Apolipoprotein E, an important player in longevity and age-related diseases

Experimental Gerontology 43 (2008) 615–622

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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

Mini Review

Apolipoprotein E, an important player in longevity and age-related diseases Lisa S. Ang 1, Rani P. Cruz 1, Alon Hendel 1, David J. Granville * Providence Heart + Lung Institute, St. Paul’s Hospital, Department of Pathology and Laboratory Medicine, University of British Columbia, 166-1081 Burrard Street, Vancouver, BC, Canada V6Z IY6

a r t i c l e

i n f o

Article history: Received 23 November 2007 Received in revised form 11 March 2008 Accepted 17 March 2008 Available online 4 April 2008 Keywords: Apolipoprotein E Aging Atherosclerosis Alzheimer’s Allele Mouse model

a b s t r a c t Numerous murine models are available for the study of the human aging process. Most of these models are based on known mutations that cause progeroid disease in humans or are involved in DNA repair and cell senescence. While these models certainly have contributed to our knowledge of age-related diseases, none adequately represent the range of human ailments involving cardiovascular and neurocognitive deterioration. In the current review, we summarize the available murine models of aging to date. We then discuss the known involvement of apolipoprotein E (ApoE) in various symptoms of the human aging process and describe the corresponding age-related phenotypes presented by the ApoE knockout mouse. Ó 2008 Elsevier Inc. All rights reserved.

1. Murine models of aging 1.1. Murine models of aging: introduction Genetically manipulated mouse models are important tools in the quest to understand the mechanisms that are involved in human aging. The use of mice is especially informative for studying aging because they are closely positioned to humans on the evolutionary scale, exhibit easy genetic accessibility, have a relative short lifespan and demonstrate many DNA maintenance mechanisms that are similar in humans (Hasty et al., 2003). However, it is important to validate each model as a genuine representation of native aging since humans manifest an array of aging phenotypes that cannot be completely recapitulated in any particular animal model. As native aging is a consequence of a large number of genetic and environmental causes, it is unlikely that a single genetic alteration in mice will include all aging phenotypes demonstrated in humans. To evaluate mouse models for human aging, it has been proposed that three criteria be taken under consideration when validating accelerated aging phenotypes in single genetically modified mice: (1) the aging phenotype should be present after development and maturation have been completed; (2) control * Corresponding author. Address: Providence Heart + Lung Institute, St. Paul’s Hospital, Department of Pathology and Laboratory Medicine, University of British Columbia, 166-1081 Burrard Street, Vancouver, BC, Canada V6Z IY6. Tel.: +604 806 9267; fax: +604 806 9274. E-mail address: [email protected] (D.J. Granville). 1 These authors contributed equally to this work. 0531-5565/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2008.03.010

population should demonstrate the phenotype in a more or less similar point in their survival curve; and (3) the genetic alteration should accelerate multiple aging phenotypes in multiple tissues (Hasty and Vijg, 2004). Taking into account these criteria, the following mouse models were found to closely reflect normal senescence mechanisms that underline native aging processes in humans (summarized in Table 1). 1.2. Murine models of aging: DNA maintenance and repair Currently, most mouse models of aging are obtained by interruptions in genes that have key roles in DNA maintenance and repair. For that reason, it is believed at large that human native aging might be a result of DNA damage accumulation and inability to preserve normal genome maintenance mechanisms, such as DNA replication and repair. An important step that initially linked between DNA maintenance genes and the development of aging phenotypes was established through studies of premature-aging syndromes in humans. Premature-aging syndromes, also known as progeroid syndromes, are hereditary genetic disorders that accelerate the normal process of aging. Werner syndrome (WS) is one of the most striking of the human progeroid syndromes, where a loss of function mutation in WRN gene, which encodes a member of the RecQ family of DNA helicases, results in premature expression of aging phenotypes such as atrophic skin, greying and loss of hair, osteoporosis, malignant neoplasm and shortened life span (Yu et al., 1996). In an attempt to replicate the premature-aging phenotypes observed in WS, Wrn-knockout mice were generated. Surprisingly, these mice were found to be essentially normal with

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Table 1 Genetically modified mouse models of human aging Gene

Aging associated diseases

Aging phenotypes

Human aging phenotypes that were not shown

References

Wrn / , Terc /

Osteoporosis, Type II diabetes, cataracts, cancer

Low body mass, hair greying, alopecia, hypogonadism, impaired wound healing, kyphosis

Atherosclerosis, skin atrophy

Chang et al. (2004)

LmnaL530P/L530P

Osteoporosis

Muscle atrophy, decreased hair follicles, thinning of the skin, reduced subcutaneous adipose tissue

