Examining the levels of ganglioside and cholesterol in cell
membrane on attenuation the cytotoxicity of beta-amyloid
peptide
Ming-Shen Lin1, Liang-Yu Chen2, Steven S.-S. Wang3, Yung Chang4 and Wen-Yih Chen1*
1
Department of Chemical and Materials Engineering, National Central University, Jhong-Li,
Taiwan 320
2
Department of Biotechnology, Ming-Chuan University, Gui-Shan, Taiwan 333.
3
Department of Chemical Engineering, National Taiwan University,
Taipei, Taiwan 10617
4
Center for Membrane Technology and Department of Chemical Engineering, Chung
Yuan Christian University, Jhong-Li, Taiwan 320
Submitted to:
Neuropeptides
*Corresponding Author
E-mail: [email protected]
Tel: (886) 3-422-7151 x 34222
Fax: (886) 3-422-5258
Αbstract
The deposition of β-amyloid (Aβ) on cell membranes is considered as one of the primary
factors in having Alzheimer’s disease (AD). Recent studies have suggested that certain
components of plasma membrane, ganglioside and cholesterol could accelerate the
accumulation of Aβ on the plasma membranes. However, the effect of cholesterol and
ganglioside (GM1) on Aβ cytotoxicity is still a controversial issue. The aim of this study was
to understand the roles of GM1 and cholesterol in AD by using PC12, a neuron-like cell. The
effects of the sequence, conformation, and concentration of Aβ on cytotoxicity were also
investigated. Monomeric Aβ could attack the plasma membrane resulting in cytotoxicity,
however, fibril Aβ was found to be less toxic. Our results showed that Aβ (1-40) was more
toxic than Aβ (25-35) and the cytotoxcity of Aβ was proportional to its concentration. Besides,
the depletion of GM1 from plasma membrane helped block Aβ-induced cytotoxicity.
Decreasing the cholesterol level by around 30% could attenuate the cytotoxicity of Aβ. These
findings validate our idea that the cholesterol could stabilize the lateral pressure derived from
the formation of GM1-Aβ complex on the membrane surface. Furthermore, both GM1 and
cholesterol are essential in mechanism of Aβ accumulation and could modulate the
cytotoxicity of monomeric Aβ.
Key words：β-amyloid (Aβ), cholesterol, ganglioside, cytotoxicity
Introduction
Alzheimer's disease (AD) is the most common form of senile dementia which affects
approximately 10% of all individuals over 65 years of age and more than 50% of those over
85 (Garber, 2001). It has created a large burden on the health care system in terms of both
services and costs of over $10B per year in the United States (Kar et al., 2004).
AD is one kind of protein conformational diseases which accompany memory and recognition
degeneration. β-amyloid (Aβ) is an amphiphilic peptide responsible for the development of
extracellular senile plaques in the brain which are considered one of the key pathological
hallmarks in AD (Wood et al., 2003). The main constituents of senile plaques are Aβ (1-40)
and Aβ (1-42), which are normally derived from the amyloid precursor protein (APP), a
constituent membrane protein in the brain (Selkoe, 2001). The conversion of nontoxic,
monomeric Aβ, to toxic Aβ rich in β-sheet structure by aggregation is considered to be the
key step in the development of AD (McLean et al., 1999; Lambert et al., 1998).
Several evidences showed that Aβ can directly induce cell death using MTT assay (Wang et
al., 2001; Wang et al., 2005). Moreover, Wirths et al. (Wirths et al., 2007) analyzed axonal
neuropathology in the brain and spinal cord of a transgenic mouse model with abundant
intraneuronal Aβ production and provided compelling evidence for axonal degeneration. In
animal model, Fu et al. (Fu et al., 2006) examined the learning and memory functions in mice
by injecting Aβ. The water maze performance demonstrated that Aβ caused impairments in
memory and cognitive ability. Such findings may relate to a certain component of cell
membrane, such as ganglioside (GM1) (Wang et al., 2001; Kakio et al., 2002; Wakabayashi et
al., 2005) and cholesterol (Wakabayashi et al., 2005; Kakio et al., 2001; Sun et al., 2005),
which induce Aβ to attack the cells leading to cell death and even dysfunction.
