Mutant presenilin 2 causes abnormality in the brain lipid profile in the development of Alzheimer's disease.

Abstract

Mutation in the presenilin 2 (PS2mt) is known to be one of factors involved in the development of Alzheimer's disease (AD). It was recently revealed that an abnormality of lipid metabolism is a phenomenon occurring in AD. Therefore, the aim of this study was to investigate the potential relationship between the mutation of PS2 and alterations of the lipid profile within the brain. The results showed there increases in the levels of cholesterol, low density lipoprotein and triglyceride, but a decrease in the level of high density lipoprotein in brain tissues expressing mutant PS2. These findings indicated that PS2mt is involved in the abnormalities of the lipid profile, which could cause or result in the development of AD.

Full text

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Arch Pharm Res Vol 29, No 10, 884-889, 2006
884
http://apr.psk.or.kr
Mutant Presenilin 2 Causes Abnormality in the Brain Lipid
Profile in the Development of Alzheimer’s Disease
Hong Nga Nguyen, Dong Ju Son, Jae Woong Lee, Dae Youn Hwang1, Young Kyu Kim1, Jeong Sik Cho1,
Ung Soo Lee2, Hwan Soo Yoo, Dong Cheul Moon, Ki Wan Oh, and Jin Tae Hong
College of Pharmacy and CBITRC, Chungbuk National University, Cheongju 361-763, Korea, 1National Institute of
Toxicological Research, Korea Food and Drug Administration, Seoul 122-704, Korea, and 2Department of Food
and Biotechnology, Chungju National University, Chungju 380-702, Korea
(Received July 19, 2006)
Mutation in the presenilin 2 (PS2mt) is known to be one of factors involved in the development
of Alzheimer’s disease (AD). It was recently revealed that an abnormality of lipid metabolism is
a phenomenon occurring in AD. Therefore, the aim of this study was to investigate the poten-
tial relationship between the mutation of PS2 and alterations of the lipid profile within the brain.
The results showed there increases in the levels of cholesterol, low density lipoprotein and trig-
lyceride, but a decrease in the level of high density lipoprotein in brain tissues expressing
mutant PS2. These findings indicated that PS2mt is involved in the abnormalities of the lipid
profile, which could cause or result in the development of AD.
Key words: Transgenic mice, Presenilin 2, Alzheimer’s disease, Cholesterol, Triglyceride,
HDL cholesterol, LDL cholesterol
INTRODUCTION
Alzheimer’s disease (AD) is a neurodegenerative
disorder characterized by the progressive deterioration of
cognition and memory. Recent studies have revealed
abnormalities in lipid metabolism in brain ageing and the
pathogenesis of late onset AD (Wellington et al., 2004;
Cutler et al., 2004; Sawamura et al., 2000). An elevated
level of plasma cholesterol is supposed to be associated
with an increased risk for the development of AD. The
major apolipoprotein (ApoE) at levels of approximately 3-5
µg/mL in the cerebrospinal fluid has been shown to affect
late onset AD (Mahley, 1998). The synthesis of ApoE is
induced in response to central nervous system injury or
disease, where it coordinates the mobilization and redistri-
bution of cholesterol in the repair and maintenance of
neuronal membranes (Ignatius et al., 1986). Furthermore,
intracellular cholesterol regulates the generation of Aβ
peptides from amyloid precursor protein, which accumulate
as amyloid plaques in the brains and cerebral blood
vessels of AD patients. It has also been suggested that
ApoE is a key mediator of Aβ metabolism (Shie et al.,
2002; Wahrle et al., 2002; Burns et al., 2003; Refolo et al.,
2001; Buxbaum et al., 2001; Ehehalt et al., 2003). Moreover,
cholesterol-lowering drugs reduce the prevalence of AD
(Refolo, 2001; Jick et al., 2000; Fassbender et al., 2001).
Inhibition of cholesterol biosynthesis was found to reduce
the amyloid burden in guinea pig and murine models of
AD (Refolo et al., 2001; Fassbender et al., 2001). There
are conflicting reports on whether the levels of plasma
lipid or lipoprotein are altered in AD patients (Pappolla et
al., 2003; Tan et al., 2003). However, several pieces of
evidence indicate the possible roles of plasma cholesterol
in the clearance of Aβ peptides, which are thought to bind
to plasma lipoproteins after crossing the blood brain
barrier in the pathogenesis of AD (Koudinov et al., 2001;
Koudinov et al., 1998). These observations suggest that
intracellular lipid metabolism in the peripheral or central
nervous system may participate in the pathogenesis of
AD.