Atherosclerosis, cancer, diabetes, cataract

Mounkes et al. (2003)

XpdR722W/R722W

Osteoporosis

Grey and brittle hair, cachexia, kyphosis

Atherosclerosis, skin atrophy, cancer, alopecia

de Boer et al. (2002)

Ku80 /

Cancer

Osteopenia, epiphyses closure, atrophic skin, hair follicle atrophy, kyphosis

Atherosclerosis, osteoporosis, muscle atrophy

Vogel et al. (1999)

Cancer, osteoporosis

Low body weight, kyphosis, impaired wound healing, thinning of the skin, impaired hair regeneration

Atherosclerosis, alopecia

Cao et al. (2003)

Trp53+/m

Osteoporosis

Weight loss, kyphosis, skin atrophy, impaired hair regeneration, impaired wound healing, muscle atrophy, decreased organ mass

Atherosclerosis, cancer

Tyner et al. (2002)

Terc /

Cancer

Grey hair, intestinal atrophy, spleen atrophy, impaired wound healing

Atherosclerosis, osteoporosis, alopecia, skin atrophy

Blasco et al. (1997) and Rudolph et al. (1999)

Klotho (kl/kl)

Atherosclerosis, osteoporosis

Hypokinesis, skin atrophy, hair loss, infertility, brain atrophy, ectopic calcification

Cancer, cataracts

Kuro-o et al. (1997)

ApoE /

Atherosclerosis, Type II diabetes, xanthomatosis

Hypokinesis, skin atrophy, hair loss, grey hair, weight loss, kyphosis, cachexia, arrested spermatogenesis

Cancer, cataracts, osteoporosis

Moghadasian et al. (2001)

Brca1(D

11/D11)

Trp53+/

minimal characteristics of premature-aging (Lombard et al., 2000). Only after deletion of the Terc gene, which encodes the RNA component in the telomerase enzyme (to be discussed later), and the generation of Wrn / , Terc / double knockout mice, were Werner-like phenotypes observed (Chang et al., 2004). These mice also exhibit many of the disease characteristics that are associated with native human aging (see Table 1). Since WRN helicase was shown to play an important role in DNA replication, recombination and repair, it is believed that the accelerating aging phenotypes observed in both the mouse model and humans are a consequent of DNA damage accumulation (recently reviewed in Brosh and Bohr, 2007). Thus, for their contribution towards the understanding of the role WRN plays in genomic maintenance and its impact on premature-aging process, Wrn / Terc / mice are often considered to be one of the more significant models in the study of age-related diseases (Chang et al., 2004). Deficiency in a number of important DNA repair mechanisms has been demonstrated to promote accelerating aging in mice. Impairment in nucleotide excision repair mechanisms has been identified in mice that carry a mutation in their Xpd gene (de Boer et al., 2002). These mice were generated as analogs for the naturally occurring mutation in patients with trichothiodystrophy, a progeroid syndrome in which patients carry a mutation in the XPD gene. This gene also encodes for DNA helicase, which is involved in both repair and transcription. Mice that carry the corresponding mutation in their Xpd gene also exhibit premature-aging symptoms that appear in adulthood, indicating that these phenotypes are not the result of aberrant development but rather reflect the mechanism of aging (de Boer et al., 2002). However, this mouse model did not present other age-related pathologies associated with native and premature-aging, such as atherosclerosis, cataracts, diabetes mellitus and cancer. BRCA1 is a protein involved in the repair of DNA doublestranded breaks through homologous recombination. Mice that carry mutations in their Brca1 gene also exhibit premature-aging phenotypes. However, to overcome embryonic lethality, these mice had to be deficient in one copy of their p53-encoding gene

in order to suppress cell senescence and apoptosis, and allow maturation of the mouse to adulthood (Cao et al., 2003). These mice experience shorter life span and present many features of premature-aging (see Table 1). As one might expect, a gain-offunction mutation in the p53-encoding gene, Trp53, also yielded an accelerated aging mouse model (Tyner et al., 2002). p53 is a tumour suppressor protein that regulates cell response to DNA damage by promoting either cell senescence or apoptosis (Levine, 1997), and its overexpression has been found to induce cellular senescence (Sugrue et al., 1997). Further studies on chromosome-maintaining genes have strengthen the idea that defective DNA repair mechanisms play a major role in promoting cell senescence and the consequent expression of aging phenotypes (Hasty et al., 2003). A mouse model with a null mutation of Ku80 gene has also exhibited accelerated aging. Ku80 (also known as Ku86 or Xrcc5) plays an important role in the repair of DNA double-strand breaks through a non-homologous end joining mechanism in association with Ku70 (Lieber, 1998). Interestingly, Ku80 has been demonstrated to be physically interacting with WRNp helicase and functionally affecting its exonuclease activity, suggesting an interrelation between several repair mechanisms that maintain genome stability. Ku80-knockout mice exhibit shortened life span and demonstrate several aging phenotypes associated with human aging (Vogel et al., 1999). These phenotypes manifest after sexual maturation and at around the same point on survival curves in comparison to their control, making Ku80-deficient mice an important model for prematureaging (Hasty et al., 2003). Increased cellular senescence and reduced replication capability have also been suggested to contribute to the aging process. As such, it has been important to examine the factors that contribute to cell senescence, or alternatively, examine factors that promote senescence escape. Tumour cells have the ability to escape senescence mostly due to the reactivation of telomerase, an enzyme that is responsible for maintaining telomere length (Blasco, 2005). Telomeres are capping structures at the end of the chromosomes that consist of DNA-bound proteins and repetitive DNA sequences

