The release of calcium from the endoplasmic reticulum induced by
amyloid-beta and prion peptides activates the mitochondrial apoptotic
Elisabete Ferreiro, Catarina R. Oliveira, and Cláudia M.F. Pereira⁎
Center for Neuroscience and Cell Biology, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal
Received 21 June 2007; revised 1 February 2008; accepted 6 February 2008
Available online 20 February 2008
In this study, we analyzed whether ER Ca2+release, induced by amyloid-
β (Aβ) and prion (PrP) peptides activates the mitochondrial-mediated
apoptotic pathway. In cortical neurons, addition of the synthetic Aβ1–40
+overload. The Ca2+released through ryanodine (RyR) and inositol
1,4,5-trisphosphate (IP3R) receptors was shown to be involved in the loss
of mitochondrial membrane potential, Bax translocation to mitochondria
and apoptotic death. Our data further demonstrate that Ca2+released
from the ER leads to the depletion of endogenous GSH levels and
accumulation of reactive oxygen species, which were also involved in the
depolarization of the mitochondrial membrane. These results illustrate
affects mitochondrial function, activating the mitochondrial-mediated
apoptotic pathway and help to clarify the mechanism implicated in
neuronal death that occurs in AD and PrD.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Alzheimer's disease; Prion disorders; Amyloid-β peptide; Prion
peptide; Apoptosis; Ca2+homeostasis; Endoplasmic reticulum; Mitochon-
dria; Oxidative stress
Alzheimer's (AD) and Prion's diseases (PrD) are neurodegen-
erative disorders that share several common characteristics,
including progressive dementia, accumulation of abnormally folded
proteins and pronounced neuronal loss. AD and PrD appear to be
caused, respectively, by the intracerebral accumulation of amyloid-
beta (Aβ), resulting from the abnormal cleavage of the amyloid
precursor protein (APP), or by the scrapie isoform of prion protein
(PrPc) (Prusiner, 1996; Wisniewski et al., 1997).
stress-initiated cell death pathway is involved in AD and PrD
(Katayama et al., 1999; Nakagawa et al., 2000; Hetz et al., 2003,
2005). The ER is an essential intracellular organelle involved in
calcium homeostasis, and in the folding and processing of proteins
(Baumann and Walz, 2001). In response to several stimuli that
perturb the normal ER function, protein misfolding occurs and
unfolded proteins accumulate in the ER (Kaufman, 1999; Pashen,
2001), activating the unfolded protein response (UPR). Under
conditions of severe or prolonged ER dysfunction, UPR can trigger
apoptotic cell death (Kaufman, 1999; Oyadomari et al., 2002).
Ca2+release from the ER is mediated by ryanodine receptors
(RyR) and inositol 1,4,5-trisphosphate receptors (IP3R) (Berridge
can capture a significant Ca2+fraction due to the close physical
proximity between the two organelles (Rizzuto et al., 1998; Csordas
mitochondrial membrane potential (Δψmit) (Duchen, 2000; Haj-
nóczky et al., 2000). Loss of Δψmitcan also result from the opening
of the mitochondrial transition pore (PTP),resulting inthe release of
apoptogenic factors, including cytochrome c (Kroemer et al., 1998).
Once in the cytosol, cytochrome c binds to the apoptosis-inducing
3 that cleaves several cellular substrates, culminating in cell death
(Zoratti and Szabo, 1995; Liu et al., 1996; Li et al., 1997). Several
proteins of the Bcl-2 family have been implicated in the
mitochondria-mediated apoptosis (Kluck et al., 1997; Yang et al.,
1997; Wei et al., 2001). The translocation of the pro-apoptotic
protein Bax to the mitochondria, where it oligomerizes and forms a
channel in the outer membrane, can promote the release of
cytochrome c (Wei et al., 2000; Cheng et al., 2001).
We have previously shown that Aβ1–40 and PrP106–126
peptides trigger apoptosis due to the early release of Ca2+from
Neurobiology of Disease 30 (2008) 331–342
Abbreviations: Aβ, amyloid-beta peptide; PrP, prion peptide; AD,
Alzheimer's disease; APP, amyloid precursor protein; ER, endoplasmic
reticulum; IP3R, inositol 1,4,5-triphosphate receptor; Δψmit, mitochondrial
membrane potential; PrD, Prion disorders; PTP, mitochondrial transition
pore; ROS, reactive oxygen species; RyR, ryanodine receptor.
⁎Corresponding author. Fax: +351 239822776.
E-mail address: email@example.com (C.M.F. Pereira).
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
ER through the RyR and IP3R (Ferreiro et al., 2004, 2006). The aim
of this study was to determine whether activation of the
mitochondria-mediated apoptotic pathway is triggered by Aβ1–40-
and PrP106–126-induced Ca2+release. Furthermore, generation of
reactive oxygen species (ROS) will be examined as a possible link
between ER Ca2+release and loss of mitochondrial function. The
evidence gathered in this study indicates that Aβ1–40 and PrP106–
126-induced ER Ca2+release affects mitochondrial function by a
mechanism that involves the generation of ROS, possibly amplify-
ing the ERstress-mediated apoptosis induced by these two peptides.
