Original Contribution

Aging sensitizes toward ROS formation and lipid peroxidation

in PS1M146L transgenic mice

Katrin Schuessel

a,

*, Claudia Frey

a

, Claudia Jourdan

a

, Uta Keil

a

, Claudia C. Weber

a

,

Franz Mu¨ller-Spahn

b

, Walter E. Mu¨ller

a

, Anne Eckert

a,b

a

Department of Pharmacology, Biocentre, University of Frankfurt, 60439 Frankfurt am Main, Germany

b

Neurobiology Research Laboratory, Psychiatry University Hospital, CH-4025 Basel, Switzerland

Received 24 June 2005; revised 13 September 2005; accepted 10 October 2005

Available online 15 November 2005

Abstract

Mutations in the presenilins (PS) account for the majority of familial Alzheimer disease (FAD) cases. To test the hypothesis that oxidative

stress can underlie the deleterious effects of presenilin mutations, we analyzed lipid peroxidation products (4-hydroxynonenal (HNE) and
malondialdehyde) and antioxidant defenses in brain tissue and levels of reactive oxygen species (ROS) in splenic lymphocytes from transgenic
mice bearing human PS1 with the M146L mutation (PS1M146L) compared to those from mice transgenic for wild-type human PS1 (PS1wt) and
nontransgenic littermate control mice. In brain tissue, HNE levels were increased only in aged (19 – 22 months) PS1M146L transgenic animals
compared to PS1wt mice and not in young (3 – 4 months) or middle-aged mice (13 – 15 months). Similarly, in splenic lymphocytes expressing the
transgenic PS1 proteins, mitochondrial and cytosolic ROS levels were elevated to 142.1 and 120.5% relative to controls only in cells from aged
PS1M146L animals. Additionally, brain tissue HNE levels were positively correlated with mitochondrial ROS levels in splenic lymphocytes,
indicating that oxidative stress can be detected in different tissues of PS1 transgenic mice. Antioxidant defenses (activities of antioxidant enzymes
Cu/Zn-SOD, GPx, or GR) or susceptibility to in vitro oxidative stimulation was unaltered. In summary, these results demonstrate that the
PS1M146L mutation increases mitochondrial ROS formation and oxidative damage in aged mice. Hence, oxidative stress caused by the combined
effects of aging and PS1 mutations may be causative for triggering neurodegenerative events in FAD patients.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Aging; Alzheimer disease; Amyloid h; Brain; Antioxidant enzyme; Lipid peroxidation; Lymphocyte; Mitochondria; Hydroxynonenal; Oxidative stress;
Presenilin; Reactive oxygen species; Transgenic mouse; Transgenic; Free radical

Alzheimer

_s disease (AD) is the most frequent form of

dementia and among the leading causes of death in the elderly.
Although the precise mechanisms by which neurodegeneration
in AD patients is triggered remain largely speculative, the past
decades of research have identified rare genetic mutations in
the amyloid precursor protein (APP) or in the presenilins PS1

or PS2, which have provided valuable insight into putative
pathogenic mechanisms. These mutations cause inheritable
familial forms of AD (FAD) showing an early onset of
cognitive symptoms—in contrast to the much more common
sporadic form of AD with onset of symptoms usually over the
age of 60, for which aging represents the main risk factor

[1]

.

Mutations in the presenilins PS1 and PS2 account for the

majority of FAD cases, and clinical onset in some carriers of
PS1 mutations is extremely early, occasionally as soon as in the
third decade of life

[2,3]

. In recent years, the number of

mutations identified in the PS genes has been ever increasing,
especially in PS1, in which more than a hundred mutations
have been found so far

[4]

. Presenilins are a component of the

g-secretase complex, which is involved in the formation of
amyloid h (Ah) from its precursor protein APP

[5]

. Several

different PS mutations have been shown to alter g-secretase

0891-5849/$ - see front matter

D 2005 Elsevier Inc. All rights reserved.

doi:

10.1016/j.freeradbiomed.2005.10.041

Abbreviations: AD, Alzheimer disease; Ah, amyloid h; APP, amyloid

precursor protein; DAF-2-DA, diaminofluorescein-2-diacetate; DHE, dihy-
droethidium; DHR, dihydrorhodamine 123; FAD, familial Alzheimer disease;
GPx, glutathione peroxidase; GR, glutathione disulfide reductase; HBSS,
Hanks

_ balanced salt solution; H

2

DCF-DA, dichlorodihydrofluorescein diace-

tate, HNE, 4-hydroxynonenal; MDA, malondialdehyde; Mio, million cells;
PS1, presenilin 1; PS2, presenilin 2; R123, rhodamine 123; ROS, reactive
oxygen species; SOD, superoxide dismutase.

* Corresponding author. Fax: [phone].

E-mail address:

[e-mail]

(K. Schuessel).

Free Radical Biology & Medicine 40 (2006) 850 – 862

www.elsevier.com/locate/freeradbiomed

processing of APP to yield higher levels of the Ah

1 – 42

isoform

[6 – 10]

and a higher production of Ah

1 – 42

correlated with an

earlier clinical onset of Alzheimer dementia in carriers of PS1
mutations

[11]

. With the exception of one deletion mutant, all

these mutations are single amino acid mutations, which are
spread across the whole length of PS1 protein. This suggests
that all PS1 mutations trigger the development of FAD via a
common toxic mechanism caused by only subtle alterations of
the protein structure, consistent with a recent report that
different PS1 mutations all cause a similar structural change in
protein conformation and interaction with APP

[12]

. Similar to

PS mutations, FAD mutations in APP result in elevated
formation of Ah species

[13,14]

. The high amounts of Ah

deposited as amyloid plaques, which are a characteristic feature
of brains from AD patients, suggest a pathological role for this
peptide and led to the development of the amyloid hypothesis
of AD

[15,16]

. It states that the increased formation and

accumulation of Ah, and especially the Ah

1 – 42

isoform,

triggers neurodegenerative events in AD patients. Therefore,
the increased formation of Ah

1 – 42

formation is proposed to be

the causative mechanism for neurotoxicity induced by pre-
senilin mutations. Consistent with this hypothesis, proapoptotic
effects are induced by expression of mutant PS1 in cell culture
systems

[17 – 19]

and transgenic mice

[20]

. Similarly, in

primary cultured neurons of transgenic rats expressing wild-
type PS1, increased apoptosis correlated with the amount of
PS1 expression

[21]

. Moreover, mice transgenic for human

PS1 with FAD mutations display increased formation of rodent
Ah

1 – 42

and neuronal degeneration

[22,23]

and show cognitive

deficits

[24]

.