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whose length is progressively reduced during each cycle of cellular replication. The effect of telomere shortening on the aging process was established through studies on Terc-deficient mice (Blasco et al., 1997; Rudolph et al., 1999). Terc is the essential RNA component of the telomerase holoenzyme. Without the ability to maintain telomere length Terc-null mice exhibited premature-aging symptoms such as hair greying, alopecia, infertility, heart dysfunction and various tissue atrophies (Leri et al., 2003; Rudolph et al., 1999). Importantly, mouse telomeres are significantly longer than in human, and the study on Terc-null mice indicated that there was no aging phenotypes present in the first generation, where only the 3rd generation began to exhibit age-related phenotypes that grew pronouncedly in later generations (Blasco et al., 1997), a fact that might limit this mouse model in its representation of native human aging. Nevertheless these findings indicates that maintenance of telomere length is required for normal tissue homeostasis in mice, and can be extrapolated to suggest that progressive telomere shortening may contribute to the aging process in humans (Blasco, 2005). Investigations of other progeroid syndromes have yielded several other aging mouse models. A mutation in LMNA, a gene encoding lamin A, was identified as the underlying cause of Hutchinson– Gilford progeria syndrome (HGPS) (Eriksson et al., 2003). Lamin A is an intermediate filament family member that makes up the nuclear lamina. This is part of the fibrous network underlying the nuclear envelope and plays a role in maintaining chromatin organization (Burke and Stewart, 2002). Mice that carry the same mutation in the LMNA gene exhibit many progeroid phenotypes such as hypoplasia, muscle atrophy, loss of hair follicles, thinning of the skin, osteoporosis and reduced life span, all of which are consistent with progeroid phenotypes in humans (Mounkes et al., 2003). Taken together, the aforementioned mouse models have been highly useful in establishing a strong link between cellular impairment of DNA up keeping and accumulation of genetic damage to the development of aging phenotypes. However, it was also been recently suggested that non-DNA maintenance genes might also have a role in promoting human native aging. 1.3. Murine models of aging: Klotho In Kuro-o et al. (1997) reported that mutation of the Klotho gene in mice produced a phenotype resembling premature-aging in humans which included reduced life span and a number of pathologies associated with human aging, such as atherosclerosis and osteoporosis. The Klotho gene encodes a novel single-pass membrane protein that shares sequence similarity with the b-glucosidase enzymes, and although it has been suggested that this membrane bound protein might be involved in regulation of antioxidant defense (Nagai et al., 2003), its exact function has yet to be determined (Kuro-o et al., 1997). However, signs of aging in these mice were presented before reproductive maturity, and common aging phenotypes such as cancer and cataracts were not observed (Kuro-o et al., 1997). Nevertheless, this report is intriguing because Klotho does not appear to have a direct role in either chromosome maintenance or DNA repair mechanisms, and although no disease equivalent has been identified in humans, it has been reported that variation at the human KLOTHO locus may contribute to survival (Arking et al., 2002). While the progeroid mouse models have certainly contributed to our understanding of the mechanisms of aging, it is important to note these models only represent a condition of accelerated aging and may not completely represent native aging in human. Although it is acknowledged that dysfunctional DNA repair, chromosomal maintenance and senescence all contribute towards the aging phenotype as we know it, the identification of a genetic factor like Klotho should serve as a basis for looking outside these

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areas in our search for the rest of the elusive effectors of the complex biological process that is aging. In search for such factors our lab has recently observed distinct age-related phenotypes in ApoE knockout mice that mimic the major phenotypes of human native aging. A population study examining telomere length as a marker for accelerating aging has associated the presence of ApoE e4 allele in individual with shorter telomeres who had higher mortality rate and exhibited common aging phenotypes such as dementia and Alzheimer (Honig et al., 2006). Hence, ApoE might have other roles that are related to the process of native aging.