Materials and methods
Neurobasal medium and B27 supplement were purchased from
Gibco BRL, Life Technologies (Scotland, UK). Trypsin, deoxyr-
ibonuclease I (DNase I), trypsin inhibitor type II-S-soybean, protease
inhibitors, phenylmethylsulfonyl fluoride (PMSF), bovine serum
albumin (BSA), dantrolene, xestospongin C, reduced glutathione
(GSH), o-Phthalaldehyde (OPT), rhodamine 123 (Rh123), D-α-
tocopherol succinate (vitamin E), Coenzyme Q10(CoQ10), carbonyl
cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and thapsi-
gargin were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). SYTO-13, propidium iodide (PI) and Fura-2/AM were
purchased from Molecular Probes (Leiden, Netherlands). The
synthetic Aβ1–40 peptide was from Bachem (Bubendorf, Switzer-
land) or American Peptides (Sunnyvale, CA, USA). PrP106–126
the Bax channel blocker ((±)-1-(3,6-Dibromocarbazol-9-yl)-3-piper-
azin-1-yl-propan-2-ol, bis TFA) were purchased from Calbiochem
(Darmstadt, Germany). Bio-Rad protein dye assay, reagents and
apparatus used in immunoblotting assays were purchased from Bio-
Rad (Hercules, CA, USA). Rabbit primary anti-bax antibody was
from Cell Signalling (Danvers, MA, USA) and mouse primary anti-
cytochrome c antibody was from BD Pharmingen (San Diego, CA,
USA). The goat anti-rabbit IgG conjugated to alkaline phosphatase,
the enhanced chemifluorescence reagent (ECF) and the polyvinyli-
dene difluoride (PVDF) membrane were obtained from Amersham
Pharmacia Biotech (Buckinghamshire, UK). The fluorescent mount-
ing medium was purchased from DakoCytomation (Carpinteria,
(St. Louis, MO, USA) or from Merck kgaA (Darmstadt, Germany).
Primary cortical neuronal cultures and experimental treatments
Primary cultures of cortical neurons were prepared from 15 to
16 days embryos of Wistar rats according to the method described by
2003). Briefly, removed cortices were dissected and placed in Ca2+-
and Mg2+-free Krebs buffer (in mM): NaCl 120, KCl 4.8, KH2PO4
1.2, glucose 13 and HEPES 10 (pH 7.4) supplemented with BSA
(0.3 mg/mL). Minced cortical tissues were washed and incubated in
Krebs solution supplemented with BSA, and containing trypsin
(0.5 mg/mL) and DNase I (0.04 mg/mL), for 10 min at 37 °C. The
digestion was stopped with Krebs buffer containing trypsin inhibitor
(type II-S) (0.75 mg/mL) and DNase I (0.04 mg/mL), followed by a
centrifugation at140×g for5 min. After washing the pellet once with
Krebsbuffer, the cells were resuspended infresh Neurobasalmedium
supplemented with 2 mM L-glutamine, 2% (v/v) B27 supplement,
penicillin (100 U/mL) and streptomycin (100 μg/mL) and were
dissociated mechanically. Cortical cells were plated on poly-L-lysine
(0.1 mg/mL) coated glass coverslips at a density of 0.10×106cells/
cm2for single cell Ca2+measurements, nuclear morphology studies
using SYTO-13/PI and TUNEL assay. For the measurement of ROS
levels and Rh123 retention, cells were plated on poly-L-lysine
(0.1 mg/mL)-coated dishes at a density of 0.10×106cells/cm2. For
immunoblotting, neurons were mounted on poly-L-lysine (0.1 mg/
mL)-coated dishes at a density of 0.45×106cells/cm2. The cultures
culture conditions, 90% of the cells are neurons as evaluated using
MAP-2 and GFAP immunoreactivity (Ferreiro et al., 2006).
Differentiated cortical neurons were treated with Aβ1–40 (0.5 μM),
PrP106–126 (25 μM) or thapsigargin (2.5 μM) in serum-free
Neurobasal medium supplemented with B27. PrP106–126 was added
from a stock solution prepared in sterile distilled water, at a concen-
tration of 1 mM. Aβ1–40 was dissolved in sterile distilled water, at
a concentration of 6 mg/mL and diluted to 1 mg/mL (231.5 μM)
with phosphatesaline buffer (PBS) and then incubated for 5–7daysto
induce fibril formation. Thapsigargin was added from a stock solution
prepared in dimethylsulfoxide (DMSO), at a concentration of 5 mM.
chosen based on previous results demonstrating that, on cortical
neurons, 0.5 μM Aβ1–40, 25 μM PrP106–126 or 2.5 μM thapsigargin
induced a maximal toxic effect (50% decrease in cell viability). Before
the addition of Aβ1–40, PrP106–126 or thapsigargin, cells were pre-
incubated for 1 h with dantrolene (10 μM) or xestospongin C (1 μM)
nuclear morphology or Western blotting studies, using SYTO-13/PI,
Aβ1–40- or PrP106–126-treated cells were pre-incubated 30 min with
Bax blocker channel (25 or 50 nM).
Measurement of ER Ca2+content
Measurement of ER Ca2+content was assessed by singlecellCa2+
Aβ1–40 (0.5 μM) or PrP106–126 (25 μM) for 1 or 24 h. Treated and
andloaded with Fura-2/AM (5 μM)supplementedwith 0.2% pluronic
acid in Krebs buffer for 40 min at 37 °C, inthe dark. Afterwards,cells
were washed 3 times in Ca2+-free Krebs buffer, supplemented with
50 μM EGTA, and the coverslip was assembled toperfusion chamber,
with 500 μL of Ca2+-free Krebs buffer, in an inverted fluorescence
microscope Axiovert 200 (Zeiss, Germany). Cells were alternately
excited at 340 and 380 nm using a Lambda DG4 apparatus (Sutter
Instruments company, Nocato, CA, USA), and emitted fluorescence
was collected with a 40× objective and was driven to a Coll SNAP
digital camera (Roper Scientific, Trenton, NJ, USA). After a baseline
was established, cells were stimulated with thapsigargin (2.5 μM final
concentration), in the absence of extracellular Ca2+, to empty Ca2+
from ER. Acquired values were processed using the MetaFluor
software (Universal Imaging Corporation, Buckinghamshire, UK).
