Báo cáo khoa học: Differing molecular mechanisms appear to underlie early toxicity of prefibrillar HypF-N aggregates to different cell types potx

Differing molecular mechanisms appear to underlie early
toxicity of prefibrillar HypF-N aggregates to different cell
types
Cristina Cecchi
1
, Anna Pensalfini
1
, Serena Baglioni
1
, Claudia Fiorillo
1
, Roberto Caporale
2
,
Lucia Formigli
3
, Gianfranco Liguri
1,4
and Massimo Stefani
1,4
1 Department of Biochemical Sciences, University of Florence, Italy
2 U.O. Hematology, Azienda Ospedaliera Careggi, Florence, Italy
3 Department of Anatomy, Histology and Forensic Medicine, University of Florence, Italy
4 Interuniversity Centre for the Study of the Molecular Basis of Neurodegenerative Diseases, University of Florence, Italy
Keywords
amyloid toxicity; apoptosis; mitochondrial
permeability transition pore opening;
prefibrillar protein aggregates; protein
misfolding and cell death
Correspondence
C. Cecchi, Department of Biochemical

Sciences, University of Florence, viale
Morgagni 50, 50134 Florence, Italy
Fax: +39 055 459 8905
Tel: +39 055 459 8320
E-mail: cristina.cecchi@unifi.it
(Received 10 February 2006, accepted
16 March 2006)
doi:10.1111/j.1742-4658.2006.05234.x
Considerable attention has been paid to the high cytotoxic potential of
small, prefibrillar aggregates of proteins ⁄ peptides, either associated or not
associated with amyloid diseases. Recently, we reported that different cell
types are variously affected by early aggregates of the N-terminal domain
of the prokaryotic hydrogenase maturation factor HypF (HypF-N), a pro-
tein not involved in any disease. In this study, we provide detailed informa-
tion on a chain of events triggered in Hend murine endothelial cells and
IMR90 fibroblasts, which have previously been shown to be highly vulner-
able or very resistant, respectively, to HypF-N aggregates. Initially, both
cell lines displayed impaired viability upon exposure to HypF-N toxic
aggregates; however, at longer exposure times, IMR90 cells recovered com-
pletely, whereas Hend cells did not. In particular, significant initial mito-
chondrial permeability transition (MPT) pore opening was found in
IMR90 cells followed by a sudden repair of membrane integrity with rapid
and efficient inhibition of cytochrome c and AIF release, and upregulation
of Bcl-2. The greater resistance of IMR90 fibroblasts may also be due to
a higher cholesterol content in the plasma membrane, which disfavours
interaction with the aggregates. In contrast, Hend cells, which have less
membrane cholesterol, showed delayed MPT opening with prolonged
translocation of cytochrome c into the cytosol. Finally, the caspase 9 active
fragment was increased significantly in both Hend and IMR90 cells; how-
ever, only Hend cells showed caspase 8 and caspase 3 activation with DNA

fragmentation. From our data, the different responses of the two cell types
to the same aggregates appear to be associated with two key events: (a)
aggregate interaction with the plasma membrane, disfavoured by a high
level of membrane cholesterol; and (b) alterations in mitochondrial func-
tionality, leading to the release of pro-apoptotic stimuli, which are counter-
acted by upregulation of Bcl-2.
Abbreviations
DCFH-DA, 2¢,7¢-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HRP,
horseradish peroxidase; HypF-N, N-terminal domain of the prokaryotic hydrogenase maturation factor; IP, iodide propidium; LDH, lactate
dehydrogenase; MAC, mitochondrial apoptotic channel; MPT, mitochondrial permeability transition; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide; PARP, poly(ADP-ribose) polymerase; PtdSer, phosphatidylserine; PVDF, poly(vinylidene difluoride); ROS,
reactive oxygen species; TFE, trifluoroethanol.
2206 FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS
The amyloidoses are a group of protein-folding dis-
eases in which specific peptides or proteins, which are
either incorrectly folded or unfolded, aggregate intra-
or extracellularly into polymeric assemblies rich in
b sheet, and are eventually deposited in tissue as amy-
loid fibrils [1,2]. Amyloid diseases include a number of
sporadic, familial or transmissible degenerative pathol-
ogies affecting either the central nervous system (Alz-
heimer’s, Parkinson’s and Creutzfeldt–Jakob diseases)
or a variety of peripheral tissues and organs (systemic
amyloidoses and type II diabetes) [1]. Since 1998, a
growing number of peptides and proteins not associ-
ated with known protein deposition diseases have been
shown to aggregate in vitro, under suitable experimen-
tal conditions, into fibrils that are indistinguishable
from those associated with pathological conditions
[3,4]. This has led to the proposal that the ability to

form amyloid assemblies can be considered a generic
property inherent in any polypeptide chain [1].
Currently, considerable attention is focused on the
cytotoxic potential of small prefibrillar protein aggre-
gates arising initially in the protein fibrillization path-
way. This cytotoxic potential appears to be higher
than that of mature fibrils [2,3]. These early assem-
blies share basic structural features that, in most cases
at least, seem to underlie the common biochemical
mechanisms of cytotoxicity [5,6]. Cells exposed to
toxic prefibrillar aggregates apparently die as a conse-
quence of apoptosis [7–9] or, less frequently, by sec-
ondary necrosis [10–13]. Recent studies have shown
that cells experiencing prefibrillar aggregates undergo
similar early biochemical modifications; these include
interaction between the aggregates and cell mem-
branes and, possibly, interaction with membrane
receptors [14–16], followed by an imbalance in the
intracellular redox status [13,15] and ion levels [1,17],
and mitochondria impairment [9,18], together with
other modifications such as lipid homeostasis. Prefi-
brillar aggregates of a number of peptides associ-
ated with amyloid diseases can also induce
mitochondrial permeability transition (MPT) pore
opening in exposed cells, allowing molecules smaller
than 1500 Da to diffuse freely between the matrix
and the cytosol [18–23]. These modifications can
result in the collapse of the transmembrane electro-
chemical gradient with loss of solutes from the mat-
rix, mitochondrial swelling, release of proapoptotic

factors such as cytochrome c and AIF, and activation
of procaspase 2, 3 and 9. Cytochrome c, in complex
with the cytosolic factor Apaf-1 activates the caspase-
dependent apoptotic pathway, whereas AIF translo-
cates to the nucleus inducing chromatin condensation
and large-scale fragmentation of DNA [23,24].
Similar modifications have also been found in cells
exposed to prefibrillar amyloid aggregates of proteins
that are not associated with disease, including the
N-terminal domain of the prokaryotic hydrogenase
maturation factor HypF (HypF-N) [5,25]. In partic-
ular, when added to the cell culture media, early
HypF-N aggregates can be internalized by the cells
[13], where they induce modifications in intracellular
free Ca
2+
and reactive oxygen species (ROS) levels
[10–13,26], reducing the potential across the inner
mitochondrial membrane. In turn, ROS trigger the
intrinsic or extrinsic apoptotic pathways [26], or in
some cases lead to cell death by necrosis [13,26]. Data
on the toxicity of HypF-N prefibrillar aggregates sug-
gest a mechanism of cell death that is possibly shared
with the prefibrillar aggregates of most peptides and
proteins [27].
Much research is currently being carried out into
molecules that are able to avoid the appearance of
misfolded proteins and their initial aggregates in tis-
sue. Notwithstanding the validity of such an approach,
better knowledge of the biochemical basis of cell

