Biochem. J. (2012) 441, 579–590 (Printed in Great Britain)
579
doi:10.1042/BJ20110749
Visualization of co-localization in Aβ42-administered neuroblastoma
cells reveals lysosome damage and autophagosome accumulation
related to cell death
Violetta SOURA*, Maris STEWART-PARKER*, Thomas L. WILLIAMS*1 , Arjuna RATNAYAKA*, Joe ATHERTON*,
Kirsti GORRINGE*, Jack TUFFIN*, Elisabeth DARWENT*, Roma RAMBARAN*, William KLEIN†, Pascale LACOR†,
Kevin STARAS*, Julian THORPE* and Louise C. SERPELL*2
*School of Life Sciences, University of Sussex, Falmer, BN1 9QG, U.K., and †Northwestern University 2205 Tech Drive, Evanston, IL 60208-3520, U.S.A.
Aβ42 [amyloid-β peptide-(1–42)] plays a central role in
Alzheimer’s disease and is known to have a detrimental effect
on neuronal cell function and survival when assembled into an
oligomeric form. In the present study we show that administration
of freshly prepared Aβ42 oligomers to a neuroblastoma (SHSY5Y) cell line results in a reduction in survival, and that
Aβ42 enters the cells prior to cell death. Immunoconfocal and
immunogold electron microscopy reveal the path of the Aβ42 with
time through the endosomal system and shows that it accumulates
in lysosomes. A 24 h incubation with Aβ results in cells that
INTRODUCTION
AD (Alzheimer’s disease) is characterized by deposition of
assembled Aβ42 [Aβ (amyloid-β peptide)-(1–42)] peptides
in extracellular amyloid plaques [1]. However, accumulating
evidence suggests that early aggregates of Aβ form small
oligomeric species that are directly involved in causing neuronal
functional disruption and cell death. In vitro, Aβ40 [Aβ-(1–40)]
and Aβ42 are able to form a number of different species from
dimers and trimers to larger ‘ADDLs’ (Aβ-derived diffusible ligands), oligomers, protofibrils and fibrils [2]. Although extensive
investigations have been performed to identify a specific ‘toxic’
species, it appears that many of the assemblies that form on the
pathway may have a toxic effect. Therefore, in the present paper,
we use the term ‘oligomers’ to describe small soluble forms of
Aβ made from freshly prepared peptide. External administration
of Aβ oligomers, but not fibrils, to tissue cultured cells results in
a reduction in cell survival [3,4], and intracellular accumulation is
thought to be via an endosomal process leading to accumulation
within lysosomes [5]. Disruption of membrane integrity has been
implicated in the toxic mechanism of Aβ oligomers [6,7], and
we have previously shown that the ability of Aβ42 to permeate
synthetic membranes declines as the peptide assembles into fibrils
[6]. Aβ oligomers have been shown to cause deterioration of LTP
(long-term potentiation) in hippocampal neurons [8,9], to have a
severe effect on memory in animal models [10] and to lead to loss
of synaptic elements [11,12], indicating that the neurons become
damaged and dysfunctional prior to cell death.
Aβ is produced by cleavage of APP (amyloid precursor
protein) by BACE (β-site amyloid precursor protein-cleaving
enzyme; β-secretase) and γ -secretase following clathrin-
have damaged lysosomes showing signs of enzyme leakage,
accumulate autophagic vacuoles and exhibit severely disrupted
nuclei. Endogenous Aβ is evident in the cells and the results
of the present study suggest that the addition of Aβ oligomers
disrupts a crucial balance in Aβ conformation and concentration
inside neuronal cells, resulting in catastrophic effects on cellular
function and, ultimately, in cell death.
Key words: Alzheimer’s disease, amyloid-β peptide-(1–42)
(Aβ42), autophagosome, lysosome, oligomer.
mediated internalization of APP from the plasma membrane
[1,13]. Endogenous Aβ42 has been observed in neuronal NT2
cells and appears to increase in amount with aging in culture [14].
In AD brains, Aβ42 has been localized to intraneuronal regions
[15], and in particular to multivesicular bodies [16]. It has also
been shown that oligomeric toxic Aβ can be generated in neuronal
cells [17–19].
Previous studies have focussed on the involvement of macroautophagy (referred to as autophagy) in the progression of
AD [20], and AD brains show a high number of autophagic
vacuoles in neuronal cells [20,21]. Flies expressing Aβ42 also
showed accumulation of autophagosomes [22]; however, the
role of autophagy remains controversial, being either protective
or pathogenic [23]. Recent work has pointed to a disruption
in the autophagosome pathway of fusion to lysosomes linked
to a mutation (or partial knockout) of the presenilin 1 gene
related to FAD (familial AD) [24]. Increasing lysosomal protease
activity has been shown to increase Aβ42 clearance from
autophagosomes, resulting in reduced cognitive deficits in an AD
transgenic mouse, indicating that autophagosomes are important
for disease symptoms [25].
In the present study we show that freshly prepared Aβ42
is internalized into neuroblastoma cells, and we visually
monitor the intracellular fate of Aβ administered at two
different concentrations with incubation time. The results of
the present study reveal that externally administered synthetic
Aβ is initially observed in clathrin-positive organelles and
later accumulates in lysosomes. The Aβ42 uptake observed is
associated with the formation of autophagosomes that may lead
to cell death. These results reveal significant damage to the
lysosomal/autophagosomal system caused by the administration
Abbreviations used: Aβ, amyloid-β peptide; Aβ42, Aβ-(1–42); AD, Alzheimer’s disease; APP, amyloid precursor protein; DAPI, 4 ,6-diamidino-2phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; FAD, familial AD; FCS, fetal calf serum; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H -tetrazolium bromide; PFA, paraformaldehyde; PI, propidium iodide; TEM, transmission electron microscopy;
TLV, TAAB low-viscosity.
1
Present address: Drexel University, Physics Department, 3141 Chestnut Street, Philadelphia, PA 19104, U.S.A.
2
To whom correspondence should be addressed (email [email protected]).
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V. Soura and others
of Aβ42, and the labelling of a lysosomal protease, cathepsin
D, shows that lysosomes appear to be leaking contents into
the cytoplasm. We have previously shown that freshly prepared
Aβ42 is able to cause significant permeation of biomimetic
membranes [26]. Observation of damaged organelles involved
in protein degradation is supported by electron microscopic
examination of acid-phosphatase-treated cells, which highlights
lysosomes and autophagosomes. Importantly, cells showing
enriched autophagosomes have invaginated nuclear envelopes,
revealing a link between accumulating autophagosomes and
cell death. In stark contrast, untreated cells remained entirely
healthy for the incubation time, with smooth densely packed
chromatin in circular nuclei. They showed no autophagosomes
and intact lysosomes. In the present study, we have followed Aβ42
internalization and accumulation within cellular compartments
over 24 h for the first time. Our results reveal that the
administration of Aβ42 alone can be responsible for the cellular
changes associated with AD.
