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5. The effect of APOE genotype on brain levels of oxysterols in young and old human APOE ε2, ε3 and ε4 knock-in mice

NIH-PA Author Manuscript NIH Public Access

NIH-PA Author Manuscript Author Manuscript

NIH-PA Author Manuscript Neuroscience. Author manuscript; available in PMC 2014 April 18.
Published in final edited form as:

Neuroscience. 2010 August 11; 169(1): 109–115. doi:10.1016/j.neuroscience.2010.04.026.

The effect of APOE genotype on brain levels of oxysterols in
young and old human APOE ε2, ε3 and ε4 knock-in mice

A.M. Jennera,#, W.L.F. Limb,e,#, M.P.E. Nga, M.R. Wenka, G. Shuia, M.J. Sharmanb,c,d,e, SE
Gandyf, and R.N. Martinsb,c,d,e,*
aNeurobiology and Ageing Research Program, Yong Loo Lin School of Medicine, 8 Medical Drive,
Singapore 117587

bSchool of Psychiatry and Clinical Neurosciences, The University of Western Australia, Nedlands,
6009, Australia

cCentre of Excellence in Alzheimer’s Disease Research and Care, Edith Cowan University, 270
Joondalup Drive, Joondalup, 6027, Australia

dSchool of Exercise, Biomedical and Health Sciences, Edith Cowan University, 270 Joondalup
Drive, Joondalup, 6027, Australia

eMcCusker Foundation for Alzheimer’s Disease Research Inc., 184 Hampden Road, Nedlands,
6009, Australia

fMt Sinai School of Medicine, Mount Sinai, New York, NY, USA

Abstract

Despite apolipoprotein E’s important role in cholesterol transport and metabolism in the brain as
well as its influence on Alzheimer’s disease, the impact of the human APOE genotype on
cholesterol metabolism in brain has not been fully examined. This study was carried out to
investigate APOE genotype effects on oxysterols measured. In this study the measurement of
cholesterol and several oxysterols in the brains of human APOE ε2, ε3 and ε4 knock-in mice at 8
weeks and 1 year of age using gas chromatography mass spectrometry (GC-MS) demonstrated no
APOE genotype or age effect on total brain cholesterol and the oxysterol 24-hydroxycholesterol.
The level of 27-hydroxycholesterol was elevated in 1 year old animals for all APOE genotypes.
Interestingly, lathosterol an indicator of cholesterol synthesis was significantly reduced in the 1
year old animals for all APOE genotypes. APOE ε4 expressing mice exhibited statistically lower
levels of lathosterol compared to APOE ε2 in both the young and old mice. Oxidized cholesterol
metabolites were significantly lower in APOE ε2 mice compared to other genotypes at 8 weeks
old. Although minimal differences were observed between APOE E3 and E4 KI mice, these

© 2010 IBRO. Published by Elsevier Ltd. All rights reserved.
*Correspondence to: Prof Ralph Martins, School of Exercise, Biomedical and Health Sciences, Edith Cowan University, 270
Joondalup Drive, Joondalup, Western Australia 6027, Australia, Ph: +61863045456, r.martins@ecu.edu.au.
#These authors contributed equally to the study.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
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Jenner et al. Page 2

findings indicate that there are some clear APOE genotype specific effects on brain cholesterol
synthesis and associated metabolic pathways, particularly in APOE ε2 KI mice.

NIH-PA Author Manuscript Keywords

NIH-PA Author Manuscript Oxysterols; APOE KI mice; Alzheimer’s disease; 24-hydroxycholesterol; 27-hydroxycholesterol