GM1 and cholesterol are two important constituents of the plasma membrane. GM1 are
abundant in neuron cells and are involved in important neurobiological events, such as
neurodifferentiation, synaptogenesis and synaptic transmission (Nagai, 1995). Cholesterol is
the most predominant sterol in the plasma membrane which is related to numerous cellular
functions such as lipid fluidity, receptor function, endocytosis, enzyme activity, etc (Liu et al.,
2000; Lundbaek et al., 1996; Chen et al., 2007). Several researches have demonstrated that
monomeric Aβ may preferentially bound to GM1 and serves as a seed on the plasma
membrane, which results in promoting aggregation of other Aβs (ChooSmith et al., 1997;
Yanagisawa et al., 1995; Wakabayashi et al., 2005). However, long-term studies have not
shown a consistent relationship between cholesterol levels in cell membrane and the
cytotoxicity of Aβ. Some researchers have proposed that lower cholesterol level could make
cell more vulnerable to the activity of Aβ (Arispe and Doh, 2006). Others have postulated the
opposite results (Wang et al., 2001; Wang et al., 2005; Schneider et al., 2006). These
contradictions prompted us to analyze the role of neuronal cholesterol and GM1 in AD.
Moreover, the study focused on the relationship between GM1 and cholesterol. It might be
crucial to understand the correlation to the mechanisms of interaction between Aβ and plasma
membrane.
The aim of this study was to understand the roles of ganglioside and cholesterol in AD by
using PC12, a neuron-like cell. The effects of the sequence, conformation, and concentration
of Aβ on the cytotoxicity were also investigated. Furthermore, the causal relationship between
GM1, cholesterol and Aβ was discussed below. Both of these findings may provide the
insights into the roles of ganglioside and cholesterol in the interaction between monomeric Aβ
or aggregated Aβ and cell membranes.
Materials and Methods
Materials. Aβ (25-35) and Aβ (1-40) were obtained from Sigma and Biosource International
(Camarillo, CA), respectively. Horse serum, fetal bovine serum, penicillin and streptomycin
were purchased from Gibco BRL (Gaithersburg, MD). Dulbecco’s Modified Eagle’s Medium
(DMEM), methyl-β-cyclodextrin (MβCD) and all other chemicals were obtained from Sigma.
Water was de-ionized at 18 MΩ and sterile filtered (0.22 μm) before usage.
Peptide preparation. Aβ (25–35) and Aβ (1–40) peptides were prepared analogously to
methods which consistently lead to peptides that are toxic to cultured cells (Lin et al., 2007).
Aβ peptides were dissolved in 1, 1, 1, 3, 3, 3-hexafluor-2-propanol (HFIP) at room
temperature. Thereafter, Aβ solution was dried by using vacuum oven, then desired
concentration was adjusted by adding serum-containing medium. The aggregated Aβ was
incubated by stirring at 50 rpm for 4 days and its conformation was identified by CD. (Fig. 1)
Cell culture and Aβ treatment. Rat pheochromocytoma cells (PC12) were cultured in DMEM
containing 5% (v/v) horse serum, 10% (v/v) fetal bovine serum, 3 mM L-glutamine, 100
units/mL penicillin, and 0.1mg/mL streptomycin in a 5% (v/v) CO2/air environment at 37℃.
For determination of cytotoxicity of Aβ peptides, PC12 were initially plated in 96-well plates
at the density of 10,000 cells/well and maintained for 12 hrs in a complete medium. Cells
were then treated with enzymes to modulate the content of cell plasma. Subsequently, the
cells were incubated with Aβ for 24 hours. The cell viability was analyzed by MTT assay.
Change in membrane cholesterol and synthesis inhibition.
