTS UL1RR029879. The authors would like to acknowledge Steve Estus, Goujun Bu, William Rebeck, and Edwin Weeber for ongoing collaborations.

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