EFAD transgenic mice as a human APOE relevant preclinical model of Alzheimer’s disease[S]

Identified in 1993, APOE4 is the greatest genetic risk factor for sporadic Alzheimer’s disease (AD), increasing risk up to 15-fold compared with APOE3, with APOE2 decreasing AD risk. However, the functional effects of APOE4 on AD pathology remain unclear and, in some cases, controversial. In vivo progress to understand how the human (h)-APOE genotypes affect AD pathology has been limited by the lack of a tractable familial AD-transgenic (FAD-Tg) mouse model expressing h-APOE rather than mouse (m)-APOE. The disparity between m- and h-apoE is relevant for virtually every AD-relevant pathway, including amyloid-β (Aβ) deposition and clearance, neuroinflammation, tau pathology, neural plasticity and cerebrovascular deficits. EFAD mice were designed as a temporally useful preclinical FAD-Tg-mouse model expressing the h-APOE genotypes for identifying mechanisms underlying APOE-modulated symptoms of AD pathology. From their first description in 2012, EFAD mice have enabled critical basic and therapeutic research. Here we review insights gleaned from the EFAD mice and summarize future directions.


Alzheimer's disease: humans to transgenic mice
In humans, Alzheimer's disease (AD) progresses over decades, resulting in synaptic dysfunction eventually leading to neuronal loss. Despite the large number of longitudinal amyloid plaques as a therapeutic target and even A as a measure of AD pathology, evident from publications (82)(83)(84)(85), press releases (http://www.thetimes.co.uk/article/ how-mice-immunity-is-hindering-research-n5fl2j5fj, https:// www.theatlantic.com/health/archive/2017/02/alzheimersamyloid-hypothesis/517185/), documentaries (http:// www.monsterinthemind.com/), and TEDx talks (https:// www.youtube.com/watch?v=6tBSFEzkk0A). One aspect of this debate is the nature of the aggregation pathways for A, particularly A42. As illustrated in Fig. 1, one paradigm for considering this assembly process is whether soluble oA is either "on" or "off" pathway to the assembly of amyloid plaques (86). Via the on pathway, oA can be pathogenic, but are structural precursors for the formation of the traditional insoluble fibrils that continue to assemble into the parallel -sheet structure of amyloid plaques also considered to be pathogenic (Fig. 1A)? Alternatively, in the off pathway assembly process, A can proceed via two separate processes, the pathogenic formation of oA or formation of the traditional insoluble fibrils that continue to assemble into amyloid plaques, considered to be benign (Fig. 1B). Whether the on or off pathway is correct has not yet been determined. However, both the vaccine trials and the imaging studies focus on amyloid plaques, not oA, as the pathogenic target. This distinction is critical as FAD mutations indicate only that increases in A42 or the increase in the A42/A40 ratio cause FAD, not that the toxic assembly form of the peptide is amyloid plaques. Indeed, a novel FAD-Tg mouse model that promotes oA formation, rather than fibrils and amyloid plaques, develops features of AD pathology, including tau hyperphosphorylation, neuroinflammation, synaptic alteration, cognitive deficits, and neuronal loss (87). For the purposes of this review, we consider that oA levels and induced signaling cascades are pathogenic, and amyloid plaques are likely, at best, less neurotoxic and more likely to be benign.

The 5xFAD mice versus EFAD mice
The EFAD mice are a tractable mouse model to study the development of a number of components of AD pathology (Tables 2, 3). Although 5xFAD mice express m-apoE, which we argue is distinct from h-apoE in the proximal mechanistic pathways that lead to AD pathology, we have included a comparison of AD pathology in 5xFAD versus EFAD mice to demonstrate, in part, the rapid onset and accelerated development of AD symptoms in the 5xFAD mice (Tables 2, 3). In addition, pathology in 5xFAD mice can be used to predict pathology in the EFAD mice. A particularly useful example is neuronal loss, which is significant by 12 months in the 5xFAD mice, predicting that in the EFAD mice neuronal loss will likely occur at >16 months. However, APOE genotype will significantly influence most components of AD pathology and analysis of these differences will illuminate APOE-modulation of AD pathology. In general, there is an APOE genotype (E4FAD > E3FAD  E2FAD), as well as an emerging sex effect (♀ > ♂) on behavioral deficits, histopathology, and neuronal viability (Table  2), as well as A42 and apoE solubility, neurotrophic factors, and neuroinflammatory cytokines, but not with APP processing (Table 3). Thus, the EFAD mice allow for the monitoring of multiple AD-related symptoms through the mouse lifespan (Tables 2, 3). An important note is that the sex and APOE4 genotype interactions are recent findings, and ♂ EFAD mice are more extensively characterized than ♀ mice. Here our goal is to detail APOE-modulated AD pathology in EFAD mice and, where applicable, ♀ versus ♂ comparisons.