vulnerability to protein aggregates may also provide
clues to possible interventions aimed at increasing the
resistance of cells to these toxic assemblies. We sought
to provide information on the chain of events that
leads to death in cells experiencing toxic aggregates by
investigating features of the apoptotic pathways trig-
gered in two different cell lines upon exposure to toxic
HypF-N prefibrillar aggregates. Although different cell
types often show similar biochemical alterations, they
are variously affected by exposure to the same toxic
protein aggregates, such that only specific cell popula-
tions are stressed [14,15,28,29]. Such differences in
vulnerability reflect the inherent ability of any cell
to disfavour aggregate interaction with the plasma
membrane, and possibly other membranes, and the
subsequent early modifications by using its specific
biochemical equipment. This equipment includes the
specific membrane lipid composition, the total anti-
oxidant defences (TAC), the efficiency of Ca
2+
extru-
sion membrane pumps and the energy load (ATP
availability).
A recent study showed large variations in the toxic
effects of HypF-N prefibrillar aggregates on a panel
of cultured cell lines [14], leading us to rank the cell
lines according to their vulnerability. This study was
carried out using murine endothelial Hend cells and
human IMR90 fibroblasts; these were chosen as
examples of cells that are very vulnerable or very

resistant to toxic HypF-N aggregates, respectively.
The different vulnerability of the cell lines was asso-
ciated with different plasma membrane cholesterol
C. Cecchi et al. Apoptotic pathways in HypF-aggregate-treated cells
FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS 2207
content, which has been shown to disfavour mem-
brane interaction with aggregates [14]. We found that
both cell lines showed early activation of a pro-
grammed cell death following exposure to the aggre-
gates; however, IMR90 cells were able to counteract
the toxic insult and recover despite initial impair-
ment. Details of the apparent differences in the spe-
cific apoptotic pathways in the two cell lines are also
discussed.
Results
Hend and IMR90 cells are differently impaired
upon exposure to toxic HypF-N aggregates
We recently reported that different cell types exposed
for 24 h to HypF-N prefibrillar aggregates are vari-
ously impaired, as assessed using the 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
cell viability assay [14]. In this study, we performed a
more detailed time-course analysis of the viability of
two cell lines when exposed to the same aggregates:
Hend endothelial cells and IMR90 fibroblasts, previ-
ously shown to be highly vulnerable and highly resist-
ant to HypF-N toxic aggregates, respectively [14].
Our analysis was carried out using a highly sensitive
test based on Resazurin reduction by mitochondrial
oxidoreductases. A significant early decrease ( 27%)

in Hend and IMR90 cell viability was evident after
3 h exposure to the toxic aggregates. However, Hend
viability was increasingly impaired over 24 h of aggre-
gate treatment, whereas IMR90 cells had recovered
completely after 24 h exposure (Fig. 1A). Hend cells
were not able to recover even at longer (48 h) expo-
sure times (data not shown). We also investigated the
reversibility of the cell damage. Hend cells were
exposed to toxic aggregates for different times, follow-
ing transfer into aggregate-free fresh medium for
24 h. Figure 1A shows that cell damage appears to be
almost completely reversible when aggregate exposure
was for relatively short lengths of time (< 6 h) fol-
lowing cell transfer into aggregate-free medium; under
these conditions, it can be assumed that cell damage
is not so great that it hinders complete recovery. At
longer exposure times (to 24 h) cell viability recovered
only partially under our experimental conditions. In
both cell lines, global cell impairment was not due to
the necrosis of individual cells. In fact, lactate dehy-
drogenase (LDH) activity, measured in the culture
media, remained substantially unchanged compared
with control cells exposed to the same amount of a
harmless monomeric soluble protein (Fig. 1A, inset).
The differences in vulnerability seen in the two cell
types is not due to differing sensitivity to the aggre-
gates in terms of dose–response; in fact, IMR90 cells,
unlike Hend cells, appeared resistant to exposure to a
wide range (0.02–20 lm) of aggregate concentrations
(Fig. 1B).

Fig. 1. IMR90 and Hend cells display different susceptibility to
damage by HypF-N prefibrillar aggregates. (A) Cell viability was
checked by the Resazurin reduction test, after supplementing the
cell media with 2.0 l
M HypF-N prefibrillar aggregates or the same
amount of soluble monomeric protein for differing lengths of time
(0.5, 1, 3, 6, 16 and 24 h). The reversibility of damage was checked
in Hend cells (Rev-Hend, dotted line) cultured for 24 h in fresh
aggregate-free medium, after exposure to aggregates for the indic-
ated times. In the inset, the unchanged levels of LDH release in
IMR90 and Hend cells after treatment for differing times with the
toxic aggregates or the same amount of the soluble monomeric
protein. (B) Cell viability in cells exposed for 24 h to varying
amounts of aggregates. Values are relative to cells treated with sol-
uble monomeric protein and are given as means ± SD. The report-
ed values are representative of three independent experiments,
each performed in triplicate. *Significant difference (P £ 0.05) ver-
sus cells treated with soluble monomeric protein. For details, see
Experimental procedures.
Apoptotic pathways in HypF-aggregate-treated cells C. Cecchi et al.
2208 FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS
Early modifications of the intracellular redox
status, free Ca
2+
and ATP levels: possible role
of membrane cholesterol
There is strong experimental evidence that oxidative
stress is one of the earliest biochemical modifications
in cells exposed to toxic prefibrillar aggregates [13–15];
therefore, we carried out a time-course confocal analy-

sis of ROS production in Hend and IMR90 cells
exposed to toxic HypF-N aggregates. As shown in
Fig. 2, intracellular ROS levels increased over time in
Hend cells, reaching a maximum at 24 h exposure,
whereas in IMR90 cells ROS levels were substantially
unchanged with respect to control values, showing
only a negligible increase. It is widely reported that in
different cell types oxidative stress matches a sharp
increase in the levels of free cytosolic Ca
2+
[1]. This is
in agreement with our time-course analysis of the
intracellular Ca
2+
content of Hend and IMR90 cells
exposed to HypF-N aggregates, plated on glass cover-
slips and fixed at various exposure times (Fig. 3).
Indeed, in Hend cells we found an increase in free
Ca
2+
that was earlier and stronger than the increase in
ROS and was followed by a partial reduction at
30 min; it then remained substantially unchanged and
higher than in controls, increasing slowly between 3
and 24 h. In contrast, in IMR90 cells the Ca
2+
increase was much smoother and smaller, and without
an early peak. The data suggest that the more vulner-
able Hend and more resistant IMR90 cells are provi-
ded, respectively, with a poorly or highly efficient

biochemical machinery that is aimed at counteracting
any uncontrolled increase in the levels of ROS and free
Ca
2+
.
The differing vulnerability of the two cell lines was
confirmed by the different changes in intracellular
ATP content upon exposure to the aggregates. The
ATP load provides cells with the energy needed to
counteract early biochemical modifications, such as
any imbalance in Ca
2+
homeostasis, induced by expo-
sure to prefibrillar aggregates and ⁄ or to sustain apop-
tosis [14]. In IMR90 cells exposed to HypF-N
aggregates for 3 h intracellular ATP was significantly
decreased (to  65% with respect to control cells
exposed to the same amount of monomeric soluble
protein), with complete energy recovery at longer incu-
bation times (Fig. 4A). A much stronger and more
prolonged ATP depletion was seen in Hend cells trea-
ted with HypF-N aggregates, which suggests more
serious mitochondrial impairment, with substantial
Fig. 2. Changes in intracellular ROS levels in
IMR90 and Hend cells as determined by
confocal analysis. Cells were exposed for
15, 30, 60, 180 min and 24 h to 2.0 l
M
HypF-N prefibrillar aggregates or to the
same amount of soluble monomeric protein