EXPERIMENTAL
Preparation of Aβ
Aβ42 HFIP (1,1,1,3,3,3-hexafluoro-2-propanol), >97 % purity
was purchased from rPeptide and was used without further
purification. To prepare ‘freshly prepared’ oligomers, the peptide
was solubilized at 1 mg/ml in HFIP >99.0 % (Fluka, Sigma–
Aldrich), vortex-mixed vigorously for 60 s and sonicated in a
50/60 Hz bath sonicator for 5 min. The solvent was removed
using dry nitrogen, and the peptidic film was vacuum desiccated
for 30 min. The dried Aβ42 was dissolved in DMSO >99.9 %
(Sigma–Aldrich) at a concentration of 1 mg/ml before being
eluted from a 5 ml HiTrap desalting column (GE Healthcare)
in 1 ml of Hepes buffer [10 mM Hepes, 50 mM NaCl, 1.6 mM
KCl, 2 mM MgCl2 · 6H2 O and 3.5 mM CaCl2 · 2H2 O (pH 7.4)],
from this point referred to as Hepes pH 7.4. The eluted peptide
was centrifuged in a 4 ◦ C-controlled Eppendorf microcentrifuge
at 16 000 g for 30 min to remove contaminants and pre-formed
fibrillar material. The supernatant was placed in a clean nonstick microcentrifuge tube and kept at 4 ◦ C until use to minimize
fibrillization. The concentration was determined using a molar
absorption coefficient of 1490 M − 1 · cm − 1 and the absorbance
was measured at a wavelength of 280 nm using an Eppendorf
Biophotometer. The peptide stocks were diluted to 10 μM or
25 μM working concentrations directly into the SH-SY5Y cells.
It has been noted that the solubilization of Aβ using HFIP can
cause neurotoxic ion flux through cellular membranes, as residues
of the solvent can cause gradual thinning of the membranes which
results in a concomitant increase in transmembrane current [27];
however, the above preparation ensures that all residual HFIP has
been evaporated and DMSO has been replaced with buffer [28].
Complete removal of DMSO and HFIP was confirmed by the
absence of characteristic solvent signals in the 1 H- and 19 F-NMR
spectra respectively [6].
To prepare Alexa Fluor® 555-tagged Aβ, the peptide was
solubilized in DMSO as described above and then vortexmixed vigorously for 60 s and sonicated for 60 s. Triethylamine
(100 mM) was added to the Aβ42 solubilized in DMSO to ensure
the deprotonation of the reactive amines. Alexa Fluor® 555
(11.3 nM; Invitrogen) dissolved in 0.22 μm-filtered water was added to the deprotonated Aβ42 and pipette mixed. The peptide and
Alexa Fluor® 555 tag were incubated at 21 ◦ C for 15 min. Then
200 μl of the tagged peptide in DMSO was added to a 2 ml ZebaTM
buffer-exchange spin column equilibrated with 10 mM Hepes
pH 7.4. Once absorbed into the column resin, a 40 μl stacker of
c The Authors Journal compilation c 2012 Biochemical Society
0.22 μm-filtered water was added to the column. The column was
spun in a 4 ◦ C-controlled Mikro 22R centrifuge (Hettich) at 1000 g
for 2 min. The eluted peptide was then centrifuged at 16 000 g
for 30 min at 4 ◦ C in a Eppendorf microcentrifuge. The peptide
concentration was corrected for the Alexa Fluor® 555 contribution
to the 280 nm absorbance as stated in the manufacturer’s protocol.
Cell lines and culture conditions
Undifferentiated human neuroblastoma SH-SY5Y cells (a gift
from the Crowther group, University of Cambridge, Cambridge,
U.K.) were supplemented with 10 % Hyclone FCS (fetal calf
serum), 1/100 L-glutamine and 1/100 penicillin/streptomycin
(Gibco).
Western blot analysis
Undifferentiated SH-SY5Y cells were seeded at a density of
3×105 cells/well on a 12-well plate. At 24 h later the cells were
treated with 10 μM or 25 μM Aβ42 at 37 ◦ C for 1, 5 and 24 h, or
starved for 3 h in serum-free medium before being lysed on ice in
100 μl of lysis buffer [50 mM Tris/HCl (pH 8.1), 5 mM EDTA,
150 mM NaCl, 1 % Triton X-100, and proteinase (Roche) and
phosphatase (cocktails 1 and 2; Sigma) inhibitors] for 15 min.
The cell lysates were then centrifuged at 14 000 g for 10 min
at 4 ◦ C and 2 mg of total protein was loaded on to a 4–20 %
Novex A Tris-glycine gradient gel. For experiments that involved
different cell fractions, the medium that the cells were growing in,
a PBS wash, the supernatant after centrifugation and finally the
pellet fraction were loaded on to the gradient gel. For experiments
that involved analysis of the 10 or 25 μM Aβ42 in the absence
of cells, the oligomeric peptides were allowed to fibrilize at
37 ◦ C in the presence of DMEM (Dulbecco’s modified Eagle’s
medium)/F12 and in the presence or absence of 10 % FCS and
then loaded on to a 4–20 % Novex A Tris-glycine gradient gel
in duplicate with 5 and 10 μl of sample. The gels were then run
for 90 min at 125 V followed by a 30 min or 12 h transfer on to
a Hybond-LFP (low fluorescent PVDF) nitrocellulose membrane
(GE Healthcare). The membranes were blocked in 10 % non-fat
dried skimmed milk for 1 h at room temperature (24 ◦ C), boiled
in PBS for 5 min (when labelling for Aβ42) and labelled with
mouse monoclonal anti-[Aβ-(1–16)] (6E10) antibody (1:2500
dilution, Signet), mouse monoclonal NU1 antibody (an antiAβ antibody; 1:1000 dilution [11]) or rabbit polyclonal anti(LC3-II) antibody (autophagosomal marker; 1:1000 dilution, New
England Biolabs) on a rocker at 4 ◦ C overnight, or anti-(αtubulin) clone DM1A mouse monoclonal IgG (1:10000 dilution,
Upstate Biotechnology) for 1 h at room temperature, followed by
polyclonal goat anti-mouse or swine anti-rabbit HRP (horseradish
peroxidase)-conjugated secondary antibodies (1:15 000 dilution,
Dako Cytomation).
Cell toxicity assays
In order to assess any toxic effect of Aβ42 oligomers on undifferentiated SH-SY5Y cells, the Vybrant
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide] cell-proliferation assay (Invitrogen) was used according
to the manufacturer’s protocol (see Supplementary material
at http://www.BiochemJ.org/bj/441/bj4410579add.htm). In addition, to determine the mode of cell death that Aβ42 conferred
on undifferentiated SH-SY5Y cells, the annexin-V-Fluos
staining kit (Roche) was used according to the manufacturer’s
protocol. SH-SY5Y cells (2×105 cells/well) were seeded on
uncoated or collagen-I-coated glass coverslips in a 24-well plate
Cellular effect of Alzheimer’s Aβ
1 day prior to the assay. The cells were treated with buffer only,
or 10 or 25 μM oligomeric Aβ42 for 1, 5 or 24 h at 37 ◦ C.
