Potential pathogenic role of Я-amyloid 1–42–aluminum complex in

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Available online at www.sciencedirect.com
The International Journal of Biochemistry & Cell Biology 40 (2008) 731–746
Potential pathogenic role of ␤-amyloid1–42–aluminum
complex in Alzheimer’s disease夽
Denise Drago a , Mikol Bettella b , Silvia Bolognin a , Laura Cendron c ,
Janez Scancar d , Radmila Milacic d , Fernanda Ricchelli a , Angela Casini e ,
Luigi Messori e , Giuseppe Tognon a , Paolo Zatta a,∗
a
e
CNR-Institute for Biomedical Technologies, Padova “Metalloproteins” Unit, Department of Biology,
University of Padova, Viale G. Colombo, 3-35121 Padova, Italy
b Department of Pharmacy, University of Padova, Italy
c Department of Chemistry, University of Padova, Italy
d Department of Environmental Sciences, Jozef Stefan Institute, Jamova 39-1000, Ljubljana, Slovenia
Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy
Received 6 August 2007; received in revised form 9 October 2007; accepted 9 October 2007
Available online 22 October 2007
Abstract
The etiopathogenesis of Alzheimer’s disease is far from being clearly understood. However, the involvement of metal ions as a
potential key factor towards conformational modifications and aggregation of amyloid is widely recognized. The aim of the present
study is to shed some light on the relationship between metal ions, amyloid conformation/aggregation, and their potential relationship
with the conformational aspects of AD. We compare the effects of ␤-amyloid1–42 and its various metal complexes (␤-amyloid–Al, ␤amyloid–Zn, ␤-amyloid–Cu, ␤-amyloid–Fe) in human neuroblastoma cells in terms of cell viability, membrane structure properties,
and cell morphology. No significant toxic effects were observed in neuroblastoma cells after 24 h treatment both with ␤-amyloid and
␤-amyloid–metals (␤-amyloid–Zn, ␤-amyloid–Cu, ␤-amyloid–Fe); on the other hand, there was a marked reduction of cellular viability after treatment with ␤-amyloid–Al complex. In addition, treatment with ␤-amyloid–Al increased membrane fluidity much more
than other ␤-amyloid–metal complexes, whose contribution was negligible. Furthermore, the cellular morphology, as observed by
electron microscopy, was deeply altered by ␤-amyloid–Al. Importantly, ␤-amyloid–Al toxicity is closely and significantly associated
with a great difference in the structure/aggregation of this complex with respect to that of ␤-amyloid alone and other ␤-amyloid–metal
complexes. In addition, ␤-amyloid, as a consequence of Al binding, becomes strongly hydrophobic in character. These findings show
a significant involvement of Al, compared to the other metal ions used in our experiments, in promoting a specific amyloid1–42 aggregation, which is able to produce marked toxic effects on neuroblastoma cells, as clearly demonstrated for the first time in this study.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Alzheimer; Aluminum; Metal ions; Membranes; Neuroblastoma; Fibrils
Abbreviations: AD, Alzheimer’s disease; A␤, ␤-amyloid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DPH, 1,6diphenyl-1,3,5-hexatriene; TMA-DPH, N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) phenylammonium p-toluenesulfonate; ETASS, electrothermal atomic absorption spectrometry; FAAS, flame atomic absorption spectrometry; TEM, transmission electron microscopy; SEM, scanning
electron microscopy; SEC, size exclusion chromatography; HFIP, hexafluoroisopropanol.
夽 This work was supported in part by grant from Italian Ministry of Research and University FIRB # RGNEO3PX83.
∗ Corresponding author. Tel.: +39 049 8276331; fax: +39 049 8276330.
E-mail address: zatta@mail.bio.unipd.it (P. Zatta).
1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2007.10.014
732
D. Drago et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 731–746
1. Introduction
Alzheimer’s disease (AD) is characterized, among
other pathological features, by amyloid plaques and
the formation of “tangles” as a peculiar specificity
of this devastating syndrome. Senile plaques (SP) are
made up mainly of ␤-amyloid (A␤) peptide accumulation in its fibrillar form, concomitantly with metal
ions accumulation and the presence of various elements
from the immuno-response system (Lovell, Ehmann,
& Markesbery, 1993; Lovell, Robertson, Teesdale,
Campbell, & Markesbery, 1998). In vitro studies have
suggested that the observed A␤ neurotoxicity might be
a consequence of an amyloid fibrillar aggregation state
(Pike, Burdick, Walencewicz, Glabe, & Cotman, 1993).
More recently it has been suggested that A␤-soluble
(A␤s) oligomers might be the principal neurotoxic agent
(Cleary et al., 2005; Dahlgren et al., 2002; Finder &
Glockshuber, 2007; Kayed et al., 2003). In this connection, enormous efforts have been made to identify
which of the various forms of A␤ found in the brains
of AD patients could be most important in inducing
the neuropathological changes and neurological clinical
symptoms that characterize this disease.
Currently, several laboratories are focusing extensive research on attempting to understand the chemical
structure/conformation, of A␤s species, as a important
element in the etiopathogenesis of AD (Deshpande,
Mina, Glabe, & Busciglio, 2006; Oddo et al., 2006). A␤s
from the cerebrospinal fluid (CSF) of AD patients, have
been shown to be neurotoxic in character at very low
concentrations, and at the same time, capable of inducing
marked alterations in neuronal long-term potentiation as
well as cognitive impairment (Lesnè et al., 2006). The
aggregation/oligomerization of A␤ has been the subject of numerous studies, mainly in vitro, employing
a variety of experimental approaches (Chen & Glabe,
2006) including the use of transgenic animals (Oddo et
al., 2006). The pivotal event in the amyloid aggregation
appears to be the protein misfolding that drives the peptides towards a ␤-sheet structure formation, which result
in the ability of amyloid to aggregate in an infinitely
propagating fashion. Such protein misfolding, associated with A␤ aggregation, is greatly affected by various
biophysical and chemical factors including metal ions
which have been found in high concentration in the core
and the rim of the SP in the AD brain (Beauchemin
& Kisilevsky, 1998; Dong et al., 2003; Lovell et al.,
1993, 1998; Miu & Benga, 2006). Metal ions have
been widely demonstrated to be implicated as potential risk cofactors in several neurodegenerative disorders
(Liu et al., 2006; Zatta, 2003). Several recent studies
reported that some metals are able to accelerate the
dynamic of A␤ aggregation, thus increasing the neurotoxic effects on neuronal cells as a consequence of
marked biophysical alterations properties of the peptide
(Bush, 2003; Ricchelli, Drago, Filippi, Tognon, & Zatta,
2005).