in mice is not without drawbacks, it is striking that EFAD mice demonstrate APOE genotype-and sex-induced loss of cognitive function similar to humans.
Extracellular amyloid and A in ♂ EFAD mice. Human data demonstrate higher levels of extracellular amyloid/ A with APOE4 compared with APOE3 (20,103,104). For our initial characterization of the pathology in the EFAD mice, we used ♂ mice aged 2-6 months ( Fig. 3) (66). Both A accumulation and thioflavine-S (Thio-S)-positive plaque deposition begin in the subiculum (SB), followed by the deep layers of the frontal cortex (CX) (Fig. 3A, B) then spreading to the outer layers of the CX and the thalamus (50, 66). As described above, introduction of h-APOE delayed extracellular A accumulation from 2 to 6 months compared with the 5xFAD mice expressing m-APOE: 5xFAD > E4FAD > E3FAD  E2FAD (Fig. 3A). Plaque morphology is also affected by APOE genotype, with diffuse plaques: E2FAD = E3FAD > E4FAD and compact plaques: E2FAD = E3FAD < E4FAD (66).
Neuronal viability (synaptic proteins) in ♀ EFAD mice. Decreases in presynaptic and postsynaptic proteins in HP and CX contribute to altered functional connectivity, suggesting n.m. n.m.
Solubility of apoE and A42 in ♂ EFAD mice. In brain homogenates, total A42 levels are 5xFAD >> E4FAD > E3FAD = E2FAD (Fig. 5A) (66). To measure the solubility of both A and apoE, particularly the apoE-lipoproteins in brain tissue, we adapted a three-step sequential protein extraction protocol using TBS, TBS + Triton X-100 (TBSX), and formic acid, producing extraction profiles for A and apoE (Fig. 5B, D) (66,67). In the A extraction profile, the 5xFAD >> E4FAD > E3FAD = E2FAD pattern was observed in the TBS and formic acid extraction fractions, while the TBSX fraction contained low levels of A42 that were comparable across the Tg-mouse strains [ Fig. 5B, adapted from (66,67)]. These biochemical results are consistent with the delay in A deposition induced by the replacement of m-APOE with h-APOE (Fig. 3A), as are the genotype effects in levels of total and soluble A42 with E4FAD > E3FAD = E2FAD (Fig. 5A, B).
The apoE extraction profile detects apoE-lipoproteins in the TBSX fraction, a nonionic detergent that releases apoE from lipoproteins without inducing the formation of new micelles, as can occur with SDS and other ionic detergents (66,67). Thus, although total levels of apoE4 < apoE3 = apoE2 in the brains of humans (103,166,167), APOE-TR mice (5,47,(166)(167)(168)(169)(170), and EFAD mice (Fig. 5C), this isoform-specific difference is only in the TBSX-apoE4 (Fig.  5D) (66). C57BL/6 WT mice do not express A42, but do express m-apoE and, so, are included for comparison with the 5xFAD mice. For total apoE, the order is the Fig. 1. A assembly pathways: oA on or off pathways for amyloid plaque deposition. A: Via the on pathway, oAs can be pathogenic, but are structural precursors for the formation of the traditional insoluble fibrils that continue to assemble into the parallel -sheet structure of amyloid plaques, also considered to be pathogenic. B: Via the off pathway assembly process, A can proceed via two separate processes, the pathogenic formation of oA or formation of the traditional insoluble fibrils that continue to assemble into amyloid plaques, considered to be benign [reproduced from (86)].  (66, 67)]. However, in the extraction fractions, m-apoE from the WT is detected in the TBSX fraction, while the m-apoE from the 5xFAD mice is not detectable, though low levels of m-apoE from both WT and 5xFAD are detected in the TBS and formic acid fractions. Thus, the presence of A42 affects m-apoE solubility, specifically eliminating TBSXm-apoE.