At the given time points, the culture medium was replaced
with Hepes medium containing Annexin V and PI (propidium
iodide) for 15 min at room temperature. The cells were rinsed
once with PBS and fixed with 4 % PFA (paraformaldehyde)
for 15 min at room temperature. The coverslips were mounted
on glass slides using prolong gold anti-fade mounting medium
with DAPI (4 ,6-diamidino-2-phenylindole; Fisher Scientific) and
images were taken using a Nikon microscope. Cell analysis was
performed using ImageJ (National Institutes of Health). More
specifically, early necrotic cells were labelled only with PI (green),
late necrotic cells with both annexin V and PI (red and green),
and apoptotic cells with annexin V only (red), whereas living
cells were coloured blue (DAPI, nuclei). Data were plotted using
GraphPad Prism software.
Immunofluorescence microscopy
Undifferentiated SH-SY5Y cells were seeded on to glass
coverslips at a density of 2×105 cells/well in a 24-well plate. The
cells were treated with buffer only, 10 or 25 μM oligomeric Aβ42
or Alexa Fluor® 555-tagged Aβ42 for 1, 5 or 24 h. Autophagy
was induced by starving the cells in DMEM/F12 without serum
for 3 h. At the given time points the cells were fixed with 4 % PFA
for 15 min at room temperature, permeabilized in 0.1 % Triton X100 for 5 min and blocked for 30 min in 2 % BSA. The proteins
of interest were then detected using the mouse monoclonal NU1
antibody [11] (1:600 dilution) (total Aβ levels), the anti-(LC3II) rabbit polyclonal antibody (1:400 dilution) (autophagosomal
marker) and the rabbit polyclonal antibodies against clathrin
heavy chain (1:500 dilution, Abcam) or cathepsin D (5 μM,
Scripps Laboratories, San Diego, U.S.A.) followed by Alexa
Fluor® 546-labelled goat anti-mouse IgG (H + L) (1:200 dilution,
Invitrogen) and Alexa Fluor® 488-labelled goat anti-rabbit
IgG (H + L) (1:200 dilution, Invitrogen). The differentiation
levels of the SH-SY5Y cells were detected using the Alexa
Fluor® -labelled anti-(α-tubulin) clone DM1A antibody (1:250
dilution, Upstate Biotechnology). Cell images were obtained
using a confocal microscope (Zeiss Cell Observer with an
Infinity multibeam confocal scanning head) and analysed using
ImageJ.
TEM (transmission electron microscopy)
Negative stain TEM
Aβ42 was incubated in cell medium, serum or Hepes buffer
at 10 and 25 μM at 37 ◦ C. Then, a 4 μl sample of Aβ42 was
withdrawn at selected time points, and negatively stained electron
microscopy grids were prepared by placing the solution on
carbon/formvar copper 400 mesh grids (Agar Scientific), blotting
and washing with filtered water, blotting and then staining with
2 % (w/v) uranyl acetate. Grids were examined using a Hitachi
7100 microscope operated at 80 kV.
Immunogold labelling TEM
Undifferentiated SH-SY5Y cells were seeded in 12-well plates
at a density of 3×105 cells/well. The cells were treated with
buffer only, or 10 or 25 μM freshly prepared Aβ42 for 1, 5 or
24 h. The cells were trypsinized using 0.25 % trypsin in PBS
(Gibco), centrifuged for 5 min at 14 000 g and fixed in 4 %
formaldehyde/0.1 % glutaraldehyde in 0.1 M mono-/di-sodium
581
phosphate (pH 7.4) for a few hours at room temperature and then
at 4 ◦ C on a rotator overnight. The following procedures were
all carried out at 4 ◦ C. The fixed pellets were then washed in
0.1 M mono-/di-sodium phosphate and dehydrated in an ethanol
series, followed by 2:1 and then 1:2 100 % ethanol/UnicrylTM
resin (British BioCell) for 30 min each. Finally the pellets were
infiltrated in complete UnicrylTM resin and light-polymerized as
described previously [29].
We used established methods to perform immunogold labelling
[30]. Briefly, a modified PBS (pH 8.2) containing 1 % BSA,
500 μl/l Tween 20, 10 mM Na-EDTA and 0.2g/l sodium azide
(termed PBS + ) was used throughout the following procedures
for all dilutions of antibodies and gold probes. Thin sections were
cut and initially blocked in normal goat serum (1:10 dilution in
PBS + ) for 30 min at room temperature (approximately 24 ◦ C),
then double-labelled with mouse monoclonal NU1 antibody
(10 μg/ml IgG) and either a rabbit monoclonal antibody against
cathepsin D (5 μg/ml IgG, Scripps Laboratories, San Diego, CA,
U.S.A.) or rabbit polyclonal antibody against clathrin heavy chain
(1:300 dilution, AbCam).
After three 2 min PBS + rinses, the sections were
immunolabelled in a mixture of 5 and 10 nm respectively goldparticle-conjugated goat anti-rabbit and goat anti-mouse IgG
secondary probes (1:10 dilution in PBS + for 1 h at room
temperature; British BioCell). Sections were subsequently rinsed
in PBS + (three 10 min washes) and distilled water (four
5 min washes). All immunogold-labelled thin sections were
subsequently post-stained in 2 % (w/v) aqueous 0.22 μm-filtered
uranyl acetate for 1 h.
TEM ultrastructural analysis
Undifferentiated SH-SY5Y cells were treated with buffer only,
or 10 or 25 μM freshly prepared Aβ42 and pelleted as described
above for immunogold labelling TEM. The pellets were fixed
in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate/HCl buffer
(pH 7.4) for 2 h at room temperature and then at 4 ◦ C on a rotator
overnight. The pellets were then post-fixed in 1 % (w/v) osmium
tetroxide in 0.1 M sodium cacodylate/HCl buffer (pH 7.4) for
2 h at room temperature before being dehydrated in an ethanol
series. After two 20 min washes in propylene oxide, the pellets
were infiltrated over several days, with a few resin changes, in
TLV (TAAB low-viscosity) resin before polymerizing at 60 ◦ C for
16 h. The pellets were then sectioned and stained with 2 % (w/v)
aqueous 0.22 μm-filtered uranyl acetate at room temperature
for 1 h.
TEM acid phosphatase assay
The assay was performed as described in [31] with a few
alterations. Briefly, undifferentiated SH-SY5Y cells were treated
with buffer only, or 10 or 25 μM freshly prepared Aβ42 and
pelleted as described above. The pellets were fixed in 2 %
glutaraldehyde in 0.1 M sodium cacodylate/HCl buffer (pH 7.4)
for 1 h at room temperature and then at 4 ◦ C on a rotator overnight.
The pellets were then incubated in 40 mM Tris-maleate buffer
(pH 5.2), containing 8 mM sodium glycerophosphate, 2.4 mM
lead nitrate and 7 % sucrose for 1 h at 37 ◦ C, before being washed
in 40 mM Tris-maleate buffer containing 7 % sucrose. They were
then post-fixed with 1 % (w/v) aqueous osmium tetroxide for 1 h
at room temperature and then dehydrated and embedded in TLV
resin as described above. Thin sections were taken and stained
with 2 % (w/v) aqueous 0.22 μm-filtered uranyl acetate at room
temperature for 1 h.