However, it remains speculative how neurodegeneration

may be mediated by Ah and whether similar events take place
during the development of AD. It has been suggested that
oxidative stress plays an important role, which is mainly based
on two observations: (1) Aging is the most important risk factor
for the development of sporadic AD and also plays an
important role in the familial forms of AD, as even in FAD
cases only very few patients show onset of cognitive symptoms
before the age of 30

[2,3]

. The phenomenon of aging has

previously been closely linked to the accumulation of oxidative
stress

[25]

, which could also be demonstrated in brains from

sporadic and familial AD patients

[26 – 28]

. (2) In accordance

with the amyloid hypothesis, numerous studies of Ah toxicity
in cell culture

[29]

or APP transgenic mice

[30,31]

have

provided evidence that Ah can induce neuronal cell death by
eliciting oxidative damage.

In order to test the hypothesis that presenilin mutations can

similarly provoke the development of FAD by inducing
oxidative stress in vivo, we analyzed several oxidative stress-
related parameters in transgenic mice expressing human PS1
with a FAD mutation. As controls, nontransgenic littermate
mice as well as mice expressing human wild-type PS1 were
employed. The comparison with PS1wt transgenic mice allows
us to detect effects that are caused specifically by the presence
of the M146L FAD mutation in PS1, as the expression of wild-
type PS1 alone has been reported to exhibit cytotoxic effects in
some cell culture models

[18,32]

. Brain tissue from these mice

was analyzed for lipid peroxidation products malondialdehyde
(MDA) and 4-hydroxynonenal (HNE) as markers of oxidative
damage. Oxidative stress results from an imbalance between
the production and the detoxification of reactive oxygen
species (ROS), and we have previously identified an impair-
ment of the antioxidant enzyme Cu/Zn-dependent superoxide
dismutase as a possible causative factor for oxidative damage
in mutant APP transgenic mice

[31,33]

. In order to test whether

a similar deficiency in antioxidant defense may be present in
PS1 transgenic mice, we assayed antioxidant enzymatic
activities of Cu/Zn-dependent superoxide dismutase (Cu/Zn-
SOD), glutathione peroxidase (GPx), and glutathione reductase
(GR). The expression of transgenic PS1 under the control of
the HMG-CoA reductase promoter leads to high expression in
brain tissue, but also in peripheral tissues from PS1 transgenic
mice

[34,35]

. Therefore, splenic lymphocytes could be isolated

and employed for a direct study of mutant presenilin effects on
ROS levels in living cells by staining with various ROS-
sensitive fluorescent dyes and selective analysis by flow
cytometry. In order to additionally assess a putative interaction
between aging and the effects of the PS1 mutation, transgenic
mice of different age groups were studied, i.e., young, middle-
aged, and aged mice (3 – 4, 13 – 15, and 19 – 22 months of age,
respectively).

Materials and methods

Animals

PS1 transgenic mice were generated as described before

[21,36]

. Mice were bred on a C57BL/6J background (Iffa

Credo, France), and for all experiments, heterozygous trans-
genic mice bearing human wild-type presenilin 1 (PS1wt) or
human presenilin 1 with the M146L mutation (PS1M146L) and
the respective nontransgenic control animals from the same
litter (non-tg) were used. Mice were maintained on a 12-
h dark – light cycle with pelleted food and tap water ad libitum.
Animals were handled according to the French and German
guidelines for animal care.

Transgene expression of either PS1wt or PS1M146L is

under the control of a HMG-CoA reductase promoter. This
results in a strong neuronal expression but also ubiquitous
expression in peripheral tissues

[34,35]

. PS1 mutations lead to

increased amyloidogenic processing of endogenous mouse
APP; however, no Ah plaques can be detected in the brains
from these mice, which seems to be due to differences in
aggregation properties of rodent compared to human Ah

[37]

.

Animals were studied at 3 – 4 (young), 13 – 15 (middle-aged),

and 19 – 22 (aged) months of age. Brain tissue and splenic
lymphocytes were studied in a total of 11 nontransgenic control,
11 PS1wt, and 11 PS1M146L transgenic mice at 3 – 4 months of
age; 27 nontransgenic control, 18 PS1wt, and 13 PS1M146L
transgenic mice at 13 – 15 months of age; as well as 27
nontransgenic control, 10 PS1wt, and 12 PS1M146L transgenic
mice at 19 – 22 months of age. An additional cohort of 7
nontransgenic and 7 PS1M146L transgenic mice were employed
at an age of 21 months for detailed analysis of splenic lym-

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

851

phocyte subgroups and apoptosis quantification. Gender was
distributed equally between the groups.

Isolation of mouse brain tissue and preparation of splenic
lymphocytes

Mice were quickly killed by cervical dislocation, and brains

were removed and dissected on ice. Cerebellum and brain stem
were removed and the forebrain was dissected midsagittally.
Both hemispheres were immediately snap frozen in liquid
nitrogen and stored at

80

-C until homogenization. Splenic

lymphocytes were prepared by mechanical dissociation of
individual spleens and erythrocyte lysis as described previously

[38]

. For determination of ROS with fluorescent dyes, live cells

were counted via trypan blue exclusion (Biochrom AG, Berlin,
Germany) in a Neubauer chamber and adjusted to 1 million
cells per ml (1 Mio/ml).

Preparation of mouse brains for determination of lipid
peroxidation products and antioxidant enzyme activities

One mouse brain hemisphere was minced in 1 ml of cold 20

mM Tris-buffered saline with 10 strokes in a Potter homog-
enizer at 1200 rpm. An aliquot of this homogenate was diluted
1:1 with Tris-buffered saline and centrifuged at 3000g and 4

-C

for 10 min and supernatants were collected and stored at

80

-C for lipid peroxidation assays. The remaining homog-

enate was centrifuged at 8500g and 4

-C for 10 min and

supernatants were collected and stored at

80

-C for determi-

nation of antioxidant enzyme activities.