In the soluble fraction, the levels of the apoE isoforms are not significantly different, while soluble A42 and oA are significantly higher with apoE4, and apoE4/A complex levels are significantly lower (Fig. 5E) (20,66). Indeed, APOE4 is characterized by increased levels of soluble A (A42 and oA) (20,66). Soluble A correlates with cognitive decline and disease severity in humans, and memory decline in FAD-Tg mice [for review see (14)]. To address the pathways that may underlie this correlation between apoE4 and increased soluble A required the development of novel reagents, including a new monoclonal antibody (mAb) specific to A (MOAB-2) (171) that enabled development of the critical oA (66) and apoE/A ELISAs (20). Importantly, lower levels of apoE lipidation and apoE/A complex in APOE4 versus APOE3 negatively correlate with soluble A levels (Fig. 5E) (20,66,172).
Lipidomic analysis in ♂ EFAD mice. Recent improvements in lipidomic technology have advanced the field to a point where it is now possible to accurately identify and quantify thousands of phospholipids (PLs) in complex biological samples with relative ease. Recently, a lipidomics study revealed that the ratios of arachidonic acid (AA) and DHA increased in several major PL classes in the blood from cognitively normal 4 carriers who converted to mild cognitive impairment/AD within 3 years (173). Longitudinal profiling of E3FAD and E4FAD mice showed that blood Fig. 3. AD histopathology in 2-to 6-month-old ♂ 5xFAD and EFAD mice. A: Total A deposition by immunohistochemistry (IHC) in 5xFAD and ♂EFAD sagittal brain sections at 2, 4, and 6 months with mAb for A (MOAB-2, red) and mAb for neurons [RNA binding protein, fox-1 homolog 3 (NeuN), green]. B: Plaque deposition: Thio-S staining in 6-month-old ♂ EFAD sagittal brain sections, quantified by percent area of CX [reproduced from (66)]. *P < 0.05 versus E4FAD. C: Neuroinflammation: IHC staining in 6 month ♂ EFAD sagittal brain sections for astrocytes (GFAP) [adapted from (72)], quantified as percent area of CX; and for reactive microglia [ionized calcium binding adaptor molecule 1 (Iba1), green] and A (MOAB-2, red), quantified as plaque-associated microglial density [reproduced from (72)]. *P < 0.05 versus E4FAD. D: pTau: IHC in 7 month ♂ EFAD CX for phosphorylated Tau pT205 and pS404 sites. Insets: 20× magnification (black box). pTau quantified in EFAD CX by Western blot, expressed as E4FAD/ E3FAD, #P < 0.05 versus E3FAD [reproduced from (75)]. Arrows for pathology: yellow = HP, red = CX. AA-and DHA-containing PL species were altered as early as 2.5 months of age. At 6 months, AA-and DHA-containing lysophosphatidylcholine species increased in blood, but decreased in the brains of E4FAD compared with E3FAD mice (173). Previous studies have shown that lysophosphatidylcholine-DHA is a preferred DHA carrier to the brain (174), and that the major facilitator superfamily domaincontaining protein 2 (mfsd2a) transports this lipid across the blood-brain barrier (BBB) (175,176). Hence, an increase in this lipid in the blood and a decrease in the brain suggests reduced transport of DHA to the brain in E4FAD mice compared to E3FAD and E2FAD mice. In addition, brain DHA transport is reduced in APOE4-TR mice compared with APOE2-TR mice, evidence for DHA transport deficiencies (177). Collectively, these studies suggest that an imbalance in AA and DHA may be due to transport deficiencies among the 4 carriers, which further contribute to the neuroinflammation associated with AD pathogenesis.

EFAD mice as a model for cerebrovascular dysfunction: a detailed example
CerVD is reemerging as a key component of AD, with unresolved questions as to the extent of CerVD and its significance to cognitive decline. Additional issues include the apparent differences between CerVD outcomes in humans and mouse models caused by APOE4, A, and sex.  (20,66,67)]. *P < 0.05 significantly less than E4FAD, #P < 0.05 significantly greater than E4FAD, ¶P < 0.05 significantly less than C57BL/6. All values are represented as mean ± SE normalized per milligram tissue (A-D) or per milligram protein (E).