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V. Soura and others
the results of annexin V survival assays, indicating that Aβ42 was
most toxic following 24 h incubation at both concentrations, but
that the 10 μM concentration reduced cell survival more than the
higher concentration after a 24 h incubation. This may be due to
the larger accumulation of toxic oligomeric peptide at the lower
concentration and the increased assembly speed at the higher
concentration, resulting in more fibrils than oligomers. Annexin
V assays showed that a higher proportion of the cells died by
a necrotic mechanism at 24 h (Figure 2B). These results were
supported by the results of MTT assays (Supplementary Figure
S1 at http://www.BiochemJ.org/bj/441/bj4410579add.htm).
Externally administered Aβ42 is internalized into SH-SY5Y cells
Figure 1 Transmission electron micrographs showing assembly of 10 μM
Aβ42 in Hepes buffer at 37 ◦ C and 5 % CO2
(A) 0 h, (B) 1 h, (C) 5 h, (D) 24 h, (E) 48 h and (F) 72 h.
TEM imaging
All of the TEM samples above were examined in a Hitachi-7100
transmission electron microscope at 100 kV and images were
acquired digitally with an axially mounted (2000×2000 pixel)
Gatan Ultrascan 1000 CCD (charge-coupled device) camera
(Gatan UK) and examined using ImageJ. The circularity and area
of the nucleus was examined and measured using a module within
ImageJ.
RESULTS
Aβ42 assembly in cell medium and buffer
Aβ42 is known to assemble rapidly and therefore the Aβ42
peptide was prepared using a method developed to ensure removal
of preformed aggregates. This method also ensured removal of
residual solvents that could affect results, since recent work has
shown that remaining HFIP solvent can be responsible for some
of the results shown for previous studies [27]. It is well established
that Aβ forms transient oligomeric species and protofibrils and
eventually mature amyloid fibrils. Therefore although it may be
possible to establish the starting state of the Aβ, it is not clear
how the structures develop within the time of a cell assay. To
investigate the behaviour of Aβ in cell medium and in buffer,
freshly prepared Aβ42 was dissolved at 10 and 25 μM and
assembly was monitored using electron microscopy. The samples
were withdrawn at selected time points and examined by negativestain electron microscopy to chart the progress of oligomerization
(Figure 1). The results reveal that the peptide assembled as
expected through small oligomeric species, rapidly to curvilinear
protofilaments by 5 h and finally amyloid-like fibrils over the
incubation period of 72 h. Electron micrographs taken of Aβ
incubated in medium were more difficult to image but appeared
to show a similar or slightly faster aggregation process (results
not shown).
Aβ42 oligomers reduce cell survival over 24 h
Cultured neuroblastoma cells were incubated with a final
concentration of 10 or 25 μM freshly prepared Aβ42 and
neurotoxicity was monitored using an annexin V assay and results
were supported by carrying out an MTT reduction assay.
‘Untreated’ cells were treated with buffer only. Figure 2 shows
c The Authors Journal compilation c 2012 Biochemical Society
Immunofluorescence confocal microscopy was used to monitor
the accumulation of Aβ42 oligomers on/in the treated
neuroblastoma cells (Figure 3) using the antibody NU1 [11],
a conformation-specific monoclonal antibody that binds to Aβ
oligomers and fibrils [11], following 1, 5 and 24 h incubation.
Cells were also labelled with a fluorescent antibody against
clathrin, since Aβ uptake has previously been shown to be
partially mediated by clathrin endocytosis [32]. The results
revealed accumulation of large Aβ42 immunoreactive areas on the
surface of the cells and Z-stacking revealed that some of the Aβ42
penetrated and was internalized into the cells (Figure 3). Untreated
cells also showed Aβ-positive staining, but this was diffuse and
did not show the accumulated signals (Figure 3). However, this did
highlight that these cells contained significant immunoreactive
endogenous Aβ, as observed previously [15,33]. In order to
confirm the presence of endogenous Aβ, we performed Western
blot analysis using the NU1 antibody on homogenized untreated
neuroblastoma cells (Figure 4). These results revealed a strong
band at 64 kDa and at approximately 50 kDa arising from Aβ
generated within these cells. Western blots were also performed
using the anti-[Aβ-(1–16)] monoclonal antibody 6E10. Untreated
cells showed a similar pattern of bands to NU1, although the
64 kDa and 50 kDa bands were not observed (Figure 4). However,
Western blot analysis of cells treated with Aβ42 at 25 μM showed
an additional 6E10 immunoreactive band at approximately 4–
5 kDa in the soluble fractions (wash, supernatant and medium),
which probably represents an SDS-soluble monomeric species.
This band was not observed in Western blots from untreated cells
and therefore this is likely to arise from exogenously added Aβ42.
Interestingly, this band became weaker with time, and by 24 h
incubation was no longer observed (Figure 4). This observation
supports the idea that Aβ42 is assembling in the presence of the
cells and, once internalized, continues to assemble to form higher
molecular mass assemblies [5].
Immunofluorescence of Aβ42 oligomer-treated cells clearly
revealed a difference between treated and untreated cells,
showing accumulation and internalization over 24 h incubation
with Aβ42 at the two concentrations (Figure 5). However,
in order to distinguish exogenous Aβ42 from endogeneous
Aβ, we used an Alexa Fluor® 555-tagged Aβ42 system.
It is possible that the fluorescent tag could cause steric
hindrance that would affect Aβ42 aggregation and therefore
we followed assembly using TEM. The results revealed that
Alexa Fluor® 555–Aβ42 was more fibrillogenic, forming a
large number of ordered fibrils by 4 h (Supplementary Figure
S2 at http://www.BiochemJ.org/bj/441/bj4410579add.htm). MTT
assays indicated that the Alexa Fluor® 555–Aβ42 (used
immediately following dissolution) caused a significant reduction
in MTT intensity compared with untreated cells (Supplementary
Figure S1B), indicating a reduction in cell survival. However, this
effect was less than the effect on cells administered with freshly
Cellular effect of Alzheimer’s Aβ
Figure 2
583
Cell toxicity assays
Comparison of the effect of freshly prepared oligomeric Aβ42 on SH-SY5Y cells with untreated cells using an annexin V assay at 1 (A) and 24 (B) h. Aβ at 25 μM (black bars) and 10 μM Aβ (grey
bars) compared with untreated (white bars).