Assay of lipid peroxidation products

Lipid peroxidation products MDA and HNE were deter-

mined by a photometrical method utilizing the lipid peroxida-
tion assay kit from Calbiochem (Schwalbach, Germany), which
is based on the method of Esterbauer and Cheeseman

[39]

. The

colorimetric reaction is a condensation of the respective
aldehyde with 1-methyl-2-phenylindole yielding chromophores
with absorption maxima at 586 nm. Both aldehydes react with
1-methyl-2-phenylindole in the presence of methanesulfonic
acid, whereas substitution of methanesulfonic acid by hydro-
chloric acid allows measurement of MDA alone due to an
irreversible cyclization reaction of hydroxyalkenals

[40]

. HNE

levels were calculated from absorbance levels obtained by
subtracting the absorbance of samples after hydrochloric acid
reaction from absorbance after methanesulfonic acid reaction.
Basal levels of MDA and HNE were assayed after incubation
of samples at 37

-C for 30 min. Stimulated MDA levels were

assayed after incubation in the presence of 50 AM FeCl

3

(VWR

International, Darmstadt, Germany).

Antioxidant enzyme activities: Cu/Zn-SOD, GPx, and GR
activity

The assay of Cu/Zn-SOD activity (EC 1.15.1.1) is based

on the method of Nebot et al.

[41]

utilizing the commercially

available Superoxide Dismutase Assay Kit from Calbiochem.
The assay of GPx activity (cytosolic GPx, EC 1.11.1.9),
based on the reaction described by Paglia et al.

[42]

with tert-

butylhydroperoxide as substrate, and the assay of GR activity
(EC 1.8.1.7), based on the method of Mizuno and Ohta

[43]

,

were performed utilizing the commercially available Cellular
Glutathione Peroxidase Assay Kit and Glutathione Reductase
Assay Kit from Calbiochem, respectively. All assays were
performed as described in more detail previously

[31]

.

Isolation of mRNA from splenic lymphocytes and RT-PCR for
detection of human PS1 expression in transgenic mice

RNA was isolated from splenic lymphocytes utilizing the

High Pure RNA Isolation Kit from Boehringer (Mannheim,
Germany) as described in the manufacturer

_s protocol. Sixty-

four nanograms of RNA was used for reverse transcriptase
PCR, and RT reactions were performed with the SuperScript II
Kit from Invitrogen (Karlsruhe, Germany) according to the
supplier

_s instructions. cDNA synthesis was performed

from RNA samples with oligo(dT) primers. PCR of PS1
DNA was conducted with the Eppendorf Master Taq Kit
(Cologne, Germany) utilizing the primers sense, 5V-TAA-
TTGGTCCATAAAAGGC-3V, and antisense, 5V-GCACAGAA-
AGGGAGTCACAAG-3V, which yields a 425-bp product
specific for human presenilin 1. PCR products were detected
after electrophoresis in ethidium-stained agarose gels (VWR
International).

Measurement of reactive oxygen species in splenic lymphocytes
with oxidation-sensitive fluorescent dyes and flow cytometry
analysis

Splenic lymphocytes were incubated at 1 Mio/ml with ROS-

sensitive dyes at 37

-C, washed twice, and resuspended for

flow-cytometric analysis with Becton – Dickinson (Heidelberg,
Germany) FACSCalibur utilizing CellQuest Pro software. A
minimum of 8000 events were recorded per single measure-
ment. Cells were analyzed immediately after staining and
always kept on ice in the dark until measurement.

Dihydrorhodamine 123 (DHR), rhodamine 123 (R123),

dichlorodihydrofluorescein diacetate (H

2

DCF-DA), and dihy-

droethidium (DHE) (5 mM stabilized solution in DMSO) were
purchased from Molecular Probes (Leiden, Netherlands).
Diaminofluorescein-2-diacetate (DAF-2-DA) (5 mM solution
in DMSO) was obtained from Calbiochem.

Basal levels of ROS were assayed immediately after

lymphocyte isolation in RPMI medium containing 5% fetal
calf serum (Sigma, Taufkirchen, Germany). Serum withdraw-
al was conducted by transferring lymphocytes to HBSS
(Sigma) buffer containing 10 mM Hepes, 1 mM CaCl

2

, and

0.5 mM MgSO

4

(all VWR International), pH 7.4. Cells were

oxidatively stimulated in HBSS buffer by addition of
hydrogen peroxide (Sigma) and incubation at 37

-C for 15

min. After stimulation, cells were washed, resuspended in
RPMI medium, and stained with ROS-sensitive dyes as
described below.

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

852

DHR is the nonfluorescent reduced form of R123. Inside the

cell, it is oxidized mainly by mitochondrial ROS to the
positively charged fluorescent rhodamine R123

[44]

. Because

the fluorescence signal obtained after staining of lymphocytes
with DHR depends on two parameters, i.e., (1) oxidation of the
dye and (2) incorporation of R123 into mitochondria,
lymphocytes were also stained with the oxidized form of
DHR, R123, to correct for possible mitochondrial defects that
would affect uptake of oxidized DHR. Furthermore, R123
staining also controls for effects of P-glycoprotein

[45]

, as

R123 is a substrate for P-glycoprotein, which is expressed in
murine lymphocytes and can affect R123 staining of cells

[46]

.

DHR and R123 were used at a final concentration of 10 and 1
A

M, respectively, and an incubation time of 15 min. H

2

DCF-

DA is hydrolyzed and oxidized by various ROS to the
fluorescent DCF

[47]

. H

2

DCF-DA does not stain mitochondria

[48]

, but is oxidized mainly by peroxides in the cytosol

[49]

.

H

2

DCF-DA was used at a concentration of 10 AM and 30 min

incubation time. Both DHR and H

2

DCF have been described to

be also oxidized by peroxynitrite

[50]

. Peroxynitrite is formed

in a reaction of the superoxide radical anion with nitric oxide

[51]

. In order to assess a possible contribution of peroxynitrite

to DHR and H

2

DCF oxidation in our studies, we also

monitored superoxide and nitric oxide levels. Superoxide
anions are readily detected by oxidation of DHE

[52]

, which

was used at a concentration of 5 AM and 30 min of incubation.
Nitric oxide levels were measured with DAF-2-DA

[53]

at a

concentration of 2.5 AM and 30 min of incubation time. In the
presence of nitric oxide within cells, DAF-2-DA is hydrolyzed
and converted to the fluorescent triazole derivate DAF-2T.

The oxidized forms of all ROS-sensitive dyes as well as

R123 could be excited with the 488 nm laser of the Becton –
Dickinson FACSCalibur flow cytometer. Emission of R123,
DCF, and DAF-2T fluorescence was detected in channel FL-1
(530-nm filter, 30-nm bandpass); emission of ethidium
fluorescence after DHE oxidation was quantified in channel
FL-2 (585-nm filter, 42-nm bandpass).