EFAD mice provide critical insight on the interactive role of APOE, ♀ sex, and A in CerVD.

Cerebrovascular leakiness and vessel coverage in ♂ and ♀
EFAD mice. Plasma protein extravasation into the brain is a key outcome of CerVD, particularly BBB capillary leakage. Here, we demonstrate that fibrinogen levels in the CX and SB in 8-month-old EFAD mice follow the order: ♀ E4FAD > ♀ E3FAD = ♂ E4FAD > ♂ E3FAD (Fig. 6A). Total vessel coverage is a complimentary outcome measure of BBB damage, and in the SB and deep CX layers follow the order: ♂ E3FAD > ♂ E4FAD  ♀ E3FAD > ♀ E4FAD [ Fig.  6B, C reproduced from (71)]. Thus, the combination of ♀ sex, APOE4, and A induce pronounced BBB deficits that likely contribute to cognitive deficits. Indeed, high fibrinogen levels have been demonstrated in AD patients that are 4 carriers (178) and induce glial activation and neuronal dysfunction. Angiogenic growth factors are important for maintaining BBB function. Recent data demonstrate that plasma levels of one angiogenic growth factor, the epidermal growth factor (EGF), follow the order: ♂ E3FAD > ♂ E4FAD  ♀ E3FAD > ♀ E4FAD (71). Importantly, peripheral EGF administration to mice prevented cognitive decline, cerebrovascular leakiness, and vessel coverage deficits in ♀ E4FAD (71). Therefore, disruption of plasma angiogenic growth factor levels is a potential downstream pathway that contributes to BBB dysfunction in EFAD mice.
Cerebral amyloid angiopathy and microbleeds in ♂ and ♀ EFAD mice. Cerebral amyloid angiopathy (CAA) and microbleeds are often described as linked pathology. Here, we present data that CAA in the CX is higher in E4FAD mice regardless of sex (upper layers > deep layers), but not the SB (Fig. 7A), consistent with other investigators (73). The CAA is present primarily in larger vessels, although some capillary CAA is observed. When assessed using triple staining confocal analysis of the larger vessels, A is found attached to the outside of laminin, in the perivascular space between brain endothelial cells and laminin, and penetrating the vessel (Fig. 7B). These data raise the important general question of how A in the brain interstitial fluid deposits as CAA. Identified interstitial fluid drainage pathways include: 1) perivascular flow along the capillaries to the arteriole/artery basement membrane and; 2) the glymphatic pathway, in which CSF enters the brain along paravascular channels surrounding small arteries, exchanges with interstitial fluid, which is then cleared along paravascular spaces of large veins. We propose that our data are consistent with apoE4-induced impaired perivascular A drainage in arterioles (179,180), which also results in capillary CAA. In human AD patients, CAA is higher with APOE4 (181). However, in APOE genotype-matched AD patients, CAA is higher in ♂ compared with ♀. While one suggestion for this apparent difference between the EFAD mice and humans is that CerVD in humans is unique (73), there are several alternative explanations. First, the ♂/♀ differences in CAA may become more apparent in aged EFAD mice. Second, apoE3 may be more protective in ♀ versus ♂ 3/4 carriers. Third, and our hypothesis, is that peripheral AD-risk factors are greater in ♂, inducing cerebrovascular and BBB dysfunction, leading to CAA.
In EFAD mice, microbleeds partially mimic fibrinogen extravasation, rather than CAA (71,73,182): ♀ E4FAD > ♀ E3FAD > ♂ E4FAD = ♂ E3FAD (Fig. 7C) (71). In AD, microbleeds are associated with ♂, higher blood pressure, lower CSF A42, and APOE4 (183). The same considerations for CAA may underlie the microbleed differences between EFAD mice and humans. In addition, microbleeds are not severe, even in ♀ E4FAD mice, and in our experience, are localized to the deeper layers of the CX. Therefore, at 8 months, microbleeds could be driven by capillary or postcapillary venule breakdown, rather than CAA-induced arteriole damage. Alternatively, APOE-modulated damage to different parts of the vascular tree may be APOE genotypeand sex-specific, eventually leading to microbleeds.