The peak amplitude of Fura-2 fluorescence (ratio at 340/380 nm) was
used to evaluate ER Ca2+content.
Western blotting analysis
Mitochondrial and cytosolic extracts were prepared at 4 °C as
followed: cells were washed with ice-cold PBS (all of the following
332E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
procedures were performed at 4 °C) and resuspended in a sucrose
buffercontaining (in mM): sucrose 250; HEPES–Na20; MgCl21.5;
KCl 10; EDTA 1; EGTA 1; DTT 1 and protease inhibitor cocktail
(containing 1 μg/mL leupeptin, pepstatin A, chymostatin and
antipain). Lysates were homogenized and centrifuged at 1000×g for
10 min. The pellet corresponding to nuclei and unlysed cells was
discarded and the supernatant was subjected to centrifugation at
10,000×g for 30 min. The pellet corresponding to the mitochondrial
fraction was resuspended in lysis buffer. TCA 15% (v/v) was added
to the supernatant and centrifuged at 15,800×g for 10 min. The
resulting pellet (cytosolic fraction) was resuspended in supplemen-
ted sucrose buffer and brought to pH 7 with NaOH. Protein
concentration in the supernatant was measured using the Bio-Rad
protein dye assay reagent. Samples were denaturated at 95 °C for
3 min in a 6× concentrated sample buffer (mM): Tris 500, DTT 600,
10.3% SDS, 30% glycerol and 0.012% bromophenol blue. Equal
protein amounts of each sample (60 μg) were separated by
electrophoresis on a 10% SDS-polyacrylamide gels (SDS-PAGE)
and electroblotted onto PVDF membranes. The identification of
proteins of interest was facilitated by the usage of a prestained
precision proteinstandard (Bio-Rad) whichwas runsimultaneously.
After the proteins were electrophoretically transferred, the mem-
branes were incubated for 1 h at room temperature (RT) in Tris-
buffer (mM): NaCl 150, Tris–HCl 25 (pH 7.6) with 0.1% Tween 20
(TBS-T) containing 5% nonfat dry milk to eliminate nonspecific
T containing 5% nonfat dry milk with a rabbit polyclonal primary
antibody against Bax (1:1000) or with mouse monoclonal primary
antibody against cytochrome c (1:250). The membranes were
washed several times and then incubated in TBS-T with 1% nonfat
anti-rabbit secondary antibody at a dilution of 1:25,000. Immunor-
eactive bands were detected after incubation of membranes with
ECF reagent for 5–10 min, on a Bio-Rad Versa Doc 3000 Imaging
System (Bio-Rad, Hercules, CA, USA).
Measurement of glutathione content
Reduced glutathione (GSH) levels were determined with
fluorescence detection, according to Moreira et al. (2005). In
brief, cells were washed with ice-cold PBS, resuspended in
perchloric acid (0.6 M) and EDTA–Na+(25 mM) and centrifuged
at 15,800×g for 2 min at 4 °C. The pellet was resuspended in
NaOH (1 M) and the protein concentration was measured using the
Bio-Rad protein dye assay reagent. The supernatant (100 μL) was
added to 1.8 mL phosphate buffer and 100 μL OPT. After mixing
and incubation at RT for 15 min, the solution was transferred to a
quartz cuvette and the fluorescence was measured at 420 and
350 nm emission and excitation wavelength, respectively. The
GSH content was calculated using a linear reduced GSH standard
curve and the values expressed as the percentage above control.
Measurement of intracellular reactive oxygen species
ROS were measured according to Bass et al. (1983), by following
the oxidation of 2′,7′-dichlorodihydrofluorescin (DCFH2-DA) to
fluorescent DCF. Plated cells, untreated or treated with Aβ1–40,
PrP106–126 or thapsigargin, in the presence or absence of CoQ10 or
vitamin E, were loaded with 5 μM DCFH2-DA in sodium medium
containing: (in mM): NaCl 132, KCl 4, NaH2PO41.2, MgCl21.4,
DCFH2-DA incubation, cells were washed and sodium medium was
was monitored for 30 min at 480 nm excitation and 550 nm emission
(with a cutoff at 530 nm), at 37 °C, using a temperature-controlled
SpectraMax Gemini EM spectrometer (Molecular Devices, Sunny-
vale, USA). The values were expressed as arbitrary units of the
increase of DCF fluorescence above control values.
Analysis of mitochondrial membrane potential
The changes in mitochondrial membrane potential (ψmit) were
estimated using the fluorescent cationic dye Rh123, which
accumulates in mitochondria as a direct function of the membrane
potential and is released upon membrane depolarization (Palmeira
et al., 1996, with some modifications). After exposure to Aβ1–40,
PrP106–126 or thapsigargin, in the presence or absence of CoQ10,
vitamin E, dantrolene or xestospongin C, the cells were incubated
Fig. 1. Aβ1–40 and PrP106–126 deplete ER Ca2+content. The fluorescence
ratio at 340/380 nm of the Ca2+-sensitive Fura-2/AM was monitored during
12 min, in the absence of external Ca2+. After 2 min, 2.5 μM thapsigargin was
(B) are presented. The peak amplitude of Fura-2 fluorescence was used to
evaluate ER Ca2+content and data from at least three different experiments,
obtained from Aβ1–40- or PrP106–126-treated neurons, were normalized to
mean±SEM.⁎⁎⁎pb0.001 with respect to control values.