Figure 3
Confocal microscopy images of SH-SY5Y cells incubated in the presence or absence of 10 and 25 μM Aβ42 for 1 and 24 h
Aβ is shown in red and clathrin is shown in green; DAPI staining for nuclei is shown in blue. Z -stacking images reveal the localization of fluorescence at sections through the cell. The images reveal
that cells treated with Aβ42 show punctate staining surrounding cells, on the membranes and within cells. In contrast, untreated cells show diffuse Aβ42 immunofluoresence.
prepared untagged Aβ42 (Supplementary Figure S1A). Confocal
microscopy was again used to monitor the effect of Alexa Fluor®
555–Aβ42 oligomers on neuroblastoma cells. Again, we observed
cell-membrane accumulation and internalization with a similar
pattern to untagged Aβ42 (Figure 5). Immunofluorescence of
clathrin showed some overlap with Aβ staining (Figure 5B)
and the co-localization analysis (Pearson correlation) revealed
significant co-localization between clathrin and Alexa Fluor®
555–Aβ42 fluorescence, especially for the cells administered with
a higher concentration of Aβ42 (25 μM). This co-localization was
observed to decrease by 24 h, suggesting that initial internalization
may be partially clathrin-mediated. This indicates that exogenous
Aβ42 oligomers accumulate in patches on the cell membrane
and some are internalized into the cells via a partially clathrinmediated endocytosis mechanism.
Externally administered Aβ42 accumulates in lysosomal
compartments
It has been shown previously that Aβ can accumulate in
lysosomal vesicles [7], so to follow the pathway of Aβ through the
endosomal pathway and possible accumulation within lysosomes,
cathespin D co-labelling with NU1 was monitored. Confocal
images of Aβ-treated cells showed some co-localization with
cathepsin D at later time points (24 h), indicating the accumulation
of Aβ in cathepsin D-positive organelles such as lysosomal
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584
Figure 4
V. Soura and others
Western blots from lysed untreated SH-SY5Y cells, showing levels of Aβ-positive bands
Incubation with NU1 [11] reveals bands at approximately 100 kDa and 64 kDa, and at approximately 56 kDa. This lowest molecular mass band may represent the 56 kDa* band [41]. Western blots
from cells treated with 25 μM Aβ42 or untreated were incubated with the commercially available antibody 6E10. A labelled low molecular mass band was apparent in treated but not untreated cells
in the soluble fraction (supernatant, wash and medium) and this decreased over the time that the cells were incubated with Aβ. This may represent the administered ‘monomeric’ Aβ42. P, pellet; SN,
supernatant; W, wash; M, medium. The molecular mass in kDa is indicated.
Figure 5
Confocal microscopy images of SH-SY5Y cells incubated in the presence of absence of 10 and 25 μM Alexa Fluor® 555–Aβ42 for 1 and 24 h
(A) Alexa Fluor® 555–Aβ is red and clathrin is shown in green. DAPI staining for nuclei is shown in blue. (B) Co-localization analysis showed a significantly higher amount of Alexa Fluor®
555–Aβ found in clathrin-immunoreactive regions after 1 h of incubation of SH-SY5Y cells with 25 μM Alexa Fluor® 555-tagged Aβ (black) compared with 10 μM (grey). This difference is no
longer evident after 24 h. Correlation by chance (rRand) is in green.
vesicles. The results shown in Figure 6 also show a clear
difference in the distribution of Aβ in untreated and treated cells
at 1 h, whereby treated cells showed punctate staining with NU1.
By 24 h, there was overlap of NU1 and anti-(cathepsin D) signals.
However, analysis did not show significant differences between
co-localization in treated and untreated cells, but comparison
of the distribution of labelling did show a difference in the two
groups.
Immunogold labelling TEM reveals internalization of Aβ and
proximity with clathrin
Confocal microscopy has indicated that Aβ42 is internalized into
the cells and may accumulate in lysosomes or late endosomes.
In order to examine internalization of Aβ42 at higher resolution
and to gain greater insights into the proximity of Aβ with the
markers for endosomes, we monitored immunogold labelling
by TEM of Aβ42 oligomer-treated cells following incubation
for 1, 5 and 24 h. This method allowed us to examine the
pathway of entry into cells, as well as allowing the localization
c The Authors Journal compilation c 2012 Biochemical Society
of Aβ within cells, at magnifications of ×10 000–20 000,
allowing conclusions to be drawn regarding the proximity of
peptides with markers. Examination of treated cells revealed
that Aβ42 was being internalized via invaginations in the cell
membrane that co-labelled with clathrin (Figures 7A and 7B).
High magnification images (Figure 7B), clearly revealed circular
labelling with both NU1 and anti-clathrin antibodies that appeared
to resemble endosomes, and these were most appreciable at
earlier time points (Figure 7B). Although this does not preclude
other mechanisms of entry into the cells, we observed the
invagination and internalization via an endosomal process
(Figures 3, 5, 7A and 7B).
Immunogold labelling TEM reveals Aβ proximity with the
lysosomal enzyme cathepsin D
To examine the pathway and eventual destination of Aβ following
incubation at 10 and 25 μM, we co-labelled with NU1 and
anti-(cathepsin D). Figures 7(A) and 7(C) show that in treated
Cellular effect of Alzheimer’s Aβ
Figure 6
585
Confocal microscopy images of SH-SY5Y cells incubated in the presence or absence of 10 and 25 μM Aβ42 for 1 and 24 h
Aβ is shown in red and cathepsin D is shown in green. DAPI staining for nuclei is shown in blue. Z -stacking images reveal the localization of fluorescence at sections through the cell. The images
reveal that cells treated with Aβ42 show punctate staining surrounding cells, on the membranes and within cells. In contrast, untreated cells show diffuse Aβ42 immunofluoresence and no apparent
co-localization of Aβ with cathepsin D.
cells Aβ is found within vesicles that label with cathepsin D,
indicating that they are lysosomes. Interestingly, large cathepsin
D-positive organelles were observed following 1 h incubation
with both concentrations of Aβ, and these accumulated and
grew to be more defined by 24 h incubation (Figure 7C). The
size of the cathepsin D-positive organelles was approximately
300–400 nm and they were observed to have a multivesicular
appearance (Figure 7D). Untreated cells showed small membranebound organelles that co-labelled with Aβ and cathepsin D, which
resembled lysosomes, suggesting that endogeneous Aβ was also
found in lysosomes (Figure 7C). These observations are consistent
with immunofluorescence results showing co-localization in both
treated and untreated cells (Figure 6). The major observation
here, however, is that the lysosomes differ significantly in their
appearance in the Aβ42-treated cells.
Comparison of the ultrastructure of Aβ42-treated and untreated
cells
Preparation of cells for immunogold labelling, via a minimal cold
fixation protocol to maximize retention of protein antigenicity,
results in a relatively poorly resolved ultrastructure. Therefore
to compare the high-resolution ultrastructure of organelles of
treated and untreated cells, we prepared the cells using a
standard double-fixation protocol, including osmium tetroxide
post-fixation. In treated cells, there were a number of significant
differences, particularly following 24 h incubation (at the time
point that correlates with highest cell death). The nuclei of
untreated cells generally had consistent smooth envelopes, were
predominantly circular, and usually possessed a single clear
nucleolus. Treated cells often exhibited highly invaginated nuclear
envelopes, fragmented/speckled heterochromatin and several
nucleoli. These differences may well indicate pre-apoptotic
events in affected cells. Measurement of the circularity of nuclei
was performed using a circularity measurement module within
ImageJ [34] and revealed that the 10 μM-treated cells had
significantly invaginated envelopes following 5 h incubation when
compared with the untreated cells at 5 h (Supplementary Figure
S3 at http://www.BiochemJ.org/bj/441/bj4410579add.htm). In
addition, treated cells showed a clear accumulation of large
vacuolar structures often found close to the nucleus. In order to
examine this phenomenon in more detail and monitor lysosome
integrity, the cells were prepared using an acid phosphatase
cytochemical assay [31] to focus on lysosomes and lysoautophagosomes [23].