EFAD mice are a vital tool for testing therapeutics
EFAD mice exhibit temporally-defined, APOE-modulated changes in outcomes for efficacy (behavior, neuronal protein levels), pharmacodynamic activity (A levels, neuroinflammation, and CerVD), and indirect targeted engagement. Thus, the activity of therapies/drug-like molecules can be assessed in prevention, treatment, and reversal paradigms. Therapies can target proximal (e.g., apoE lipidation) or downstream processes (e.g., neuroinflammation) that are disrupted by APOE4 and the detrimental interaction of ♀, APOE4, and A (e.g., sex hormone based). Further, whether a therapy is specific for APOE4, A, or is applicable for all groups (APOE genotype, sex, ±Α) can be determined by incorporating E2FAD, E3FAD mice, ±FAD mutations (EFAD, EFAD-NC). Thus far, pharmacological and nonpharmacological therapies targeting apoE-lipidation, general neuroprotection, CerVD, and sex hormone pathways have been tested in EFAD mice.

Targeting apoE4 lipidation
Lower lipidation/lipoprotein-associated levels in apoE4 was targeted in ♂ E4FAD mice using retinoid X receptor (RXR) agonists. The history of RXR agonists in the context of AD has been extensively reviewed elsewhere (184)(185)(186)(187)(188)(189)(190)(191)(192). Briefly, key issues center on whether RXR agonists increase apoE levels or lipidation (via increasing ABCA1 levels), the effect of human apoE4 and the duration of treatment. In ♂ E4FAD mice, short-term RXR agonist treatment (5.75-6 months) increased ABCA1 levels, apoE4 lipoprotein-association/lipidation, and apoE4/A complex, decreased soluble A, and increased PSD95 in the HP (172). However, RXR agonists induced no beneficial effects in ♂ E4FAD using a prevention protocol (5-6 months) and actually increased soluble A levels in ♂ E3FAD and ♂ E4FAD CX with the short-term protocol, possibly the result of systemic hepatomegaly. These data support RXR agonists to address

Nonpharmacological treatments
Additional nonpharmacological treatments tested in EFAD mice include EGF targeting cerebrovascular dysfunction (71) and 17- estradiol (E 2 ) treatment of ovariectomized (OVX) ♀ EFAD mice. E 2 decreased soluble A42 levels in ♀ E3FAD and ♀ E4FAD mice. However, insoluble A levels increased in ♀ E4FAD mice (194). Therefore, the activity of E 2 may be dependent on the relative impact of extracellular and soluble A on AD-induced neurodegeneration, with the results consistent with the hypothesis that soluble oA is toxic, while amyloid plaques are relatively benign (Fig. 1).

POTENTIAL DISADVANTAGES OF THE EFAD MOUSE MODEL
EFAD mice share weaknesses common to all FAD-Tg mice, including questions regarding the relevance of FAD transgene-induced pathology to sporadic AD, particularly during aging. The comparison of rodent to human aging is also a construct with inherent limitations based on differences in species, and strain differences among mice. Thus, it is useful to evaluate whether FAD-Tg mice can mimic aspects of aging and AD pathology. A major issue with h-APP-Tg mice is that their 2 year life-span may not be sufficient to observe the development of AD pathology (195). As with most FAD-Tg models, AD-related pathology, particularly A deposition, develops prior to middle age, which does not model the human condition. These concerns are mitigated to some extent in the EFAD by two factors. First, based on the genetic background of EFAD mice [(B6SJLF1×C57BL/6) from 5xFAD (50) × (C57BL/6) from APOE-TR (32)], we estimate that 10-14 months will represent middle age and 18 months will represent old age (https://www.nia.nih.gov/research/dab/aged-rodent-colonieshandbook/strain-survival-information) (196). Specifically, with the known survival rates for the background strains of the EFAD mice: 1) 5xFAD have a 75% survival rate at 16 months (197); and 2) C57BL/6 and APOE-TR have 75% survival rate at 24 months for ♂ and 22 months for ♀ (196). Thus, the EFAD mice have 75% survival at 20 months for ♂ and 19 months for ♀, making our target "old age" 18 months. Although specific measures of AD pathology in the EFAD are significant by 6 months, pathology continues to develop until at least 18 months, the oldest EFAD mice we have examined thus far (data not shown). Second, the EFAD-NC littermates provide both a comparison to the EFAD mice and a complementary approach to the address functional questions about APOE in the absence of FAD-induced pathology.