and then fixed with 2.0% paraformaldehyde.
ROS were determined by incubating
exposed cells for 10 min in the presence of
the redox fluorescent probe and measuring
the fluorescence of DCFH-DA. The data are
reported as a proportion of the values deter-
mined at time 0 and are expressed as
means ± SD of four experiments, each car-
ried out in duplicate. *Significant difference
(P £ 0.05) versus cells treated w ith soluble
monomeric protein. For details, see Experi-
mental procedures.
C. Cecchi et al. Apoptotic pathways in HypF-aggregate-treated cells
FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS 2209
recovery to starting values at longer exposure times
(16 and 24 h). In any case, basal ATP levels were sig-
nificantly higher in untreated IMR90 fibroblasts than
in untreated Hend cells.
Despite controversy about the role of blood choles-
terol levels and neuronal membrane cholesterol content
in the pathogenesis of amyloid diseases [30], our recent
investigation on a wide range of cell lines supports the
increasingly accepted idea that membrane lipid compo-
sition is a key biochemical feature affecting protein
aggregation, interactions between aggregates and cells,
and the response of cells to the presence of aggregates
[14,31]. According to our previous data, the differing
extent of alterations in ROS and free Ca
2+
in IMR90

and Hend cell lines exposed to HypF-N aggregates
was inversely correlated with membrane cholesterol
content. In particular, we found a significantly higher
level of basal cholesterol in the resistant IMR90 cells
(14.9 ± 1.6 lgÆmg
)1
of protein; P £ 0.05) than in the
vulnerable Hend cells (9.0 ± 1.2 lgÆmg
)1
of protein).
Cholesterol may increase the resistance of membranes
to the destabilizing effects of aggregates by reducing
the interaction between the membrane and the aggre-
gates or by changing membrane fluidity [31]. These
effects provide a possible explanation for the different
responses of the two exposed cell lines in terms of
increases in free Ca
2+
and ROS.
Aggregate-induced stress is associated with
typical apoptotic features
It is widely reported that protein aggregates are able
to interact with cell membranes thus impairing funda-
mental cellular processes, and eventually resulting in
apoptotic or, less frequently, necrotic cell death [1]. In
our previous study on a panel of different cell lines,
aggregate-induced cellular stress was associated with
typical apoptotic features rather than with a necrotic
pattern [14]. A distinct feature of apoptotic cells is the
exposure of phosphatidylserine (PtdSer) on the outer

membrane surface. PtdSer, normally found in the
inner membrane leaflet, flips to the outer leaflet during
the early stages of apoptosis [32]. We used annexin
V-FITC and propidium iodide (PI) double labelling to
detect PtdSer externalization and membrane integrity
in Hend and IMR90 cells exposed to HypF-N aggre-
gates. A larger fraction of Hend cells, with respect to
IMR90 cells, was double stained with high annexin-V
and low PI positivity, indicating, in the former, a pro-
gressive apoptotic (PtdSer exposure) rather than a
necrotic (membrane rupture) outcome (Table 1). In
contrast, neither cell line when treated with the mono-
meric soluble HypF-N displayed annexin V-FITC or
PI binding until 24 h exposure. Only in a minority of
cells exposed to the HypF-N aggregates was a high
Fig. 3. Changes in intracellular free Ca
2+
lev-
els in IMR90 and Hend cells determined by
confocal analysis. Cells were exposed for
15, 30, 60, 180 min and 24 h to 2.0 l
M
HypF-N prefibrillar aggregates or to the
same amount of soluble monomeric protein
and then fixed with 2.0% paraformaldehyde.
Intracellular free Ca
2+
levels were determ-
ined by incubating the exposed cells for
15 min in the presence of the fluorescent

dye Fluo-3AM. The data are reported as a
proportion of the values determined at time
0 and are expressed as means ± SD of four
experiments each carried out in duplicate.
*Significant difference (P £ 0.05) versus
cells treated with soluble monomeric pro-
tein. For details, see Experimental proced-
ures.
Apoptotic pathways in HypF-aggregate-treated cells C. Cecchi et al.
2210 FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS
positivity to both annexin-V and PI observed, suggest-
ing a very low percentage of plasma membrane rup-
tures (Table 1). The data agree with those reported in
Fig. 1A (inset) showing a substantial lack of LDH
release from both Hend and IMR90 cells exposed to
aggregates.
Mechanisms of apoptotic death in exposed cells
We then sought to explain the different degree of
recovery in the two cell lines shown in Fig. 1A. We
therefore analysed the mitochondrial status and some
apoptotic markers in our cellular models exposed for
24 h to toxic HypF-N aggregates. It has recently been
reported that b-amyloids gradually impair mitochond-
rial structure and function via changes in membrane
viscosity, energy load, ROS production and cyto-
chrome c release [33]. One well-described consequence
of aggregate toxicity is induction of the MPT, a
Ca
2+
-dependent process characterized by the opening

of pores in the inner mitochondrial membrane and by
ATP depletion [34]. Figure 4B shows changes in the
fluorescence of mitochondria loaded with calcein in the
presence of Co
2+
, a method that allows detection of
MPT [31]. The presence of HypF-N aggregates in the
cell culture media resulted in a large initial (at 15 min)
decrease in calcein fluorescence in IMR90 mitochon-
dria due to MTP opening, followed by a rapid (at
30 min), almost complete, recovery of membrane integ-
rity. In contrast, Hend cells showed a delayed progres-
sive decrease in calcein fluorescence. It therefore
appears that in IMR90 cells, mitochondria are initially
heavily affected by the aggregates, but they are able to
recover rapidly; whereas in Hend cells mitochondrial
involvement is delayed, but is progressively more
severe and without any possibility of recovery. The
data may explain the more severe loss of ATP load
seen in Hend cells compared with IMR90 cells exposed
to the toxic aggregates (Fig. 4A).
It has been reported that MPT opening triggers the
release of cytochrome c from mitochondria, which, in
turn, activates procaspase 9 and then the effector casp-
ases that amplify programmed cell death [23]. Under
these conditions, other mitochondrial proteins, inclu-
ding AIF can be released [24]. The early apoptotic
steps in either cell line exposed to the toxic HypF-N
aggregates were investigated using a time-course analy-
sis of cytochrome c and AIF translocation. As shown

in Fig. 5A, in Hend cells cytochrome c was signifi-
cantly released into the cytosol at 30 min exposure and
was maintained at significantly higher levels than con-
trols up to 24 h exposure. In contrast, IMR90 cells
showed earlier and sharper cytochrome c translocation
to the cytosol followed by recovery to basal levels
(Fig. 5A). Significant early, although delayed with
respect to cytochrome c, AIF translocation from the
Fig. 4. Time course of ATP levels in exposed cells and determin-
ation of MTP opening. (A) ATP levels were assessed in Hend and
IMR90 cells exposed to 2.0 l
M aggregated HypF-N for 0.5, 1, 3, 6,
16 and 24 h (means ± SD) or to the same amount of soluble mono-
meric protein. (B) MTP opening was assessed by measuring chan-
ges in mitochondrial calcein fluorescence intensity. After exposure
to 2.0 l
M HypF-N aggregates, IMR90 and Hend cells were coloaded
with calcein and CoCl
2
. Quantitative data are reported as the means
± SD of the flow cytometer analysis of treated cells with respect to
cells treated with the same amount of soluble monomeric protein,
assumed to be 100%. The values shown are averages of three indep-
endent experiments. *Significant difference (P £ 0.05) versus cells
treated with soluble monomeric protein. For details, see Experimen-
tal procedures.
Table 1. Annexin V assay. The data are reported as a per cent of
the value determined in the total population and are means ± SD
of three independent experiments.
Time