Visualization of lysosomes in Aβ42-treated and untreated cells
Lysosomes have previously been highlighted as being affected in
AD and in FAD transgenic animals [20,24]. For the first time,
acid phosphatase assays were performed to highlight differences
between treated and untreated cells (Figure 8). Untreated cells
showed acid phosphatase-positive organelles, consistent with
lysosomes, and these were small, round and membrane-bound,
c The Authors Journal compilation c 2012 Biochemical Society
586
Figure 7
V. Soura and others
Immunogold labelling TEM images of SH-SY5Y cells incubated in the presence or absence of Aβ42 at 10 and 25 μM for 1, 5 and 24 h
(A) Aβ is labelled with 10 nm and clathrin labelled with 5 nm secondary-antibody-conjugated gold particles. Clathrin co-localized with Aβ in small vesicles that resemble endosomes. (B) Enlarged
electron micrograph showing the co-labelling with the NU1 and anti-clathrin antibodies. Circular vesicles highlighted with black arrows are likely to represent endosomal vesicles. This image shows
cells that have been incubated with 10 μM Aβ42 for 1 h. (C) The Aβ is labelled with 10 nm and cathepsin D is labelled with 5 nm secondary-antibody-conjugated gold particles. Cathepsin D
co-localized with Aβ in lysosomes and also larger vacuoles that may represent autophagosomes. The density of labelling is more obvious after a 24 h incubation. (D) Enlarged electron micrograph
showing the co-labelling with NU1 and anti-cathepsin D antibodies showing a large multivescicular body containing Aβ and cathepsin D. This image shows cells that had been incubated with
25 μM Aβ42 for 24 h.
and often densely and evenly stained. This was in stark contrast
with treated cells that had more numerous and larger acid
phosphatase-stained organelles. However, compared with those
in the untreated cells, these lysosomes often had relatively
undefined boundaries and sharply speckled staining. In addition,
autophagosomes were numerous and often appeared close to the
nucleus. It was also clear that cells showing autophagosomes
also had invaginated nuclear envelopes. These autophagosomes were never observed in untreated cells and were more
commonly found in treated cells that appeared to be deteriorating
at 24 h following treatment with Aβ. Careful re-evaluation of
cathepsin D co-labelled electron microscope sections revealed that
cathespsin D was often found around lysosomes, and not always
inside them, suggesting that these lysosomes may be leaking. This
was supported by the observation of poorly resolved lysosomes
in the treated cells (Figures 7C, 7D and 8).
Confocal microscopy was performed in order to monitor the
level of LC3-II expression, as a marker for autophagosome
formation and accumulation. Figure 9(A) showed an increased
punctate level of LC3-II staining that correlated with punctate
c The Authors Journal compilation c 2012 Biochemical Society
Aβ staining in treated cells, which did not appear in images of
untreated cells. In order to ‘induce’ autophagosome formation in
cells as a positive control, the cells were starved. Interestingly,
these starved cells that had not been treated with Aβ, showed a
significant increase in Aβ levels similar to those of 10 μM-treated
cells, suggesting that Aβ may play a role in mediating autophagosome formation (Figure 9B). The analysis of immunofluorescence
images showed that Aβ-treated cells had significantly higher
Aβ immunofluorescence and LC3 fluorescence than untreated
cells (Figures 9B and 9C). Co-localization of LC3 and Aβ
was observed in treated and starved cells, but not in untreated
cells (Figure 9D). Alexa Fluor® –Aβ42 showed increasing
LC3 levels with incubation time (Supplementary Figure S4 at
http://www.BiochemJ.org/bj/441/bj4410579add.htm).
Internalization of Aβ42 to primary hippocampal neurons
To assess the distribution of Aβ in a primary neuronal
system, mature hippocampal cells were treated with either
Alexa Fluor® 555-tagged or untagged Aβ42 peptides. Alexa
Cellular effect of Alzheimer’s Aβ
Figure 8
587
Electron microscopy images of acid phosphatase-stained SH-SY5Y cells incubated in the presence or absence of 10 or 25 μM Aβ42 for 1 and 24 h
Acid phosphatase stains lysosomes and lyso-autophagosomes. Representative areas showing lysosomes and autophagosomes are shown.
Fluor® 555–Aβ42 was incubated with neurons for 24 h and
the cells imaged at 2 and 24 h (Supplementary Figure S5 at
http://www.BiochemJ.org/bj/441/bj4410579add.htm). Aβ42 was
administered to neurons and the cells were fixed after 1
and 24 h and labelled with NU1 (Supplementary Figure S6
http://www.BiochemJ.org/bj/441/bj4410579add.htm). Both live
imaging and fixed preparations showed the initial distribution
of Alexa Fluor® 555–Aβ or Aβ as small discrete puncta visible
across the axons and dendrites, and on the soma, changing to larger
intracellular aggregates over a 24 h time period (Supplementary
Figures S5 and S6). Although it is currently unclear whether
these aggregates localize to specific intracellular organelles, these
observations support the idea of gradual Aβ accumulation in
susceptible neurons.
DISCUSSION
The results of the present study have revealed that oligomeric Aβ
is able to enter neuroblastoma cells and that this may be visualized
using not only light microscopy, but also electron microscopy. The
intracellular accumulation of Aβ administered at two different
concentrations was monitored. Although these concentrations do
not directly correlate with concentrations measured in cerebral
spinal fluid in AD, the exact local concentration of Aβ around
and within neurons is yet to be established. In addition, the
accumulation and damage caused by AD pathology is a very longterm process, and in our model system we aimed to investigate
changes induced within a relatively short time frame of 24 h.
Fluorescent-tagged Aβ has allowed us to differentiate between
endogenous Aβ and administered Aβ. We note that there is a
significant reduction in cell survival following incubation with
Aβ for 24 h, and that this correlates with an observation of
accumulation of Aβ inside cells within endosomes (co-labelled
with clathrin) and lysosomes (co-labelled with cathepsin D), and
with the appearance of autophagosomes around damaged nuclei.
Acid phosphatase staining, which highlights lysosomes and autophagosomes, reveals that Aβ-treated cells have a very different
appearance compared with untreated cells, with nuclei that appear
damaged associated with apparently damaged lysosomes and
significant numbers of autophagosomes around the nucleus.
