Subido por ali akbar Meratan

Membrane Integrity and Amyloid Cytotoxicity- A Model Study Involving Mitochondria and Lysozyme Fibrillation products

J. Mol. Biol. (2011) 409, 826–838
Contents lists available at
Journal of Molecular Biology
j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
Membrane Integrity and Amyloid Cytotoxicity:
A Model Study Involving Mitochondria and
Lysozyme Fibrillation Products
Ali Akbar Meratan, Atiyeh Ghasemi and Mohsen Nemat-Gorgani⁎
Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, 1417614411 Tehran, Iran
Received 26 January 2011;
received in revised form
12 April 2011;
accepted 17 April 2011
Available online
4 May 2011
Edited by S. Radford
hen egg white lysozyme;
membrane permeabilization;
Recent findings implicate that fibrillation products, the protein aggregates
formed during the various steps leading to formation of mature fibrils,
induce neurotoxicity predominantly in their intermediate oligomeric state.
This has been shown to occur by increasing membrane permeability,
eventually leading to cell death. Despite accumulating reports describing
mechanisms of membrane permeabilization by oligomers in model
membranes, studies directly targeted at characterizing the events occurring
in biological membranes are rare. In the present report, we describe
interaction of the original native structure, prefibrils and fibrils of hen egg
white lysozyme (HEWL) with mitochondrial membranes, as an in vitro
biological model, with the aim of gaining insight into possible mechanism of
cytotoxicity at the membrane level. These structures were first characterized
using a range of techniques, including fluorescence, size-exclusion
chromatography, dynamic light scattering, transmission electron microscopy, dot blot analysis and circular dichroism. HEWL oligomers were
found to be flexible/hydrophobic structures with the capacity to interact
with mitochondrial membranes. Possible permeabilization of mitochondria
was explored utilizing sensitive fluorometric and luminometric assays.
Results presented demonstrate release of mitochondrial enzymes upon
exposure to HEWL oligomers, but not native enzyme monomer or mature
fibrils, in a concentration-dependent manner. Release of cytochrome c was
also observed, as reported earlier, and membrane stabilization promoted by
addition of calcium prevented release. Moreover, the oligomer–membrane
interaction was influenced by high concentrations of NaCl and spermine.
The observed release of proteins from mitochondria is suggested to occur by
a nonspecific perturbation mechanism.
© 2011 Published by Elsevier Ltd.
*Corresponding author. E-mail address:
[email protected]
Abbreviations used: HEWL, hen egg white lysozyme;
ThT, thioflavin T; ANS, 1-anilino-naphthalene 8-sulfonate;
SEC, size-exclusion chromatography; DLS, dynamic light
scattering; TEM, transmission electron microscopy; MDH,
malate dehydrogenase; GDH, glutamate dehydrogenase;
CS, citrate synthase; AK, adenylate kinase; MAO,
monoamine oxidase; EDTA, ethylenediaminetetraacetic
0022-2836/$ - see front matter © 2011 Published by Elsevier Ltd.