Despite these limitations, EFAD mice are the only wellcharacterized FAD/h-APOE-Tg mouse model with an extensive and growing provenance. Consistent with human AD patients, E4FAD mice develop pathology in a number of APOE genotype-, sex-, and age-dependent pathways.
EFAD mice are a tractable mouse model to study a number of AD-related outcomes, including changes in behavior, A deposition, tau pathology, neuroinflammation, and neuronal viability (Table 2), as well as apoE lipidation and A solubility (Table 3). These mice also allow for study of the interactions among AD risk factors, including age, APOE genotype, and sex.

Using EFAD mice as a model of aging and development of AD pathology
Understanding the interaction and dominance of APOE genotype versus sex with aging. Identification of the interactions between APOE genotype and sex are critical to understanding both aging and the development of AD pathology. Making predictions requires identification of the dominant risk factor in a given comparison, APOE genotype or sex: 1) The levels of A and amyloid deposition, as well as soluble A levels are higher in ♀ versus ♂ in several FAD-Tg mice (Tg2576, APP/PS1, 3xTg-AD) (64,(198)(199)(200)(201)(202)(203), as well as the EFAD mice (Table 3) (73). 2) In APOE-Tg mice, cognitive deficits are greater in ♀ APOE4 versus APOE3 [for review (21,(204)(205)(206)(207)(208)(209)]. In EFAD mice, behavioral deficits are E4FAD > E3FAD and ♀ > ♂ in 6-and 8-month-old mice (Fig. 2, Table 2). 3) In humans, lifetime AD risk, cognitive decline and accumulation of A is ♀ > ♂ in 4 carriers. These data suggest that the greatest risk for AD is with ♀ APOE4 carriers (6-13, 102, 210-213). These observations introduce a reoccurring theme in this field of research: which risk factor is dominant in its effects on AD pathology: APOE genotype or sex, and does this change with age? Based on cognition and AD-related histopathology, our general predicted order for AD pathology, with the addition of heterozygous E3/4FAD, is: ♂ E3FAD < ♀ E3FAD < ♂ E3/4FAD < ♀ E3/4FAD < ♂ E4FAD < ♀ E4FAD (the effect of the APOE2 genotypes are discussed separately). However, the dominant risk factor in a given comparison, APOE genotype or sex, is unclear. In general, the key comparisons for establishing the dominant effects of APOE versus sex will be determined by heterozygous E3/4FAD mice versus homozygous E3FAD and E4FAD mice, as established by age and AD pathology. For example, A deposition, neuroinflammation, and tau pathology in ♂ E4FAD versus ♀ E3/4FAD will predict a dominant risk factor: APOE4 if ♂ E4FAD shows the greatest pathology, or ♀ sex if ♀ E3/4FAD has the greater pathology. How this relative risk changes with age is critical. As well, using the EFAD-NC we can determine the effect of APOE versus sex interactions on normal aging.
Understanding trajectories, cliffs, and therapeutic windows. Multiple measures of AD pathology during aging will inform two critical components that indicate the relative contribution of risk factors, APOE or sex, and how their contributions are altered along the trajectory of the disease: 1) "Cliffs" or tipping points suggest a clear dominant risk factor: ♀ sex or APOE. For example, while ♀ E4FAD mice exhibit the greatest behavioral deficits and A pathology at both 6 and 8 months, ♀ E3FAD ≈ ♂ E4FAD at 8 months (71), suggesting a cliff or tipping point where ♀ sex is dominant compared with APOE genotype. However, unlike humans, as the ♀ EFAD mice age, they maintain 45-80% E 2 levels and normal uterine weight (214)(215)(216)(217)(218), which may produce an interesting phenotype at older ages with the scale tipping toward the dominance of APOE genotype. This change can be compared with OVX ± E 2 replacement. 2) Therapeutic windows are periods during which specific components of AD pathology are differentially affected by APOE or ♀ sex, allowing us to design and test specific therapeutic targets in preclinical studies using prevention or reversal paradigms.