(h)
IMR90
Apoptotic
cells (%)
Necrotic
cells (%)
Hend
Apoptotic
cells (%)
Necrotic
cells (%)
0 0.60 ± 0.15 0.81 ± 0.57 2.69 ± 1.92 0.28 ± 0.10
0.5 0.35 ± 0.09 0.90 ± 1.09 8.50 ± 2.12 0.98 ± 0.26
1 0.60 ± 0.15 1.22 ± 1.54 7.70 ± 3.92 1.63 ± 1.51
3 3.10 ± 1.02* 0.83 ± 0.46 16.86 ± 4.22* 0.93 ± 0.31
6 1.60 ± 0.77 1.22 ± 1.01 20.20 ± 5.87* 0.73 ± 0.53
24 1.70 ± 0.87 2.20 ± 2.36 23.51 ± 5.04* 0.60 ± 0.48
*P £ 0.05 versus cells treated with soluble monomeric protein.
C. Cecchi et al. Apoptotic pathways in HypF-aggregate-treated cells
FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS 2211
mitochondria to the nuclear fraction was also seen
in IMR90 cells at up to 3 h exposure, whereas in
Hend cells AIF did not appear to be involved in the
apoptotic response (Fig. 5B).
It is well known that cytochrome c can activate clea-
vage of procaspase 9 into its active fragment by form-
ing a complex with the cytosolic factor Apaf-1 [23].
We therefore measured the levels, in the total homo-
genates, of active caspase 9, a marker for the activa-
tion of the intrinsic apoptotic pathway. A sharper

increase in caspase 9 active fragment was seen in
IMR90 cells than in Hend cells at early exposure times
(Fig. 6A). However, in IMR90 cells, caspase 9
returned to control levels after 3 h of treatment,
whereas in Hend cells the caspase 9 content appeared
to increase slowly, reaching significant activation only
after 24 h exposure to the aggregates (Fig. 6A). This
agrees with data relative to the cytochrome c translo-
cation in both cell lines up to 24 h exposure (Fig. 5A).
In contrast, levels of the caspase 8 active fragment, a
marker of the extrinsic apoptotic pathway, were signifi-
cantly increased after 20 min and from 1 to 16 h of
treatment in Hend cells, whereas in IMR90 cells
caspase 8 remained at control levels (Fig. 6B). This
agrees with data showing a lower interaction of the
aggregates with the plasma membrane in IMR90 cells
than in Hend cells, possibly due to the different choles-
terol content.
A
B
Fig. 5. Time course of cytochrome c and
AIF translocation in exposed cells. (A) Cyto-
chrome c (16 kDa) compartmentalization
was quantified in the cytosolic fraction of
IMR90 and Hend cells exposed to 2.0 l
M
HypF-N aggregates or to the same amount
of soluble monomeric protein for differing
lengths of time. Tubulin was used as a load-
ing control. (B) AIF (57 kDa) compartmental-

ization was assessed in the nuclear
fractions of IMR90 and Hend cells exposed
to 2.0 l
M HypF-N prefibrillar aggregates or
to the same amount of soluble monomeric
protein for differing lengths of time.
Histones were used as loading controls.
Quantitative data are reported as means ±
SD of the densitometric analysis of treated
cells with respect to cells treated with
soluble monomeric protein, assumed to be
100%. Values shown are averages of three
independent experiments. *Significant
difference (P £ 0.05) versus cells treated
with soluble monomeric protein. For details,
see Experimental procedures.
Apoptotic pathways in HypF-aggregate-treated cells C. Cecchi et al.
2212 FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS
Early caspase 3 activation followed by a reduction
and further increase at longer exposure times (16 and
24 h) was seen in Hend cells (Fig. 7A). This possibly
reflects the initial activation, in these cells, of
caspase 8, followed by later activation of caspase 9. In
IMR90 cells only moderate activation of caspase 3 was
found as a consequence of the modest activation of
caspase 9 and the lack of activation of caspase 8
(Fig. 7A). It is known that the activated caspase 3
fragment may cleave poly(ADP-ribose) polymerase
(PARP; EC 2.4.2.30), which functions primarily as a
DNA damage sensor in the nucleus [35]. Accordingly,

we found that, in Hend cells, early caspase 3 activation
triggered cleavage of PARP (data not shown), resulting
in a significant decrease of PARP activity early and
late during aggregate treatment (0.5, 16 and 24 h)
(Fig. 7B). In contrast, exposed IMR90 cells showed
significant activation of PARP resulting in the
enhancement of its DNA repair function. Data on the
caspase active fragments and the different impairment
of mitochondria were further confirmed by the analysis
of the antiapoptotic factor Bcl-2 in either exposed cell
line. Interestingly, in IMR90 cells Bcl-2 was signifi-
cantly and progressively upregulated up to 1 h of
aggregate treatment and persisted at the highest levels
until 16 h of treatment, whereas it was significantly
reduced in Hend cells at 3 and 24 h of treatment
(Fig. 8). Consequently, Bcl-2 levels were significantly
Fig. 6. Time course of caspase 8 and
caspase 9 translocation. (A) The levels of
caspase 9 active fragment (37 kDa) were
achieved in the total homogenates of
IMR90 and Hend cells exposed for differing
times to 2.0 l
M HypF-N prefibrillar aggreg-
ates or to the same amount of soluble
monomeric protein. (B) The levels of cas-
pase 8 active fragment (43 kDa) were
determined in total homogenates of IMR90
and Hend cells exposed for varying times to
2.0 l
M HypF-N prefibrillar aggregates or to

the same amount of monomeric soluble pro-
tein. Tubulin was used as a loading control
in (A) and (B). Quantitative data are reported
as the means ± SD of the densitometric
analysis of treated cells with respect to cells
treated with soluble monomeric protein,
assumed to be 100%. Values shown are
averages of five independent experiments.
*Significant difference (P £ 0.05) versus
cells treated with soluble monomeric pro-
tein. For details, see Experimental proced-
ures.
C. Cecchi et al. Apoptotic pathways in HypF-aggregate-treated cells
FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS 2213
higher in IMR90 cells than in Hend cells at all expo-
sure times and increased by > 100% in cells exposed
for 1–3 h. Finally, IMR90 and Hend cells exposed to
HypF-N aggregates displayed a typical DNA fragmen-
tation pattern as evaluated in terms of enrichment of
histone-associated oligonucleosomes released into the
cytoplasm. As expected from the susceptibility scale
and from the extent of caspase 3 activation, a greater
increase was found in Hend cells (224 ± 35%) than in
IMR90 cells (116 ± 18%) after 24 h exposure to the
toxic aggregates.
Discussion
It is known that only specific cell types are impaired in
tissues facing amyloid deposits [29,36] and that cell
stress eventually leads to cell death by apoptosis or, in
some cases, to secondary necrosis [12,37]. We previ-