According to some authors (Bush, 2003; House et
al., 2004), zinc (Zn), copper (Cu) and iron (Fe) are found
markedly concentrated in the cerebral A␤ deposits, leading to the final formation of A␤ aggregation. It is worth
noticing that in human brains and in amyloid transgenic
mice the chelation of these metal ions could reverse the
A␤ peptide aggregation dissolving amyloid aggregates
(Cherny et al., 2001, 1999). Moreover, in the past years,
many hostilities rejected the possible role of aluminum
(Al) in the aetiology or pathogenesis of Alzheimer’s
disease and this issue has never been resolved properly. However, since long time Al concentration in the
brain of Alzheimer’s disease patients has been analytically well established (Beauchemin & Kisilevsky, 1998;
Candy et al., 1986; Good, Perl, Bierer, & Schmeidler,
1992; Walton, 2006). Recently, the controversial issue of
the role played by Al in the aetiology of Alzheimer’s disease has been renewed following numerous experiments,
albeit with conflicting results (Munoz, 1998; Zatta, 1993;
Zatta, Lucchini, Van Rensburg, & Taylor, 2003). Thus,
the possible link between Al and AD remains on the
other hand, still controversial along with many other
hypotheses on AD aetiology (see alzforum.com), but
at the same time of great current interest (Bala Gupta
et al., 2005; Miu & Benga, 2006; Walton, 2006; Zatta,
2006).
The complexity of defining the etiopathogenesis of
AD clearly demonstrates that, in spite of numerous interesting results obtained so far, our navigation in the vast
sea of AD remains fogbound. Recent studies, from our
laboratory and others, have clearly demonstrated that of
the various metal ions, Al appears to be the most efficient
cation in promoting A␤ aggregation in vitro increasing A␤ neurotoxicity dramatically (Kawahara, Kato, &
Kuroda, 2001; Kawahara, Muramoto, Kobayashi, Mori,
& Kuroda, 1994; Ricchelli et al., 2005). Furthermore,
the marked involvement of Al in human A␤ aggregation,
compared to that observed for the rat A␤–Al complex in
terms of increased toxicity in endothelial cells, clearly
showed the peculiarity of the effects of the human A␤–Al
complex (Drago et al., 2007).
The aim of this paper is to shed some light on the
relationship between metal ions, amyloid conformation/aggregation, and their potential relationship with the
conformational aspects of AD. Our findings show for the
first time that each single metal ion (Zn, Fe, Cu, Al) can
affect A␤ oligomerization in a specific way. Finally, we
also describe how A␤–metal complexes can contribute
to membrane dysfunctions and neurodegeneration.
2. Materials and methods
2.1. Chemicals
Synthetic A␤1–42 was purchased from Biosource
(Camarillo, CA, USA). Al(C3 H5 O3 )3 , CuSO4 , ZnCl2 ,
FeCl3 , acridine orange (AO) and propidium iodide (PI)
were obtained from Sigma–Aldrich (St. Louis, MO).
Hexafluoroisopropanol (HFIP), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,6diphenyl-1,3,5-hexatriene (DPH) and N,N,N-trimethyl4-(6-phenyl-1,3,5-hexatrien-1-yl) phenylammonium ptoluenesulfonate (TMA-DPH) were obtained from
Sigma–Aldrich.
2.2. Cell cultures
SH-SY5Y human neuroblastoma cells were purchased from ECACC (European Collection of Cell
Culture, Salisbury, UK). SH-SY5Y human neuroblastoma cells were maintained in Dulbecco’s modified
Eagle’s medium (MEM):F-12 (1:1) with l-glutamine
and 15 mM Hepes (Gibco, Carlsbad, CA, USA) at
37 ◦ C with 5% CO2 in a humidified atmosphere
(90% humidity). The medium was replaced every 2
days. Penicillin (100 units/ml; Gibco) and streptomycin
(100 ␮g/ml; Gibco), 15% fetal bovine serum (FBS;
Sigma–Aldrich) and MEM non-essential amino acids
(100×; Sigma–Aldrich) were added to the medium.
0.25% trypsin–EDTA solution and phosphate buffered
saline (PBS) were obtained from Sigma–Aldrich.
2.3. Preparation of Aβ–metal complexes
1.0 mg of synthetic A␤1–42 was dissolved to 1 mM in
hexafluoroisopropanol for 40 min at room temperature.
After this incubation, the A␤1–42 solution was separated
into aliquots in microcentrifuge tubes. Hexafluoroisopropanol was removed under vacuum in a Speed Vac
(Sc110 Savant Instruments) and lyophilized peptide film
was stored desiccated at −20 ◦ C. This treatment with
HFIP was repeated three times in order to destroy the
peptide’s pre-existing structure during synthesis. All
work with HFIP was done in a chemical fume hood
with adequate protection. Immediately prior to use, the
HFIP-treated aliquots were carefully and completely resuspended in distilled water to a concentration of 50 ␮M
(modified protocol from Dahlgren et al., 2002).
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The A␤–metal complexes were prepared by 24h dialysis against different metal solutions (Al(C3 H5 O3 )3 ,
CuSO4 , ZnCl2 , FeCl3 ) at T = 4 ◦ C using Spectra/Por®
Float-A-Lyser® tubes (Spectrum Labs) with 1000
molecular weight cut offs (MWCO). Al(C3 H5 O3 )3 was
used instead of Al inorganic salts in order to improve
the metal-soluble concentrations (Bala Gupta et al.,
2005). Then, A␤–metal complexes were dialysed against
water (three water changes) for 24 h in order to remove
the excess of metals not bound to the peptide. The
same treatment was also performed with A␤ alone.