333E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
with Rh123 (1 μM) in sodium medium containing: (in mM): NaCl
132, KCl 4, NaH2PO41.2, MgCl21.4, glucose 6, HEPES–Na 10,
CaCl21, pH 7.4, for 45 min at 37 °C, in a SpectraMax Gemini EM
spectrometer (Molecular Devices, Sunnyvale, USA). The fluores-
cence signals of the dye remaining trapped were monitored at
505 nm excitation and 525 nm emission. At the end of the 45 min
incubation, FCCP (1.5 μM) and oligomycin (0.25 μg/mL) were
added in order to establish the maximum retention of Rh123. The
difference between the fluorescence monitored before and after the
addition of FCCP and oligomycin was used to evaluate the ψmit.
The results were expressed as percentage of increase or decrease
above control fluorescence.
Assessment of neuronal injury
After exposure of cortical cells to Aβ1–40 (0.5 μM) or PrP106–
126 (25 μM), in the absence or in the presence of a Bax channel
blocker (25 or 50 nM), neuronal injury was assessed as described in
Ferreiro et al. (2004) using the fluorescent probes SYTO-13/PI.
Control and treated neurons were incubated with SYTO-13 (3.8 μM)
Axiovert 200 fluorescence microscope (Zeiss, Germany). All
experiments were performed in duplicate, and a minimum of 300
expressed as the percentage (%) of the total number of cells in the
microscope field. Cell death was also analyzed by TUNEL staining,
performed using an in Situ Cell Death Detection Kit, Fluorescein
(Roche Applied Science, Mannheim, Germany) according to the
manufacturer's directions. Cells were washed in PBS buffer (pH 7.4)
and were fixed with 4% paraformaldehyde for 30 min at RT. The
slides were then immersed in 0.1% Triton X-100, supplemented with
0.1% sodium citrate in PBS, for 2 min, on ice, to permeabilized the
cells. After washing, slides were incubated in a mixture of the
Finally, slides were washed with PBS and prepared with DakoCy-
tomation Fluorescent mounting solution on a microscope slide for the
visualization in an Axiovert Microscope 200 (Zeiss, Thornwood, NY,
USA). DNAse pretreated cells were used as a positive control. The
photographs were taken at a magnification of 400×.Total number and
each coverslips. The results were presented as the percentage (%) of
the total number of cells.
Results are expressed as means±SEM of the number of exper-
iments indicated in the figure captions. Statistical significance was
performed using an analysis of variance (ANOVA), followed by
Fig. 2. Aβ1–40 and PrP106–126 reduce ΔΨmit. Protection by antioxidants, IP3R and RyR inhibitors. Mitochondrial membrane potential (ΔΨmit) was evaluated
by the capacity of mitochondria from cortical neurons to take up the fluorescent cationic dye Rh123. Cells were incubated with Aβ1–40 (0.5 μM), PrP106–126
(25 μM) or thapsigargin (2.5 μM), in the absence or in the presence of CoQ10 (1 μM), vitamin E (13 μM), dantrolene (10 μM) or xestospongin C (1 μM), for 6
(A) or 24 h (B). The results, expressed as the percentage of the baseline membrane potential of control cortical neurons, are the means±SEM of values
corresponding at least to 3 experiments, each value being the mean of duplicate assays.⁎pb0.05;⁎⁎pb0.01 with respect to control values.#pb0.05;##pb0.01;
###pb0.001 with respect to Aβ, PrP or thapsigargin addition.
334E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
Dunnett’s post-hoc tests for multiple comparisons or by the unpaired
two-tailed Student’s t-test. A pb0.05 value was considered statis-
Aβ1–40 and PrP106–126 deplete ER Ca2+content
Several studies link ER stress and apoptosis to the perturbation of
shown that the synthetic Aβ1–40 and PrP106–126 peptides
significantly increase in cytosolic Ca2+levels that was prevented in
the presence of dantrolene and xestospongin C, which inhibit the
and IP3R, respectively (Ferreiro et al., 2006) In order to further
investigate whether ER Ca2+levels are altered by both peptides, ER
Ca2+content was analysed by single cell Ca2+imaging in Aβ1–40-
and PrP106–126-treated cortical neurons. Control cells and cells
treated with Aβ1–40 (0.5 μM) or PrP106–126 (25 μM) for 1 or 24 h,
were loaded with the Ca2+sensitive fluorescent dye Fura-2/AM. ER
Ca2+content was evaluated indirectly measuring Fura-2 fluorescence
in the absence of external Ca2+, before and after the addition of
thapsigargin, a SERCA-Ca2+ATPase inhibitor, which depletes ER
stores. Representative traces of Fura-2 fluorescence ratio at 340 and
380 nm are presented in Figs. 1A and B. Basal values of Fura-2
fluorescence ratio, measured before thapsigargin addition, were
(Figs. 1A and B), demonstrating that cytosolic Ca2+levels are altered
by both peptides (Figs. 1A and B), accordingly with our previous
published results (Ferreiro et al., 2006).
were calculated and the results normalized to ER Ca2+content of
untreated cortical neurons (Fig. 1C). After 1 h incubation, Aβ1–40
(0.5 μM) induced a significant decrease in neuronal ER Ca2+
content, which persisted at 24 h. Similar results were obtained in
cortical neurons treated with PrP106–126 (25 μM) for 1 or 24 h
(pb0.001) (Fig. 1C). These results show that Aβ and PrP peptides
affect ER Ca2+homeostasis in cortical neurons, inducing a
substantial reduction in ER Ca2+content, which subsequently leads
to the increase of cytosolic Ca2+levels.