To enrich the cholesterol content of the membrane, PC12 cells were incubated in a
cholesterol-enriched medium (Arispe and Doh, 2006). Water-soluble cholesterol was first
dissolved in de-ionized water at a concentration of 10 mg/mL and added to the culture
medium. Cells were incubated for 2 h in the media containing soluble cholesterol and washed
with cholesterol-free media before the addition of Aβ. Under these conditions, the cellular
cholesterol level was increased to 30% versus that in untreated cells. The cholesterol content
of the PC12 surface membranes was decreased by methyl-β-cyclodextrin. The plated PC12
cells were incubated for 3 h in serum-free medium and then treated with 10 mM MβCD in
serum-free medium for 60 min at 37 °C. The MβCD-containing medium was then removed
from cells and replaced with fresh, serum-containing medium, with the peptide for the MTT
toxicity assays. MβCD has been demonstrated to specifically remove cellular cholesterol
(Wang et al., 2001; Wakabayashi et al., 2005; Sun et al., 2005).Under these conditions, the
cellular cholesterol level was reduced to 30%-40% of that in untreated cells (Sun et al., 2005;
Arispe and Doh, 2006). Alternatively, the plated PC12 cells were treated with 0.2 μg/ml
filipin complex or 1 μM compactin in medium and incubated for 48 h at 37 °C prior to the
peptide addition for the toxicity assays. Filipin has been reported to form complexes with
cholesterol, and compactin has been demonstrated to inhibit cholesterol production (Wang et
al., 2001). Control cells were treated identically except for the presence of peptide.
Sialic acid depletion and ganglioside synthesis inhibition
Membrane-associated sialic acids from gangliosides and cell surface glycoproteins were
removed from cells analogous to the established procedures (Wang et al., 2001). The plated
PC12 cells were incubated for 3 h in a serum-free medium, and then treated with 11.7
milliunits of V. cholerae neuraminidase and 3.3 milliunits of A. ureafaciens neuraminidase in
a serum-free medium for 1 h at 37 °C prior to the toxicity assays of the peptide or epinephrine
addition. To inhibit cellular ganglioside re-synthesis, PC12 cells were treated with 20 μM
fumonisin B1 in medium and incubated for 48 h at 37 °C prior to the peptide addition for the
toxicity assays. Fumonisin B1 has been reported to inhibit cellular ganglioside synthesis.
Control cells were treated identically except for the presence of peptide.
MTT reduction assay. The mitochondrial dehydrogenase activity that reduces 3-(4,
5-dimethyithiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was used to determine
cellular redox activity, an initial indicator of cell death, in a quantitative colorimetric assay.
PC12 cells cultured in 96-well plates at a density of 10,000 cells/well were exposed to
β-amyloid peptide at various concentrations for 24 h. Thereafter, PC 12 cells were incubated
to the growth medium with MTT (5 mg/ml) for 4 h at 37℃. The medium was then aspirated
and the MTT reduction product, formazan, was dissolved in dimethyl sulfoxide (DMSO) and
quantified spectrophotometrically at 540 nm. MTT reduction was expressed as percentage of
control ± S.E.M. from at least five independent experiments.
Results
The effects of sequence, conformation and concentration of Aβ on the cytotoxicity
The cytotoxic effects of Aβ (25-35) and Aβ (1-40) on PC12 cells are shown in Fig. 2. The
unobvious cytotoxicity of Aβ (25-35) and Aβ (1-40) were observed at low Aβ concentrations;
on the contrary, a dramatic increase in cytotoxicity was seen at higher Aβ concentrations. As
the concentration of Aβ (1−40) was elevated to 20 μM and 40 μM, the MTT values were
decreased to 65% and 37%, respectively. Nevertheless, the MTT values were kept at around
80% in 20 μM and 40 μM of Aβ (25-35). The MTT values of Aβ (25-35) and Aβ (1-40)
showed no noticeable difference at the concentration below 10 μM. The results on
cytotoxicity induced by monomeric and aggregated Aβ are shown in Fig. 3. The aggregated
Aβ has almost no toxic effect even at higher concentration of Aβ (1-40). However, the
cytotoxicity was proportional to the concentration of monomeric Aβ of both Aβ (25-35) and
Aβ (1-40). Therefore, we can conclude that the higher concentration of monomeric Aβ (1−40)
could increase the risk for cell damage. For the subsequent studies, we therefore only focus on
MTT-sensitive Aβ (1-40) with 20 μM and 40 μM concentration in the monomeric form.