It is now generally accepted that protein misfolding leading to aggregation is a key pathogenic
feature of various amyloid-related disorders, including Alzheimer's disease, Parkinson's disease,
Huntington's disease, type II diabetes and prion
diseases.1,2 Ordered protein aggregates, often referred to as amyloid fibrils, are commonly found in
these diseases. Moreover, recent findings have
indicated that the ability to form amyloid fibrils is
Membrane Integrity and Amyloid Cytotoxicity
a generic property of all proteins rather than a
characteristic feature of only those associated with
pathological conditions.1–5 Formation of amyloid
fibrils by various proteins and polypeptides precedes by the occurrence of metastable, partially
folded oligomeric intermediates often referred to as
protofibrils, 6–8 which have been found to be
cytotoxic.2,9–12 Moreover, the toxic activity of prefibrillar oligomers may be due to the fact that they
share a common structure, suggesting that they may
also share a common mechanism of toxicity.13 The
fact that different amyloids arise from either the
cytosolic or the extracellular protein points to the
plasma membrane as a potential primary target
that is accessible to both compartments.2,11,14,15
Although there appears to be a consensus on
membrane permeabilization by amyloid oligomers,
it is disputed whether this is due to the discrete
channel formation or to a nonspecific perturbation of
bilayer integrity, since evidence for both possibilities
has been obtained. 14–20 In addition to plasma
membrane as a primary target, internal organelles,
such as mitochondria, may also be affected.21 Recent
reports indicate that mitochondrial dysfunction may
play a critical role in the development of neurodegeneration in a number of pathological
conditions.22,23 Accumulation of β-peptide and αsynuclein has been shown to occur with predominant localization in the inner-membrane
structures.24,25 Of the various consequential events,
cell death is often attributed to an increase in
mitochondrial membrane permeability.26,27 Lysozyme, more specifically hen egg white lysozyme
(HEWL), is one of the most extensively studied
proteins whose structure and physiochemical properties have been well characterized.28 With a high pI
of 11,29 the protein bears a net positive charge over a
broad pH range and has a high affinity for anionic
and neutral phospholipids, both at low and high
ionic strengths.30 In addition to its lipid-binding
properties, it was found to induce release of the
aqueous contents of uncharged vesicles, presumably
as a result of its insertion into the lipid bilayer,
causing disruption.30,31 HEWL has been shown to
abundantly form well-defined amyloid fibrils under
in vitro conditions, making it an ideal model for the
study of amyloid aggregation.32 Additionally, it is
homologous to human lysozyme, the variants of
which have been shown to form amyloid fibrils,
implicated in hereditary systemic amyloidosis.33
Although a large body of evidence suggests membrane perturbation as a primary mechanism of
toxicity in neurodegenerative diseases, most of
such conclusions have been based on studies
involving phospholipid model systems. In the
present study, mitochondria isolated from rat brain
were used as an in vitro model to examine the
possible destructive effects of HEWL oligomers and
fibrils. It is proposed that the organelle with its well-
characterized membranes consisting of various
biologically active components, combined with its
exceptional biochemical composition and compartmental diversity, may provide an extremely useful
model system for biophysical studies related to
mechanism of cytotoxicity at the membrane level,
leading to cell death.
Results and Discussion
Membrane disruption by amyloid oligomers is
often considered as a primary mechanism of toxicity
in neurodegenerative disorders, but the mechanism
by which these structures eventually cause cell
dysfunction and death is not clearly understood.
One of the problems associated with these investigations is related to the fact that the oligomeric
species are often unstable, making detailed structural analyses difficult.34 It is generally believed that
enhancement of hydrophobicity initiated by protein
misfolding provides these structures with the ability
to interact with membranes, resulting in loss of
membrane integrity and thereby cytotoxicity.9,35–38
Furthermore, such hydrophobicity-based toxicity
mechanism has been shown to be shared by
bacterial toxins and viral proteins.39 In addition to
hydrophobic interactions, electrostatic forces involving charged residues in protein structures and
charged or polar lipid molecules have been concluded to provide the second major interactions in
the association process.40
Two mechanisms of membrane disruption have
been recently proposed. The first argues that
membrane permeabilization is induced by formation of specific, discrete ion channels that may be
inhibited by specific channel blockers,18–20 and the
second suggests involvement of a nonspecific
mechanism of membrane perturbation in the
absence of unitary conductance.14–17 HEWL is one
of the best-characterized proteins whose amyloidogenesis, in particular, has been extensively studied
under in vitro conditions.32,41–43 In the present
study, interaction of HEWL oligomers and fibrils
on mitochondrial membranes was investigated.
Oligomeric and not fibrillar structures have been
found to have the capacity to cause release of
mitochondrial enzymes.