Aβ1–40 and PrP106–126 reduce ΔΨmitand increase ROS levels.
Protection by antioxidants, IP3R and RyR inhibitors
Increased intracellular free Ca2+overload can lead to the
excessive mitochondrial Ca2+uptake, reduce the mitochondrial
membrane potential (Δψmit) and disrupt electron transport,
resulting in an increased production of the reactive free radical
superoxide anion in neurons (Luetjens et al., 2000).
The changes in Δψmitinduced by Aβ1–40 and PrP106–126
peptides were measured using the fluorescent cationic dye Rh123,
which is known to be selectively taken up by mitochondria and
evaluate the involvement of ER Ca2+release in the perturbation of
Δψmit, cortical neurons were pre-incubated with dantrolene, an
inhibitor of RyR, or xestospongin C, an inhibitor of IP3R, before the
addition of Aβ1–40 and PrP106–126 peptides. Furthermore, results
in cells with thapsigargin, a known ER stress inducer, in the absence
or in the presence of dantrolene or xestopongin C. In addition, the
effect of two well-known antioxidants, CoQ10 and vitamin E, was
tested to evaluate whether reactive oxygen species (ROS) produc-
tion upon ER Ca2+release perturbs the Δψmit. As shown in Figs. 2A
126 (25 μM), or thapsigargin (2.5 μM), leads to a significant drop of
Fig. 3. Aβ1–40 and PrP106–126 increase the intracellular reactive oxygen
species (ROS) levels. Protection by CoQ10 and vitamin E. Cortical neurons
were treated with Aβ1–40 (0.5 μM), PrP106–126 (25 μM) or thapsigargin
(2.5 μM) for 6 h, in the absence or in the presence of CoQ10 (1 μM) or
vitamin E (13 μM). Intracellular ROS levels were determined using the
DCFH2-DA fluorescent probe. The results are the means±SEM of values
corresponding at least 3 experiments, each value being the mean of duplicate
assays.⁎pb0.05;⁎⁎⁎pb0.001 with respect to control values.#pb0.05;
##pb0.01 with respect to Aβ, PrP or thapsigargin addition.
Fig. 4. GSH levels are increased in neurons treated with Aβ1–40 or
PrP106–126. Protection by dantrolene and xestospongin. Cortical neurons
were treated for 24 h with Aβ1–40 (0.5 μM) (A) or PrP106–126 (25 μM)
(B), in the absence or in the presence of dantrolene (10 μM) or xestospongin
C (1 μM). GSH content, expressed as percentage of control values, are the
means±SEM of values corresponding at least to 3 experiments, each value
being the mean of duplicate assays.⁎pb0.05;⁎⁎⁎pb0.001 with respect to
control values.#pb0.05;##pb0.01 with respect to Aβ or PrP addition.
335E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
we can also depict that dantrolene (10 μM) and xestospongin C
(1 μM) protected cells against Aβ1–40-, PrP106–126- and
thapsigargin-induced decrease of Δψmit, since no alteration of
Rh123 was observed under these conditions, demonstrating the
involvement of ER Ca2+release in the perturbation of the Δψmit. In
addition, CoQ10 (1 μM) and vitamin E (13 μM) also afforded
protection against these toxic elements, revealing that ROS are
involved in the altered Δψmitin response to Aβ1–40, PrP106–126 or
thapsigargin. CoQ10, vitamin E, dantrolene or xestospongin c alone
did not alter the Δψmit(Figs. 2A and B).
in ROS production induced by Aβ1–40 and PrP106–126 (Ferreiro
a positive control. In cortical neurons treated for 6 h with Aβ1–40
suppressed by CoQ10 and vitamin E (Fig. 3). These results confirm
that the effect of CoQ10 and vitamin E on the loss of Δψmitupon
exposure to Aβ1–40, PrP106–126 or thapsigargin is correlated with
the suppression of ROS production.
Aβ1–40 and PrP106–126 reduce GSH levels mediated by ER Ca2+
Reduction of Δψmithas been linked to depletion of reduced
glutathione (GSH) (Macho et al., 1997), the most abundant
antioxidant in the cell. Aβ1–40 (0.5 μM) or PrP106–126 (25 μM)
Fig. 5. Aβ1–40 and PrP106–126 induce Bax translocation to mitochondria. Involvement of ER Ca2+released through IP3R and RyR. Cortical neurons were
treated with Aβ1–40 (0.5 μM) (A) or PrP106–126 (25 μM) (B), for 6 or 24 h, in the absence or in the presence of dantrolene (10 μM) or xestospongin C (1 μM).
Bax levels in cytosolic- and mitochondria-enriched fractions were detected as a 20-kDa band by Western blot analysis, using a specific monoclonal antibody. To
determine the significance of the differences in expression levels, data from at least three different experiments were analyzed by densitometry (graphs). Data
were expressed as the mean±SEM.⁎⁎⁎pb0.001 with respect to control values.#pb0.05;##pb0.01 with respect to Aβ or PrP addition.