GM1 blocks Aβ-induced cytotoxicity
GM1 was shown to affect the behavior of monomeric Aβ aggregated on the surface of the
liposomes in our previous kinetic studies (Lin et al., 2007). Therefore, the influence of GM1
on Aβ-induced cytotoxicity must be evaluated. To improve the accuracy, the inhibition of the
GM1 re-synthesized from cells was performed by the addition of fumonisin B1 after depletion
of sialic acid in GM1. As shown in Fig. 4, the reduction in sialic acid content results in
inhibition of cytotoxicity induced by Aβ. In addition, the viability of cell decreases either in
the presence of both sialic acid and cholesterol, or sialic acid alone, indicating that GM1 -- not
either of these two -- is an essential factor in inducing the attack of Aβ on the cell.
Cholesterol modulates the Aβ-induced cytotoxicity
To determine the possible involvement of membrane cholesterol in the interaction between
Aβ and plasma membrane, the modulation of cholesterol content on the plasma membrane
was carried out to analyze the effect of cholesterol on cytotoxicity. Similar to the above
investigation, the inhibition of the cholesterol re-synthesized from the cell was achieved by
the addition of compactin and filipin after extracting the cholesterol by MβCD. Cholesterol is
the predominant sterol in the plasma membrane and is necessary to stabilize the conformation
of plasma membrane expected for cellular function (Liu et al., 2000; Lundbaek et al., 1996;
Chen et al., 2007). Apoptosis or even death may be inevitable due to the excessive extraction
of cholesterol from the surface plasma of cultured PC12 cells, which could affect subsequent
studies. Therefore, the verification of the cell is necessary after the removal of cholesterol.
Experiments were performed to determine the time limits for the safe use of 10 mM MβCD
on PC12 cells. Beyond certain time limit, 10 mM MβCD could affect the viability of PC12.
After exposure of over 60 minutes, 10 mM MβCD became toxic to cells (Fig. 5). Therefore,
to simultaneously perform cholesterol extraction and keep the cell viability over 90% of the
control cells, cells were treated with 10 mM MβCD for 60 minutes.
The effects of cholesterol levels on Aβ-induced cytotoxicity are shown in Fig. 6. After
incubation with 40 μM monomeric Aβ (1-40) for 24 hours, the MTT values for cells with
decreased cholesterol contents was 0.57 and 0.28 for those with increased cholesterol contents.
It implied that reducing cholesterol from plasma membrane could diminish cytotoxicity
induced by monomeric Aβ; inversely, higher cholesterol content would cause higher
cytotoxicity. Besides, there were no significant difference in the effect of cholesterol levels on
the cytotoxicity of aggregated Aβ as evident by MTT values that were all in the range of 91%.
Discussion
In our previous liposomal studies, the analysis of bio-molecular interactions between the
β-amyloid and liposomes was characterized by surface plasmon resonance (SPR) and
isothermal titration calorimetry (ITC) (Lin et al., 2007). During Aβ adsorb to the plasma
membrane, both cholesterol and GM1 have essential roles. The present study was able to
verify the previous assumption based on in vitro data that reveals that the reduction of
cholesterol and GM1 levels also affected the interaction between Aβ and cell plasma. Recent
studies suggested that Aβ may not be toxic when deposited in amyloid plaques; instead, its
neurotoxic properties might be attributed to the oligomeric or prefibrillar Aβ aggregates
(McLean et al., 1999; Lambert et al., 1998). This notion was supported by our observation
that monomeric Aβ could cause significant cytotoxicity. Synapse failure before neuron death
has been considered to be caused by the accumulation of Aβ oligomers rather than amyloid
fibrils (Kayed et al., 2003). Therefore, cell functions might degrade as monomeric Aβ
aggregate to form oligomer. Consistent with our previous kinetics data, monomeric Aβ has a
high affinity constant for interaction with the cell membrane, which implies that there is
easier adsorption of monomeric Aβ.