Oligomer characterization
The structural and morphological features
of HEWL oligomers have been investigated by
employment of a range of techniques, including
fluorescence [(thioflavin T (ThT), 1-anilino-naphthalene 8-sulfonate (ANS) and acrylamide quenching)],
size-exclusion chromatography (SEC), dynamic
light scattering (DLS), transmission electron microscopy (TEM), circular dichroism (CD) and dot blot
analysis. ThT, a fluorescent dye specific for amyloid
structures, was used for monitoring the kinetics of
HEWL fibril formation. The experimental data
points fit well to a sigmoidal curve, indicating the
presence of an initial lag phase of about 5 days,
followed by a growth phase that reaches a plateau at
about 12 days of incubation (Fig. 1a). In the course of
oligomer formation (Fig. 1b), hydrophobic sites
became increasingly exposed, as monitored by
binding of ANS, leading to fluorescence enhancement, with a pronounced blue shift of the spectrum
from 528 nm to 485 nm (Fig. 1b). Further incubation
resulted in shifting of the spectrum toward shorter
wavelengths by not more than 5 nm (data not
shown). Structural flexibility was determined by
dynamic quenching experiments, using acrylamide
as a neutral fluorescence quencher. A significant
enhancement in the Stern–Volmer constant was
observed at 5 days of incubation (Fig. 1b), suggesting an increase in flexibility upon oligomer formation. Continuation of the fibrillation process
coincided with a decrease in flexibility/more com-
Membrane Integrity and Amyloid Cytotoxicity
pact structure (Fig. 1b). The amount and molecular
mass of the oligomeric species formed upon 5 days
of incubation were determined as presented in Fig.
2a, where two peaks corresponding to prefibrillar
oligomeric species (14 min , consisting of about 15%
of total protein) and monomer (24 min) are evident.
The void peak containing HEWL oligomers
appeared slightly after thyroglobulin (670 kDa),
used as one of the molecular mass standards,
suggesting an average molecular mass of about
600 kDa (N 40 monomers). The actual mass of these
prefibrillar species could be smaller than what
appears.44 Particle dimensions and distributions of
HEWL monomer and oligomers were extracted
from DLS experiments. Both DLS runs and TEM
images indicated formation of two main oligomeric
species (Fig. 2b and c). However, only one peak
corresponding to larger oligomeric structures was
suggested by the SEC chromatograms, presumably
due to the more transient nature of the smaller
aggregates.45 Upon 15 days of incubation, welldefined mature fibrils with typical amyloid morphology were formed, with no oligomer being
visible (Fig. 2c). Moreover, formation of HEWL
oligomers was found to take place well before any
marked increase in ThT fluorescence (Fig. 1a), with
transition from the predominantly α-helical structure in lysozyme monomers to a partially unfolded
form (Fig. 2d). Our results suggest that structural
flexibility and exposure of hydrophobic surfaces are
two remarkable characteristics of HEWL oligomers,
indicating a major conformational rearrangement in
the protein structure upon incubation under the
denaturing conditions employed, eventually leading to fibril formation. Furthermore, the A11
antibody that is frequently used for recognition of
the conformational epitopes specific for oligomeric
aggregates binds to the HEWL oligomers, initiated
from polypeptides of diverse primary structures
(Fig. 2e).13
Release of mitochondrial enzymes upon
interaction with HEWL fibrillation products
Fig. 1. HEWL amyloid fibrillation upon incubation at
1 mM concentration in 50 mM glycine buffer (pH 2.2) at
57 °C. (a) Kinetics of amyloid formation as monitored by
ThT fluorescence. (b) Size distribution upon oligomerization (□) and ANS binding (■) are indicated on the
left-hand y-axis, and changes in the Stern–Volmer
constant with time of incubation (○) are indicated on
the right-hand y-axis. ANS fluorescence at 12 days of
incubation is also indicated as one single point. The inset
shows Stern–Volmer plots at different times of incubation of the native monomer: zero time (●), day 3 (○),
day 5 (□) and day 7 (■).
Mitochondrial membrane permeabilization was
characterized using sensitive fluorometric and
luminometric assays. Initial experiments suggested
that the oligomeric species formed upon 5 days of
incubation of HEWL under the denaturing conditions were very effective in causing substantial
release of mitochondrial enzymes. This was not
unexpected since earlier results indicated that the
oligomers were both flexible and hydrophobic (Fig.