336 E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
with Aβ or PrP peptides in the presence of dantrolene (10 μM) or
xestospongin C (10 μM), GSH levels were similar to those observed
in the control, demonstrating that RyR- and IP3R-mediated ER Ca2+
in cortical neurons (Figs. 4A and B). Dantrolene or xestospongin C
per se did not affect GSH levels (Figs. 4A and B).
Aβ1–40 and PrP106–126 induce cytochrome c release due to the
translocation of Bax to the mitochondria
opening of the mitochondrial permeable transition pore (PTP)
(Kroemer et al., 1998). Bax, a cytosolic Bcl-2 family protein, can
translocate to mitochondria wherein it oligomerizes and inserts into
the outer mitochondrial membrane, causing permeabilization and
release of apoptogenic factors from the intermembrane space
(Breckenridge and Xue, 2004). Both processes can contribute to
the loss of Δψmit. We previously showed that both Aβ and PrP
peptides induce the release of cytochrome c in cortical neurons both
peptides could induce the translocation of Bax to the mitochondria
by a mechanism involving the release of Ca2+from ER. To inves-
tigate this possibility, we isolated mitochondrial and cytosolic
fractions from untreated or Aβ- or PrP-treated neurons and analyzed
Bax levels by Western blotting. Furthemore, we also analysed the
contribution of ER Ca2+release by pre-incubating cells with
dantrolene or xestospongin C. Both Aβ1–40 (0.5 μM) and PrP106–
126 (25 μM) increased Bax levels in the mitochondrial fraction at 6
and 24 h ofincubation (Figs. 5A and B)and concomitantly decreased
Bax levels in the cytosolic fraction (Figs. 5C and D). In addition,
both dantrolene and xestospongin C prevented the translocation of
Bax to the mitochondria in treated cells (Fig. 5). These results further
implicate ER Ca2+, released through IP3R and RyR, in the mito-
chondrial dysfunction induced by Aβ and PrP peptides.
Fig. 6. Bax channel blockerprevents cytochrome c release induced by Aβ1–
40 and PrP106–126. Cortical neurons were treated for 6 h with 5 μM Aβ1–
40, in the absence or in the presence of 50 nM of Bax channel blocker (A) or
with 25 μM PrP106–126, in the absence or in the presence of 25 nM of Bax
channel blocker (B). Cytochrome c levels in mitochondria- and cytosolic-
enriched fractions were detected as a 15-kDa band by Western blot analysis.
Graphs represent the differences in expression levels from at least three
different experiments, analyzed by densitometry. Data were expressed as the
mean±SEM.⁎pb0.05;⁎⁎pb0.01 with respect to control values.#pb0.05;
##pb0.01 with respect to Aβ or PrP addition.
Fig. 7. Bax channel blocker reduces DNA fragmentation induced by Aβ1–
40 and PrP106–126. The number of cells exhibiting fragmented DNA was
measured by fluorescence microscopy using SYTO-13 and PI staining in
cortical neuronal cultures treated for 24 h with 0.5 μM Aβ1–40, in the
absence or in the presence of 50 nM of Bax channel blocker (A) or with
25 μM PrP106–126, in the absence or in the presence of 25 nM of Bax
channel blocker (B), as described in the Materials and methods section. The
results, expressed as the percentage of the total number of cells, are the
means±SEM of values corresponding at least to 3 experiments, each value
being the mean of duplicate assays.⁎pb0.05 with respect to control values.
#pb0.05 with respect to Aβ or PrP addition.
337E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
To ascertain that cytochrome c is released through the Bax
channel formed in the outer mitochondrial membrane, the levels of
this apoptogenic factor was analysed by Western blotting in
mitochondrial and cytosolic fractions isolated from Aβ- or PrP-
treated cortical neurons in the presence of a Bax channel blocker
or PrP106–126 (25 μM), cytochrome c levels decreased in the
mitochondria, increasing the cytosolic content. This effect was
prevented by the Bax channel blocker (Figs. 6A and B), implicating
this channel in the release of cytochrome c from mitochondria upon
Aβ or PrP treatment (Figs. 6A and B). Similar results were obtained
in cells treated with PrP106–126 and Aβ1–40, in the presence or
absence of Bax channel blocker for 24 h (data not shown).
To further analyse if Bax translocation to mitochondria was
essential for Aβ- and PrP- induced apoptosis, cells were incubated
with the Bax channel blocker and the number of apoptotic cells
Fig. 8. Aβ1–40 and PrP106–126 induce an increase in the number of TUNEL-positive cells. Protection by dantrolene. Cortical neurons were treated for 24 h
with Aβ1–40 (0.5 μM) or PrP106–126 (25 μM), in the absence or in the presence of dantrolene (10 μM) and TUNEL assay was performed as described in the
Materials and methods section. Representative phase contrast, fluorescent (TUNEL-positive cells are green fluorescent) and merged images (400×
magnification)arepresented. (Graph) Thenumbersof TUNEL-positive cells areexpressed as the percentage of the total number of cellsandare the means±SEM
of values corresponding at least to 3 experiments.⁎⁎⁎pb0.001 with respect to control values.##pb0.01 with respect to Aβ or PrP addition.
338 E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
was quantified by fluorescence microscopy after SYTO-13/PI
labelling. After 24 h of incubation with Aβ1–40 (0.5 μM) or
PrP106–126 (25 μM), the number of apoptotic cells significantly
increased when compared to control values (Figs. 7A and B). In
both cases, the Bax channel blocker reduced the number of
apoptotic cells to values similar to the control (Fig. 7). In the case
of PrP peptide, 25 nM of Bax channel blocker effectively reduced
the number of apoptotic cells (Fig. 7B), whereas 50 nM were
required to observe a similar effect against Aβ-induced cell death
(Fig. 7A). Both concentrations of Bax channel blocker did not
show any toxic effects per se (Figs. 7A and B).