We note that the level of immunoreactive endogenous Aβ
in untreated cells is surprisingly high. However, there is a
striking difference between untreated and Aβ-treated cells at
both light and electron microscope levels. At early time points,
Aβ accumulates on the cell membrane and is internalized,
and this internalization is also observed when tagged Aβ
is administered to cells, allowing us to distinguish between
exogenously administered Aβ and endogenous Aβ already
present. Internalization may be partly mediated by clathrin, since
we observed by TEM clathrin-positive endosomes with Aβ
labelling in Aβ42-treated cells (Figures 7A and 7B). Confocal
microscopy using Alexa Fluor® 555–Aβ also revealed colocalization of clathrin and Aβ (Figure 5). Previously clathrinmediated endocytosis has been implicated in Aβ internalization
[32], and inhibition of the endosomal process has been shown
c The Authors Journal compilation c 2012 Biochemical Society
588
Figure 9
V. Soura and others
Immunofluorescence confocal microscopy images showing the LC3 levels in treated and untreated cells
(A) Staining for Aβ (red) and LC3 (green) following incubation of SH-SY5Y cells with 10 and 25 μM Aβ42 at 1, 5 and 24 h compared with untreated and untreated starved cells (at 1 h). The results
are shown graphically for the intensity of fluorescence from Aβ immunolabelling (B) and LC3 labelling (C), and the correlation is shown in (D). Comparison of 25 μM Aβ42 (black bars), 10 μM
Aβ42 (grey bars) and untreated (white bars). Untreated starved cells are shown at 1 h only (striped bars). Correlation by chance (rRand) is in green.
to partially reduce Aβ-induced toxicity [35]. Immunogold
microscopy revealed Aβ in endosomes and lysosomes in both
untreated and treated cells. However, the treated cells appear
to have distorted nuclear envelopes and dense accumulations of
Aβ in lysosomes. In particular, autophagosomes are found in
treated cells at later time points, but not observed in untreated
cells.
We have used an antibody against cathepsin D as a marker
for lysosomes. Cathepsin D is a lysosomal aspartyl protease
that has been suggested to be involved in clearance of Aβ
c The Authors Journal compilation c 2012 Biochemical Society
through cleavage of Aβ and aggregates [36]. Our results from
TEM immunogold studies reveal co-localization within lysosomal
vesicles, supporting the view that cathepsin D plays a major role in
the clearance of Aβ peptide species. Elevated levels of cathepsin
D have been observed in AD senile plaques and the cerebrospinal
fluid of AD patients [37]. Recent work has revealed evidence for
cathepsin D leakage from lysosomes in transgenic mice with the
Osaka APP mutation and NU1 labelling revealed accumulation
of Aβ oligomers in lysosomes, as well as in mitochondria and
endoplasmic reticulum [38].
Cellular effect of Alzheimer’s Aβ
Recent work by the Nixon group has revealed that autophagosomes accumulate in PS1 mutant cells, and that de-acidification
of the lysosomes and autophagosomes appears to accompany
deterioration of the cells [24]. Previously, it has been shown that
autophagosomes contain APP and all the secretases necessary to
produce Aβ [39]. It has also been shown that autophagosome
accumulation is a particular hallmark of AD brain cells [20,39],
and that the accumulation of intermediates involved in the later
stages of autophagic clearance are a feature of AD pathology
and related neurodegenerative diseases, and are linked directly
with neuronal apoptosis through rapid large-scale autophagic
vacuole accumulation [40]. Amelioration of lysosomal protease
dysfunction in a transgenic AD mouse revealed reduction in
Aβ accumulation and also reduction in deficits in learning and
memory, supporting the view that autophagosome accumulation
is important in the disease process [25].
The results of the present study lead us to suggest that
the observed increase in autophagosomes in cells and also the
accumulation observed in AD brains [20] can be correlated
with increased Aβ following exogenous administration. The
internalization of Aβ and the observed pathway through
the endosomal system suggests a disruption to a delicate
balance between autophagosome formation and autophagosome
degradation in neuronal cells, resulting in an observed increase in
autophagosome accumulation and decreased cell survival. The
autophagosomes observed are positively stained for cathepsin
D as well as LC3, suggesting that they are autophagosomes
following fusion with lysosomes. These were observed at
relatively early time points following incubation with Aβ42
and appeared to accumulate with time. In healthy cells,
autophagosomes are cleared following fusion with lysosomes
by which lysosomal enzymes complete the degradation of
the contents of the autophagosome. Under the conditions
of treatment used in the present study, the autophagosomes appear
to accumulate and are not degraded. We hypothesize that this
is due to disruption of the lysosomal environment, possibly by
permeation of the lysosomal membrane. Lysosomal damage has
been reported previously [7], and this idea is supported by our
recent work in which we show that Aβ42 is able to permeate
and damage biomimetic vesicles and bilayers [26]. Any small
change in the permeability of lysosomes could lead to catastrophic
changes in the internal pH of the lysosome, leading to inactivation
of lysosomal enzymes and accumulation of autophagosomes.
Aβ oligomers are known to affect membrane integrity [6] and
have been shown previously to affect the integrity of lysosomal
membranes leading to leakage and also a change in internal pH
[7]. This in turn would be expected to directly affect the activity
of lysosomal enzymes, such as cathepsin D, whose activity is
optimum at low pH values. It has been shown previously that
inhibitors of lysosomal proteases lead to increased accumulation
of autophagosomes. We suggest that the increased level of
intracellular toxic Aβ leads to cell death by disrupting lysosomes
and their proteases, leading to autophagosome accumulation that
cannot be cleared. Acid phosphatase staining and immunogold colabelling with cathepsin D indicates the possibility that lysosomal
membranes are being disrupted. It is likely that leakiness of
lysosomes would not only allow potentially dangerous proteases
to leak out into the cytoplasm, but that the pH of the lysosomes
and lysoautophagosomes will equilibrate with the cytosol, thus
significantly reducing the efficiency of the remaining proteases.
Conclusions
The present study reveals, for the first time, that administration of
exogenous Aβ oligomers results in damaged and degenerating
589
neuroblastoma cells that accumulate autophagosomes and
damaged lysosomes. It cannot be excluded that endogenous Aβ
is being recruited by exogenous oligomeric material and is also
accumulating in autophagosomes. These findings support the view
that autophagosome accumulation is pathogenic and related to
degeneration of the cells.
AUTHOR CONTRIBUTION
Violetta Soura and Maris Stewart-Parker designed and carried out the experiments,
and analysed the data. Joe Atherton, Roma Ramaran and Elisabeth Darwent conducted
experiments and collected data. Thomas Williams developed and prepared tagged Aβ.
Arjuna Ratnayaka and Kevin Staras provided expertise in experimental work on primary
neurons. Kirsti Gorringe and Jack Tuffin conducted data analysis. William Klein and
Pascale Lacor provided the NU1 antibody, and advised on the experimental approach and
writing of the paper. Julian Thorpe provided expertise in experimental preparation, image
collection and analysis of electron microscopy data, and contributed to writing the paper.
Louise Serpell managed the project, critically assessed the data and wrote the paper.
ACKNOWLEDGEMENTS
We thank Nadia Lovegrove, Stuart Rulten, Sally Wheatley, Roger Phillips, Vincenzo Marra,
Majid Hafezparast, George Kemenes, Damian Crowther, George Banting and Aidan Doherty
for their valuable help in the experimental work and writing of this paper.