Aliquots of A␤ and different A␤–metal complexes
were taken at 48 h incubation time, after dialysis, for
observation by electron microscopy, for metal detection
by atomic absorption (electrothermal atomic absorption spectrometry, ETAAS, or flame atomic absorption
spectrometry, FAAS) and size exclusion chromatography.
2.4. Transmission electron microscopy (TEM) and
scanning electron microscopy (SEM)
2.4.1. TEM of Aβ–metal complexes
Aliquots of A␤ and different A␤–metal complexes
were absorbed onto glow-discharged carbon-coated butwar films on 400-mesh copper grids. The grids were
negatively stained with 1% uranyl acetate and observed
at 40,000× by TEM (Fei Tecnai 12). All experiments
were carried out at 10 ␮M peptide concentration.
2.4.2. SEM of human neuroblastoma cells
SH-SY5Y human neuroblastoma cells were seeded
onto glass cover slips and treated with A␤ and different
A␤–metal complexes at 0.5 ␮M peptide concentration
for 24 h. After this incubation, the cells on glass cover
slips were fixed with formaldehyde pH 7.4 and dehydrated in a graded ethanol series. Then, the samples were
critical point dried with CO2 in an HCP-2 Hitachi 2 Critical Point Dryer and gold-coated for examination under a
XL30 ESEM scanning electron microscope. The working pressure was 4.2–4.3 bar and the temperature was
5 ◦ C.
2.5. Atomic absorption measurements
To each sample (A␤ and different A␤–metal
complexes at 50 ␮M) was added 200 ␮l HNO3 for mineralization and, after 24 h at 70 ◦ C, the samples were made
up a final volume of 1 ml with distilled water. Before
measurement, all samples were adequately diluted with
MilliQ water. Al, Fe and Cu were determined by electrothermal atomic absorption spectrometry (ETAAS)
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on a Hitachi (Tokyo, Japan) Z-8270 polarized Zeeman
atomic absorption spectrometer.
Zn was determined by flame atomic absorption
spectrometry (FAAS) using a Varian (Mulgrave, Victoria, Australia) Spectra AA 110 instrument, in an
air–acetylene flame.
Calibration standards were prepared in the same acid
(nitric acid) concentration as was used in measured
samples. Before measurement, elements were diluted
1:5, 1:10 and 1:2, using matrix matched standards (the
addition of acid was the same as that in the standards).
The reproducibility of measurements (three subsequent determinations of each sample) was greater than
2% for all elements determined.
2.6. Size exclusion chromatography (SEC)
Size exclusion chromatography was performed on
AKTA HPLC system with a P-900 pump and a variable wavelength P900 UV detector (GE Healthcare,
Italy). Unicorn software was used to analyse data. Chromatographic separations of A␤ and different A␤–metal
complexes were performed in 30 mM Tris/HCl and NaCl
150 mM (pH 7.4) at a flow rate of 0.5 ml/min on a
Zorbax GF 250 column (Agilent Technologies, Wilmington, DE). GF-250 columns are recommended for
the size separation of water-soluble macromolecules
having molecular weights from 400,000 to 4000 Da.
Samples of A␤ and different A␤–metal complexes
were injected at 50 ␮M peptide concentration and
detected by UV absorbance at 215 nm. The column
was equilibrated with at least three column volumes of
elution buffer and then calibrated with six molecular
weight standards: bovine serum albumin (67,000 Da),
avian ovalbumin (43,000 Da), bovine carbonic anhydrase (29,000 Da), ribonuclease A (13,700 Da), equine
cytochrome C (12,400 Da) and insulin from bovine pancreas (11,466 Da as a dimmer). The chromatograms
shown are representative of the results from the two
separate experiments.
2.7. Mass spectrometry
Spectra of the A␤1–42 and A␤–Al complex (50 ␮M)
were recorded on an LTQ Orbitrap High-resolution mass
spectrometer (Thermo, San Jose, CA) just after addition of 0.5% Formic Acid. The instrument was equipped
with a conventional ESI source. The working conditions
were the following: sample flow rate was 3 ␮l/min, spray
voltage was 2.6 kV, capillary voltage was 20 V and capillary temperature was kept at 403 K. Sheath gas was
set at 10 (arbitrary units), the sweep gas and auxiliary
gas were kept at 0 (arbitrary units). For spectra acquisition a nominal resolution (at m/z 400) of 60,000 was
used.
2.8. Cell viability assay
MTT assay was performed with SHSY5Y cells plated
onto 96-well plates (at a density of 8 × 104 cells per
well, to confluency, in 100 ␮l medium containing 15%
FBS per well). The day after this plating, the culture medium was replaced with the same medium with
2% FBS containing A␤1–42 or A␤–metal complexes
at 0.5 ␮M peptide concentration. The cells were incubated with different A␤ or A␤–metal complexes for
24 h. The assay was also performed in the presence of
different metals (Al, Cu, Zn, Fe) in a range of concentrations of 5–100 ␮M. At the end of incubation, 10 ␮l of
MTT (5 mg/ml) was added to each well and the incubation continued for an additional 3 h. The MTT solution
was carefully decanted off, and formazan was extracted
from the cells with 100 ␮l of acidic isopropanol (0.04 M
HCl in absolute isopropanol) in each well (Shearman,
Hawtin, & Tailor, 1995).
Colour was measured with a 96-well ELISA plate
reader (Microplate SPECTRAmax® at 550 nm). All
MTT assays were repeated nine times.
2.9. Fluorescence anisotropy
The stock solution of fluorescent probe TMA-DPH
was prepared by dissolving the probe in dimethylsulfoxide (DMSO; Sigma–Aldrich) at a final concentration of
2 mM, while the probe DPH was dissolved in tetrahydrofuran (THF; Sigma–Aldrich) at a final concentration
of 1 mM. SH-SY5Y cells (4 × 105 cells/ml) were centrifuged, washed three times with PBS and re-suspended
in PBS. Cells suspended in PBS were labelled with the
fluorescent probes TMA-DPH or DPH at room temperature for 10 and 20 min, respectively, i.e. the requisite
time to obtain a stationary fluorescence equilibrium. The
final concentration of both probes was 2 ␮M (Kuhry,
Duportail, Bronner, & Laustriat, 1985). Probe incorporation was assessed by recording the fluorescence
intensity in a Perkin-Elmer LS-50B spectrofluorimeter fitted with an automated polarization accessory
(Perkin-Elmer, Monza, Italy). The non-polar DPH and
its cationic derivative, TMA-DPH, intercalate into lipid
membranes and their fluorescence polarization is related
to the reorientation of their long axes, making them sensitive to the angular re-orientation of the acyl chains of
surrounding lipids (Lentz, 1993). After the incubation
of the fluorescent probes, the cells were treated with A␤
and different A␤–metal complexes at 0.5 ␮M peptide
concentration and the fluorescence anisotropy was followed for 20 min. All fluorescence measurements were
carried out at 37 ◦ C.