For quantification of mitochondrial ROS in lymphocyte

subgroups, cells were double stained with DHR and anti-CD3 – ,
anti-CD4 – , or anti-CD8 – PE antibodies (all from Becton –
Dickinson), and double stained cells were gated according to
their fluorescence signals in channels FL-1 (R123 signal) and
FL-2 (PE signal).

Apoptosis measurement in splenic lymphocytes

The apoptotic status of lymphocytes was quantified with the

DNA dye 7-actino-antimycin (7-AAD; Molecular Probes) as
described by

[54]

. 7-AAD stains cells that have lost membrane

integrity. For quantification of apoptosis, 1 Mio/ml splenic
lymphocytes were incubated with 6 AM 7-AAD for 15 min at
room temperature. Afterward, cells were washed, resuspended
in phosphate-buffered saline (PAA, Co¨lbe, Germany), and
immediately analyzed by flow cytometry. Apoptotic cells were
gated and quantified based on the signal

_s forward scatter and

7-AAD fluorescence detected in FL-3 (650-nm filter)

[55]

.

Lymphocyte subsets were identified by double staining with

FITC-conjugated anti-CD3 antibody and fluorescence detec-
tion in FL-1 or PE-conjugated anti-CD4 antibody and
fluorescence detection in FL-2. All antibodies were purchased
from Becton – Dickinson.

Chemicals

NH

4

Cl, KHCO

3

, hydrochloric acid, ethanol, and chloro-

form were purchased from VWR International. All aqueous
solutions were prepared with deionized and filtered water
(Millipore).

Protein content

Protein levels in brain homogenates were determined

according to the method of Lowry et al.

[56]

using Bio-Rad

Protein Assay solutions (Munich, Germany) and bovine serum
albumin (Sigma – Aldrich) as standard.

Statistics

Student

_s t test was calculated for comparison between two

groups. For simultaneous estimation of the effects of two
parameters, two-way ANOVA was calculated. For calculation
of correlation between two parameters assayed in samples from
the same animal, linear regression assuming Gaussian distri-
bution was performed. All calculations were done with Prism
GraphPad software and p

 0.05 was assessed as significant.

All data in bar graphs are represented as means + SEM.

Results

Increased oxidative damage in brains from aged PS1M146L
transgenic mice

Brain tissue from nontransgenic littermate control mice as

well as PS1wt and PS1M146L transgenic mice was studied for
the levels of lipid peroxidation products MDA and HNE as
markers of oxidative damage. Whereas no differences in lipid
peroxidation levels were found in young (3 – 4 months) or
middle-aged (13 – 15) mice, increased levels of HNE could be
detected in aged PS1M146L mice (19 – 22 months) (see

Fig.

1

A). Interestingly, HNE levels were not increased in PS1wt

transgenic mice, indicating that merely the transgenic PS1
protein does not induce oxidative damage. Rather, the presence
of the M146L mutation is responsible for increased lipid
peroxidation. Of note, whereas HNE levels were increased,
MDA levels were not different between the mouse models
(data not shown), suggesting that predominantly HNE is
formed in brains from these mice.

As impaired antioxidant defenses can account for ROS

accumulation and oxidative damage, we analyzed enzymatic
activities of Cu/Zn-SOD, GPx, and GR in brains from these
mice. However, none of these enzyme activities was impaired in
PS1M146L transgenic mice in any age group analyzed (data not
shown). As an additional indicator of antioxidant capacity, lipid
peroxidation in brain homogenates was stimulated with ferric

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

853

iron in vitro. Again, no difference in the formation of MDA could
be observed between nontransgenic and PS1 transgenic mice
(

Fig. 1

B). Hence, antioxidant activity is seemingly not impaired

by the presence of mutant PS1, indicating that oxidative damage
in brains from PS1M146L mice stems from overproduction
rather than from impaired detoxification of ROS.

Increased ROS levels in splenic lymphocytes expressing mutant
PS1

In order to analyze ROS production in PS1 transgenic mice

in more detail, ROS levels were measured directly in living
cells from these mice. To this purpose, splenic lymphocytes
were isolated from nontransgenic and PS1wt and PS1M146L
transgenic mice and analyzed for expression of PS1 mRNA by
RT-PCR. The expression of transgenic human PS1 could be
detected in splenic lymphocytes from PS1wt or PS1M146L
transgenic mice (see

Fig. 2

), which confirms previously

published results showing that the HMG-CoA reductase

promoter leads to strong expression in brain tissue but also
in peripheral cells

[34,35]

. Hence, these cells could be used to

conveniently study ROS levels directly by flow cytometry.

Similar to the situation in brain tissue from PS1 transgenic

mice, only cells from aged transgenic mice bearing the
PS1M146L mutation displayed increased ROS levels as
measured by increased oxidation of DHR (see

Fig. 3

A). Of

note, DHR oxidation was not increased in lymphocytes from
PS1wt transgenic mice, indicating that this effect is specifically
caused by the presence of the FAD mutation PS1M146L.

In order to specify putative sources and/or types of ROS,

splenic lymphocytes were stained with further ROS-sensitive
dyes. Apart from DHR, which detects mainly mitochondrial
ROS production, H

2

DCF-DA for indication of cytosolic

peroxides and DHE detecting superoxide radicals were
employed. Additionally, confounding factors like incorporation
of oxidized DHR into mitochondria or oxidation of DHR and
H

2

DCF-DA by peroxynitrite

[50]

were controlled for by staining

with R123, i.e., the oxidized form of DHR, and DAF2-DA, a
nitric oxide-sensitive dye, respectively. The results demonstrate
that mainly mitochondrial ROS production (142.1% DHR
oxidation relative to nontransgenic controls, see

Fig. 3

A) and

Fig. 1. Lipid peroxidation products in brains from aged mice (19 – 22 months of
age) transgenic for human wild-type presenilin 1 (PS1wt; n = 9 – 10) or human
mutant presenilin 1 (PS1M146L; n = 10) and nontransgenic age-matched
littermate controls (non-tg, n = 18 – 20). (A) Levels of HNE (nmol/mg protein)
are increased in brains from PS1M146L transgenic mice compared to PS1wt
mice, *p < 0.05, Student

_s t test. (B) Levels of MDA (nmol/mg protein)

formation after incubation of brain homogenates with 50 AM FeCl

3

for 30 min

at 37

-C. Differences between groups are not statistically significant.