Conversely, the majority of familial AD is supposed to
be caused by mutations within the presenilin genes,
although the involved mechanism remains to be fully
understood (Mori et al., 2002; Deng et al., 1996; Janicki et
al., 1997; De Sarno et al., 2001). Neuronal cells expressing
Correspondence to: Jin Tae Hong, College of Pharmacy, Chung-
buk National University, 48, Gaeshin-dong, Heungduk-gu, Cheon-
gju, Chungbuk 361-763, Korea
Tel: 82-43-261-2813, Fax: 82-43-268-2732
E-mail: jinthong@chungbuk.ac.kr

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Mutant Presenilin 2 Causes Abnormality in the Brain Lipid Profile in the Development of Alzheimer’s Disease885
N141I mutant PS2 were revealed to significantly increase
apoptotic cell death compared to those expressing wild
PS2 or untransfected control (Mori et al., 2002; Deng et
al., 1996; Janicki et al., 1997). The lack of memory in PS2
transgenic mice, particularly in cases of mutant PS2
transgenic mice, also suggests the involvement of the
PS2 gene in the neuronal degeneration associated with
AD (Hwang et al., 2002; Lee et al., 2006).
Therefore, the aim of this study was to investigate the
possible relationship between the mutation of PS2 and the
alteration of the brain lipid profile, which may be accom-
panied with neuronal cell death in the development of AD.
MATERIALS AND METHODS
Mutant and wild PS2 transgenic mice
Transgenic mice expressing wild PS2 (PS2wt-Tg) and
mutant PS2 (PS2mt-Tg), described elsewhere (Hwang et
al., 2002), were used in this study. Transgenic and age-
matched control mice were handled in an accredited
Korea FDA animal facility in accordance with the AAALAC
International Animal Care policies. Mice were housed in
cages under a strict light cycle (light on at 06:00 and off at
18:00), and given a standard irradiated chow diet (Purina
Mills, St. Louis, MO) at libitum. The mice were maintained
in a specified pathogen-free state.
Determination of triglyceride
The brains of the mice were removed immediately after
exsanguinations, and frozen on dry ice. Chloroform-
methanol (1:1) extracts of the brains were dried under
nitrogen gas and re-extracted with chloroform–methanol
(2:1). The lower phase was evaporated, and the residue
extracted with isopropanol prior to analyses. Plasma was
separated by centrifugation at 1,500 rpm for 20 min at
4oC. The levels of triglycerides were measured with an
enzymatic colorimetric kit obtained from Roche for use
with the Cobas Mira Chemstation (Boehringer Mannheim
Corp., Germany). The triglycerides in the sample were
hydrolyzed to glycerol and fatty acids using lipoprotein
lipase. The glycerol was then phosphorylated to glycerol-
3-phosphate using glycerol kinase and subsequently
catalyzed with glycerol oxidase to form dihydroxyacetone
phosphate and hydrogen peroxide. The hydrogen peroxide
was then reacted in the presence of peroxidase to form a
chromogen, with the increases in the absorbance measured
at 405 nm being proportional to the triglycerides concen-
tration. The amount of triglyceride was express in units of
mg/dl.
Determination of total cholesterol
The total cholesterol was measured using an enzymatic
kit obtained from Roche for use with the Cobas Mira
Chemstation (Boehringer Mannheim Corp., Germany).
This method follows a two step approach. In the first step,
cholesterol is desterified by the action of cholesterol
esterase, and subsequently exposed to the action of
cholesterol oxidase. This second step was coupled to a
chromogen (color-forming compound), which can be
measured using a spectrophotometer, with the increases
in the absorbance due to the chromogen at 405 nm being
proportional to the cholesterol concentration in the sample.
The amount of total cholesterol was expressed in units of
mg/dl.