ously reported that the vulnerability of different cell
lines to toxic HypF-N prefibrillar aggregates appears
to be related to intrinsic biochemical features of the
cells [14]. We also provided data suggesting that the
choice between an apoptotic and a necrotic outcome
depends on the timing and severity of mitochondria
impairment [26]. In this study, we investigated the
apoptotic pathways activated in two different cell lines,
Hend and IMR90, chosen as examples of cells that are
highly vulnerable or highly resistant to insult by toxic
prefibrillar aggregates, respectively. The differing sus-
ceptibility to the damage by the aggregates was not an
artefact due to a different dose–response in each cell
line, as shown by the substantial resistance of IMR90
cells to much higher amounts of aggregates than those
impairing Hend cells. Both cell lines appeared signifi-
cantly stressed after 3 h exposure to the aggregates. At
this time, cell damage appeared substantially reversible
even for the most heavily affected Hend cells; however,
at longer exposure times cell recovery was increasingly
less complete, indicating a progressive deterioration
in cell viability. At longer exposure times, IMR90 cells
recovered completely despite early activation of the
apoptotic programme, whereas a significant fraction of
Fig. 7. Time course of caspase 3 activation
and PARP activity. (A) Levels of caspase 3
active fragment (11 kDa) were determined
in total homogenates of IMR90 and Hend
cells after differing exposure times to
2.0 l

M HypF-N prefibrillar aggregates or to
the same amount of soluble monomeric pro-
tein. Tubulin was used as a loading control.
(B) PARP activity was assessed on purified
nuclear samples on the basis of its auto-
poly(ADP-ribosylation) level in Dot Blot an-
alysis. Quantitative data are reported as the
means ± SD of the densitometric analysis
of treated cells with respect to cells treated
with soluble monomeric protein, assumed
to be 100%. The values shown are aver-
ages of three independent experiments.
*Significant difference (P £ 0.05) versus
cells treated with soluble monomeric pro-
tein. For details, see Experimental proced-
ures.
Apoptotic pathways in HypF-aggregate-treated cells C. Cecchi et al.
2214 FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS
Hend cells underwent apoptotic death at 24 h expo-
sure. Therefore, the differing vulnerability seen in the
two cell lines following 24 h exposure to the aggregates
appears to be associated with the greater ability of
IMR90 cells to counteract the early biochemical modi-
fications underlying activation of the apoptotic path-
way, rather than an effect of a lower sensitivity to
similar amounts of aggregates.
Severe alterations in many biochemical parameters,
including intracellular redox status, energy load and
free Ca
2+

homeostasis [2], as well as membrane lipid
composition [14,38], appear to be key factors in
favouring cell impairment or resistance to the toxic
aggregates of peptides and proteins either associated
[1,39] or not associated with amyloid diseases [13,14].
It is also well known that protein prefibrillar aggre-
gates can interact with the plasma membrane of
exposed cells inducing modifications in the lipid or
proteolipid structure, or self-assembling into pores thus
inducing alterations in membrane selective permeabil-
ity [3]. In this scenario, it is conceivable that cells
endowed with higher basal antioxidant defences and
efficient Ca
2+
pumps are better suited to resist any
increase in free Ca
2+
(or other ion) and the conse-
quent biochemical modifications [14].
We found that the highly vulnerable Hend cells
exposed to HypF-N toxic aggregates displayed earlier
and greater increases in both intracellular ROS and
free Ca
2+
when compared with the more resistant
IMR90 cells. The early Ca
2+
increase may induce
ROS overproduction by speeding up oxidative metabo-
lism to supply energy for the increased activity of the

membrane Ca
2+
pumps [38]. The resulting oxidative
stress may subsequently favour entry of Ca
2+
into the
cell with endoplasmic reticulum stress and mitochond-
rial impairment eventually targeting the cell for apop-
totic death [40,41]. Resistance of IMR90 to aggregate
damage was previously found to be significantly rela-
ted to the high efficiency of these cells in counteracting
early modifications of the intracellular free Ca
2+
and
redox status [14]. Under our experimental conditions,
both exposed cell lines displayed ATP depletion sup-
porting mitochondria involvement; however, Hend
cells, endowed with a lower basal energy load, showed
much more serious and prolonged loss of ATP than
IMR90 cells, indicating that the former were less suited
to counteracting ion balance derangement, which may
explain their higher vulnerability to apoptotic death.
The higher resistance of IMR90 fibroblasts to toxic
insult by the aggregates may also result from a signifi-
cant upregulation of Bcl-2. Such an antiapoptotic fac-
tor acts as an endogenous inhibitor of MPT pore
opening and mitochondrial apoptotic channel (MAC)
formation by Bax and Bak [42,43], resulting in the
release of proapoptotic factors such as AIF and cyto-
chrome c [1,23] and inhibition of the proteolytic pro-

cessing of AIF [44]. Interestingly, nuclear AIF was
unchanged in Hend cells, suggesting that it is not
involved in the apoptotic cascade. The partial release
of cytochrome c not associated with AIF release found
in Hend cells agrees with previous data on infrared-
irradiated human fibroblasts [45]. AIF was significantly
increased in the nuclei of IMR90 cells after 3 h expo-
sure, where it matched, although in a delayed manner,
cytochrome c release. However, the release of AIF and
cytochrome c was not sustained at longer exposure
times, where upregulation of Bcl-2 occurred. The latter
could disassemble MAC, the proposed channel allow-
ing cytochrome c to translocate to the cytosol [43],
thus explaining the complete recovery in mitochondrial
function, which is also supported by the recovery in
ATP levels, and hence cell viability.
As pointed out above, both exposed cell lines dis-
played early translocation of cytochrome c from the
mitochondria to the cytosol. However, cytochrome c
release was much higher and decreased rapidly in
IMR90 cells, whereas in Hend cells it increased pro-
gressively up to 24 h exposure. Once released from the
Fig. 8. Time course of Bcl-2 expression in exposed cells. Bcl-2
(25 kDa) expression was determined in the mitochondrial fraction
of IMR90 and Hend cells exposed to 2.0 l
M HypF-N granular aggreg-
ates or to the same amount of soluble monomeric protein for dif-
fering lengths of time. Prohibitin was used as a loading control.
Quantitative data are reported as the means ± SD of the densito-
metric analysis of treated cells with respect to cells treated with

soluble monomeric protein, assumed to be 100%. Values shown
are the averages of three independent experiments. *Significant
difference (P £ 0.05) versus cells treated with soluble monomeric
protein. For details, see Experimental procedures.
C. Cecchi et al. Apoptotic pathways in HypF-aggregate-treated cells
FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS 2215
mitochondria, cytochrome c, in association with Apaf-1,
is involved in the activation of caspase 9. Indeed, both
Hend and IMR90 cells showed a significant increase
in the caspase 9 active fragment, however, the latter
occurred earlier and was higher in IMR90 cells than in
Hend cells, where a sharp increase in caspase 9 activa-
tion was observed after 24 h exposure. Moreover, the
extrinsic apoptotic pathway triggered by caspase 8
cleavage was activated only in Hend cells after just
20 min exposure to aggregates. It is known that activa-
tion of the effector caspase 3 occurs downstream of
caspase 8 and caspase 9 cleavages in response to differ-
ing apoptotic stimuli; once activated, caspase 3 can
activate caspase 9 directly in a feedback loop, and
caspase 8 indirectly [46]. Indeed, in Hend cells exposed
to HypF-N aggregates the increase in caspase 3 active
fragment was earlier and sharper than in IMR90 cells
and was probably responsible for the late activation of
caspase 8 in Hend cells. It seems likely, therefore, that
the different response of either cell line to the same
toxic insult can be traced to, among others, a differing
interaction between the aggregates and the plasma
membrane, resulting in differing activation of the
apoptotic extrinsic pathway.

