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
Trang 1toxicity of prefibrillar HypF-N aggregates to different cell types
Cristina Cecchi1, Anna Pensalfini1, Serena Baglioni1, Claudia Fiorillo1, Roberto Caporale2,
Lucia Formigli3, Gianfranco Liguri1,4and Massimo Stefani1,4
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.
Trang 2The 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] Amyamy-loid 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 Ca2+ 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 Ca2+ 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
Trang 3content, 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.
Trang 4Early modifications of the intracellular redox
status, free Ca2+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 Ca2+[1] This is
in agreement with our time-course analysis of the
intracellular Ca2+ 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
Ca2+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 Ca2+ 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
Ca2+ 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 Ca2+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 with soluble
monomeric protein For details, see
Experi-mental procedures.
Trang 5recovery 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 cholescholes-terol 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 Ca2+ 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 Ca2+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 Ca2+ 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.
Trang 6positivity 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
Ca2+-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 Co2+, 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.
Trang 7mitochondria 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.
Trang 8Early 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.
Trang 9higher 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.
Trang 10Hend 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 Ca2+ 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 Ca2+ pumps are better suited to resist any
increase in free Ca2+ (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 Ca2+ when compared with the more resistant IMR90 cells The early Ca2+ increase may induce ROS overproduction by speeding up oxidative metabo-lism to supply energy for the increased activity of the membrane Ca2+ pumps [38] The resulting oxidative stress may subsequently favour entry of Ca2+ 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 Ca2+ 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 inhibifac-tor 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.