NIH-PA Author Manuscript Apolipoprotein E (apoE) functions as an important carrier protein and plays a prominent role
in the transport and metabolism of cholesterol, triacylglycerols (TAG), and phospholipids
among various cells of the body (Lin et al., 1986, Mahley, 1988). It exists in three major
isoforms: apoE2, apoE3 and apoE4, with frequencies of 7-8%, 77-78% and 14-15%
respectively in the general population (Cedazo-Minguez and Cowburn, 2001), which result
from the expression of the ε2, ε3 and ε4 alleles of the APOE gene (Zannis et al., 1981).
APOE has been shown to be the major apolipoprotein in the central nervous system, where it
plays a key role in neurobiology, and is also an important mediator of cholesterol and lipid
transport in the brain (Adibhatla and Hatcher, 2008).The ε4 allele of APOE is recognised
universally as the major genetic risk factor for Alzheimer’s disease (AD). APOE ε4 in
particular is strongly associated with familial and sporadic late-onset AD (LOAD)
(Strittmatter and Roses, 1996).

The brain is the most cholesterol-rich organ in the body (~25% of total amount) and must
synthesize its own cholesterol de novo, since cholesterol cannot pass the blood brain barrier
(BBB) (Björkhem et al., 1998, Björkhem et al., 2006). Consequently, regulation of
cholesterol synthesis and metabolism in brain is largely independent of changes in the
periphery (Björkhem et al., 2006). However, there is increasing evidence that changes in
cholesterol metabolism plays a role in the pathogenesis of many neurodegenerative
disorders, including AD and that brain cholesterol metabolites may act as clinically relevant
biomarkers (Björkhem et al., 2006).

Although cholesterol itself is unable to enter the brain from the circulation in significant
amounts, side-chain oxidised cholesterol metabolites also known as oxysterols are able to
freely pass the BBB (Björkhem et al., 2006, Vaja and Schipper, 2007). Oxysterols including,
7β-hydroxycholesterol and 7-ketocholesterol, are formed as a result of oxidative damage to
cholesterol (Figure 2). They can be measured in trace amounts in human tissue (Iuliano et
al., 2003, Lee et al., 2008) and are elevated in diseases that involve oxidative stress
including AD. Since there is considerable evidence for oxidative damage during
neurodegeneration and cholesterol comprises a major lipid target in brain (~2% w/w) these
oxysterols have the potential of being important biomarkers of oxidative damage in this
tissue.

In spite of apoE’s important role in cholesterol transport and the growing evidence that
altered cholesterol metabolism in the brain contributes to the pathogenesis of AD, the impact
of different APOE genotype on brain cholesterol metabolism is poorly understood. To date,
investigations performed on oxysterols mainly concentrated on 24-OH Chol and 27-OH
Chol. It is also interesting to note that despite the critical role APOE plays in the
pathogenesis of AD, no oxysterols have yet been studied in APOE knock-in mice. In this

Neuroscience. Author manuscript; available in PMC 2014 April 18.

Jenner et al. Page 3

NIH-PA Author Manuscript study, we have used gas chromatography mass spectrometry (GC-MS) with heavy isotope
dilution to reliably and sensitively measure the level of cholesterol precursors, metabolites,
plus oxidative damage products in brains from APOE knock-in mice expressing different
human apoE isoforms.

Experimental Procedures

Animals

NIH-PA Author Manuscript Male APOE knock-in (KI) mice, homozygous for human APOE ε2, ε3 and ε4 were
originally obtained from Taconic (Germantown, NY, USA) and a colony derived and
maintained at the Animal Resources Centre (ARC, Perth, Western Australia). These mice
have been described previously (Piedrahita et al., 1992, Sullivan et al., 1997, Sullivan et al.,
1998, Knouff et al., 1999). The mice were maintained on standard rodent chow (rat and
mouse cubes, Specialty Feeds, Glen Forrest, WA, Australia) and kept until either 8 weeks or
1 year old. Food and water were accessible ad libitum. This study was conducted in
accordance with the Australian code of practice for the care and use of animals for scientific
purposes [National Health and medical research Council (NHMRC) 2004], and the
experimental protocols were approved by the University of Western Australia Animal Ethics
Committee.