We have previously shown that HypF-N prefibrillar
aggregates interact with the plasma membrane of Hend
cells more extensively than with the membrane of more
resistant cell lines, apparently due to a different lipid
composition, including cholesterol [14]. In this study,
we confirmed the dependence of cell resistance on
membrane cholesterol content, which is considerably
higher in IMR90 cells than in Hend cells. Therefore,
according to our previous results, we expect a reduced
interaction between the aggregates and the plasma
membrane in IMR90 cells compared with Hend cells.
In IMR90 cells, reduced membrane fluidity with
increased resistance to the destabilizing effects of the
aggregates can also be hypothesized. These considera-
tions may explain the lack of activation of the apop-
totic extrinsic pathway and cell recovery after the
initial insult; they also agree with recent findings indi-
cating that cholesterol can modulate membrane-associ-
ated Ab fibrillogenesis and neurotoxicity, and that
decreasing the fluidity of brain lipid bilayers reduces
the interaction of Ab40 with the bilayer surface and
insertion of the latter inside the bilayer itself [47].
Caspase 3 activation targeted exposed Hend cells to
apoptotic death. In these cells, the amount of histone-
associated oligonucleosomes released into the cytoplasm
confirmed a significant increase in DNA fragmentation,
in agreement with the severe impairment of cell viability
and the increases in ROS and active caspase 3 fragment.
Caspase 3 activation resulted in PARP cleavage and
inactivation. In contrast, IMR90 cells appeared more

resistant to DNA oxidative attack, possibly because of
their higher basal antioxidant capacity [14] and ⁄ or
reduced damage and permeabilization of the cell mem-
brane by the aggregates.
Overall, the data support differing scenarios for the
responses of Hend and IMR90 cells to the toxic aggre-
gates. These differences can, at least in part, be traced
to differing involvement of the plasma membranes
with the aggregates (Fig. 9). In Hend cells, reduced
membrane cholesterol content favours interaction of
the aggregates with the plasma membrane [14] leading
to membrane destabilization and permeabilization. The
structural and biochemical modifications of the plasma
membrane result in early and transient increases in
cytosolic free Ca
2+
that appears sufficient to trigger
the extrinsic apoptotic pathway. In this case, the meta-
bolic efficiency of the mitochondria appears to be
impaired at late exposure times, possibly following the
oxidative stress accompanying the initial high energy
demand to counteract the altered membrane perme-
ability. However, mitochondria do not release large
amounts of proapoptotic factors, nor is the antiapop-
totic Bcl-2 upregulated, and activation of the effector
caspase 3 appears to result mainly from the early acti-
vation of caspase 8.
In IMR90 cells, whose plasma membranes are richer
in cholesterol, the extrinsic pathway does not appear
to be activated. Nevertheless, early involvement of the

mitochondria is apparent in this cell line, with a signifi-
cant but transient release of cytochrome c and, slightly
later, of AIF, suggesting that a signal arising from the
plasma membrane may trigger transient MAC organ-
ization in the mitochondria outer membrane. At the
same time, a significant but transient activation of
caspase 9 was seen with subsequent recovery to basal
values (Fig. 9). The ability of these cells to recover
after an initial insult can be tentatively traced to the
higher resistance of a plasma membrane rich in choles-
terol to the destabilizing effects of the aggregates, and
to the early upregulation of Bcl-2, possibly counteract-
ing the initial formation of MAC in the outer mito-
chondrial membrane.
Experimental procedures
Materials
All reagents were of analytical grade or the highest purity
available. Unless stated otherwise, chemicals were pur-
chased from Sigma (Milan, Italy). HypF-N was expressed
and purified as previously described [25]. In brief, the
domain was made to aggregate upon incubation for 48 h at
Apoptotic pathways in HypF-aggregate-treated cells C. Cecchi et al.
2216 FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS
0.3 mgÆmL
)1
protein concentration in 50 mm acetate buffer,
pH 5.5 in the presence of 30% (v ⁄ v) trifluoroethanol (TFE)
at room temperature. Under these conditions, granular
aggregates about 4–8 nm wide and 20–60 nm long are
formed; the presence of such aggregates in the preparations

used for the experiments was checked by transmission elec-
tron microscopy (not shown).
Cell culture and HypF-N-aggregate treatment
Human IMR90 pulmonary fibroblasts were obtained from
the ATCC (Manassas, VA), murine endothelium cells
(Hend) were a kind gift of F. Bussolino (University of
Turin, Italy). IMR90 and Hend cells were cultured in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented
with 1.0 mm glutamine and antibiotics and 10% fetal
bovine serum, (FBS) (Mascia Brunelli, Milan, Italy) or
10% FBS (Sigma), respectively. Cell cultures were main-
tained and used for experiments at  90% confluence in a
5% CO
2
humidified atmosphere at 37 °C.
Aliquots of solutions containing prefibrillar HypF-N
aggregates were centrifuged, dried under N
2
to remove the
TFE when necessary, dissolved in the appropriate cell
media at 200 lm and immediately added to cultured cells at
2.0 lm final concentration. No microscopic differences in
aggregate structures were observed following dilution in the
cellular culture medium. Cells were incubated for differing
times in the presence of prefibrillar aggregates or of mono-
meric soluble protein as a control.
Cell viability assay and the reversibility
of cell damage
The cytotoxicity of the HypF-N aggregates was assessed
using the CellTiter-Blueä Cell Viability Assay (Promega,

Milan, Italy) based on the reduction of the indicator dye
Resazurin into Resurfin by viable cells. Cells were plated
on 96-well plates ( 1.0 · 10
4
cells per well) and after
24 h HypF-N prefibrillar aggregates (2.0 lm) were added
to fresh culture media for differing times (0.5, 1, 3, 6,
16, 24 h). Hend and IMR90 cells were also exposed to
various concentrations (0.02, 0.2, 2.0 or 20 lm) of prefi-
brillar HypF-N aggregates and to the soluble monomeric
protein for 24 h. To analyse the reversibility of cell
impairment the cells were incubated with culture medium
containing 2 lm prefibrillar aggregates for differing times
(0.5, 1, 3, 6, 16, 24 h), washed, and cultured in fresh cul-
ture medium for 24 h. After aggregate treatment, 100 lL
CellTiter-Blueä reagent solution in RPMI (1 : 6) was
added to each well at, shacked for 10 s and incubated at
37 °C for 1 h. Sample fluorescence was measured by
using a Fluoroscan Ascent FL (Thermo Electron Cor-
poration, Vantaa, Finland) with 544 nm excitation and
590 nm emission wavelengths, after subtracting the aver-
age fluorescence values of the culture media background
[48]. Cell viability was expressed as per cent increase of
Resazurin reduction in cells treated with prefibrillar
aggregates respect to the cells treated with the soluble
protein (assumed to be 100%).
Fig. 9. Representative flow-chart of the
molecular events underlying cell impairment
upon exposure to HypF-N prefibrillar aggreg-
ates. Membrane cholesterol content,