2. Apolipoprotein E (ApoE) and the major phenotypes of aging in humans 2.1. ApoE: polymorphism and function In humans, cardiovascular disorders and dementias are amongst the most common ailments that are associated with the aging process. Mostly through population studies, it has emerged that ApoE polymorphism is a common risk factor for the pathogenesis of these age-related diseases (Siest et al., 1995). As such, it would be of great interest to further explore the role of ApoE in human aging. Apolipoproteins are critical components of the plasma lipoprotein complexes that function as the body’s lipid shuttle system, transporting dietary lipids absorbed from the intestines to the liver, and endogenously synthesized lipids from the liver to metabolic, secretory and storage sites around the body. The apolipoproteins are the only protein component of the lipoprotein complex and generally serve to maintain the structural integrity of the lipoproteins but are also known to act as cofactors in enzymatic reactions and as ligands for lipoprotein receptors (Eichner et al., 2002). In particular, ApoE is critical for the formation and stability of chylomicrons, very low density lipoprotein (VLDL) (Eichner et al., 2002) and high density lipoprotein (HDL) (Mahley, 1988). Chylomicrons are formed by intestinal mucosa to transport exogenous lipids to the liver and adipose tissues where they are internalized by receptor-mediated endocytosis. VLDL mainly transports endogenously synthesized triglycerides, phospholipids, cholesterol and cholesteryl esters from the liver to peripheral tissues while HDL is involved in the redistribution of cholesterol between the liver and various cell types (Mahley, 1988). ApoE is synthesized mainly by the liver but is produced to some extent in other organs and tissues, most notably the brain, but also in the spleen, kidneys and macrophages (Elshourbagy et al., 1985; Mahley, 1988). In humans, the APOE gene is polymorphic, with at least seven variants identified (Corbo et al., 1999; Hallman et al., 1991; Hubacek et al., 2005; Iron et al., 1995; Scacchi et al., 2003; Siest et al., 1995; Utermann, 1987). The vast majority of population studies have focused on the three most common alleles – e2, e3 and e4. The resulting six possible genotypes can be ranked in order from most to least common: e3/e3, e4/e3, e3/e2, e4/e4, e4/e2, e2/e2 (Davignon et al., 1988). The polymorphism arises from cysteine-arginine interchanges at residues 112 and 158 in the receptor binding region of the polypeptide chain. These variations confer different low density lipoprotein (LDL)-receptor binding affinities upon each isoform, with e3 and e4 binding with similar affinities while e2 binding with 1 percent of the affinity of e3 and e4 isoforms (Siest et al., 1995). The regulation of circulating cholesterol levels is highly dependent on the specific interaction between ApoE and the LDL-receptors (Brown and Goldstein, 1986). Therefore, carriers of the e2 allele are less efficient at forming and removing VLDLs and chylomicrons from the blood to the liver because of its defective binding affinity, resulting in low levels of plasma cholesterol, upregulation