1b) and, therefore, of favorable characteristics for
efficient interaction with a phospholipid bilayer.
Release of malate dehydrogenase (MDH) occurred
via a fast phase (b 3 min) followed by a slower
increase, reaching a maximum value in about 10 min
of treatment (Fig. 3a, inset). Interaction of HEWL
Membrane Integrity and Amyloid Cytotoxicity
Fig. 2. Structural and morphological characterization of HEWL oligomers. (a) Gel-filtration chromatogram upon
incubation for 5 days, showing two distinguished peaks corresponding to prefibrillar oligomers and monomers. The
arrows indicate elution volumes of the standards [thyroglobulin, 670 kDa (1); γ-globulin, 158 kDa (2); ovalbumin, 44 kDa
(3); myoglobin, 17 kDa (4); and vitamin B12, 1.35 kDa (5)]. (b) Hydrodynamic radii of the HEWL oligomers as estimated by
DLS. Two oligomeric species with sizes of 46 nm and 180 nm and the original monomeric protein are indicated. (c) TEM
images of oligomers upon 5 days of incubation and fibrils (after 15 days). (d) Far-UV CD spectra of HEWL corresponding
to monomer (●), 5-day-old oligomers (■) and 15-day-old fibrils (▲). (e) Recognition of HEWL oligomers by the A11
antibody: HEWL monomers, oligomers and fibrils were spotted on a nitrocellulose membrane and probed with the
oligomer-specific antibody A11. The antibody showed binding only to the oligomers.
monomer, oligomer and fibrils with mitochondrial
membranes was then investigated. A number of
mitochondrial enzymes located at different compartments including monoamine oxidase (MAO)
(embedded in the mitochondrial outer membrane),
adenylate kinase (AK) (located in the inter membrane space), citrate synthase (CS), MDH and
glutamate dehydrogenase (GDH) (located in the
mitochondrial matrix) were examined upon addition of protein structures to mitochondrial suspensions and following the procedure outlined under
Materials and Methods. To reduce experimental
errors, we carried out all assays at the same time
(day, hours) with the same protein samples while
correcting for binding of the “soluble” matrix
enzymes to membrane structures after disruption
of mitochondria (see Materials and Methods for
details). Substantial release of all of the enzymes
tested was observed upon interaction of the oligomers with mitochondria, except for GDH (Fig. 3a
and b), in a concentration-dependant manner (Fig.
4). The increase of oligomer concentration or the
incubation time appeared ineffective in causing
GDH release (data not shown). While some release
was detected related to monomers, fibrils were
totally ineffective (Fig. 3a and b). Results were
highly reproducible when different batches of the
oligomeric species prepared at different times were
tested except for MAO (Fig. 3a). Release as low as
10%, up to a maximum of 40%, was obtained (based
on activity determination, see Materials and
Methods) with different batches of oligomer preparations (see Fig. 3a). These large variations in
activity may be due to the fact that MAO is a
phospholipid-requiring enzyme,46 and lipid depletion upon release/solubilization may occur to
different extents, leading to variations in catalytic
activity. These results clearly demonstrate that
HEWL oligomers may cause profound destabilization of mitochondria, resulting in its permeabilization. Similar observations were made on release of
cytochrome c (Fig. 5), in accord with earlier
reports.47,48 It appears that high degrees of flexibility
and hydrophobicity commonly reported for oligomeric species38 and confirmed in the present study
may provide these structures with the capacity to
interact with membranes.49 A growing body of
evidence strongly implicates the importance of
Membrane Integrity and Amyloid Cytotoxicity
Fig. 4. Dependency of enzyme release on oligomer
concentration. Releases of MDH (●), CS (■) and MAO (□)
are indicated on the left-hand y-axis and that of AK (○) is
indicated on the right-hand y-axis. Further details are
described in the text.
flexible structure. 50,51 Subsequently, loss of the
ability to cause permeabilization occurs (Fig. 3a
and b), which coincides with loss of the ability to
bind to A11 antibody (Fig. 2e).