Understanding the function of APOE2. The majority of the published data on the EFAD mice have used ♂ mice 8 months. As ♂ and ♀ mice are aged from 10 to 14 to 18 months, sex and APOE genotype interact to induce significant differences in various components of AD pathology (data not shown). As well, all of our work thus far has been with APOE +/+ /5xFAD +/ mice. As the APOE heterozygous genotypes are investigated (2/3, 2/4, 3/4), the influence of APOE genotype and sex interactions can be fully defined. In studying the APOE2 genotypes, it is important to keep in mind that if there are functional differences among 2/2, 2/3, and 2/4, it will likely go unidentified in all but the largest human cohort studies. This is because most studies will be underpowered for significance because of the low frequency of the 2 alleles [estimated: 2/2 at 0.4%, 2/3 at 8.8%, and 2/4 at 1.5% (5,6)]. This effect is exacerbated if the APOE2 genotypes are further stratified by age, AD status, and sex, resulting in the apparently contradictory literature for this field. However, heterozygous genotypes of APOE2 mice can be bred to reach significance via power analysis for any variable in comparison to heterozygous genotypes of APOE3 and APOE4. Indeed, the study of 2/2, 2/3, and 2/4 is perhaps a more subtle model to study the protective effects in both a normal (EFAD-NC) and AD (EFAD) cohort of mice. These results are key for identifying how the genotypes of APOE2 may cause differential effects in the context of being protective factors, for example, does 2/4 behave more like the risk 4 or the protective 2. These studies will provide new insights into how APOE2 imparts healthy brain aging and reduces AD risk, leading to diagnostic biomarkers and identification of therapeutic targets.

Using EFAD to identify environmental risk factors in AD pathology
About 98% of the human AD cases are sporadic with only half the cases linked to APOE4 and other genetic loci identified by genome-wide association study, suggesting the presence of other genetic or environmental risk factors and, thus, the potential interaction between genetic and environmental risk factors (88,(219)(220)(221)(222)(223)(224)(225)(226)(227). Thus, while APOE4 is the major genetic risk factor for AD, a number of environmental or lifestyle risk factors, have also been identified (228)(229)(230)(231)(232)(233). Two examples are given below.
Effect of high fat diets on AD. Epidemiological studies in humans consistently show an interaction between obesity and dementia/increased AD risk (228,(234)(235)(236)(237)(238)(239), though the interaction with sex remains controversial (240)(241)(242)(243). High fat diet-induced obesity accelerates AD pathology in FAD-Tg mice (244)(245)(246)(247)(248) and impairs cognition in APOE4-TR mice (249). However, the interaction among obesity, APOE genotype, and sex in modulating development of AD pathology is poorly understood (250,251). EFAD mice are a relevant model to address this question and the importance of lifestyle risk factors and their association with APOE in a genotype-and sex-dependent manner.
Effect of particulate air pollutants. The role of particulate air pollutants in accelerating cognitive impairment has been established in human (252)(253)(254)(255) and WT mouse studies (256). Exposure to particulate air pollutants increased A deposition, amyloid plaques and soluble oA in ♀ E4FAD compared with ♀ E3FAD mice (257). This increased susceptibility of ♀ 4 carriers to the neurotoxicity of particulate air pollutants provides evidence for interactive effects among genetic and environmental risk factors.

Using EFAD as a therapeutic model
Repurposing cardiovascular disease drugs. As discussed above, we previously demonstrated that in EFAD mice, induction of ABCA1/ABCG1 with RXR agonists increased apoE4 lipoprotein-association/lipidation, decreased soluble A, and increased PSD95 in the HP (172). However, treatment induced severe hepatomegaly, limiting RXR agonism for AD treatment. Approaches for targeting apoE lipoprotein-association/lipidation in the brain without the use of RXR agonists emerged as a promising alternative as the major enzymatic and lipid transport activities involved in the peripheral system are also expressed in the brain . The lipoprotein-association/lipidation of apoE in the brain parenchyma is the result of intercellular lipoprotein maturation and remodeling (263,274,(280)(281)(282)(283)(284)(285)(286)(287)(288)(289)(290)(291). Current strategies include directly targeting ABCA1 activity with an apoE mimetic peptide in the EFAD to evaluate its effect on apoE levels or apoE4 lipoprotein-association/lipidation and reduction of AD pathology.