Chemicals

Oxysterol standards 7β– hydrocholesterol, and 7-keto cholesterol were obtained from Sigma
(St. Louis, MO, USA), lathosterol, 27-OH Chol and β-sitosterol were from Steraloids,
(Newport, RI, USA ; 7β– hydroxycholesterol-d7, β-sitosterol-d7, lathosterol-d4 and 7-

ketocholesterol-d7 were purchased from CDN Isotopes (Quebec, Canada); 27-OH Chol-d5,

24-OH Chol and 24-OH Chol-d7 were from Medical Isotopes, Inc (Pelham, NH, USA). All

standards obtained were of the highest purity (>95%).

NIH-PA Author Manuscript Sample preparation

Total brain homogenate (approximately 30mg of brain tissue in 0.5 ml PBS at 4°C) obtained
from respective APOE ε2/ε3/ε4 KI mice was added to 2.5ml Folch solution
(chloroform:methanol 1:3 containing 0.005% BHT) (4°C) in a 10ml centrifuge tube and
incubated for 15minutes with gentle mixing, before centrifugation at 1,000g for 10minutes.
The lower organic layer was collected in a clean glass container and after addition of
internal standards (80ng of 7β– hydrocholesterol-d7, 120ng of 27-OH-Chol-d5, 400ng of
lathosterol-d4, 80ng of 7-ketocholesterol-d7, 400ng of 24-OH-Chol-d7 and 400ng of β-
sitosterol-d7) was evaporated under a stream of nitrogen. Hydrolysis was performed to
measure the total (free & esterified) forms of oxidized lipids. 1ml of PBS and 1ml of 1M
potassium hydroxide (prepared in 100% methanol) was added to the dried lipid extract.
Hydrolysis was performed at 23°C for 15 h with gentle agitation. 1.35ml of 80mM formic
acid (pH 4.5) was added to the tube and then neutralized with 0.25ml of 2.5M HCl. The
mixture was filtered with 25mm nylon disc (60 μm pore size).

Neuroscience. Author manuscript; available in PMC 2014 April 18.

Jenner et al. Page 4

NIH-PA Author Manuscript Extraction and derivatization of oxysterols

Mixed anion-exchange SPE columns were preconditioned with 2ml methanol; followed by
2ml of 20mM formic acid pH 4.5. The hydrolyzed samples were loaded onto the columns.
These columns were washed with 2ml of 2% ammonium hydroxide and then with 2ml of
40% methanol/formic acid pH 4.5. After the wash, 2ml of hexane and then 2ml of ethyl
acetate/hexane (30:70) was added to elute cholesterol and oxysterols. The eluted samples
were evaporated under a stream of nitrogen. The aliquots were derivatised with 25μl
acetonitrile and 25μl BSTFA and 1% TMCS for an hour at room temperature. The samples
were evaporated under a stream of nitrogen and later reconstituted in 35μl of undecane prior
to injection into the GC-MS.

NIH-PA Author Manuscript Analysis of oxysterols by gas chromatography-mass spectrometry

For oxysterols measurement, the derivatized samples were analysed using an Agilent 5975
inert XL mass selective detector. Helium was used as the carrier gas at a flow rate of 0.8ml/
min, derivatised samples (1μl) were injected splitless into the GC injection port (280°C).
Column temperature was increased from 160°C to 300°C at 40°C/min after 1 min at 160°C
then held at 300°C for 6 mins. Selective ion monitoring was performed using electron
ionization mode at 70eV (with ion source maintained at 230°C and the quadrupole at 150°C)
to monitor one target ion and 2 qualifier ions selected from each compound’s mass spectrum
to optimize sensitivity and specificity. Quantitation of oxysterols was achieved by relating
its peak area of target ion to its corresponding internal standard peak. Careful minimisations
of artifactual oxidation to cholesterol during the analytical procedure, anaerobic and lower
temperature hydrolysis conditions that have been reported to minimize oxidation were used
(Iuliano et al., 2003).

Statistical analyses

Means and standard deviations were calculated for all variables using conventional methods.
A Kruskal-Wallis ANOVA by ranks for nonparametric data was used to evaluate significant
differences between the groups of animals. All data were analyzed using Matlab version 6.5,
Massachusetts, United States of America.