735
The excitation and emission wavelengths for TMADPH were 356 and 428 nm, respectively. For DPH, the
corresponding wavelengths were 360 and 471 nm. The
fluorescence anisotropy (r) was obtained from the fluorescence intensities parallel (Ivv ) and perpendicular (Ivh )
Fig. 1. Transmission electron microscopy (TEM) of A␤ and A␤–metal complexes. Electron micrographs of human A␤1–42 in the absence (A) and in
the presence of different metal ions: Al (B), Cu (C), Zn (D) and Fe (E). A␤–metal complexes were prepared by 24 h dialysis (T = 4 ◦ C) against metal
solution. The incubation of A␤–metal complexes was carried out for 48 h with three water changes in order to eliminate the unbound metal. Before
dialysis, A␤1–42 was pre-treated with hexafluoroisopropanol for 40 min at room temperature in order to destroy the pre-existing peptide structure.
The peptide concentration was 10 ␮M. Scale bars, 200 nm.
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to the direction of polarization of the excitation light
using the following equation (Van der Meer, 1988):
r=
Ivv − Ivh G
Ivv + 2Ivh G
Student–Newman–Keuls t-test as post-hoc test. A value
of p < 0.05 was considered statistically significant.
3. Results
where G is an instrumental correction factor.
3.1. Characterization of Aβ and Aβ–metal
complexes by transmission electron microscopy
2.10. Analysis of cell death by fluorescence
microscopy: acridine orange/propidium iodide
double staining
Morphological study of apoptosis and necrosis were
carried out by means of acridine orange and propidium iodide staining as previously described (Martin
& Leonardo, 1994) after 24 h treatment with A␤ and
A␤–Al (0.5 ␮M). Cells were seeded on glass cover
slips, and stained with 4 ␮g/ml acridine orange (AO) and
4 ␮g/ml propidium iodide (PI). Within 30 min, the cells
were examined by fluorescence microscopy (Leica DM
5000 B microscope) with 480 and 520 nm filters and
photographed using a Leica DCF 300 FX camera. An
apoptotic index was determined by nuclear condensation
and segmentation, and by plasma membrane integrity
using the two fluorescent dyes mentioned above. AO
was used to characterize chromatin condensation and PI
to characterize membrane integrity. AO is a membrane
permeable marker that stains nuclei green; PI binds to
DNA, stains nuclei red and is mainly taken up by cells
with lost membrane integrity.
Controls (green nuclei), early apoptotic (DNA condensed, green nuclei), late apoptotic (DNA condensed,
orange nuclei) and necrotic (red nuclei) cells were analysed. At least 300 cells were counted in total five
independent experiments.
2.10.1. Statistical analysis
The experimental data were expressed as a percentage
with respect to control values and were presented as the
mean ± S.D. of, at least, four separate experiments. Statistical analysis was performed by ANOVA followed by
Samples of A␤ and various A␤–metal complexes (see
Section 2) were studied by TEM at relatively low peptide concentration (10 ␮M) (Fig. 1). After 48 h dialysis
at 4 ◦ C, many short and irregular protofibrillar structures
were present in the A␤ sample as the consequence of selfaggregation and few fibrils were observed (Fig. 1A). By
contrast, A␤–Al complex was characterized by a large
population of small oligomeric aggregates (Fig. 1B).
A␤–Cu complex, showed few aggregates, bigger than
those observed for A␤–Al (Fig. 1C; see scale bar).
Electron micrographs of A␤–Zn complex showed few
aggregates and unstructured filaments (Fig. 1D). Finally,
A␤–Fe complex promoted the formation of some filaments with very small aggregates (Fig. 1E).
3.2. Analytical determination of various metal ions
(Al, Zn, Cu, Fe) in samples of Aβ and Aβ–metal
complexes using atomic absorption measurements
To measure the metal content in A␤ and in different A␤–metal complexes, and to exclude possible metal
contamination in the synthetic peptide, each sample was
assessed by ETAAS and FAAS at 50 ␮M peptide concentration. Tables 1 and 2 show the metal content of A␤ with
respect to the blank sample. No analytically detectable
metal contamination was ascertained in the synthetic
A␤1–42 peptide. By contrast, the presence of metal ions
in different A␤–metal complexes was established analytically. The level of different metal ions in A␤–metal
complexes were very similar with a 422.65 ␮M for Al in
A␤–Al sample and 377.50, 274.53 and 251.29 ␮M for
A␤–Cu, A␤–Fe and A␤–Zn, respectively. These metal
Table 1
Detection of different metal ions in A␤ and A␤–metal complexes by ETAAS and FAAS
Samples (50 ␮M)
Metals deterimantion
Al (ng/ml)
Cu (ng/ml)
Fe (ng/ml)
Zn (ng/ml)
A␤
A␤–Al
A␤–Cu
A␤–Fe
A␤–Zn
Blank
Al, Cu, Fe, Zn
Al
Cu
Fe
Zn
Al, Cu, Fe, Zn
80 ± 2
11,500 ± 300
–
–
–
97 ± 3
8.0 ± 0.2
–
24,000 ± 700
–
–
9.0 ± 0.2
56 ± 2
–
–
15,400 ± 500
–
67 ± 2
74 ± 2
–
–
–
16,500 ± 500
68 ± 2
The metal content for A␤ and A␤–metals samples was measured after 48 h dialysis at 4 ◦ C. Al, Fe and Cu were determined by ETAAS whereas Zn
was determined by FAAS.