Fig. 3. Mitochondrial enzyme release upon interaction
with HEWL monomer, oligomer and fibrils. (a) Release of
MDH (white bars), CS (light-gray bars), GDH (black bars)
and MAO (dark-gray bars), expressed as percentage of the
maximum observed upon treatment with Triton X-100
(0.1%) and (b) AK release expressed relative to buffer. The
inset represents rapid release of MDH upon incubation of
the protofibrillar structures with mitochondria. The
minimum time taken to test for enzyme release following
the procedure described in Materials and Methods was
3 min. Additional details are described under Materials
and Methods. *, p b 0.05; **, p b 0.01.
hydrophobic nature of protein aggregates in their
cytotoxicity.35–37 Accordingly, the results presented
here may be explained in terms of interaction of
some exposed hydrophobic and flexible oligomer
components with the corresponding structures in
the phospholipid bilayers of mitochondria, thereby
inducing membrane disruption leading to release of
cytochrome c and other proteins, such as those
tested in the present investigation. Furthermore, it is
proposed that any condition (such as medium
temperature and pH, salt or protein/ligand concentration) that may induce protein–protein interactions leading to formation of (hydrophobic/flexible)
aggregate assemblies could potentially be cytotoxic.
As fibrils become sufficiently long (∼ 12 days of
incubation), lateral fibril–fibril interactions may
occur, leading to eventual burial of hydrophobic
patches (as indicated here by decrease in ANS
fluorescence, see Fig. 1b), in a more compact, less
Effect of calcium on enzyme release
from mitochondria
Divalent cations, including calcium, have been
found very effective in causing stabilization of
biomembranes. 52–54 The effect of calcium on
membrane permeabilization was tested in the
present study. As indicated in Fig. 6a, release of
MDH and AK from mitochondria was very
efficiently prevented when mitochondrial membrane components were provided with the opportunity to interact with this counterion. There are
many reports suggesting that divalent ions such
as calcium may stabilize biological membranes by
surface charge reduction,53,54 thereby protecting
them from destructive effects of some viruses and
toxins.55 Phospholipid vesicle permeabilization
induced by wild-type and mutant forms of
protofibrillar α-synuclein has also been shown to
be diminished in the presence of calcium.56 The
Fig. 5. Release of cytochrome c from mitochondria
upon interaction with HEWL fibrillation products.
Maximum release by treatment with 0.1% Triton X-100
is also indicated. Details are described under Materials
and Methods.
Membrane Integrity and Amyloid Cytotoxicity
distributed in the inner mitochondrial membrane,
being preferentially oriented toward the matrix
side.57 Membrane stabilization may also be a
consequence of an increase in interaction of the
oligomers with negatively charged membrane
proteins. Membrane condensation afforded by
the presence of calcium could increase the energy
required for insertion of the HEWL oligomers into
the phospholipid bilayer and/or membrane disruption, thereby reducing permeabilization.56 This
observation clearly suggests that factors affecting
the natural biophysical properties of cellular
membranes, such as its flexibility/fluidity, may
influence its permeabilization. A clear mechanism
for this observation would undoubtedly require
extensive investigations.
Effect of salts and polyamines
Use of 100 mM or higher concentrations of NaCl
appreciably diminished (about 15–20%; data not
shown) release. The polyamine spermine was more
effective (Fig. 6b), further demonstrating the role of
electrostatic forces on interaction of oligomers with
mitochondria. In addition to this capacity, spermine
was found appreciably more effective than salt,
presumably due to its multivalent nature and/or
membrane stabilization effect.58
Fig. 6. Effect of calcium (a) and spermine (b) on release
of enzymes from mitochondria upon interaction with
HEWL oligomers. Mitochondrial suspensions at 1 mg/ml
protein concentration were incubated with various concentrations of calcium or spermine on ice for 10 min,
followed by the procedure described under Materials and
Methods to assay for release of enzymes. Releases of MDH
(●) and AK (○) are indicated on the left-hand and on the
right-hand y-axes, respectively, in (a), and those of MDH
(white bars) and CS (dark-gray bars) are shown in (b). *,
p b 0.05; **, p b 0.01.