Results

NIH-PA Author Manuscript All sterols except 25- OH Chol were significantly detected in all brain samples. Significant

age-associated increases in 27-OH Chol levels were observed for all three APOE genotypes.
It was noted that the increase in APOE ε2 KI mice was significantly higher (P<0.01) in 1
year old APOE ε2 KI mice when compared to 8 weeks old APOE ε2 KI mice (Figure 1a).
Significant increases in 27-OH Chol were also observed in APOE ε3 (P<0.05) and APOE ε4

(P<0.05) KI mice when compared to their respective 8 weeks old APOE KI matched mice
(Figure 1a). There was a stronger statistical age-related difference in APOE ε2 KI mice as

compared to the other genotypes but this is predominantly due to the lower variance in the
APOE ε2 KI mice. No significant differences were noted in the level of 24-OH Chol across

the various APOE KI mice (Figure 1b). There was also no significant difference with age in

the respective APOE KI mice for 24-OH Chol levels.

Neuroscience. Author manuscript; available in PMC 2014 April 18.

Jenner et al. Page 5

NIH-PA Author Manuscript Significant increases in 7-β hydroxycholesterol, β-sitosterol and 7-keto cholesterol (P<0.01)
were observed in 1year old APOE ε2 KI mice as compared to 8 weeks old APOE ε2 KI
NIH-PA Author Manuscript mice. However, the level of lathosterol (P<0.01) significantly decreased with age in APOE
ε2 KI mice. A significant age-dependent increase in β-sitosterol (P<0.05) level was
observed in APOE ε3 KI mice. In aged APOE ε4 KI mice, significant increases in 27-OH
Chol (P<0.05) levels were observed. Lathosterol level (P<0.01) significantly declined with
age in APOE ε4 KI mice.

There were significant differences in 7-β hydroxycholesterol (P<0.01) and 7-keto
cholesterol (P<0.01) when 8 weeks old APOE ε2 KI mice were compared to other age-
matched APOE KI mice (Table 1A). However, lathosterol level was only significantly
higher in 8 weeks old APOE ε2 KI mice (P<0.05) compared to age-matched APOE ε4 KI
mice (Table 1A). The level of lathosterol was significantly decreased in 1 year old APOE ε4
KI mice as compared to age-matched APOE ε2 KI mice (Table 1B). Although significant
age dependent increases in 27-OH Chol levels were observed in mice expressing either of

the three apoE isoforms (Figure 1), no significant difference was observed between APOE

genotypes in these aged matched APOE KI mice (Table 1A & B). The level of lathosterol

declined with age in mice expressing either, apoE2, apoE3 or apoE4 isoforms. Levels of
lathosterol were highest in APOE ε2 KI mice at both ages. No differences in the lathosterol
levels were observed between the APOE ε3 KI and APOE ε4 KI mice. There were no
significant differences between APOE ε3 and APOE ε4 KI mice under the current
experimental conditions used in this study.

NIH-PA Author Manuscript Discussion

This is the first study looking at the influence of APOE genotypes on oxysterol levels in the
brains of APOE KI mice. Oxysterols, in particular 24-OH Chol, 25-OH Chol and 27-OH
Chol are able to freely pass through the BBB. 24-OH Chol is the major neuronal cholesterol
metabolite catalysed by CYP46A1 (mainly expressed in brain) (Björkhem et al., 1998,
Björkhem et al., 2006) (Figure 2). The conversion of cholesterol into 24-OH Chol in the
brain is responsible for maintaining cholesterol homeostasis and the removal of excess
cholesterol from the brain.