Fig. 2. Expression of human presenilin mRNA in lymphocytes from PS1
transgenic mice. Total RNA was isolated from splenic lymphocytes and
subjected to RT-PCR with oligo(dT) primers, and human presenilin 1 cDNA
sequences were amplified by PCR with primers specific for human presenilin 1.
Lane 1, PS1 PCR positive control (DNA isolated from PS1 transgenic mouse);
lanes 2, 3, 5, and 8, samples from nontransgenic animals; lane 4, PS1M146L
transgenic mouse; lanes 6 and 7, PS1wt transgenic mouse.

Fig. 3. Increased ROS formation in PS1M146L transgenic mice. Splenic
lymphocytes expressing the transgenic protein were isolated and stained with
different ROS-sensitive fluorescent dyes and analyzed by flow cytometry. Data
are presented as mean fluorescence intensity levels (MFI) relative to the means
of nontransgenic control mice (100%). n = 20 nontransgenic (non-tg), n = 10
PS1wt, and n = 11 PS1M146L transgenic mice were studied. (A) Mitochondrial
ROS formation (oxidation of DHR to R123) is increased in cells from
PS1M146L mice (142.1% relative to nontransgenic controls). *p < 0.05,
Student

_s t test, PS1M146L vs non-tg. (B) Cytosolic ROS formation (oxidation

of H

2

DCF-DA to DCF) is increased in cells from PS1M146L mice (120.5%

relative to nontransgenic controls). **p < 0.01, Student

_s t test, PS1M146L vs

non-tg. (C) Nitric oxide levels (oxidation of DAF-2-DA to DAF-2-T) are not
significantly different between groups. (D) Mitochondrial uptake of R123 is
slightly increased in cells from PS1M146L mice (107.2% relative to
nontransgenic controls). *p < 0.05, Student

_s t test, PS1M146L vs non-tg.

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

854

to a lesser extent cytosolic ROS production (120.5% H

2

DCF

oxidation relative to nontransgenic controls, see

Fig. 3

B) are

increased in cells from PS1M146L mice. Of note, oxidation of
DHE (data not shown) and DAF-2-DA-staining (

Fig. 3

C) were

unchanged, indicating that the oxidation of DHR and H

2

DCF is

unlikely to be confounded by increased levels of peroxynitrite.
Furthermore, increased staining of cells with DHR seems to be
specific for oxidation of the dye, because staining with R123 as
control for dye retention due to mitochondrial uptake was only
slightly increased in PS1M146L cells (

Fig. 3

D). Although this

difference was significant, the extent of R123 staining of cells
was much lower (107.2% relative to controls) compared to the
extent of DHR oxidation in cells from PS1M146L mice. Hence,
these results indicate that mainly mitochondrial and secondarily
cytosolic ROS are increased by the PS1M146L mutation,
whereas formation of nitric oxide and superoxide as well as
severe impairment of mitochondrial membrane integrity as
sources of ROS are negligible.

Importantly, the observed alterations in brain tissue HNE

and peripheral ROS levels are specific for the PS1M146L
mutation, because no differences were observed in any of the
tested parameters between nontransgenic mice and PS1wt
mice. This allows also for a direct comparison of PS1M146L
transgenic mice with nontransgenic littermate control mice in
the following experiments.

As an additional indicator of antioxidant metabolism, ROS

production in cells from these mice was also studied after
stimulation with hydrogen peroxide in vitro. Although DHR
oxidation was increased in PS1M146L-expressing cells from
aged mice (see

Fig. 4

A), this reflects the basal increased ROS

production and not an additional effect of oxidative stimula-
tion, indicating that cells from the different transgenic animals
react similarly upon ROS accumulation. This observation
supports our findings on brain tissue that oxidative damage
in PS1M146L transgenic mice is caused by overproduction
rather than by impaired detoxification of ROS.

Interestingly, in middle-aged mice (13 – 15 months of age),

basal ROS levels were unaltered (

Fig. 4

B), but DHR oxidation

was increased in cells from PS1M146L mice after serum
withdrawal (

Fig. 4

C). Serum withdrawal represents a mild stress

condition, indicating that the presence of an additional stimulus
can trigger the ROS-elevating effects of the PS1M146L
mutation. Therefore, the effect of the PS1 mutation in middle-
aged mice is evident only after a secondary insult, whereas in
aged mice basal levels of ROS are already increased.

ROS formation correlates with apoptosis in CD4

+

T cells

Previous studies by our group on lymphocytes from AD

patients found that a different vulnerability toward cell death
exists in specific cell types. Accordingly, we analyzed lympho-
cyte subpopulations, i.e., the CD3-positive pool (T lymphocytes
only) and the CD4

+

and CD8

+

subpool of T cells, for a putatively

different accumulation of ROS. In general, the ROS-elevating
effect of the PS1 mutation was similar in all lymphocyte
subgroups studied, indicating that the presence of mutant PS1
leads to increased oxidative stress independent of the cell type

(see

Fig. 5

A). However, differences could be detected between

CD4

+

and CD8

+

cells in that DHR oxidation was increased in

CD4

+

cells compared to CD8

+

, which was independent of the

transgene. Hence, CD4

+

cells accumulate higher ROS levels.

Apoptosis levels in the same cells also indicated that CD4

+

cells

react more sensitively toward this type of cell death. Although
differences failed to reach statistical significance (see

Fig. 5

B),

these results confirm previous findings from our group

[35]

.

Moreover, the levels of DHR oxidation correlated with the
amount of apoptosis in CD4

+

cells from the same animal,

indicating that ROS accumulation and apoptotic cell death are
interrelated (see

Fig. 5

C).

Correlation of mitochondrial ROS levels in lymphocytes with
HNE levels in brain tissue

Although peripheral cells from AD patients have been

widely used for studies of putative pathogenic cell death
mechanisms, evidence that relevant findings in these cells can
be transferred to the situation in human brain tissue could not
be provided so far—as noninvasive measures of apoptotic cell
death or oxidative stress in human brain tissue in vivo are
unavailable. In order to test whether measurement of ROS
levels in peripheral cells can be used as an indicator of

Fig. 4. Susceptibility of PS1-expressing splenic lymphocytes toward secondary
insults. ROS formation was quantified by oxidation of DHR in cells from
nontransgenic controls (non-tg) and PS1M146L transgenic mice (PS1M146L)
in (A) aged animals (19 – 22 months of age) and (B and C) middle-aged
animals (13 – 15 months of age). Data are presented as mean fluorescence
intensity levels (MFI) relative to the means of age-matched nontransgenic
control mice (100%). (A) Cells from aged mice react similarly upon serum
withdrawal and additional stimulation with rising concentrations of hydrogen
peroxide for 15 min at 37

-C. Two-way ANOVA: p < 0.01, effect of

PS1M146L transgene; p < 0.0001, effect of hydrogen peroxide stimulation;
interaction n.s. Serum withdrawal *p < 0.05, Student

_s t test, PS1M146L (n =

7) vs non-tg (n = 7). (B) In cells from middle-aged mice, basal levels of ROS
(DHR oxidation) are not significantly different between nontransgenic (n =
22) and PS1M146L transgenic (n = 9) mice. (C) After serum withdrawal for
15 min, more ROS are formed in cells from middle-aged PS1M146L
transgenic mice compared to nontransgenic mice. *p < 0.05, Student

_s t test

PS1M146L (n = 8) vs non-tg (n = 21).