Table 2
Detection of different metal ions in A␤ and A␤–metal complexes by
ETAAS and FAAS
Samples (50 ␮M)
Al (␮M)
A␤
A␤–Al
A␤–Cu
A␤–Fe
A␤–Zn
0
422.65
Cu (␮M)
0
Fe (␮M)
0
Zn (␮M)
0.092
377.509
737
molecular weight peaks with respect to the elution of A␤
and A␤–Al. In addition, those peaks, present a shoulder that could signify a non-homogeneity of the eluted
compounds. This finding appears to be consistent with
the TEM micrographs where the aggregates of A␤–Cu,
A␤–Zn and A␤–Fe are larger and more heterogeneous
than those of A␤ and A␤–Al.
274.531
251.291
Metal content for A␤ and A␤–metals samples expressed in ␮M. The
blank value was subtracted from the final results.
concentrations were reduced 100-fold in the A␤–metal
complexes used in different cellular experiments.
3.3. Characterization of Aβ and Aβ–metal
complexes by SEC
To define the oligomerization of A␤ and to obtain
information on the conformation of A␤–metal complexes as observed by TEM, an analytical approach
is required to resolve monomeric/oligomeric status of
A␤ under non-denaturating/non-disaggregating conditions. According to the experience of our laboratory
and that of others, SEC appears to be appropriate to
this purpose. A␤ and various A␤–metal complexes were
analysed at 50 ␮M peptide concentration immediately
after the preparation as described in the experimental procedures. As reported in Fig. 2, A␤ and A␤–Al
eluted as a symmetrical peaks (∼12 kDa). It is important to notice that A␤ and A␤–Al co-eluted, but with
a relatively different area of the eluted peak. A␤–metal
complexes (A␤–Cu, A␤–Zn, A␤–Fe), eluted as higher
3.4. Mass spectrometry
Additional ESI MS studies were performed to assess
the formation of the A␤1–42 –Al(III) complex. In the
reported experimental conditions the predominant peak
in the multicharged ESI MS spectra of both A␤1–42 and
its aluminium complex is the one corresponding to the 5+
charged state (data not shown). The deconvoluted highresolution ESI MS spectra of either A␤1–42 alone or its
aluminium-treated sample are reported in Fig. 3. In the
absence of aluminium (Fig. 3A) the main peak centered
at ∼4513 Da corresponds to the A␤1–42 peptide while a
less intense peak at 4529 Da most likely corresponds to
a portion of A␤1–42 in which oxidation of Met-35 has
occurred.
In the presence of aluminium (Fig. 3B), together
with the peaks at 4513 and 4529, an additional heavier cluster at about 4537 is observed, attributed to a
A␤1–42 –Al(III) complex in which the binding stoichiometry between A␤1–42 and Al is 1:1. These conclusions
arise from a careful analysis of the high-resolution
data. Fig. 4A shows the m/z range covering the peaks
corresponding to the most abundant 5+ charge state.
Clustered at around 903 m/z, 906 m/z and 908 m/z are
the various isotopic peaks of the A␤1–42 , A␤1–42 –Met
oxidised and A␤1–42 –Al(III) species, respectively. The
difference between the monoisotopic peaks of the free
amyloid and the aluminium complex is ∼24 Da. However, since the monoisotopic A␤1–42 –Al(III) has three
protons less, the actual mass difference is ∼27 Da,
which is close to the nominal mass of an aluminium
ion. Remarkably, the obtained experimental data perfectly match theoretical expectations (Fig. 4B), thus
confirming our hypotheses on the chemical nature of the
complex.
3.5. SEM: morphological alteration of SH-SY5Y
cells treated with Aβ and Aβ–metal complexes
Fig. 2. Size exclusion chromatography (SEC) of A␤ and A␤–metal
complexes. A␤–metal complexes were analysed using size exclusion
chromatography with a Zorbax GF 250 column. A␤ and A␤–Al coeluted with an approximate molecular weight of 12,000 Da. The gelincluded peak elutes at 13.08 ml, while the gel-excluded peak elutes at
7.3 ml. Elution positions of molecular weight standards are indicated
by arrows. Molecular masses are indicated in kDa.
Cellular morphological alteration after treatment with
A␤ and different A␤–metal complexes (0.5 ␮M peptide concentration) was also considered using an SEM
approach. Again, it was clearly demonstrated that A␤–Al
induced a deep and marked modification at the level of
738
Fig. 3. ESI MS deconvoluted spectra of A␤1–42 (A) and A␤1–42 –Al(III) complex (B).
the cellular bi-layer (Fig. 5B and C) with respect to the
controls (Fig. 5A).
3.6. MTT assay on SH-SY5Y cells treated with Aβ
and Aβ–metal complexes
Experiments on cellular vitality show the toxicity of
A␤–Al complex (0.5 ␮M peptide concentration) with a
significant decline in MTT reduction (Fig. 6A), whereas
treatment with A␤ and various A␤–metal complexes
showed no effect. It is worth noting that exposure of neu-
roblastoma cells to Al, Cu, Zn, Fe at 5–100 ␮M range did
not alter the cellular redox activity with respect to control. Importantly, metal concentrations were 10–20-fold
higher than A␤ peptide concentration (Fig. 6B–E).
3.7. Cellular membrane fluidity in the presence of
Aβ and Aβ–metal complexes using fluorescence
anisotropy
Fluorescent dyes represent a useful tool for the determining of membrane dynamics. DPH is a fluorescent
739
Fig. 4. Comparison between the observed (A) and theoretical (B) high-resolution spectra of 5+ charge state of A␤1–42 and A␤1–42 –Al(III) complex.
probe that intercalates predominantly between the acyl
chains of fatty acids in the membrane hydrocarbon core.
TMA-DPH, on the other hand, is a cationic fluorescent
aromatic hydrocarbon that anchors at the lipid–water
interface of the plasma membrane lipid bi-layer and
remains at the level of the hydrophilic head groups of
membrane phospholipids (Kuhry et al., 1985). In an
effort to further investigate the interaction of A␤ and different A␤–metal complexes with the plasma membrane,
anisotropy measurements were performed on human
neuroblastoma cells after the addition of 0.5 ␮M peptide
concentration.