No significant differences were noted in this study for 24-OH Chol, comparing either age-
effect or APOE genotype. This may be due to the lack of any AD-like pathology in these
APOE KI mice under the experimental conditions employed in this study. Levels of 24-OH
Chol measured in AD patients are correlated to brain atrophy as measured by magnetic
resonance imaging (Leoni, 2005) and this cholesterol metabolite has been reported to be
elevated in the cerebrospinal fluid (CSF) and serum of AD patients (Lütjohann et al., 2000,
Papassotiropoulos et al., 2002). The lack of significant differences in total brain 24-OH Chol
between the different animal groups in our study may indicate that there is no APOE
genotype specific effects in KI mice on this cholesterol metabolic pathway, including any
effect on CYP46A1 activity where there is an absence of environmental challenges such as a
high-fat/high-cholesterol diet. Thus, the levels of 24-OH Chol in our study were independent
of APOE genotype under the conditions employed. It is possible that potential changes in
some specific brain regions were masked by total brain homogenization. Others have

Neuroscience. Author manuscript; available in PMC 2014 April 18.

Jenner et al. Page 6

NIH-PA Author Manuscript previously reported changes of 24-OH Chol in brain regions (Heverin et al., 2004), plasma
(Lütjohann et al., 2000), and CSF (Papassotiropoulos et al., 2002). However, there was a
NIH-PA Author Manuscript lack of consistent findings between reports. Lutjohann et al., 2000 concluded in their study
that elevated plasma 24-OH Chol levels in AD patients were not significantly influenced by
NIH-PA Author Manuscript APOE genotype, whilst Papassotiropoulos et al., (Papassotiropoulos et al., 2002) reported a
positive correlation between 24-OH Chol CSF levels and APOE ε4 alleles in 32 AD
patients. However, it could be argued that these different findings may be explained by 24-
OH Chol levels varying depending on the stage of AD progression. The levels of 24-OH
Chol increase in the initial stages of AD as excess cholesterol is released from cholesterol-
rich cell membranes that are destroyed during the process of neurodegeneration (Lütjohann
et al., 2000, Papassotiropoulos et al., 2002), but as more neurons expressing 24S-
hydroxylase die i.e. in advanced AD, the conversion to 24-OH Chol and efflux from the
brain is less efficient (Papassotiropoulos et al., 2002). While the effects of APOE genotype
are unclear, Abildayeva et al., 2006 demonstrated that 24-OH Chol induces APOE gene
expression and increases apoE protein levels in primary rat and mouse astrocytes in a dose
dependent manner. Significant correlation between apoE levels and 24-OH Chol levels were
observed in the cerebrospinal fluid (CSF) of mild cognitive impairment (MCI) and AD
patients (Shafaati et al., 2007). Thus apoE expression is clearly influenced by 24-OH Chol
levels irrespective of APOE genotype.

27-OH Chol is formed in multiple organs by sterol hydroxylase (CYP27A1) and can influx
across the BBB into the brain from peripheral circulation (Figure 2). We observed
significant age associated increases in 27-OH Chol levels in all three APOE genoytpes,
which may reflect an increased uptake of 27-OH Chol into the brain of 1 yr old mice, or a
reduction in its metabolism. Leoni et al., (2002) demonstrated significant accumulation of
27-OH Chol in the brain of AD patients. The brain is known to have low levels of CYP27A1
consistent with negligible endogenous 27-OH Chol production as opposed to high levels of
24-OH Chol in this organ (Lütjohann et al., 1996). Consistent with our study, significant
influx of 27-OH Chol from peripheral circulation into the brain had also been observed in
humans (Heverin et al., 2005) and mice (Heverin et al., 2004). In the brain, 27-OH Chol has
been demonstrated to be metabolized by a combination of 3 different enzymes in a novel
metabolic route forming 7α-hydroxy-3-oxo-4-cholestenoic acid and subsequently efficiently
eliminated from the human brain (Meaney et al., 2007). This metabolic route likely explains
why low 27-OH Chol levels in brain were detected in our study, in common with previous
analytical studies of both mouse and human brain (Heverin et al., 2004, Heverin et al.,
2005). Consequently this oxysterol has been hypothesized to provide a link between
peripheral hypercholesterolemia and AD, since there is good correlation between plasma
levels of cholesterol and 27-OH Chol (Björkhem et al., 2006). There was no association
between APOE genotype and brain cholesterol levels in this study.