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of liver LDL-receptors and higher levels of circulating triglycerides. Carriers of the e2 allele are also slower at clearing dietary lipids from the bloodstream when compared to e3 and e4 carriers (Weintraub et al., 1987). 2.2. ApoE: susceptibility to cardiovascular disease Cardiovascular disease (CVD), and in particular, the atherosclerotic disorders, are the chief cause of illness, disability and death worldwide. The American Heart Association estimates that over 29% of deaths globally can be attributed to CVD and that these numbers will only continue to rise because of the aging populations of many developed nations. In North America, CVD accounts for over 40% of deaths in persons over the age of 65 (Lakatta, 2002), thus risk factors, such as abnormally high cholesterol, that contribute to the pathogenesis of atherosclerosis, hypertension and other vascular disorders, are also significant issues in the context of aging. Polymorphism of the APOE gene plays a significant role in the alteration of cholesterol levels, and are currently one of the most well-documented risk factors for development of atherosclerotic disease, thus it is conceivable that ApoE is important in cardiovascular aging. Most population allele frequency studies have been done in the context of lipid and cholesterol profiling since Utermann et al. (1975) first observed that ApoE polymorphism could be the underlying cause for Type III Familial Hyperlipoproteinemia (Utermann et al., 1975). They later reported that the e2 allele was significantly more frequent in patients with hypertriglyceridemia, and that the e4 allele was significantly more frequent in patients with hypercholesterolemia (Utermann et al., 1984). Both hypercholesterolemia and hypertriglyceridemia are classic risk factors that significantly contribute to the pathogenesis of CVD. Since then, dozens of studies involving over 150,000 healthy and diseased cases have been implemented to determine the effects of ApoE polymorphism on serum lipid levels and coronary disease outcomes. Conclusions have varied with some studies reporting highly significant contribution and others reporting none (refer to Song et al., 2004). Differences in study design, sampling methods, defined disease endpoints, adjustment for other cardiovascular disease risk factors and statistically insignificant sample size often made it difficult to determine with any certainty the true effects of ApoE polymorphism on cardiovascular risk. However, a number of meta-analyses have been conducted in an attempt to quantitatively assess the data that is available from multiple studies in a comprehensive manner. The largest meta-analysis by Bennet’s group covered (Bennet et al., 2007) the association of ApoE genotypes with both lipid levels and CHD risk in 121 studies. Total cholesterol and LDL cholesterol levels demonstrated approximately linear relationships with ApoE genotypes in order from lowest to highest: e2/e2, e2/e3, e2/e4, e3/e3, e3/e4, e4/e4. e2/e2 homozygotes were shown to have the highest triglyceride level, which confirms previous observations regarding lipid clearance but e2/e3 and e2/e4 genotypes did not have significantly higher triglyceride profiles compared with non-e2 carriers. It was concluded that e2 carriers have up to 20% reduced risk for CHD. The precise mechanisms by which the presence of a single e2 allele could confer such advantage are not well understood although it was suggested that a more efficient binding capacity for heparin and HDL might enhance reverse cholesterol transport (Bennet et al., 2007). This conflicts with traditional views that implicate e2 in hyperlipoproteinemia; a condition that is characterized by increased levels of triglycerides and premature cardiovascular disease. It is clear that explaining the differential effects of this allele on CHD risk will probably be more complex than initially thought. While most of the cardiovascular impact of ApoE polymorphism has been associated with dysfunctional lipoprotein clearance and lipid metabolism, it has been observed that e4 allele carriers tend

to have higher fasting glucose levels and more rapid changes in glucose levels over time (Craft et al., 1999; Scuteri et al., 2005; Stiefel et al., 2001). Scuteri et al. (2005) suggested that higher glucose levels can lead to higher rates of ApoE glycation. This is significant because advanced glycation end products (AGEs) have been implicated in numerous age-related disorders including diabetes mellitus, diabetes-accelerated atherosclerosis, nephropathy, retinopathy, Alzheimer’s disease (AD) and other neurodegenerative disorders (Cooper, 2004; Feng et al., 2007; Forbes et al., 2004; Laffont et al., 2002; Lassila et al., 2004; Sasaki et al., 1998). 2.3. ApoE: susceptibility to dementia and Alzheimer’s disease Alzheimer’s disease (AD) is a disabling, degenerative disorder that affects over 24 million people worldwide (Ferri et al., 2005). It is the most common form of dementia and prevalence rates of AD have been estimated to double every 5 years between the ages of 65 and 85 (Hsiung et al., 2004). AD is a slow, progressive disease that is manifested in memory loss, cognitive dysfunctions and other neuropsychiatric symptoms (Lahiri et al., 2004). It can be inherited or sporadic, and is usually arbitrarily classified as earlyonset (before age 65) and late-onset (after age 65) disease. AD is pathologically characterized by selective neuronal cell death, the deposition of amyloid-b (Ab) peptide in senile plaques and the formation of neurofibrillary tangles in the brains of afflicted individuals (Barrow et al., 1992; Haas et al., 1997; Lahiri et al., 2003). ApoE is the main apolipoprotein synthesized by the brain (Elshourbagy et al., 1985) and is a component of the lipoproteins found in cerebrospinal fluid (Pitas et al., 1987). In 1991, it was reported that both the plaques and the neurofibrillary tangles were immunoreactive for ApoE (Namba et al., 1991) and it was also demonstrated that ApoE forms stable complexes with the amyloid-b precursor protein (Haas et al., 1997). In 1993, a study of familial late-onset AD reported that e4 allele frequency in patients was 0.50 compared with 0.16 for age-matched controls (Strittmatter et al., 1993). Amongst autopsy-confirmed sporadic cases, the allele frequency was 0.40 and highly significant when compared with controls (Saunders et al., 1993). Furthermore, in a study on the effects of gene-dose on risk and age-of-onset, it was observed that in families with histories of late-onset AD, the risk for AD increased from approximately 20% to over 90% and mean age-ofonset decreased from 84 to 68 years with increasing number of e4 alleles (Corder et al., 1993). Subsequent clinical and population studies have been mostly in concurrence with these observations. A meta-analysis of ApoE polymorphism and AD conducted in 1999 (Rubinsztein and Easton, 1999) concluded that e4 was associated with a significantly higher risk of early-onset AD with an OR of 4.86 (3.61–6.54, 95% CI) and also late-onset AD with an OR of 3.20 (2.93–3.49, 95% CI). e2 carriers had a much reduced odds ratio of 0.60 (0.31–1.14) in early-onset cases and 0.68 (0.58–0.80) in late-onset cases. This corroborated results by a previous meta-analysis by Farrer et al. (1997) and has been confirmed by another meta-analysis by Bertram et al. (2007) working with the AlzGene database. Whilst AD is the most common form of dementia, it also appears that the e4 allele confers a significant risk for acquiring other types of dementias in addition to AD. In a meta-analysis that included dementias other than AD, it was found that the frequency of the e4 allele was higher in patients with AD (24.6%) and mixed dementia (MD) (22.2%) compared to age-matched controls (10.1%). Patients with vascular dementia (VD) and non-AD/nonVD (NAVD) dementia did not have significantly higher allele frequencies (8.1% and 11.8%, respectively) (Bang et al., 2003). The odds ratio for acquiring any non-AD dementia was 1.3 (1.1–1.5, 95% CI) for e4 heterozygote carriers and 3.7 (2.6–5.2, 95% CI) for e4 homozygote carriers.