The fluorescence anisotropy values of TMA-DPH
in neuroblastoma cells revealed a significant increase
in membrane fluidity (as detected by the decrease of
anisotropy intensity) after treatment with A␤ and, more
markedly, with A␤–Al with respect to the control. These
data indicated that A␤ and A␤–Al perturbed the lipid
tail/polar heads border areas of the cell membrane. No
significant changes were obtained after treatment with
other A␤–metal complexes (A␤–Cu, A␤–Zn, A␤–Fe).
Control with different metal solutions, at a concentration 10-fold higher than that of the peptide, revealed no
effect whatsoever with regard to membrane fluidity in
these regions of the lipid bi-layer (Fig. 7A).
A significant decrease in fluorescence anisotropy with
both A␤ and A␤–Al was also observed using DPH with
respect to the other A␤–metal complexes. This effect was
consistent with an enhancement of fluidity in the lipid
core region of the plasma membrane. In that case, the
greater decrease in fluorescence anisotropy was detected
in the presence of A␤. In addition, A␤–Cu produced an
increased lipid packing density, which is coherent with
a rigidification of the lipid core region of the plasma
740
membrane. Finally, A␤–Zn and A␤–Fe revealed no significant changes as well as controls with different metal
solutions at a concentration 10-fold higher than that of
the peptide (Fig. 7B).
In conclusion, the effects of A␤–Al on the fluidity of
neuroblastoma cells at the lipid water interface (TMADPH anisotropy) were quantitatively higher than those
of the membrane hydrocarbon core (DPH anisotropy),
indicating weaker effects at this membrane region.
The opposite behaviour was observed using only A␤.
Considering all A␤–metal complexes utilized in our
experiments, only A␤–Al was able to produce major
effects in terms of increasing membrane fluidity.
3.8. Determination of apoptosis and necrosis:
acridine orange/propidium iodide double staining
The morphological changes and different fluorescence of fluorochrome in cells were used to distinguish
living, apoptotic and necrotic cells after treatment with
A␤ and A␤–Al (Fig. 8). Living cells had normal shaped
nuclei with green chromatin. In early apoptosis, acridine orange entered the cell with propidium iodide
exclusion and cells had shrunken green nuclei with
chromatin condensation; in late apoptosis, with loss of
membrane integrity, both dyes entered the cell and the
nucleus appeared bright orange. A␤–Al-exposed cells
(see arrows, Fig. 8C) showed typical features of late
apoptosis including dense nuclear condensation and cell
shrinkage compared with A␤ (Fig. 8B) and control
(Fig. 8A). These morphological changes did not occur
after treatment with A␤ alone except for some cells
in early apoptosis. The results shown are representative of five independent experiments (Fig. 9). Data were
expressed as the total number of apoptotic cells (early
plus late) as a percentage of the total cell number. Cell
magnification 40×.
4. Discussion
Fig. 5. Scanning electron microscopy (SEM) of SH-SY5Y cells. Electron micrographs of untreated neuroblastoma or A␤–metal complexes
(A) and neuroblastoma treated for 24 h with A␤–Al complex (B and
C). The peptide concentration was 0.5 ␮M. Alterations in the cellular
membrane after A␤–Al treatment are clearly shown (B and C).
A␤ aggregation and accumulation are crucial aspects
of the etiopathogenesis of Alzheimer’s disease. A growing body of evidences points to the role of relatively
small soluble oligomers as the pivotal element in the
pathogenic event. To understand the pathophysiology
of AD, it is thus crucial to clarify the role of A␤ and
the dynamics of various conformational states concomitantly with the profile of the disease in terms of its clinical
and histopathological evolution. The aggregation of A␤
proceeds through several steps, starting with dimers,
then spherical oligomers, protofibrils, and eventually the
insoluble fibrillar status (Demuro, Mina, Kayed, Milton,
741
Fig. 7. Fluorescence anisotropy of TMA-DPH (A) and DPH (B) in
SH-SY5Y neuroblastoma. A decrease in anisotropy, which reflects
an increase in membrane fluidity, was obtained with A␤ and A␤–Al
complex according to the fluorescent probe used in the experiment.
A␤–metal complexes concentration was 0.5 ␮M. Data were presented
as a percentage with respect to control values. Similar results were
obtained in four independent experiments. Error bars indicate the
mean ± S.D.; ** p < 0.01 compared with control; ◦◦ p < 0.01 compared
with A␤; ◦ p < 0.05 compared with A␤; ˆˆp < 0.01 compared with
A␤–Zn, A␤–Cu, A␤–Fe; §§ p < 0.01 compared with A␤–Al.
& Parker, 2005). To assess the pathological role of A␤, a
clear understanding of the conditions that drive the peptide assembly from one conformational state to another is
essential. Any single change that alters the conformation
of A␤ is most likely to affect its biological activity as well
(Stine, Dahlgren, Krafft, & LaDu, 2003). In this rather
complex and still, at least partially, unresolved scenario,
metal ions seem to play an important compelling role in
A␤ aggregation, as reported by our laboratory (Drago et
al., 2007; Ricchelli et al., 2005) as a very crucial aspect
for A␤ neurotoxicity.
Protein–metal ion interactions have been shown to
contribute to many neurodegenerative disorders, such
as AD, Parkinson’s disease, Creutzfeldt–Jakob disease
and amyotrophic lateral sclerosis (ALS) (Sigel, Sigel,
Fig. 6. Cytotoxicity assay in SH-SY5Y cells. Redox activity in
SH-SY5Y cells after treatment with human A␤ and other A␤–metal
complexes. The metal-free and metal-complexed peptide concentration was 0.5 ␮M. Neuroblastoma redox activity was measured by MTT
assay. A significant decrease in cellular viability was obtained with
A␤–Al complex (A). MTT assay was also performed with different
metal solutions at 5–100 ␮M concentration range. The metal concentrations were 10–20-fold higher than A␤ peptide concentration (B–E).
Similar results were obtained in four independent experiments. Error
bars indicate the mean ± S.D.; * p < 0.05 compared with control.