Besides, the length of amino acid sequence and the concentration were two important factors
that greatly affect the aggregation rate. The longer the sequence of Aβ, the higher the
potential it was for forming the toxic conformation due to the presence of the more
hydrophobic amino acid in the C-terminal. The probability of intermolecular collision could
increase at higher Aβ concentration, accelerating the formation of certain toxic and
subsequent cell death. Thus, the significant cytotoxicity was seen in longer sequence and
higher concentration such as 40 μM Aβ (1-40).
So far, the exact mechanisms of AD are still not fully addressed and may be one of the factors
resulting in the non availability of efficient drugs. However, increasing evidence indicated
that the neuron cell membrane is important in the mechanism of Aβ cytotoxicity. Studies have
reported that membrane constituents, cholesterol and GM1, could alter the affinity of Aβ for
plasma membranes (Selkoe, 2001; Selkoe, 1999; Hardy, 1997). Monomeric or oligomeric Aβ
could aggregate on the cell membrane by electrostatic attractive force due to the negative
charge of sialic acid in GM1. The presence of membrane could accelerate Aβ aggregation. In
addition, GM1-Aβ complex that was formed as a seed initiates a chain reaction to promote
other Aβ to accumulate on the plasma membrane (Yanagisawa et al., 1995). Therefore,
decreasing the amount of sialic acid by neuraminidase could effectively reduce Aβ
cytotoxicity (Wang et al., 2001). Unfortunately, synaptic plasma membranes have a relatively
high concentration of GM1 with respect to other cellular membranes, and the loss of synapses
is highly correlated with the degree of AD(Iqbal and Grundke-Iqbal, 2002).
Cholesterol has demonstrated that it could modulate Aβ cytotoxicity. However, long-term
studies have not observed a consistent relationship between cholesterol levels in cell
membrane and the cytotoxicity of Aβ. In this study, changes in cholesterol levels in plasma
membranes were correlated to Aβ cytotoxicity. A decrease of around 30% cholesterol level
could reduce Aβ cytotoxicity; likewise, an increase of around 30% cholesterol level could
induce higher Aβ cytotoxicity. From in vitro studies, Aβ might be induced to form fibril and
cholesterol aggregation causing “phase separation” at high cholesterol level membranes (Ji et
al., 2002), which rendered the cell so unstable that apoptosis or cell death was inevitable.
Furthermore, as the steady state levels of all peptides in vivo are a direct consequence of the
balance between their anabolism and catabolism, peptide accumulation can arise not only
from increased production but also from less frequent breakdown. Cholesterol is related with
Aβ generation and Aβ clearance (Carson and Turner, 2002). Arispe and Doh (Arispe and Doh,
2006) revealed that PC12 cells become resistant to the cytotoxic action of Aβ when incubated
in a medium that enriches cholesterol levels of the surface membrane. These adverse findings
were seen at lower Aβ concentration. As such, the accumulation rate of seed formation is
reduced when the catabolic rate on the cell membrane is higher than the accumulation rate.
This results in catabolization of Aβ on the plasma membrane to reduce cytotoxicity. However,
in this study at higher concentration, monomeric Aβ aggregation on the plasma membrane
readily acted as seeds that increased the accumulation rate, resulting in cytotoxicity by large
quantity of Aβ accumulated on the plasma membrane. This may explain the differences in our
results as compared to those reported in past literature.