Aβ- and PrP-induced apoptosis is dependent on ER Ca2+release
Finally, to confirm the importance of ER Ca2+release in the
neuronal death induced by Aβ and PrP peptides, cortical cells were
pre-incubated with dantrolene, which prevents the release of Ca2+
from the ER through RyR. Thereafter, apoptotic death was
examined by the TUNEL assay. Aβ1–40 (0.5 μM) and PrP106–
126 (25 μM) induced a 2-fold increase in the number of TUNEL-
positive cells, which was prevented by dantrolene (Fig. 8). These
results further support the involvement of ER Ca2+release in the
neuronal death that occurs upon Aβ and PrP treatment.
Our results demonstrate that, in cortical neurons, Aβ1–40 and
PrP106–126 peptides activate the mitochondria-mediated apoptotic
pathway, by a mechanism involving the release of Ca2+from
endoplasmic reticulum (ER) through channels associated with
ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors
(IP3R). The addition of Aβ1–40 (0.5 μM) or PrP106–126 (25 μM) to
cortical neuronal cultures depleted ER Ca2+stores, leading to the
increase of cytosolic Ca2+levels. Moreover, mitochondrial function
was impaired by these peptides, which induced a significant reduction
in mitochondrial membrane potential (Δψmit). This effect was
significantly attenuated by inhibitors of Ca2+release through channels
associated to RyR and IP3R, implicating ER Ca2+release in Aβ- and
PrP-induced Δψmit loss. Furthermore, Aβ and PrP altered Bax
localization, increasing the levels of this pro-apoptotic protein in the
mitochondria. Bax channel formation was also shown to be essential
for Aβ- and PrP-induced cytochrome c release and cell death. Finally,
we provide evidence that clearly implicates the generation of reactive
protection by lowering the levels of reduced glutathione (GSH).
Accumulation of unfolded proteins due to the perturbation of
ER Ca2+homeostasis can activate the unfolded protein response
(UPR) and, consequently, the ER stress-induced apoptosis pathway
(Kaufman, 1999; Pashen, 2001). UPR and ER stress have been
shown to occur both in AD and PrD and to be involved in the
neuronal death that occurs in both diseases (Hoozemans et al.,
2005; Hetz et al., 2003). ER is the major Ca2+storage organelle in
the cell that can be released by both electrical and chemical cell
stimulation (Bootman and Lipp, 2002; Berridge et al., 2000;
Verkhratsky and Petersen, 2002), through two types of Ca2+release
channels, the IP3R and the RyR. Ca2+is released from the IP3R in
response to agonists of phospholipase C (PLC)-coupled receptors.
Activation of PLC results in cleavage of PtdIns(4,5)P2, leading to
the liberation of diacylglycerol and Ins(1,4,5)P3(IP3) that binds to
IP3R (Finch and Turner, 1991). Ca2+released from IP3R further
stimulates the release of Ca2+through RyR, a process known as
calcium-induced calcium release (CICR). It was previously shown
that the presence of the AD-linked presenilin 1 (PS1) mutation
potentiates IP3-evoked Ca2+transients (Stutzmann et al., 2004).
More recently, RyR activation triggered by Ca2+released from
IP3R was demonstrated to be responsible for the exaggerated ER
Ca2+signals observed in the neurons of PS1 knock-in and 3xTg-
AD mice, further supporting the involvement of CICR through
RyR in AD pathology (Stutzmann et al., 2006).
Perturbation in Ca2+homeostasis, possibly through the early
formation of membranar Aβ or PrP Ca2+channels, may represent an
initiating mechanism during Aβ and PrP toxicity that can be
extended to the ER as a death message (Bhathia et al., 2000;
Simakova and Arispe, 2006; Lin et al., 1997). Further intracellular
Ca2+overload, due, in particular, to its release from the ER, can lead
to the excessive Ca2+uptake into mitochondria. This effect can be
related to the close physical proximity between the ER and mito-
chondria (Rizzuto et al., 1998; Csordas et al., 1999). The massive
Ca2+influx into mitochondria collapse mitochondrial membrane
potential Δψmit, leading to cell death (Duchen, 2000; Hajnóczky
et al., 2000). We report here, that Aβ1–40, PrP106–126 and
thapsigargin, an inhibitor of endoplasmic SERCA Ca2+-ATPase and
known ER stressor, induce Δψmitloss involving the release of ER
Ca2+through IP3R and RyR. In 2002, Boya et al. had already
the N-linked glycosylation, brefeldin A, inhibitor of ER–Golgi
permeabilization and loss of Δψmit. Reduced Δψmitcan also result
from the opening of the mitochondrial transition pore (PTP). High
intramitochondrial Ca2+levels and generation of reactive oxygen
species (ROS) both contribute to PTP opening. Indeed, Ca2+-
ROS (reviewed in Orrenius et al., 2007). We have previously
provided evidence that ROS accumulation is an early event during
Aβ1–40- and PrP106–126-induced apoptosis (Ferreiro et al., 2006).