Determination of high density lipoprotein
The high density lipoprotein (HDL) was measured using
a HDL direct kit obtained from Roche for use with the
Cobas Mira Chemstation (Boehringer Mannheim Corp.,
Germany). After elimination of the cholesterol in non-HDL
lipoproteins, a second reaction mix was added and
incubated for 10 min at 37oC, where the HDL cholesterol
is selectively exposed to the action of cholesterol esterase
and cholesterol oxidase in a color forming reaction, which
can be monitored spectrophotometrically by measuring
the absorbance at 405 nm. The results were expressed in
units of mg/dl.
Determination of low density lipoprotein
Low density lipoprotein (LDL) was directly measured
with a LDL cholesterol kit obtained from Roche for use
with the Cobas Mira Chemstation (Boehringer Mannheim
Corp., Germany). After the elimination of solubilized non-
LDL lipoprotein particles, a second detergent specific for
the solubilization of the LDL fraction was added in the
presence of a chromogenic coupler for the detection of
LDL cholesterol. The enzyme reaction in the presence of
the coupler produces color proportional to the LDL
cholesterol concentration. The results were expressed in
units of mg/dl.
RESULTS
Effect of the PS2 mutation upon the total choles-
terol in PS2 transgenic mice brains
To study whether the mutation of presenilin 2 is involved
in the abnormality of lipid metabolism in AD, PS2 transgenic
mice were generated as an in vivo AD model. The results
showed that the cholesterol level in all the brains of the
PS2mt-Tg mice was higher than in the brains of non-Tg
and PS2wt-Tg mice at all the ages investigated (Fig. 1).
No change in the total cholesterol in the brain tissues
between 2 and 6 months of age (young mice) was
observed. The increases in the cholesterol levels in
PS2mt-Tg and PS2wt-Tg were clearer in the brains of
older. As shown in Fig. 1, the total cholesterol levels in

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886H. N. Nguyen et al.
non-Tg and PS2wt-Tg brains were about 94 mg/dl at 6
months of age, increase to approximately 96 mg/dl at 12
months and to 112±8 mg/dl (non-Tg) and 124±10 mg/dl
(PS2wt-Tg) at 18 months. Particularly, the minimum
cholesterol level (65.3±1.2 mg/dl) in non-Tg brains was
detected at 12 months (Fig. 1). Similar results were detected
in the brains of PS2mt-Tg mice. At 2 and 6 months of age,
the total cholesterol in PS2mt-Tg samples was about 107
mg/dl, but increase to 139±26 and 148±3.2 mg/dl at 12
and 18 months, respectively (Fig. 1). The total cholesterol
contents were always higher in PS2mt-Tg brains compared
to PS2wt-Tg (1.1 fold increase at 2 and 6 months, 1.4 fold
increase at 12 months and 1.2 fold increase at 18 months),
and especially higher than in non-Tg brains (1.1 fold in-
crease at 2 and 6 months, 2.1 fold increase at 12 months
and 1.3 fold increase at 18 months) (Fig. 1).
Decrease in HDL level in mutant PS2 transgenic
mice brains
The results showed that the level of HDL decreased in
the brains of PS2wt-Tg and PS2mt-Tg mice, particularly in
the PS2mt-Tg compared to non-Tg mice. The changes of
the levels of HDL in PS2wt-Tg, PS2mt-Tg or non-Tg
brains seemed to be independent of age. The levels of
HDL in non-Tg brains (about 60±6 mg/dl) did not signi-
ficantly increase from 2 to 18 month of age (Fig. 2).
Similar results were observed in the brains of PS2wt-Tg
mice (45±5 mg/dl) as well as PS2mt-Tg mice, but an
unusual exception was shown in the brains of 6 month old
PS2mt-Tg mice [showing a minimum HDL value (30.6±2
mg/dl) (Fig. 2)].
Increase in LDL levels in mutant PS2 transgenic
mice brains
The LDL levels were increased in all the brains of
PS2mt-Tg mice compared to non-Tg and PS2wt-Tg mice
(Fig. 3). In young mice, increases in the level of LDL
occurred not only in PS2mt-Tg, but also in PS2wt-Tg and
non-Tg mice brains. The highest level was shown in the
brains of 6 month old mice. However, in older mice, the
LDL levels were only slightly higher in the brains of
PS2mt-Tg mice compared to non-Tg mice, while the LDL
levels in the brains of PS2wt-Tg mice were equal to those
of non-Tg mice. The LDL level in non-Tg brains was
increased from 15.3±3 mg/dl at 2 months to 23.3±8 mg/dl
at 6 months, but decreased to 11.6±1.8 and 14.7±1.7 mg/dl
at 12 and 18 months of age, respectively (Fig. 3). Similar
results were obtained in the brains of PS2wt-Tg mice.