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2.4. ApoE: cognition and longevity Because cognitive function is an important diagnostic factor in the identification of dementia, there has been interest in determining the effect of e4 allele status on cognitive impairment in both demented and non-demented individuals. Results have been inconsistent due to numerous factors, including limited statistical power, age of participants, differences in assessment of cognitive ability, as well as inclusion of preclinical dementia cases. A meta-analysis of 38 studies (Small et al., 2004) on various cognitive domains determined that e4 carriers scored modestly but significantly poorer in the areas of global cognitive functioning, episodic memory and executive functioning compared to e3/e3 genotypes. Interestingly, e2 carriers appeared to perform better than controls in global cognitive functioning. The overall conclusion was that ApoE genotype does exert some small influence on cognitive function over the course of healthy aging but applies only to certain domains of cognitive abilities and the mechanisms involved are as yet unclear. As with any study of a genetically-associated age-related disease, naturally the next step has been to determine the effect of the polymorphism on longevity. With the advent of improved standards of sanitation and health care and the subsequent increase in life expectancies, the prospect of an aging society has rekindled the interest to determine the biological basis for human lifespan. The increasing number of centenarians has also allowed us a glimpse into the possible genetic and biochemical factors responsible for such longevity. Because of its involvement in three of the four most common causes of mortality in the Western world – coronary artery disease, cerebrovascular disease and AD, the ApoE gene has been the most widely examined genetic marker amongst centenarian population studies (Asada et al., 1996a,b; Choi et al., 2003; Louhija et al., 1994; Panza et al., 2004, 1999; Schachter et al., 1994). Most studies have reported that e4 frequencies appear to be lower in centenarians than in the general or middle-aged population and some have observed that e2 frequency is higher. There are limitations to using centenarian studies to determining the effect of ApoE polymorphism on longevity; the most obvious being the physiological and genetic complexity of life itself and the multitude of environmental factors that cannot be controlled for in observational studies on human subjects. The effects of ApoE polymorphism must be considered as part of a larger gene program and recent studies seem to implicate the entire genetic pathway involved in cerebral and peripheral cholesterol and lipoprotein homeostasis, which also has ramifications for vascular disorders (Carter, 2007). Links between atherosclerotic and neurodegenerative diseases have been noted before (Carter, 2007; Casserly and Topol, 2004; de la Torre, 2004; Hofman et al., 1997; Iadecola, 2003; Newman et al., 2005; van Oijen et al., 2007). Many forms of dementia have a significant vascular component and share multiple common risk factors involving the same cholesterol/lipoprotein pathway as atherosclerosis. It has also been reported that cerebrovascular atherosclerosis and ischemia feature significantly and also contribute to the neuropathology and cognitive deficits observed in AD (Roher et al., 2003; Snowdon et al., 1997). As discussed earlier, the true effect of ApoE polymorphism on CVD has been hard to determine based on the conflicting results of various studies and the rather complex nature of the disease to begin with. This is also true for AD where although possession of an e4 allele is a firmly established risk factor for developing AD, it is not necessary or sufficient to acquire AD (Bertram et al., 2007; Corder et al., 1993; Llorca et al., 2008). Furthermore, while the likelihood of acquiring dementia increases with age, several studies have suggested that e4 carrier status as a risk factor for developing AD loses potency with age (Breitner et al., 1999; Farrer et al., 1997). Here, the raw data may not be providing the entire picture however and changes in lipo-

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protein/cholesterol levels and functions probably affect these systems in a subtly differential manner where effects in one can mask effects in the other (Carter, 2007; Pappolla et al., 2003). It has also been suggested that a significant percentage of e4 carriers develop CHD which could theoretically lead to high incidence of fatal myocardial infarction that they never reach sufficient age for dementia/AD to manifest (Kuusisto et al., 1994; Roses, 1996). So while ApoE polymorphism represents a significant risk factor for dementias and cardiovascular disease, evidently more work is needed to clarify the true role of ApoE in the pathogenesis of these age-related disorders. The inherent limitations of population studies in humans mean that further mechanistic enquiries will need to be answered through the use of animal models of disease.