Cerebrovascular dysfunction (CerVD).
Many of the planned treatment strategies that target either the proximal or downstream processes modulated by APOE and sex will likely also target CerVD. Proximally, directly targeting the structural and functional deficits of apoE4 may ameliorate detrimental changes that cause CerVD (190,(292)(293)(294). Targeting downstream signaling pathways or the soluble mediators produced by APOE-modulated activated glia (astrocytes and microglia) and pericytes may ameliorate CerVD, or prevent the risk with a subsequent additional hit, such as peripheral inflammation and high fat diets (292). Further, brain endothelial cells are often overlooked as a direct therapeutic target. The advantages of this target include: 1) Brain penetration is not required; 2) Peripheral risk factors will likely initially target brain endothelial cells rather than cells in the brain and; 3) As highlighted by the EGF treatment study, as brain endothelial cells play a central role in the homeostasis of the CNS, targeting brain endothelial cells may induce a pronounced beneficial effect on cognition. Currently, the ability of EGF to reverse cognitive and cerebrovascular deficits is under evaluation.
Neuroinflammation. Epidemiological studies targeting peripheral inflammation for AD indicate APOE-dependent lowering of AD risk due to nonsteroidal anti-inflammatory drugs (NSAIDs), with a beneficial effect for 4 resulting in initiation of AD Anti-inflammatory Prevention Trial (ADAPT) (295)(296)(297)(298)(299)(300)(301)(302)(303)(304). However, ADAPT failed and led to more criticism for evaluating the role of neuroinflammation in AD. It remains unclear whether targeting AD-relevant neuroinflammation receptor pathways is beneficial, detrimental, or not effective. For example, data from FAD-Tg mice provide evidence for beneficial (25,(305)(306)(307) and detrimental effects from TLR4 inhibition (308)(309)(310)(311). Inflammatory receptors may function differently depending on stage of AD pathology and APOE genotype, necessitating prevention and treatment protocols. EFAD mice are an ideal model to investigate this interplay between neuroinflammation and neurodegeneration that result in cognitive behavioral impairments, and for identifying the appropriate timing and targets involved in AD-associated neuroinflammation. Currently, EFAD are being evaluated with a prevention and reversal paradigm trial with a small TLR4 antagonist to evaluate its effect on AD pathology.
Selective estrogen mimics and selective estrogen receptor modulators. E 2 is key for ♀ vulnerability to APOE4-induced AD risk and pathology: OVX-induced loss of circulating E 2 in premenopausal women (312)(313)(314)(315)(316) and FAD-Tg mice (201,317,318) causes cognitive deficits that can be reversed by E 2 and estrogen therapy (ET), and in FAD-Tg mice, the OVX-induced increase in amyloid deposition is also reversed with ET (319, 320). However, the timing of ET in relation to the risk of AD in naturally menopausal women is a critical factor due to the apparent opposing outcomes based on early versus late menopause treatment (321). The controversial outcomes associated with timing could be addressed with the development of safe ET alternatives for the prevention and treatment of AD, potentially specific for the APOE genotype of patient. Based on the need for ET alternatives, we plan to study selective estrogen receptor modulators (322)(323)(324)(325), or selective estrogen mimics (326) in ±OVX ♀ EFAD mice.

SUMMARY
Given the prevalence of AD and the repeated failure of clinical trials, it is critical to develop Tg-mouse models to understand the mechanisms driving the trajectory of AD, identify early-stage biomarkers, and test preclinical therapeutic targets. EFAD mice mimic a range of AD-related pathologies, including cognitive decline, region-specific A and plaque deposition, progressive neuroinflammation, reduced synaptic viability, and cerebrovascular dysfunction. EFAD mice provide insight into the specific pathways and mechanisms that underlie APOE-and sex-dependent modulation of AD pathology. A complete characterization of the EFAD mice with age will enable an understanding of how the interaction among the greatest AD risk factors modulates AD-related pathology, specifically age, APOE genotype, and sex. Consistent with the underlying principles of personalized medicine, only when we understand these interactions can we begin to design therapeutic approaches for the prevention and treatment of AD.