There were increases in the levels of LDL in the brains of
PS2wt-Tg mice from 26.6±8.5 mg/dl at 2 months to
36.3±7.4 mg/dl at 6 months, but decreases to 11.6±2 and
16±1.5 mg/dl at 12 and 18 months of age, respectively
(Fig. 3). The LDL levels in the brains of PS2mt-Tg mice
were always higher than in those of non-Tg and PS2wt-
Tg mice. The levels of LDL in PS2mt-Tg brains were
increased from 28.6±8 mg/dl at 2 months to 54±9.8 mg/dl
at 6 months, but then decreased to a consistent level of
27±8 mg/dl at 12 and 18 months of age (Fig. 3).
Fig. 1. Total cholesterol levels in the brain tissues of mice: non-Tg,
PS2wt-Tg and PS2mt-Tg at all ages; 2, 6, 12 and 18 months. The data
represent the mean ± SEM (bars) values determined from three
independent experiments (n=6). *significant difference from non-Tg
mice, #significant difference from 2 month old mice.
Fig. 2. High density lipoprotein (HDL) cholesterol levels in mice brain
tissues: non-Tg, PS2wt-Tg and PS2mt-Tg at all ages; 2, 6, 12 and 18
months. The data represent the mean ± SEM (bars) values determined
from three independent experiments (n=6). *significant difference from
non-Tg mice, #significant difference from 2 month old mice.

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Mutant Presenilin 2 Causes Abnormality in the Brain Lipid Profile in the Development of Alzheimer’s Disease 887
Alteration of triglyceride levels in mutant PS2
transgenic mice brains
The triglyceride levels in the brains of Tg mice were
increased compared to those in non-Tg mice at all ages,
particularly in PS2mt-Tg mice (Fig. 4). In the brains of non-
Tg mice, there were increases in the levels of triglyceride
from 80±14.7 mg/dl at 2 months to 100±2.3 mg/dl at 6
months, and to 118±5.6 mg/dl at 18 months of age (Fig.
4). Similarly, the triglyceride levels in the brains of PS2wt-
Tg mice were also increased from 92.5±3.7 mg/dl at 2
months to 121.6±7.5 and 135.6±8 mg/dl at 12 and 18
months, respectively (Fig. 4). At 2 months of age, the
triglyceride level in the brains of PS2mt-Tg mice was
89.7±7.8 mg/dl, but at 6 months the level was 138.3±8.8
mg/dl, which increased to 168±19.3 mg/dl at 18 months
(Fig. 4). The triglyceride levels were higher in old mice,
with the increase appearing to be age-dependent in the
brains of PS2wt-Tg and PS2mt-Tg mice (Fig. 4).
DISCUSSION
The lack of memory in PS2 transgenic mice, particularly
in mutant PS2 transgenic mice, is correlated with the
increased expression of PS2 wild (PS2wt) and PS2
mutant (PS2mt) types in the cortex and hippocampus, as
described in a previous study (Hwang et al., 2002). The
involvement of the PS2 mutation in the abnormality of lipid
metabolism in AD is unclear, although a clue that
intracellular cholesterol transport alters the PS localization
in neuronal cells suggests the possibility of a cholesterol-
dependent trafficking PS (Runz et al., 2002). In this study,
how mutant PS2 influences the lipid profile in an AD
model of the PS2 transgenic mice brains was investigated.
An increase in the amount of total cholesterol accompany-
ing the increase in the levels of triglyceride occurred in the
brains of PS2mt-Tg compared to normal mice indicates
that the mutant PS2 may be involved in these abnormalities
of the lipid profile. Moreover, the age-dependent increases
in the levels of triglyceride and cholesterol were clearer in
PS2mt-Tg than in non-Tg mice, which address the
abnormal effect of mutant PS2 in the lipid metabolism of
the cells.