742
Fig. 9. Apoptosis as assessed by fluorescent microscopy after staining
with acridine orange and propidium iodide. SH-SY5Y neuroblastoma
cells were treated with A␤ and A␤–Al complex (0.5 ␮M). The results
are mean of five independent experiments ± S.D.; ** p < 0.01 compared
with control; ◦ p < 0.05 compared with A␤. Data were expressed as a
total number of apoptotic cells (early and late apoptosis) as a percentage
of the total cell number.
Fig. 8. Acridine orange and propidium iodide double staining of
SH-SY5Y neuroblastoma (A) after treatment with A␤ (B) and A␤–Al
complex (C). SH-SY5Y neuroblastoma cells were treated with A␤
and A␤–Al complex (0.5 ␮M). Living cells have normal shaped nuclei
with green chromatin (L). Early apoptotic cells have shrunken green
nuclei with chromatin condensation (EA), whereas late apoptotic cells
had condensed nuclei that were brightly stained with propidium iodide
and appeared orange-red (LA). A␤–Al-exposed cells exhibited typical
& Sigel, 2006; Zatta, 2003). Our previous studies
on the effects of metal ions on the PrP and ataxin
conformational structures are highly significant since
the aggregational properties of the prion and ataxin
molecules have been shown to exhibit surprising analogies with A␤ in the presence of various metal ions
(Kenward, Bartolotti, & Burns, 2007; Ricchelli et al.,
2006, 2005, 2007; Sasson & Brown, 2003).
In vitro studies to define the structure and toxicity
of different conformational states of A␤, in the presence and absence of metal ions need to be based on
procedures that consistently produce fully characterized
structural populations. According to Stine et al. (2003),
the aggregation state in commercial amyloid, as used
by most laboratories, is not controlled by the manufacturers, who only guarantee the chemical purity, but not
the conformational homogeneity. Therefore, removal of
pre-existing structures using HFIP in lyophilized stocks
of A␤ is required for controlled aggregation studies, as
widely reported in the literature (Dahlgren et al., 2002;
Demuro et al., 2005; Stine et al., 2003). The solubilization and aggregation protocols herein reported (see
Section 2), showed consistent and reproducible results
for A␤ and A␤–metal complexes as observed by TEM
(Fig. 1).
Electron microscopy analysis showed that A␤–Al
was characterized by a large population of small
oligomers (Fig. 1B), which could be responsible for
the significant toxicity on neuroblastoma cells in terms
features of late apoptosis including dense nuclear condensation and
cell shrinkage (see arrows). Similar results were obtained in five independent experiments; magnification: ×40. (For interpretation of the
references to colour in this figure legend, the reader is referred to the
web version of the article.)
of alteration of cell morphology (Fig. 5), decrease in
cell viability (Fig. 6), increase in membrane fluidity
(Fig. 7) justified by its high hydrophobicity (Ricchelli
et al., 2005), and possible increase of late apoptosis
(see arrows, Fig. 8C). Al seemed somehow to be able
to “freeze” the oligomeric state of A␤, stabilizing this
assembly with respect to the conformations obtained for
other A␤–metal complexes.
With regard to A␤ assembly (Fig. 1A), the
cellular toxic effects produced were significantly
less pronounced than those obtained with A␤–Al
(Figs. 6 and 7A). Recently, it has been demonstrated
that when aluminium is bound to amyloid, forming
a stable metallorganic complex, the molecule surface
hydrophobicity dramatically increases as a consequence
of metal-induced conformational changes, favouring
misfolding/aggregation phenomena and lypophilicity
significantly (Ricchelli et al., 2005). As a consequence
of a higher hydrophobicity, with respect to A␤ alone
A␤–Al reduced its capillary sequestration increasing its
permeability through the blood brain barrier as recently
demonstrated by Banks, Niehoff, Drago, and Zatta
(2006). Additionally, the aggregation of both human and
rat amyloid in the presence of aluminium is more pronounced than that obtained with amyloids alone and the
morphology of the two aggregation types was very different. This finding was closely linked to the different
amino acid sequence of human and rat amyloid with
a consequently different cellular toxicity produced by
human and rat A␤–Al complexes (Drago et al., 2007).
The different aggregational behaviour of rat and human
amyloids in the presence of Al emphasized the close
relationship between the A␤ aggregates’ morphology
and their cell toxicity. By contrast, other A␤–metal complexes (A␤–Cu, A␤–Zn, A␤–Fe) showed the formation
of bigger agglomerates (Fig. 1C–E) unable to produce
any kind of cellular toxic effects (Figs. 6 and 7).
Taken together, all these results confirmed, once
again, the strong link, widely reported in the literature,
between A␤ conformation structure and toxicity.
The crucial role of A␤ oligomers and, in this current investigation, of A␤–Al complex in promoting
neurotoxic effects was also assessed (Dahlgren et al.,
2002; Demuro et al., 2005; Deshpande et al., 2006).
Our results appeared to be particularly important when
compared with previous publications where a higher
amyloid concentration (20 or 100 ␮M) was required
(Awasthi, Matsunaga, & Yamada, 2005; Boyd-kimball,
Sultana, Mohmmad-Abdul, & Butterfield, 2004; Datki
et al., 2003), much more indeed than 0.5 ␮M of A␤ as
used in our protocols. This aspect is worthy of particular attention because in physiological conditions the
743
brain concentration of A␤ might be at a nanomolar
level.
The presence of various metal ions in A␤–metal
complexes were confirmed by the analytical detection
using ETAAS and FAAS (Tables 1 and 2). The binding
sites of metal ions to the amyloid have been suggested
by different experimental approaches (Miura, Suzuki,
Kohata, & Takeuchi, 2000; Stellato et al., 2006; Vyas
& Duffy, 1995). Particularly, the presence of Al and its
binding to amyloid was confirmed by high-resolution
ESI mass spectrometry experiments as clearly shown in
Figs. 3 and 4. The use of ESI MS to study the binding
of metal ions with the A␤1–42 peptide has been recently
considered by Jiang et al. (2007). Of particular interest
is our finding of the presence of a portion of both A␤
and A␤–Al in which oxidation of Met-35 has occurred.