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

855

oxidative stress in brain tissue, we analyzed a possible
correlation between DHR oxidation in peripheral lymphocytes
and HNE levels in brain tissue as markers of oxidative stress in
PS1 transgenic mice and littermate controls. As shown in

Fig.

6

, a significant and positive linear correlation was found

between DHR oxidation in splenic lymphocytes and HNE
levels in brain tissue homogenates in these animals. Hence,
measurement of oxidative stress levels in peripheral cells can
be successfully used as an indicator of central nervous system
oxidative status in our animal model.

Discussion

Oxidative stress in PS1M146L mice is caused by
overproduction rather than insufficient detoxification of ROS

Our results demonstrate the presence of oxidative stress in

PS1M146L transgenic mice as assessed by increased HNE

levels as a marker for lipid peroxidation in brain tissue as well
as increased ROS levels in splenic lymphocytes expressing the
mutant human transgenic protein. Interestingly, the effect of the
FAD mutation is dependent on the natural aging process, as
only aged mice display increased ROS levels and oxidative
damage. This is in close analogy to the situation in humans, in
which carriers of these mutations live without cognitive
disabilities until the disease strikes after some decades

[57]

.

Hence, PS1M146L transgenic mice can represent a suitable
and valuable model to study FAD-relevant pathogenic mechan-
isms in vivo.

Oxidative damage in our PS1M146L transgenic mouse

model parallels previous studies on PS1M146V knock-in mice,
in which synaptosomes display increased levels of oxidatively
modified proteins

[58]

and hippocampal neurons show

increased vulnerability toward Ah-induced cell death mediated
by superoxide radical formation

[20]

. The selectively increased

HNE but not MDA levels in brains from PS1M146L transgenic
mice are furthermore in close analogy to previously published
results from our group on Thy1-APP transgenic mice, a mouse
model of Alzheimer disease based on expression of human
APP with FAD mutations. In brain tissue from these mice only
HNE and not MDA levels were similarly elevated

[31]

, and

mitochondrial dysfunction could be demonstrated

[59]

. In

addition, mitochondrial dysfunction and increased levels of
HNE-modified protein were also reported in a different
PS1M146L transgenic mouse model

[60,61]

. These findings

in animal models furthermore correlate well with a study of
lipid peroxidation levels in blood samples from AD patients, in
which only HNE and not MDA levels were specifically
increased compared to nondemented controls

[62]

. These

results suggest that increased HNE levels are a specific marker
for lipid peroxidation processes in consequence to mitochon-
drial dysfunction and oxidative stress in AD patients, and this
marker can also be detected in relevant transgenic mouse
models of the disease. As several cytotoxic properties have
been described for HNE

[63]

, it is feasible that HNE can cause

neurodegeneration and cognitive deficits, which were de-
scribed in mutant PS1 transgenic mice

[24]

.

The causal mechanism for oxidative stress in PS1M146L

transgenic mice may not stem from an impairment of

Fig. 6. In aged mice (19 – 22 months of age), ROS levels (DHR oxidation) in
splenic lymphocytes are positively correlated with brain tissue levels of lipid
peroxidation (HNE). n = 18 nontransgenic controls (non-tg), n = 10 PS1wt
mice, and n = 9 PS1M146L mice were studied. Correlation calculated by
linear regression in all samples (n = 37): p < 0.05, Pearson

_s correlation

coefficient r

p

= 0.3376.

Fig. 5. Increased ROS formation (DHR oxidation) correlated with apoptotic cell
death induced by the PS1M146L mutation in CD4

+

T lymphocytes from aged

mice (19 – 22 months of age). (A) The PS1M146L mutation increases ROS
formation (DHR oxidation) independent of the cell type. Increased mean
fluorescence intensity (MFI) after staining with DHR was detected in CD3

+

cells

(total T cells) and in the subpools of CD4

+

and CD8

+

T cells from PS1M146L

mice. CD4

+

T cells accumulate higher ROS levels compared to CD8

+

T cells.

Two-way ANOVA in CD4

+

and CD8

+

cells: p < 0.01, effect of PS1M146L

transgene; p < 0.0001, difference between CD4

+

and CD8

+

subsets; interaction

n.s. *p

 0.05, Student

_s t test, PS1M146L (n = 7) vs non-tg (n = 7) in the same

subset (CD3

+

, CD4

+

); (*)p = 0.07, Student

_s t test, PS1M146L (n = 7) vs non-tg

(n = 6) in CD8

+

subset; ++p < 0.01, Student

_s t test, CD4

+

cells vs CD8

+

cells

from nontransgenic mice. (B) Percentage of apoptotic cells in splenic
lymphocytes from non-tg (n = 7) and PS1M146L transgenic mice (n = 7). Both
CD3

+

and CD4

+

T lymphocytes from PS1M146L mice exhibit increased levels

of apoptosis, but differences are not statistically significant. (C) Correlation
between DHR oxidation and percentage of apoptotic cells in lymphocyte samples
isolated from the same individual animal. Correlation calculated by linear
regression: PS1M146L transgenic mice (n = 7), p < 0.05, Pearson

_s correlation

coefficient r

p

= 0.8134; non-tg mice (n = 7), p = 0.05, Pearson

_s correlation

coefficient r

p

= 0.7466; all (non-tg and PS1M146L mice, n = 14), p < 0.01,

Pearson

_s correlation coefficient r

p

= 0.7548.

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

856

antioxidant defenses but rather from an increased production of
ROS. This is supported by our observations that activities of
several antioxidant enzymes in brain tissue—Cu/Zn-dependent
superoxide dismutase, glutathione peroxidase, and glutathione
reductase—are unchanged. Additionally, in vitro stimulation of
lipid peroxidation in brain homogenates by addition of ferric
iron, which induces hydroxyl radical formation via Haber –
Weiss and Fenton reactions

[64]

, did not lead to a different

extent of MDA formation in PS1 transgenic animals and
controls. Additionally, no exacerbation of ROS formation
could be detected in splenic lymphocytes after oxidative
stimulation with hydrogen peroxide, which leads to hydroxyl
radical formation via Fenton reactions and mitochondrial
damage

[65]

. Hence, an impairment of antioxidant defense

mechanisms as underlying causal mechanism for oxidative
stress in PS1M146L mice seems unlikely.