3. The ApoE knockout mouse as a model of aging 3.1. Cardiovascular aging in ApoE-KO mice Atherosclerosis, although commonly associated with human progeroid syndromes, is an aging phenotype that none of the progeroid mouse models exhibit. Since the development of ApoE knockout (ApoE-KO) mice in 1992 (Piedrahita et al., 1992; Zhang et al., 1992), many valuable age-related cardiovascular research questions have been answered. ApoE deficiency leads to the accumulation of cholesterol ester-enriched particles in the blood. When ApoE-KO mice are fed a high fat Western diet, as they age they develop fatty streaks and fibroproliferative lesions sooner than on a regular chow diet; thus this model has become a powerful and widely accepted tool in cardiovascular research. The cellular composition of the atherosclerotic lesions in ApoE-KO mice is similar to that observed in humans, consisting of macrophages, T cells and smooth muscle cells, as the presence of oxidized lipoproteins (Nakashima et al., 1994). The low density lipoprotein receptor knockout mouse (LDL-R KO) is another model of atherosclerosis that is commonly used. These mice require an addition of cholate in their diets to develop atherosclerosis and their plaques are often not as complex as ApoE-KO mice, thus do not represent complex human plaques as well as ApoE-KOs. This progression of fatty streaks to complex plaques represents another hallmark of aging that we feel can discriminate between these two atherosclerotic models when studying aging. 3.2. Blood–brain barrier permeability and Alzheimer’s disease (AD) An early study demonstrated that ApoE is involved in working memory and ApoE-KO mice display cholinergic insufficiencies (Masliah et al., 1997). Testing different alleles of ApoE, an important experiment showed that in ApoE-KO mice, infusions with the ApoE allele e3 or e4 improved learning capacity in a task the mice had been trained to perform, thus suggests a therapeutic effect of ApoE (Masliah et al., 1997). These findings may link why susceptibility towards developing age-related disorders, such as AD, are associated with ApoE allelic differences. A very recent study has demonstrated that in ApoE-KO mice, blood–brain barrier permeability is associated with the age of the mice (Hafezi-Moghadam et al., 2007). In this study, the C57 control mice showed that the blood–brain barrier increased in permeability with increasing age. More dramatically, in aged-matched ApoE-KO mice, the permeability rate was increased 3.7 times. 3.3. Other evidence supporting ApoE-KO mice as a model of accelerated aging Our laboratory has observed that ApoE-KO mice, in addition to the well-documented cardiovascular disease, exhibit other

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Fig. 1. Comparison of 36-week-old C57Bl/6 wild-type mouse to an age-matched ApoE-KO mouse after 30 weeks on a high fat ‘Western’ diet. (A, C and E) are images from a C57Bl/6 mouse after a 30 weeks high fat diet. (B, D and F) are images from an ApoE-KO mouse from a C57Bl/6 background after a 30 weeks high fat diet. (A and B) show the exterior appearance of the C57Bl/6 mouse and ApoE-KO mouse, respectively. Note the differences in hair loss, conformation and presence of cutaneous xanthomas. (C and D) show the aortic roots stained with oil red O from the mice where atherosclerotic plaques first appear. Note the presence of the plaques on the ApoE-KO aortic root (stained in red). (E and F) show the skin sections of the mice. Note the presence of cholesterol deposits and immune infiltration in the ApoE-KO mouse as well as the decrease in hair follicles.

age-related phenotypes. These phenotypes are observed regardless of diet, though on a high fat diet the characteristics described occur at a faster rate. As the mice age, hair follicle loss, impaired hair regeneration and greying begin much earlier than the C57 controls (Fig. 1). An earlier analysis of ApoE-KO mice observed that mice also showed arrested spermatogenesis and atrophy of the seminiferous tubules (Moghadasian et al., 1999).