The mass shift of 16 Da between the two main peaks
(4513 and 4529) has been recently described by Chen
and Cook (2007) in the mass spectrum of A␤1–40 and
has been attributed to oxidative degradation prior to or
during analysis by ESI MS.
The structural characterization of A␤ oligomers is
a challenge, since the A␤ amphipathic properties and
the strong tendency to self-aggregate complicates both
the characterization of structure and function (Dahlgren
et al., 2002). In this connection, some results in the
literature appear to be inconsistent, due to the different experimental conditions and analytical methods
used (Bitan, Lomakin, & Teplow, 2001). The choice
of a suitable chromatographic column and the chromatographic behaviour of A␤ and A␤–metal complexes
appeared to be extremely important (Fig. 2). First of
all, A␤ eluted with an apparent molecular weight of
∼12 kDa and this finding was consistent with the size
exclusion chromatography performed recently by Chen
and Glabe (2006). These authors demonstrated that the
trimer/tetramer formation correlated well with the faster
nucleation kinetics of A␤1–42 with respect to A␤1–40 ,
suggesting that these small oligomers may be important for nucleation. The greater resistance of trimers
to denaturation supported the hypothesis that trimers
are the fundamental starting point of the A␤ assembly
unit in vivo (Lesnè et al., 2006; Townsend, Shankar,
Mehta, Walsh, & Selkoe, 2006). In addition, A␤ coeluted with A␤–Al, confirming a very similar molecular
weight in the presence of aluminium, but with different biophysical properties. However, a more pronounced
chromatographic peak was detected for A␤–Al and this
finding was consistent with the larger population of small
oligomers observed for A␤–Al by electron microscopy
(Fig. 1A and B). On the contrary, A␤–Zn, A␤–Cu,
A␤–Fe were characterized by higher molecular weights
744
and by an apparent high heterogeneity in A␤ assemblies shown by peaks not well resolved (Fig. 2). Further
investigations are still in progress to improve the quality
of the A␤ and A␤–metal complex structural characterization. The importance of these results is supported by
the lack of data about the characterization of A␤–metal
complexes in comparison to A␤ alone.
Several studies have suggested that the initial pathophysiology induced by A␤ might involve alterations
in membrane structure (Kagan, Hirakura, Azimov,
Azimova, & Lin, 2002; Kremer, Pallitto, Sklansky, &
Murphy, 2000; Lau et al., 2006). Soluble A␤ peptides could interact with cellular membranes and it
has been suggested that they affect membrane integrity
leading to apoptosis (Demeester et al., 2000). Physicochemical interaction of A␤ oligomeric species with
membrane domains, including changes in fluidity, can
be a determining factor for triggering the mechanisms
of neurotoxicity. Changes in the membrane fluidity,
for instance, have been associated with dysfunctions of
membrane receptors, ionic channels and transport proteins (Szollosi, 1994). Given that A␤ is generated in a
membrane environment and its pathological behaviour
may be due to interactions with membranes, understanding the physical nature of A␤/membrane interactions is
important for deciphering the biological role of A␤ and
A␤–metal complexes.
An extensive literature supports the view that plasma
membrane might be one major target of A␤ toxicity (Ambroggio et al., 2005; Curtain et al., 2003;
Demuro et al., 2005; Lau et al., 2006; Muller, Kirsch,
& Eckert, 2001; Qi et al., 2005). One explanation for
the A␤–membrane interaction might be that amyloid
aggregates accumulate predominantly in the extracellular compartment, close to the plasma membrane, during
the disease process. A detailed study on the permeabilization of the lipid bi-layer by soluble A␤ oligomers
demonstrated that these types of A␤ assembly were
responsible for a generalized increase in membrane
conductance that might represent the common primary
mechanism of pathogenesis in amyloid-related neurodegenerative disorders (Kayed et al., 2004). Kremer et al.
(2000) showed a correlation of A␤ aggregation size and
hydrophobicity with decreased bi-layer fluidity of model
membranes. Moreover, Muller et al. (2001) demonstrated clearly that A␤ peptides specifically disturb the
acyl-chain layer of cell membranes in a very distinct
fashion. By contrast, membrane properties at the level of
the polar heads of the phospholipids bi-layer at the interface with membrane proteins were much less affected. In
our investigation, A␤ and A␤–Al were able to produce
a strong increase in membrane fluidity in neuroblastoma
cells cultures, to different extents (Fig. 7). A␤–Al promoted a greater increase in membrane fluidity mostly in
the lipid tail/polar heads border areas of cell membrane
with respect to the other A␤–metal complexes (Fig. 7A).
These results appear to be consistent with the major alteration produced by A␤–Al in the cellular morphology
(see SEM experiments in Fig. 5). The amyloidogenesis
occurring in AD is strongly associated with cell membrane, considering that ␤-amyloid peptides derive from
sequential cleavage of the transmembrane amyloid precursor protein by two membrane-bound proteases, the
␤- and ␥-secretase. As a result, APP processing by these
enzymes might be affected by the hydrophobic environment and thus directly or indirectly by membrane
fluidity (Gamerdinger, Clement, & Behl, 2007). Finally,
no effects on membrane structure were detected in the
presence of A␤–Zn, A␤–Fe or with metal ions alone
except for a rigidification in the core of the lipid bi-layer
with A␤–Cu (Fig. 7B).
In conclusion, the in vitro experiments reported in
this study provide a unique insight into the role of
metal ions, particularly Al–A␤ complex, in affecting A␤
oligomerization. Experiments that take into account A␤
conformational differences in the presence of various
metal ions, particularly aluminium, and the consequent
neurotoxic effects would appear to be extremely important; they could provide a better biological understanding
for developing successful therapeutics for the treatment
of AD. Our findings show for the first time the crucial
importance of the complex formation of A␤ and Al in
affecting some fundamental cellular functions. Of particular interest is our finding of the complete absence of
membrane biophysical alterations produced by the metal
ion alone, even at a higher concentration, with respect to
the complex A␤–Al (0.5 ␮M). The data herein presented
support the relevant importance of A␤–Al neurotoxicity in the aetiology of AD, suggesting that aluminum,
when complexed to A␤, could be one of the potential
multifactorial events characterizing this disease.
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Potential pathogenic role of Я-amyloid 1–42–aluminum complex in

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