increases in intracellular free Ca
2+
and ROS,
mitochondrial status, cytochrome c and AIF
release, Bcl-2 expression, and caspase 8
and caspase 9 activation support different
scenarios in the response of Hend and
IMR90 cells to the same toxic aggregates.
C. Cecchi et al. Apoptotic pathways in HypF-aggregate-treated cells
FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS 2217
Assessment of necrotic cells
The presence of necrotic cells was assessed by assaying the
activity of LDH, a typical necrotic marker released to the
cell culture medium after rupture of the plasma membrane.
LDH activity was measured in the culture media of cells
exposed 0.5, 1, 3, 6, 16, 24 h to the HypF-N aggregates by
using the LDH assay kit (Roche Diagnostics) at 490 nm,
after blank subtraction at 595 nm.
Confocal measurement of intracellular ROS
Intracellular ROS were measured in intact cells treated with
5.0 mm ROS-sensitive fluorescent probe DCFH-DA using a
confocal Bio-Rad MCR 1024 ES scanning microscope (Bio-
Rad, Hercules, CA) equipped with a Kr ⁄ Ar laser source
(15 mW) as previously described [14]. Briefly, the cells were
cultured on glass coverslips, exposed for differing times to
2.0 lm HypF-N prefibrillar aggregates (monomer protein
concentration) and loaded with the dye upon incubation
for 20 min at 37 °C with DCFH-DA added to the culture
media. Then the cells on coverslips were fixed with 2.0%
paraformaldehyde for 10 min, mounted on glass and ana-

lysed by confocal microscopy. Twenty optical sections
(512 · 512 pixels) were taken for each examined sample
through the depth of the cells with a thickness of 1.0 lmat
intervals of 0.8 lm and then projected as a single composite
image by superimposition.
Confocal analysis of calcium transients
Levels of free cytosolic Ca
2+
in Hend and IMR90 cells were
imaged at differing exposure times as reported previously
[14]. Briefly, the cells were incubated with serum-free DMEM
containing 0.1% BSA, 10 lm (final concentration) Fluo3-AM
as the fluorescent calcium indicator, 0.1% dimethylsulfoxide
and Pluronic acid F-127 (0.01% w ⁄ v) for 10 min. The cells
were washed, fixed with 2.0% paraformaldehyde for 20 min,
mounted on glass and examined by confocal analysis. Cell
fluorescence was monitored at 488 nm excitation by collecting
the emitted fluorescence with a Nikon Plan Apo X60 (Nikon,
Florence, Italy) oil-immersion objective through a 510 nm
long-wave pass filter confocal microscope.
Assay of cholesterol content in the cell-surface
membrane
Cholesterol in the surface membrane of untreated cells was
assayed as previously described [49]. Briefly, 2.0 · 10
6
cells
were washed twice and resuspended in phosphate buffer,
pH 7.5, containing 310 mm sucrose. Cells were incubated at
37 °C for 2 h with cholesterol oxidase (2.5 UÆmL
)1

) and
phospholipase C (0.2 UÆmL
)1
in 1.3 mm CaCl
2
) to make
cholesterol in the intact membranes available for cholesterol
oxidase [50]. Then, 600 lL of Dole Reagent (78% isopropa-
nol, 20% heptane, 2.0% H
2
O) was added to the reaction
mixture, followed by 300 lL of heptane to stop the reaction
and to reduce the background by lipid extraction. Total
oxidized cholesterol was assayed as cholest-4-en-3-one at
235 nm in the upper phase obtained upon vortexing and
centrifuging for 10 min at 2000 g and 4 °C. Controls consis-
ted of cells supplemented with all the reagents except choles-
terol oxidase. Cholesterol content was determined by
comparison with a reference curve built by reacting differing
amounts (1–50 lg) of cholesterol dissolved in isopropanol.
Intracellular ATP assay
ATP determination was performed using a highly sensitive
bioluminescence assay (Kit HS II, Roche Diagnostics,
Mannheim, Germany) by which extremely low concentra-
tions of ATP can be detected on the basis of the ATP
dependency of the light emission of the luciferin oxidation
catalysed by luciferase [51]. Briefly, IMR90 and Hend cells,
plated on P60 wells (5.0 · 10
5
cells per plate), were exposed

to fresh media supplemented with 2.0 lm HypF-N prefibril-
lar aggregates for differing time periods (0.5, 1, 3, 6, 16,
24 h). The cells were harvested and resuspended in the dilu-
tion buffer provided with the kit, following three freeze–
thaw cycles. The lysis buffer was added to each sample in a
1 : 1 ratio and the samples were stored 5 min at room tem-
perature and finally centrifuged at 750 g for 15 min at
4 °C. Total protein content was measured in the superna-
tant according to the method of Bradford [52]. ATP meas-
urement were carried out on this fraction by using a
luminometer Lumat LB 9507 (EG & G Berthold, Bad
Wildbad, Germany).
Annexin V-FITC and PI labelling
Annexin V-FITC and PI labelling (Bender MedSystems,
Vienna, Austria) were used to detect the externalization of
PtdSer during the apoptotic progression in Hend and
IMR90 cells exposed to the 2.0 lm HypF-N aggregates for
differing lengths of time [33]. To measure the number of
apoptotic cells, HypF-N-treated cells were washed twice
with NaCl ⁄ P
i
and resuspended in binding buffer (10 mm
Hepes ⁄ NaOH, pH 7.4, 140 mm NaCl, 2.5 mm CaCl
2
)at
a density of 5.0 · 10
5
cellsÆmL
)1
. Then, 2.5 lgÆmL

)1
of
annexin V-FITC (PharMingen, San Diego, CA) and
0.2 lgÆmL
)1
PI (Sigma) were added. Within 60 min the
labelled cells were determined using a FACScalibur flow
cytometer (Becton Dickinson, Milan, Italy).
Measurement of MPT pore opening
MPT was measured by calcein fluorescence according to the
method of Petronilli et al. [34,53] with minor modifications.
Apoptotic pathways in HypF-aggregate-treated cells C. Cecchi et al.
2218 FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS
In brief, calcein ⁄ AM freely enters the cell and becomes
fluorescent upon de-esterification. Coloading of cells with
cobalt chloride quenches the fluorescence in the cell except
in mitochondria, because cobalt cannot cross mitochondrial
membranes. During induction of the MPT, cobalt can enter
the mitochondria where it is able to quench calcein fluores-
cence. Thus, decreased mitochondrial calcein fluorescence
can be taken as a measure of the extent of MPT induction.
At differing times of exposure to 2.0 lm HypF-N prefibrillar
aggregates, cell cultures were washed in HBSS (10 mm
Hepes buffer, pH 7.4, containing 144 mm NaCl, 2.0 mm
CaCl
2
, 1.0 mm MgCl
2
, 5.0 mm KCl and 10 mm glucose)
and incubated for 20 min at 37 °C in HBSS containing