ApoE-KO mice have also demonstrated shortened life spans. Moghadasian et al. (2001) had observed that 35% of ApoE-KO mice died suddenly after 18 months (Moghadasian et al., 2001). We have also observed that on a high fat diet, these mice must be euthanized after 7 months of age due to the exhibition of cachexia, severe cutaneous xanthomas, dramatic loss of subcutaneous fat and low body mass, kyphosis and hypokinesis. Comparison with the

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current accelerated aging models is summarized in Table 1. These characteristic aging phenotypes support the notion that ApoE-KO mice are a useful mouse model for aging. 4. Conclusions As the evidence suggests, ApoE undeniably plays a role in the human aging process although the finer details have yet to reveal themselves. While population level studies have revealed much about the role of ApoE polymorphism in risk calculations, ethical and logistical reasons make necessary the use of animal models to answer any questions of a mechanistic nature. In the meantime, more research is required to establish the ApoE-KO mouse as an aging model. For example, possible bone density loss in the ApoE-KO mouse has not been determined yet. Other areas that require attention include the role of ApoE in cancer, rheumatoid arthritis and macular degeneration. An extensive survey of the ApoE-KO mouse organs as it ages should be performed to determine other aging phenotypes. In conclusion, the ApoE-KO model of aging exhibits many of the features that are associated with accelerated aging and should be considered as a useful tool for aging studies in the future. Acknowledgements This work was funded in part through Grants from the Canadian Institutes for Health Research (CIHR) (D.J.G.) Heart and Stroke Foundation (H&SF) (D.J.G.), and the Michael Smith Foundation for Health Research (MSFHR) (D.J.G.). L.S.A. is a recipient of a CIHR Canada Graduate Scholarships Master’s Award. R.P.C. is a recipient of a H&SF Doctoral Research Award and a British Columbia Innovation Council Scholarship. D.J.G. holds a Canada Research Chair in Cardiovascular Biochemistry and is a MSFHR Scholar. References Arking, D.E., Krebsova, A., Macek Sr., M., Macek Jr., M., Arking, A., Mian, I.S., Fried, L., Hamosh, A., Dey, S., McIntosh, I., Dietz, H.C., 2002. Association of human aging with a functional variant of klotho. Proc. Natl. Acad. Sci. USA 99, 856– 861. Asada, T., Kariya, T., Yamagata, Z., Kinoshita, T., Asaka, A., 1996a. Apolipoprotein E allele in centenarians. Neurology 46, 1484. Asada, T., Yamagata, Z., Kinoshita, T., Kinoshita, A., Kariya, T., Asaka, A., Kakuma, T., 1996b. Prevalence of dementia and distribution of ApoE alleles in Japanese centenarians: an almost-complete survey in Yamanashi Prefecture, Japan. J. Am. Geriatr. Soc. 44, 151–155. Bang, O.Y., Kwak, Y.T., Joo, I.S., Huh, K., 2003. Important link between dementia subtype and apolipoprotein E: a meta-analysis. Yonsei Med. J. 44, 401–413. Barrow, C.J., Yasuda, A., Kenny, P.T., Zagorski, M.G., 1992. Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer’s disease. Analysis of circular dichroism spectra. J. Mol. Biol. 225, 1075–1093. Bennet, A.M., Di Angelantonio, E., Ye, Z., Wensley, F., Dahlin, A., Ahlbom, A., Keavney, B., Collins, R., Wiman, B., de Faire, U., Danesh, J., 2007. Association of apolipoprotein E genotypes with lipid levels and coronary risk. J. Am. Med. Assoc. 298, 1300–1311. Bertram, L., McQueen, M.B., Mullin, K., Blacker, D., Tanzi, R.E., 2007. Systematic meta-analyses of Alzheimer disease genetic association studies: the AlzGene database. Nat. Genet. 39, 17–23. Blasco, M.A., Lee, H.W., Hande, M.P., Samper, E., Lansdorp, P.M., DePinho, R.A., Greider, C.W., 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34. Blasco, M.A., 2005. Mice with bad ends: mouse models for the study of telomeres and telomerase in cancer and aging. EMBO J. 24, 1095–1103. Breitner, J.C., Wyse, B.W., Anthony, J.C., Welsh-Bohmer, K.A., Steffens, D.C., Norton, M.C., Tschanz, J.T., Plassman, B.L., Meyer, M.R., Skoog, I., Khachaturian, A., 1999. APOE-epsilon4 count predicts age when prevalence of AD increases, then declines: the Cache County Study. Neurology 53, 321–331. Brosh Jr., R.M., Bohr, V.A., 2007. Human premature aging, DNA repair and RecQ helicases. Nucleic Acids Res. 35, 7527–7544. Brown, M.S., Goldstein, J.L., 1986. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47. Burke, B., Stewart, C.L., 2002. Life at the edge: the nuclear envelope and human disease. Nat. Rev. Mol. Cell Biol. 3, 575–585.

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