FUNDING
This work was supported by Alzheimer’s Research UK; Biotechnology and Biological
Science Research Council [grant number BB/E009042/1]; and the Wellcome Trust [student
internship]. K.S. is supported by the Wellcome Trust [grant number WT084357MF]; and
Biotechnology and Biological Science Research Council [grant number BB/F018371]. W.
K. and P. L. would like to acknowledge funding from an AA Zenith Award and the National
Institutes of Health [grant numbers RO1 AG029460 and RO1 AG022547].
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doi:10.1042/BJ20110749
SUPPLEMENTARY ONLINE DATA
Visualization of co-localization in Aβ42-administered neuroblastoma
cells reveals lysosome damage and autophagosome accumulation related
to cell death
Violetta SOURA*, Maris STEWART-PARKER*, Thomas L. WILLIAMS*1 , Arjuna RATNAYAKA*, Joe ATHERTON*,
Kirsti GORRINGE*, Jack TUFFIN*, Elisabeth DARWENT*, Roma RAMBARAN*, William KLEIN†, Pascale LACOR†,
Kevin STARAS*, Julian THORPE* and Louise C. SERPELL*2
*School of Life Sciences, University of Sussex, Falmer, BN1 9QG, U.K., and †Northwestern University 2205 Tech Drive, Evanston, IL 60208-3520, U.S.A.
Figure S1 Comparison of the effect of freshly prepared, oligomeric Aβ42 (A) and Alexa Fluor® 555-tagged Aβ42 (B) to untreated SH-SY5Y cells, using the
MTT reduction assay
The Vybrant MTT cell proliferation assay (Invitrogen) was used according to manufacturer’s protocol. Briefly, 105 undifferentiated cells/well were seeded on 96-well plates. The cells were treated with
buffer only, or 10 or 25 μM oligomeric Aβ42 or Alexa Fluor® 555-tagged Aβ42 for 1, 5 and 24 h at 37 ◦ C. At the given time points, 12 mM MTT solution was added and the cells were incubated
for a further 2 h at 37 ◦ C. The resulting formazan precipitate was dissolved with 50 μl of DMSO and the fluorescence was measured using a plate reader at 540 nm with a 620 nm reference filter. The
untreated cell reading at each time point was set to 100 % redox activity and the treated cell readings were converted into a percentage. The redox activity gives an indication of cell survival.
Figure S2
(C) 24 h
Progression of Alexa Fluor® 555–Aβ42 assembly, incubated in Hepes at 82 μM, by TEM at (A) 0 h, immediately following dissolution, (B) 4 h and
When compared with non-tagged Aβ, it appears that assembly to form fibrils is faster and large numbers of amyloid-like fibrils are visible at only 4 h incubation.
1
2
Present address: Drexel University, Physics Department, 3141 Chestnut Street, Philadelphia, PA 19104, U.S.A.
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
V. Soura and others
Figure S3
A significant difference at 5 h showing invagination of the nuclear envelope in cells treated with 10 μM and 25 μM Aβ42
Electron micrographs showing the ultrastructure of treated and untreated cells were examined using ImageJ and measurements performed to measure the circularity of the nuclear envelope
Figure S4 Fluorescence confocal microscopy images showing staining for Alexa Fluor® 555–Aβ (red) and immunofluorescence against LC3 (green) following
incubation of SH-SY5Y cells with 10 and 25 μM Alexa Fluor® 555–Aβ42 at 1 and 24 h compared with untreated cells
The images show a general increase in LC3 levels with increasing incubation time.
EXPERIMENTAL
Hippocampal neuron cell culture
Dissociated hippocampal cultures were prepared from P0–P1 rats
by plating neurons on an astrocyte feeder layer, and maintained in
BME (Eagles basal medium) with 20 mM glucose, 10 mM Hepes
buffer, 1 mM sodium pyruvate, 1 % Glutamax, 2 % FCS and
2 % B27 supplement. Mature hippocampal neurons were used for
c The Authors Journal compilation c 2012 Biochemical Society
experiments after 10 days in vitro. Experiments were performed
in accordance with the UK-Animal (Scientific Procedures) Act
1986 and complied with local institutional regulations. Cells
were imaged in external bath solution {137 mM NaCl, 5 mM
KCl, 2.5 mM CaCl2 , 1 mM MgCl2 , 10 mM D-glucose, 5 mM
Hepes, 20 μM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione)
and 50 μM AP5 [D( − )-2-amino-5-phosphonovaleric acid]}
at 25 ◦ C.
Cellular effect of Alzheimer’s Aβ
Labelling and imaging of cultured neurons
Hippocampal cultures were treated with freshly prepared Alexa
Fluor® 555–Aβ42 to a final concentration of 1 μM. Imaging
of living cells was carried out with an Olympus BX61WI
microscope using a ×60, 1.0 NA (numerical aperture) dipping
objective. Excitation and emission filters used were 556/20 and
624/40. Image analysis was performed using ImageJ on raw
unfiltered images and DIC (differential interference contrast)
image overlaid with fluorescence image to form a composite.
The images were all scaled to the same level of contrast and
filtered.
Immunofluorescence
Aβ42 oligomers were prepared as described in the Experimental
section of the main text and incubated with 10 day cultured
dissociated hippocampal neurons for 24 h at a final concentration
of 5 μM. Untreated cells were treated with an equal amount of
Hepes. After 1 and 24 h, hippocampal cultures were fixed with
4 % PFA in PBS at room temperature. The preparations were
washed in PBS and quenched in 50 mM NH4 Cl for 15 min and
permeabilized in 0.2 % Triton X-100 in PBS and then washed
again. Prior to immunostaining the cells were blocked using 0.2 %
donkey serum in PBS for 20 min at room temperature, and cells
were incubated with the NU1 antibody (1:600 dilution) overnight,
followed by a secondary incubation with goat anti-mouse Alexa
Fluor® 568 (Invitrogen, 1:500 dilution) for 45 min. Coverslips
were mounted in fluorescence mounting medium (DAKO) for
imaging. Images were taken using Olympus BX61WI microscope
using a stack height of 8 μm and step width of 0.25 μm. Images
were inspected using ImageJ and the images were all scaled to
the same level of contrast and filtered.
Figure S5 Immunofluorescence microscopy showing the accumulation of
Alexa Fluor® 555–Aβ42 in primary hippocampal neurons at (A) 2 and (B)
24 h following administration
The images show composites of DIC images overlaid with fluorescence signal at 555 nm (red).
Figure S6 Fluorescence microscopy showing immunofluorescence for NU1
antibody labelling following Aβ42 administration to primary hippocampal
neurons incubations of 1 h (A) and 24 h (B) compared with NU1 labelling of
untreated cells (C)
Received 27 April 2011/26 September 2011; accepted 29 September 2011
Published as BJ Immediate Publication 29 September 2011, doi:10.1042/BJ20110749
c The Authors Journal compilation c 2012 Biochemical Society