Cholesterol and triglyceride are known to be required
for the formation and maintenance of cell membrane
permeability and fluidity, as well as for cellular signaling
and other forms of cellular biosynthesis (Simons et al.,
2000; Galbete et al., 2000). Therefore, alterations to the
cholesterol and triglyceride contained within cells may
reflect an abnormality in the cell membrane properties
influencing the other forms of metabolism concerned,
including Aβ generation and the signaling transmission of
cells. In support of this notion, another study has reported
that the cholesterol levels were increased in a vulnerable
brain region, but not in a non-vulnerable brain region in
AD patients (Cutler et al., 2004). It has been suggested
that psychoactive drugs increase the triglyceride change
in the membrane fluidity and receptor function (Diebold et
al., 1998). There is accumulating data supporting the
Fig. 3. Low density lipoprotein (LDL) cholesterol levels of mice brain
tissues: non-Tg, PS2wt-Tg and PS2mt-Tg at all ages; 2, 6, 12 and 18
months. The data represent the mean ± SEM (bars) values determined
from three independent experiments (n=6). *significant difference from
non-Tg mice, #significant difference from 2 month old mice.
Fig. 4. Triglyceride levels of mice brain tissues: non-Tg, PS2wt-Tg and
PS2mt-Tg at all ages; 2, 6, 12 and 18 months. The data represent the
mean ± SEM (bars) values determined from three independent experi-
ments (n=6). *significant difference from non-Tg mice, #significant
difference from 2 month old mice.

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888H. N. Nguyen et al.
hypothesis that alterations in cholesterol levels influence
the development of AD by affecting the formation and
distribution Aβ within cholesterol rich membranes (Runz
et al., 2002; Subasinghe et al., 2003; Refolo et al., 2000).
Furthermore, the decrease in the levels of HDL as opposed
to the increase in levels of LDL, which are supposed to be
‘good and bad cholesterol’ for cells, respectively, occurred
in the brains of PS2mt-Tg mice, demonstrating that mutant
PS2 may cause cellular degeneration via alteration of the
lipid transport mechanism in cells. The HDL level is
consistent at all ages, suggesting HDL may be a stable
component in the cell structure and not an age dependent
agent. Thus, alternations in the level of HDL could influence
the function of brain or cause damage to neuronal cells.
Mutant PS2 may influence the maintenance of the levels
of HDL, which may also be involved in the risk of
developing AD (Michikawa, 2003). In contrast, LDL may
play a role in age related cell activities during the life of
cells, which alter the levels of LDL in the brains of non-Tg
and PS2wt-Tg mice at all ages investigated. Our results
have also demonstrated the existence of a potential LDL
level regulation mechanism during each term of cell life. In
the case of cells expressing mutant PS2, the LDL levels
were always higher, suggesting mutant PS2 may also
affect the membrane fluidity leading to disturbance of the
membrane function, including APP processing, which is
involved in the development of AD (Irizarry et al., 2004).
Overall, the level of LDL was higher in the brains of
PS2mt-Tg mice, but the increase in the level of LDL was
higher in relatively young (6 months old) than older mice.
The reason for this is not clear, and it may not be signi-
ficant, since there was much variation among the animals,
and the corresponding HDL level was proportionally
decreased at the same time. Remarkably, the levels of
total cholesterol, triglyceride and LDL, which are supposed
to be risk factors for AD (Tan et al., 2003; Refolo et al.,
2001), in the brain tissues of mice expressing PS2mt
were always higher than in normal brains, and also age
dependent, indicating the noteworthy contribution of
PS2mt to neurodegeneration during ageing.
In conclusion, this study elucidated the involvement of
PS2 in the lipid profile of the brain. The mutation of PS2
alters the lipid metabolism, which may disturb the
regulation of lipid mechanism in neuronal cells, and in turn
influence the neuronal function, rendering the lack of
memory in PS2mt transgenic mice. These findings, there-
fore, could be useful in the development of an appropriate
therapeutic intervention for targeting mutant PS2-induced
AD cases.
ACKNOWLEDGEMENTS
This work was supported by the Regional Research
Centers Program of the Ministry of Education & Human
Resources Development in Korea.
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