1.0 mm calcein ⁄ AM and 1.0 mm cobalt chloride. Follow-
ing cobalt quenching, cultures were washed with HBSS
and then analyzed using a flow cytometer FACScalibur
(Becton Dickinson) with 488 nm excitation and 590 nm
emission filters.
Subcellular fractionation
Subcellular fractionation was achieved by the cytosol ⁄ mito-
chondria fractionation kit (Oncogene Research Products,
San Diego, CA). The homogenized samples were treated as
indicated by the manufacturer. Briefly, after treatment cells
were harvested and resuspended in the cytosol extraction
buffer supplemented with 1.0 mm dithiothreitol and a mix
of proteases inhibitors. Plasma membrane rupture was
achieved by three freeze–thaw cycles, the samples were then
centrifuged at 750 g for 10 min at 4 °C. The resultant pellet,
representing the nuclear fraction, was resuspended in 20 mm
Hepes buffer, pH 7.4, containing 250 mm sucrose, 2.0 mm
EGTA, 1.0 mm EDTA and sonicated twice for 5 s in ice.
The supernatant was centrifuged at 10 000 g for 30 min at
4 °C and the pellet, containing the mitochondrial fraction,
resuspended in the mitochondria extraction buffer supple-
mented with 1.0 mm dithiothreitol and a mix of protease
inhibitors. The supernatant of the centrifugation was repre-
sentative of the cytosolic fraction. Nuclear, mitochondrial
and cytosolic fractions were used to assess Bcl-2, cyto-
chrome c and AIF compartmentalization in both cell lines.
Another set of experiments was carried out on samples
of cell homogenates. Cells were harvested and resuspended
in 20 mm Tris ⁄ HCl buffer, pH 8.0, containing 2.0 mm
EDTA, 1.0% Triton X-100, 10% glycerol, 137 mm NaCl,

6.0 m urea, 0.2 mm phenylmethanesulfonyl fluoride,
10 lgÆmL
)1
aprotinin and leupeptin. After three freeze–
thaw cycles, the samples were sonicated twice for 5 s in ice
and then centrifuged at 14 000 g for 10 min at 4 °C. The
homogenates were used to assess the content of caspase 3
(the 11 kDa active fragment), caspase 8 (the 43 kDa active
fragment) and caspase 9 (the 37 kDa active fragment).
Total protein content in the nuclear and cytosolic fraction
and in cell homogenates was measured according to Brad-
ford [52].
Western blot analysis
To assess the intracellular levels of various apoptotic mark-
ers, equal amounts of cellular fractions were diluted in
Laemmli sample buffer and boiled for 5 min. Samples
containing  50 lg of proteins were run on SDS ⁄ PAGE
and then blotted onto poly(vinylidene difluoride) (PVDF)
Immobilio-P Transfer Membrane (Millipore Corp., Bed-
ford, MA). The native form of the mitochondrial Bcl-2
(25 kDa) was determined in blots of 12% SDS ⁄ PAGE gels
using anti-(Bcl-2) monoclonal sera (Santa Cruz Biotechno-
logy, San Diego, CA). The cytosolic fractions of cyto-
chrome c (16 kDa) were quantified in blots of 15%
SDS ⁄ PAGE gels by using mouse anti-(cytochrome c)
monoclonal sera (Oncogene Research Products). After
washing, the membranes were incubated with peroxidase-
conjugated anti-(mouse Ig) secondary sera for 1 h and the
immunolabelled bands were detected using a SuperSignal
West Dura (Pierce, Rockford, IL).

The nuclear fractions of AIF (57 kDa) were determined
in blots of 12% SDS ⁄ PAGE gels by using anti-AIF poly-
clonal sera (Santa Cruz Biotechnology). The caspase 3
(11 kDa active fragment) content in the total homogenate
fractions was determined by 15% SDS ⁄ PAGE, western
blotting and blot incubation with anti-(caspase 3) polyclon-
al sera (Santa Cruz Biotechnology). Determination of
caspase 8 and caspase 9 fragments was carried out in cell
homogenates run in 12% SDS ⁄ PAGE, and blotted, by
using rabbit anti-(caspase 8) and anti-(caspase 9) polyclonal
sera (Chemicon Int., Temecula, CA), respectively. After
washing, membranes were incubated with peroxidase-conju-
gated anti-rabbit secondary antibodies for 1 h and the
immunolabelled bands were detected using a SuperSignal
West Dura (Pierce).
All band densities were measured as densitometric unitsÆ
lg
)1
total proteins using the image analysis and densito-
metric program quantity one (Bio-Rad, Milan, Italy). For
each band of interest, values relative to cells treated with
soluble HypF-N were taken as 100%, whereas the values
relative to cells treated with the prefibrillar aggregates were
calculated as a percentage of the control within the same
blot. b-Tubulin (for homogenate and cytosolic fraction)
(Santa Cruz Biotechnology), prohibitin (for mitochondria)
(Abcam Ltd, Cambridge, UK) and histones (for nuclei)
(Chemicon Int.) were used to normalize the samples for equal
amount of protein loading.
PARP activity measurement

PARP activity was assessed by an immunodot blot which
detects poly(ADP-ribosylated) proteins [54]. Aliquots of
nuclear suspensions were diluted in 0.4 m NaOH containing
10 mm EDTA and loaded onto a Hybond N
+
nylon mem-
brane (Amersham Life Science, UK). The membrane was
washed once with 0.4 m NaOH, blocked in NaCl ⁄ P
i
–MT
C. Cecchi et al. Apoptotic pathways in HypF-aggregate-treated cells
FEBS Journal 273 (2006) 2206–2222 ª 2006 The Authors Journal compilation ª 2006 FEBS 2219
(NaCl ⁄ Pi, pH 7.4, containing 5.0% notfat dried milk and
0.1% Tween 20) and then incubated overnight with anti-
poly(ADP-ribose) polyclonal sera (96-10-04, Alexis, San
Diego, CA). The membrane was washed with NaCl ⁄ P
i
–MT
and incubated for 30 min with peroxidase-conjugated anti-
(rabbit IgG) (Amersham Life Science). At the end of the
incubation the blot was washed with NaCl ⁄ P
i
–MT followed
by chemiluminescence analysis. The membrane was subse-
quently immunoblotted with anti-histone sera (Chemicon
Int.) to assess that equal amounts of proteins were loaded.
Band (dot) densities were expressed as densitometric unitsÆ
lg
)1
protein (the constant protein amount applied on

Hybond N
+
nylon membrane) using the program for image
analysis and densitometry quantity one software (Bio-
Rad). Cells treated with soluble HypF-N were taken as
100%, whereas the values relative to HypF-N aggregate
treated cells were calculated as a percentage of the control
within the same immunodot blot.
DNA fragmentation analysis
DNA fragmentation was determined using an immuno-
metric method (Cell Death Detection ELISAPLUS, Roche
Diagnostics) according to the manufacturer’s instructions.
Briefly, after 24 h exposure to HypF-N prefibrillar aggre-
gates, 20 lL of the cell homogenates were placed in a
streptavidin-coated microtitre plate and incubated with
a mixture of biotinylated anti-histone sera, peroxidase-
labelled anti-DNA sera and incubation buffer (1% BSA,
0.5% Tween, 1 mm EDTA in NaCl ⁄ P
i
) for 2 h. After a
washing step, the retained peroxidase-linked complexes
were incubated with ABTS for 10 min, resulting in colour
development proportional to the number of nucleosomes
captured in the antibody sandwich. DNA fragmentation
was expressed as the enrichment of histone-associated
mono- and oligonucleosomes released into the cytoplasm
by measuring the absorption at 405 nm. The enrichment
factor was proportional to the number of apoptotic cell
present in the population.
Statistical analysis

All data were expressed as mean ± SD. Comparison
between the different groups were performed by anova fol-
lowed by Bonferroni’s t-test. A P-value < 0.05 was set as
significant.
Acknowledgements
We thank Daniele Nosi and Francesca Toni for techni-
cal advice. This study was supported by grants from
the Italian MIUR (project numbers 2003054414-002
and 2005054147-001) and from Fondazione Cassa di
Risparmio Pistoia e Pescia (project number 2004,0213).
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