In contrast, impairment of antioxidant enzymes could be

identified in previous studies in PS1M5 mice bearing mutant
human presenilin with five different FAD mutations

[34]

and in

Thy1-APP transgenic mice

[31]

. Thy1-APP transgenic mice

accumulate high levels of Ah with aging, and the presence of
multiple FAD mutations in mutant PS1 shows additive effects
on Ah formation

[66]

. Therefore, in PS1M5 and in Thy1-APP

transgenic mice, high levels of Ah may lead to impairment of
antioxidant defenses—possibly via an interaction with trace
elements essential for antioxidant activity

[33]

. In contrast,

lower levels of Ah formation may not be sufficient to elicit
significant impairment of antioxidant defenses, and such a
deficit was also not identified in PS1M146L mice. Neverthe-
less, we found markers of oxidative stress in PS1M146L
transgenic mice, suggesting that already low levels of Ah can
have deleterious effects—apart from the impairment of
antioxidant enzymes.

Of note, PS1M146L mice do not develop amyloid plaques

in brain tissue with aging—similar to other mutant PS1
transgenic mice

[22,67]

. Therefore, the presence of Ah

deposits is not necessarily a prerequisite for triggering
oxidative damage. This is in accordance with studies on human
brain tissue, which suggest that oxidative stress is a very early
event in AD patients—whereas amyloid plaques form at later
stages

[68,69]

. Also, oxidative damage could be identified in

Thy1-APP transgenic mice from as early as 3 months of age

[31]

. These findings support the notion that already low levels

of Ah can result in oxidative stress—even in the absence of
amyloid plaques. Moreover, neurodegeneration was identified
in mutant PS1 transgenic mice not showing amyloid plaque
deposition

[22,23]

and in transgenic mice expressing mouse

amyloid h

[70]

, suggesting that the formation of mouse

amyloid h itself does not require the deposition of amyloid
plaques for provoking deleterious effects.

Aging sensitizes toward the effect of the PS1 mutation

Of note, the aging process sensitizes toward the deleterious

effects of the PS1 mutation: whereas no differences in HNE
levels were found in brain tissue from young (3 – 4 months) or
middle-aged animals (13 – 15 months), aged PS1M146L

transgenic mice (19 – 22 months) displayed increased HNE
levels. This is paralleled by the alterations observed in splenic
lymphocytes from these mice: whereas no differences in ROS
levels were observed in cells derived from young animals,
neither under basal nor under stimulated conditions, lympho-
cytes from middle-aged PS1M146L animals showed unaltered
basal ROS levels but increased ROS levels after serum
deprivation, which represents a mild stress condition leading
to apoptosis induction in lymphocytes

[71,72]

. Finally, in cells

from aged animals, even basal levels of ROS were increased.
Thus, the aging process seems to sensitize toward the ROS-
elevating effect of the PS1M146L mutation. Of note, the
presence of PS1wt in transgenic mice did not alter levels of
lipid peroxidation products in brain tissue nor ROS levels in
lymphocytes, suggesting that oxidative stress is specifically
caused by the presence of the M146L mutation. It can be
speculated that the aging process triggers the effect of the PS1
mutation. A slight but not significant elevation of mitochon-
drial ROS levels was observed in murine splenic lymphocytes
during aging (data not shown), and other groups have reported
increased levels of oxidative damage in lymphocytes from aged
rodents and humans

[73,74]

. Thus, an elevation of ROS levels

with aging may lower the threshold of oxidative homeostasis,
leading to exaggerated ROS accumulation in PS1M146L-
expressing cells from aged mice.

Specific elevation of mitochondrial and cytosolic ROS but not
superoxide radicals caused by the PS1 mutation

As various ROS are produced in living cells and can interact

in complex reactions, we additionally stained lymphocytes with
several different ROS-sensitive dyes for an estimation of the
exact type and the source of ROS that accounts for increased
DHR oxidation caused by PS1M146L. The increased oxidation
of DHR (142.1% relative to controls) is indicative of elevated
mitochondrial ROS levels

[44,75]

. Furthermore, cytosolic ROS

levels measured by oxidation of H

2

DCF-DA

[48]

were also

increased in PS1M146L mice (120.5% relative to controls).
However, superoxide levels as detected by DHE oxidation and
nitric oxide levels as assessed by DAF-2-DA oxidation were
not altered, suggesting that peroxynitrite is not a major
contributor to the increased DHR and H

2

DCF-DA oxidation

in lymphocytes from PS1M146L transgenic mice.

The relatively higher rate of DHR oxidation (142.1%

relative to controls) compared to H

2

DCF-DA oxidation

(120.5% relative to controls) suggests that ROS are primarily
produced by mitochondria in PS1M146L-expressing cells.
Mitochondrial ROS formation may be a consequence of
deleterious effects of Ah on mitochondria as described
previously

[59,76]

, which is supported by findings that Ah

and both APP and active g-secretase complexes containing PS1
can localize to mitochondria

[77 – 79]

. Mitochondrial ROS

formation in PS1M146L transgenic mice is in close analogy to
other reports of mitochondrial toxicity and oxidative stress in
several AD mouse models based on APP mutations

[31,59,80,81]

and double transgenic APP/PS1 mice

[82]

.

Moreover, mitochondrial dysfunction was identified in cybrids

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

857

bearing mitochondrial DNA from AD patients

[83]

. These

findings and our observation that mitochondrial ROS formation
can also be detected in PS1M146L transgenic mice add further
importance to hypotheses proposing mitochondrial dysfunction
as central to the pathogenesis of AD

[84,85]

.

Mitochondrial membrane potential was not disrupted in

PS1M146L cells, as R123 uptake into mitochondria was even
slightly but significantly increased. This is consistent with
previous findings from our group in which PC12 cells
transfected with FAD mutant APP display an increased
mitochondrial membrane potential

[59]

, which can be consid-

ered a compensatory reaction to deleterious effects of Ah on
mitochondria. Furthermore, cultured cells from AD patients
bearing PS1 mutations do not show a decreased mitochondrial
membrane potential

[86]

. The extent of increased R123

incorporation into mitochondria at 7.2% relative to controls
is, however, not sufficient to explain the 42.1% increase in
DHR fluorescence. Therefore, the major part of increased DHR
fluorescence must be due to oxidation of the dye.