Lathosterol, a precursor of cholesterol (Figure 2) and established biomarker of de novo
synthesis of cholesterol (Heverin et al., 2004, Lütjohann et al., 2004) was significantly lower
in APOE ε4 KI mice brain, compared to APOE ε2 mice at both 8 wk and 1 yr old. Such an
effect of APOE allele on brain lathosterol has not been reported previously and it is not
known by what mechanisms apoE may exert this influence. APOE ε4 may down-regulate or

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Jenner et al. Page 7

NIH-PA Author Manuscript APOE ε2 up-regulate the lathosterol pathway in various cell types, possibly by differences
in the type and quantity of oxysterols that are transported by these different apoE isoforms.
NIH-PA Author Manuscript Different oxysterols are known to have a variety of influences on cholesterol synthesis that
is usually dependent on their specific binding to receptors that regulate the transcription of
NIH-PA Author Manuscript cholesterol synthetic enzymes (Vaja and Schipper, 2007).

Since the level of brain cholesterol was not significantly different between the APOE KI
mice, we can conclude that the effect on the lathosterol pathway does not limit the rate of
cholesterol synthesis and/or that alternative synthetic pathways leading to cholesterol
synthesis are able to compensate, for example the parallel synthetic pathway via
desmosterol. In mice Lutojohann et al., (2002) reported that brain lathosterol after 3 months
of age increase whilst desmosterol decreases, but another study in mice found an age
dependent decline between 2 and 10 months of age in brain desmosterol and lathosterol
(Valenza et al., 2007). Similar to the latter study, the levels of lathosterol in our APOE KI
study exhibited a decrease with age across all three APOE genotypes, although, this decline
was only significant in APOE ε2 and APOE ε4 KI mice.

Examination of cholesterol synthesis in human hippocampi revealed an age dependent
decrease in lathosterol, but levels of desmosterol and cholesterol were unaffected (Thelen et
al., 2006). Further analytical investigations are required to confirm the potential of
lathosterol as a possible differentiation biomarker between apoE2 and apoE4 isoforms
during aging in animals and humans. Based on our data that APOE ε4 KI mice continued to
have significantly lower brain lathosterol at older ages, coupled with strong evidence for an
age dependent decline in brain lathosterol. We therefore speculate that the association of AD
with aging and apoE4 may be related to earlier critical decreases in lathosterol levels in AD
brains. Further studies will determine whether this effect on lathosterol is selectively
increased in APOE ε4 KI mice in response to AD-associated environmental risk factors such
as a high fat diet.

Significant age related increases in brain levels of β-sitosterol were also detected in the
APOE KI mice in this study. This dietary derived phytosterol is not synthesized in brain but
transported in plasma and may pass the BBB more readily during aging in APOE KI mice.
In particular APOE ε2 KI mice had larger age dependent increases, although this was
predominantly due to lower levels of sterols at a younger age compared to APOE ε3 and
APOE ε4 KI mice. Further investigation is required to determine if β-sitosterol may
contribute to the protective role apoE2 is thought to play in the pathogenesis of AD.
Previous studies also detected a significant increase in β-sitosterol in APP23 transgenic mice
(β-amyloid precursor protein carrying the Swedish mutation), but not wild-type mice
(Lütjohann et al., 2002).

Due to the fact that APOE ε4 is a major risk factor as well as overwhelming evidence
supporting the role of oxidative stress and accumulation of damage in AD amongst the
APOE KI mice, one specific aim of our analysis was to measure biomarkers of oxidative
damage to cholesterol (7-ketocholesterol and 7-β hydroxycholesterol). Basal levels of each
oxysterols in brain measured in this study, representing approximately 2-5/104 molecules of
cholesterol have not been reported previously and were higher than our previous