Few researches reported on the relationship between GM1, cholesterol, and Aβ, though GM1
and cholesterol were considered as key factors in AD. Wakabayashi et al. (Wakabayashi et al.,
2005) reported that cells with decrease GM1 block the interaction of Aβ and makes the
distribution of GM1 less uniform, resulting in a few of the Aβ accumulation on the
cholesterol-depleted cell membrane, as observed from the confocal laser microscopy. We also
utilized the NIMA trough with fluorescence microscopy to observe the aggregation behavior
in GM1 and cholesterol that were induced by Aβ (data not published). These and previous
findings supported our notions (Fig. 7) as follows: Firstly, monomeric Aβ preferentially binds
to and accumulates on GM1-rich domain in plasma membrane. Secondly, the Aβ-GM1
complex attracts other GM1 to form GM1 cluster. Finally, as the GM1 cluster incurred on the
membrane it causes the lateral pressure to rise; cholesterol is then recruited by the cell to form
the raft-like structure to stabilize the pressure and the conformation of the membrane.
Therefore, GM1 and cholesterol is necessary in Aβ adsorption process. Understanding the
insight of the relationship between GM1, cholesterol, and Aβ might prove useful in
developing medicines and strategies aimed to cure AD.
Conclusion
The apparent discrepancy inspired us to analyze not only the roles of GM1 and cholesterol in
AD but also the quantitative relationships among GM1, cholesterol, and Aβ. The results
reported here demonstrated that the GM1- and cholesterol-decreasing treatment could
attenuate the Aβ cytotoxicity. Besides, the semi-quantitative relationship in the interactions
among GM1, cholesterol, and Aβ might be a critical clue to the development of therapeutic
strategy in AD.
Acknowledgments
This work was supported by the National Science Council of the Republic of China via grant
(for W.Y. Chen) under the contract No. NSC-95-2221-E-008-086.
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Figure Legend
Fig. 1 CD spectra of 20μM Aβ(1-40) incubated in phosphate buffer, pH7 at 37oC.
Fig. 2 Effect of the concentration of Aβ on cell viability.
Fig. 3 Effect of the conformation of Aβ on cell viability. Treatment 1: 20μM Aβ(1-40);
Treatment 2: 40μM Aβ(1-40); Treatment 3: 20μM Aβ(25-35); Treatment 4: 40μM Aβ(25-35)
Fig. 4 Effect of GM1 and cholesterol depletion on the viability of fresh Aβ(1-40) treated
PC12 cell. Treatment 1: control; Treatment 2: GM1 depletion; Treatment 3: GM1 and
cholesterol depletion
Fig. 5 Effect of 10mM MβCD extract on cell viability assayed by MTT test.
Fig. 6 Effect of the level of cholesterol on fresh Aβ(1-40)−treated PC12 cells. Treatment 1:
40μM Aβ(1-40); Treatment 2: 20μM Aβ(1-40); Treatment 3: 40μM aggregated Aβ(1-40);
Treatment 4: 20μM aggregated Aβ(1-40)
Fig. 7 Schematic presentation of the interactions among GM1, cholesterol and Aβ.
Fig. 1
Fig. 2
1.2
Aβ(1-40)
Aβ(25-35)
MTT Reduction
1.0
0.8
0.6
0.4
0.2
0.0
0
1
5
10
Αβ [μΜ]
20
40
Fig. 3
1.2
MTT Reduction
1.0
0.8
0.6
0.4
0.2
0.0
1
Fresh
Aggregated
2
3
Treatment
4
Fig. 4
1.2
MTT Reduction
1.0
20uM
40uM
0.8
0.6
0.4
0.2
0.0
1
2
Treatment
3
Fig. 5
120
MTT Reduction [%]
100
80
60
40
20
0
10
30
60
90
120
time of exposure to MβCD [min]
300
Fig. 6
1.2
MTT Reduction
1.0
increased chol.
normal cell
decreased chol.
0.8
0.6
0.4
0.2
0.0
1
2
3
Treatment
4
Fig. 7