Functional relevance of mitochondrial ROS (DHR oxidation)
for apoptotic cell death in CD4

+

lymphocytes from PS1M146L

mice

As previous studies from our group and others had

identified a higher vulnerability of the CD4

+

lymphocyte

subset to oxidative stress and apoptosis during aging in
PS1M146L transgenic mice and in AD patients

[35,87,88]

,

we analyzed specific T lymphocyte subsets from transgenic
mice for a putatively different formation of ROS. The effects of
the PS1 mutation were not different in CD4

+

or CD8

+

lym-

phocyte subsets, suggesting that the transgene leads to similar
effects independent of the cell type. However, we could
observe that DHR oxidation was specifically higher in the
CD4

+

lymphocyte subset compared to CD8

+

lymphocytes, and

DHR oxidation was positively correlated with cell death in
CD4

+

cells. Hence, in CD4

+

lymphocytes, increased mito-

chondrial ROS formation may trigger apoptosis. This is in
good accordance with the important role of mitochondria as the
central executioners in apoptotic cell death signaling

[89,90]

.

Mitochondrial damage can trigger apoptosis—probably via
glutathione depletion

[91]

—followed by oxidation of mito-

chondrial thiols and loss of mitochondrial membrane potential

[92]

, providing a functional link between increased mitochon-

drial DHR oxidation in CD4

+

lymphocytes and apoptosis.

Although it is often difficult to determine whether ROS

initiate apoptosis or are a by-product of the apoptotic process,
our results suggest that increased ROS levels are a trigger of
apoptotic cell death in lymphocytes in our experiments. First,
only viable lymphocytes, not apoptosing cells, were gated by
flow cytometry analysis for quantification of ROS levels.
Second, a decrease in mitochondrial membrane potential has
been consistently reported to result in overproduction of
superoxide radicals

[93,94]

. However, we did not detect

increased superoxide formation in PS1M146L-expressing cells
as evidenced by unaltered DHE oxidation. Furthermore, R123
uptake into mitochondria, which depends upon maintained

mitochondrial membrane potential, was not impaired. For these
reasons, it is unlikely that increased DHR oxidation observed
in splenic lymphocytes from PS1M146L transgenic mice is a
secondary event due to loss of mitochondrial membrane
integrity and apoptotic processes. Rather, increased ROS
formation can be considered the trigger for apoptosis in
PS1M146L-expressing CD4

+

lymphocytes.

Use of peripheral cells to study effects in brain tissue

So far, numerous studies have been conducted on peripheral

cell models like lymphocytes, platelets, fibroblasts, or olfactory
neurons from Alzheimer disease patients, in which increased
apoptosis and markers of oxidative damage could be identified

[35,83,88,95 – 100]

. These results suggest that inherent Alzhei-

mer-specific properties of cells exist—independent of the cell
type—which can become evident also in the periphery.
Moreover, cybrid cell lines containing mitochondrial DNA
derived from platelets of AD patients show mitochondrial
defects and increased ROS production

[101]

, suggesting that it

is the mitochondria from AD patients which harbor the relevant
defects. As any viable cell contains mitochondria, peripheral
cells may therefore be used to monitor pathogenic events in
brain tissue. However, evidence for such a correlation between
peripheral cells and central nervous system tissue in humans is
currently lacking. Our data in PS1M146L transgenic mice
show that DHR oxidation in splenic lymphocytes was
positively correlated with HNE levels in brain tissue from
each individual animal. Although direct assessment of HNE
levels in splenic lymphocytes was precluded due to the need
for high tissue amounts, the correlation between peripheral
ROS and brain tissue HNE levels suggests that oxidative stress
in brain tissue can be mirrored in peripheral cells. This adds
further importance and relevance to the numerous studies
conducted on peripheral cell models in neurodegenerative
diseases so far. Moreover, as DHR oxidation detects increased
mitochondrial ROS levels, it can be speculated that mitochon-
drial ROS formation underlies the formation of HNE in
PS1M146L mice. This would be in analogy to previous studies
from our group on Thy1-APP transgenic mice as a different
mouse model of AD. In these mice, similarly increased levels
of HNE

[31]

, reactive oxygen species

[80]

, and mitochondrial

dysfunction

[59]

could be detected.

Summary of mutant PS1-induced effects

In summary, our results on changes in oxidative stress

parameters observed in brain tissue and peripheral cells support
the hypothesis of oxidative toxicity caused specifically by the
mutant PS1M146L compared to PS1wt transgenic or nontrans-
genic mice. Moreover, oxidative toxicity of the PS1M146L
mutation is evident only in aged animals, arguing for an
additive effect of aging—the most important risk factor for the
development of sporadic AD—and the FAD mutation on
elevated sensitivity toward ROS accumulation. In brain tissue,
increased lipid peroxidation as assessed by elevated HNE
levels could be observed in aged PS1M146L mice. In parallel,

K. Schuessel et al. / Free Radical Biology & Medicine 40 (2006) 850 – 862

858

mitochondrial ROS were 42.1% and cytosolic ROS 20.5%
higher in PS1M146L cells relative to cells from nontransgenic
littermate control mice, pointing to mitochondria as the main
source of ROS. Increased mitochondrial ROS formation may
trigger apoptotic cell death in vulnerable cells, as DHR
oxidation and levels of apoptosis were positively correlated
in CD4

+

cells. The mechanism of ROS accumulation in

PS1M146L transgenic mice is probably related to increased
ROS formation but not impaired detoxification, as we observed
neither an impairment of antioxidant enzyme activities in brain
tissue nor a different susceptibility toward oxidative stimuli in
brain tissue and in lymphocytes.

In conclusion, our results support the hypothesis that the

combined effects of aging and the presence of PS1 mutations
can lead to oxidative stress, which may be a causative event for
triggering neurodegeneration in familial AD patients. Further
studies will be conducted to enlighten the exact mechanisms
underlying the oxidative stress cascade in this model.

Acknowledgments

We thank Dr. Christian Czech (Hoffmann – La Roche AD,

Basel, Switzerland) and Dr. Laurent Pradier (Sanofi-Aventis,
Vitry-sur-Seine, France) for providing the PS1 transgenic
mouse models. This work was supported by grants from
Alzheimer Forschung Initiative (No. 01808) and from Hes-
sisches Kultusministerium (K.S.).

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