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Jenner et al. Page 8

NIH-PA Author Manuscript measurements in plasma or other tissues of mice (unreported data not shown). This is likely
due to the fact that compared to other tissues the brain has the highest oxygen consumption
NIH-PA Author Manuscript and level of mitochondrial and oxidative metabolism that generates increased levels of
reactive oxygen species (ROS). Also, higher cholesterol levels in brain represent an
NIH-PA Author Manuscript increased target for attack by reactive oxygen species. Our study shows that oxysterol
biomarkers at 8 weeks old were reduced in apoE2 KI mice compared to apoE4 KI mice and
apoE3 KI mice. A greater significant age dependent increase in cholesterol oxidative
damage was observed in apoE2 KI mice than apoE3 KI mice and apoE4 KI mice. Age
dependent increases in oxidative stress and damage to brain have been frequently reported in
animals and humans (Barnham et al., 2004) and have contributed to the oxidative damage
hypothesis of ageing, particularly age dependent neurodegeneration. APOE isoforms have
also been demonstrated to have effects on periphery and vasculature. The low density
lipoproteins (LDL) concentration and mass are highest in the APOE ε4 KI mice
homozygotes compared to the other genotypes, while their concentration is lowest in APOE
ε2 KI mice (Murdoch et al., 2007). Similarly, the presence of APOE ε2 allele has a lower
incidence of vasoconstriction compared to that of APOE ε4 allele (Paris et al., 1998).
However, it is unclear exactly how such mechanisms may influence brain cholesterol and
oxysterols since evidence illustrates that blood lipoproteins are unable to cross the blood
brain barrier.

To summarise, our data illustrates that APOE genotype, particularly APOE ε2, does have
significant effects on cholesterol synthesis and metabolism as well as cholesterol oxidative
damage, which is also influenced by ageing. In our study, APOE genotype does not
influence total brain cholesterol levels. In addition we observed minimal differences
between APOE ε3 and ε4, in contrast to APOE ε4 known elevated risk in AD. Our findings
are similar to previous work (Bandaru et al. 2007) suggesting that APOE ε4 is not
associated with disturbances in brain lipids without an underlying neurological disorder,
such as Alzheimer’s disease. Although the APOE gene is the strongest genetic factor
associated with AD, APOE KI mice are not exactly an AD model as they do not accumulate
beta-amyloid plaques. However we believe that other physiological stress factors such as a
high fat/high cholesterol diet and/or hormonal depletion may also be required (in addition to
age) in order to observe significant differences in cholesterol and oxysterols between APOE
ε3 and ε4 that influence AD development. The mechanisms regulating the brain cholesterol
metabolome and their influence on development of AD have yet to be fully elucidated.
Interestingly, the accumulation of 27-OH Chol in the brain of ageing mice from this study
was also found to be the most significant change in the oxysterol profile in the brains of AD
patients (Leoni et al., 2002). In this study, no significant differences were observed in 24-
OH Chol. However, there are contradicting reports in the literature of 24-OH Chol in
humans. Lutjohann et al., (2000) concluded in their study that elevated plasma 24-OH Chol
levels in AD patients were not significantly influenced by APOE genotype, whilst
Papassotiropoulos et al., (2002) reported a positive correlation between 24-OH Chol CSF
levels and APOE ε4 alleles in 32 AD patients. The effect of ageing and apoE on cholesterol
synthesis and metabolism in the brain, reinforce previous studies highlighting that
disturbance of cholesterol turnover is associated with increased risk of AD. Changes in
cholesterol intermediate levels may be clinically relevant and potentially serve as diagnostic

Neuroscience. Author manuscript; available in PMC 2014 April 18.

Jenner et al. Page 9

NIH-PA Author Manuscript markers. Further studies are necessary to decipher the exact importance and functional
contribution of apoE and cholesterol intermediates/metabolites that may lead to the
development of neurodegeneration.