Herein, we showed that Aβ1–40-, PrP106–126- and thapsigargin-
ROS formation, implicating ROS in the reduced Δψmit. The
increment in ROS levels observed upon treatment with thapsigargin
indicates that ER stress alters the equilibrium between ROS
formation and the defence mechanisms of the cell. In fact,
thapsigargin was previously shown to reduce GSH levels (Romero
et al., 1997), which constitutes the first-line defence in the cellular
antioxidant system. Aβ1–40 and PrP106–126 also induced the
depletion ofGSHcontentthatwaspreventedwhen ERCa2+released
through IP3R and RyR was inhibited.
Several proteins of the Bcl-2 family differentially affect
mitochondrial function. One of these proteins is the pro-apoptotic
protein Bax. In the presence of an pro-apoptotic stimuli, Bax
translocates from the cytosol to the mitochondria, where it
oligomerizes and forms a channel, leading to the permeabilization
of the outer mitochondrial membrane, and to the release of
cytochrome c (Wei et al., 2000; Cheng et al., 2001). Aβ1–40 and
PrP106–126 induced the translocation of Bax to the mitochondria,
which was prevented by dantrolene and xestospongin C, demon-
strating that ER Ca2+release through IP3R and RyR is involved in
this process. We have previously shown that Aβ1–40 and PrP106–
126 induce cytochrome c release from mitochondria in cortical
neurons (Ferreiro et al., 2006) and now we demonstrate that the
mitochondrial channels formed by Bax are implicated in the release
339E. Ferreiro et al. / Neurobiology of Disease 30 (2008) 331–342
of cytochrome c and also in the apoptotic cell death that occur upon
Aβ or PrP treatment. Moreover, we have recently shown that
overexpression of the anti-apoptotic Bcl-2 protein protects against
Aβ and PrP toxicity (Ferreiro et al., 2007), further implicating Bcl-2
family proteins in apoptosis induced by these amyloidogenic
peptides. Previous reports have shown that ER stress-induced cell
death requires the permeabilization of the mitochondrial membrane
and that mouse embryonic fibroblasts lacking the pro-apoptotic
proteins Bax and Bak become resistant to apoptosis induced by
tunicamycin and brefeldin A (Boya et al., 2002). In addition, Bax
ER Ca2+depletion, processing of the ER-resident caspase-12 and
Bax translocation to the mitochondrial membrane, suggesting that
changes in the redox status may be determinant for Bax-mediated
cell death (Honda et al., 2004).
Once released from mitochondria, cytochrome c binds to the
apoptosis-inducing factor, mediating caspase-9 activation, which in
turns activates caspase-3 and subsequently promotes apoptosis
(Desagher and Martinou, 2000). Our previous work demonstrated
that Aβ and PrP peptides release cytochrome c from mitochondria,
activating caspase-9 and -3. Cytochrome c was also found to bind
release, potentiating mitochondrial and cytosolic Ca2+overload and
further cytochrome c release (Boehning et al., 2003). This
mechanism could also be operating under the conditions tested in
the work here presented.
and PrD-associated Aβ and PrP peptides was supported by data
obtained in our laboratory and by others. Aβ was shown to decrease
the activity of several enzymatic complexes of mitochondrial
respiratory chain (Pereira et al., 1999). More recently, Aβ was
demonstrated to accumulate within mitochondria and such accumu-
lation was shown to be associated to mitochondrial dysfunction
(Caspersen et al., 2005; Reddy, 2006). Aβ can also interact with Aβ-
cell death (Lustbader et al., 2004). Moreover, the accumulation of
full-length amyloid precursor protein (APP) was demonstrated to
cause mitochondrial dysfunction and impaired energy metabolism
(Anandatheerthavarada et al., 2003). Finally, functional mitochon-
dria were previously shown to be required for Aβ toxicity (Cardoso
et al., 2001), revealing that mitochondria plays a fundamental role in
the cell death that occurs in AD. In mitochondria of scrapie (PrPSc)-
infected mice was reported the decrease of Δψmitand energy me-
tabolites (ATP/ADP ratio) (Lee et al., 2000). The toxic sequence of
PrPSc, PrP106–126, has also been previously shown to induce
apoptosisthrough rapid depolarization of mitochondrial membranes,
with subsequent cytochrome c release and caspases activation
(Forloni et al., 1993; O'Donavan et al., 2001; Agostinho and
Oliveira, 2003). Recent results by our group obtained in mitochon-
drial DNA-depleted cells also demonstrate that functional mitochon-
PrP peptides (Ferreiro et al., 2008 and unpublished data). The
mechanism that is behind Aβ and PrP toxicity is not fully elucidated.
The results of the present study, together with our own previously
be hypothesized that Aβ and PrP peptides induce the release of ER
Ca2+through IP3R and RyR, leading to the perturbation of ER Ca2+
homeostasis and subsequently to ER stress. As a consequence of ER
Ca2+depletion, cytosolic Ca2+levels are enhanced, ROS accumulate
and Bax translocates to mitochondria. These events can cooperate to
affect mitochondrial function, leading to cell death. Due to the close
proximity between ER and mitochondria, Ca2+can enter mitochon-
dria and compromise the normal functioning of this organelle. ROS
accumulation as a consequence of GSH depletion can further affect
mitochondrial function leading to Δψmitloss. Bax translocates to
mitochondria and induces cytochrome c release, leading to the
activation of caspase-9 and -3 and finally cell death.
In conclusion, the results of our study provide evidence that a
close relationship between the ER and the mitochondria occurs
during Aβ- and PrP-induced cell death. We propose that compounds
that regulate Ca2+homeostasis, by inhibiting its release from ER,
combined with antioxidants, could be therapeutical tools to fight the
neuronal death that occurs in AD and PrD.
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