NIH-PA Author Manuscript Acknowledgments

NIH-PA Author Manuscript Wei Ling Florence Lim is a recipient of a Scholarship for International Research Fees and University International
Stipend from University of Western Australia. This work was supported by a program grant from the NIH (2 P01
AG010491-10A2) to RNM and SEG. Andrew Jenner is grateful to the NMRC Singapore for support through the
grant (R-183-000-155-214/133). MRW, RNM and MJS were funded by the National Medical Research Council
(R-183-000-224-213). MRW was also funded by the Singapore National Research Foundation under CRP Award
No. 2007-04, the Biomedical Research Council of Singapore (R-183-000-211-305).

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Abbreviations

NIH-PA Author Manuscript 24-OH Chol 24S-hydroxycholesterol
27-OH Chol 27-hydroxycholesterol
25-OH Chol 25-hydroxycholesterol
APOE apolipoprotein E
AD Alzheimer’s Disease
HMG-CoAR 3-hydroxy-3-methyl-CoA reductase
BBB blood-brain barrier
Aβ amyloid beta
GC-MS gas chromatography mass spectrometry
BHT butylated hydrotoluene
SPE solid phase extraction
BSTFA N,O-Bis(trimethylsilyl)trifluoroacetamide
TMCS trimethylchlorosilane
LOAD Late-onset Alzheimer’s disease
ROS reactive oxygen species

NIH-PA Author Manuscript

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Jenner et al. Page 12

NIH-PA Author Manuscript

NIH-PA Author Manuscript Figure 1.
Comparison of A) 27-OH cholesterol levels, and B) 24-OH cholesterol levels in APOE ε2,
NIH-PA Author Manuscript ε3 and ε4 KI mice aged 8 weeks old and 1 year old. Values are calculated as means ± SEM
of 5 animals. * P<0.05 8weeks old vs. 1 year old, ** P<0.01 8 weeks old vs. 1 year old.

Neuroscience. Author manuscript; available in PMC 2014 April 18.

Jenner et al. Page 13

NIH-PA Author Manuscript

NIH-PA Author Manuscript Figure 2.
Oxysterol Synthesis Pathway

NIH-PA Author Manuscript

Neuroscience. Author manuscript; available in PMC 2014 April 18.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Table 1 Jenner et al.
Comparison of oxysterol levels in A) 8 week old and B) 1 year old APOE ε2, ε3 and ε4 KI mice. Values are calculated as means ± SEM of 5 animals.

APOE ε2 8 weeks (A) APOE ε4 APOE ε2 1 year (B) APOE ε4
APOE ε3 APOE ε3

lathosterol 21.82 ± 1.39 o 18.12 ± 1.66 17.19 ± 0.77 15.66 ± 0.26 oo 13.80 ± 0.99 12.60 ± 0.92

7-b OH-cholesterol 0.67 ± 0.10 ** oo 2.61 ± 0.35 2.67 ± 0.32 2.71 ± 0.18 2.87 ± 0.37 3.58 ± 0.51

Neuroscience. Author manuscript; available in PMC 2014 April 18. b-sitosterol 3.64 ± 0.29 3.57 ±0.40 4.14 ± 0.34 5.56 ±0.13 5.24 ± 0.31 5.57 ± 0.60

24-OH cholesterol 18.53 ±0.71 18.70 ± 0.97 18.24 ± 1.03 19.50 ± 1.37 18.74 ± 1.11 17.86 ± 0.84

7-keto cholesterol 2.60 ±0.51 ** oo 8.33 ±1.01 7.34 ± 0.58 8.70 ±1.29 9.20 ± 1.21 11.56 ± 1.83

27-OH cholesterol 0.56 ± 0.03 0.52 ± 0.04 0.55 ± 0.03 0.66 ± 0.01 0.63 ±0.03 0.69 ± 0.03

*P<0.05 APOE ε2 KI mice vs. APOE ε3 KI mice,

** APOE ε2 KI mice vs. APOE ε3KI mice,
P<0.01

o APOE ε2 KI mice vs. APOE ε4 KI mice,
P<0.05

ooP<0.01 APOE ε2 KI mice vs. APOE ε4KI mice. No significant differences were detected in APOE ε3 KI mice vs. APOE ε4 KI mice at 8 weeks of age.

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