lipid and protein components of membranes
repulse one another due to negative electrical
charges, resulting in decreasing membrane
stability. 54 It appears therefore that, in the
presence of a divalent ion such as calcium, charge
neutralization may eventually lead to membrane
condensation and stabilization, presumably by
lowering repulsive interactions.53,54 The possibility
for such a mechanism related to the role of
calcium in the present study is reinforced by the
presence of negatively charged phospholipids
(cardiolipin, phosphatidyl serine and phosphatidyl
inositol) in mitochondrial membranes.57 Of these,
cardiolipin, with a double anionic charge, has a
unique role in mitochondria, both structurally and
functionally. Moreover, this phospholipid, together with phosphatidyl inositol, is asymmetrically
Membrane permeabilization induced by
oligomers coincided with significant
mitochondrial aggregation
The presence of 20 μM oligomeric species induced
mitochondrial aggregation as evident by pronounced increases in the 90° light scattering of
mitochondrial suspensions (Fig. 7). Incubation with
HEWL monomer and fibrils also caused aggregation, but to lower extents (oligomers ≫ monomer N fibril; data not shown). Polyamines and salts
were much more effective in preventing aggregation
as compared to influencing permeabilization (Fig. 7),
suggesting that membrane disruption may occur in
the absence of appreciable aggregation.
Oligomer-induced mitochondrial membrane
permeabilization mechanism
Mitochondrial membrane perturbation leading to
enzyme release appears to have two major characteristics. First, release is fast (about 90% release in
the first few minutes) and independent of mitochondrial molecular masses: HEWL oligomers cause
release of mitochondrial proteins of various sizes,
ranging from 12 kDa (cytochrome c) to 102 kDa (CS).
Second, release of mitochondrial enzymes is never
complete, as maximum release (60% of that caused
by total disruption by treatment with 0.1% Triton X100; see Fig. 4) is observed at 30 min of incubation
Fig. 7. Effect of NaCl (a) and spermine (b) on
mitochondrial aggregation induced by HEWL oligomers.
Mitochondrial homogenates at a final concentration of
1 mg/ml were incubated with various concentrations of
NaCl [0 mM (●), 250 mM (■) and 500 mM (□)] or
spermine [0 mM (●), 25 mM (■) and 50 mM (□)] for
10 min on ice followed by addition of HEWL oligomers
(20 μM). Light scattering of the final suspensions was then
determined by DLS. Light scattering of mitochondrial
homogenates in the presence of 500 mM NaCl (▲) or
50 mM spermine (▲) is also indicated as controls. See
Materials and Methods for further details.
with the oligomers used at 25 μM concentration.
Furthermore, prolonged incubation (up to 120 min)
with use of higher oligomer concentrations (up to
50 μM) did not increase release (data not shown).
Two mechanisms of membrane permeabilization
have been suggested to explain the disruptive effects
of oligomers upon interaction with phospholipid
membranes. The first is described as a nonspecific
detergent-like perturbation event, and the second is
thought to involve formation of ion channels.14–20
The observations described in the present study are
more in line with the former mechanism, which
would support a size-independent rapid disruption,
while the latter would necessitate a size-selective
process. The fast nonselective release of mitochondrial enzymes observed in the present study
suggests generation of large defects as confirmed
Membrane Integrity and Amyloid Cytotoxicity
by leakage of high-molecular-weight mitochondrial
enzymes. Several proteins are known to form
morphologically compatible ion-channel-like structures in phospholipid vesicles, with inner diameters
between 1 nm and 2 nm, which, if occurred in a cell,
would destabilize cellular ionic homeostasis and
thereby induce cell death.59 Such pores, however,
would only allow small molecules and metabolites
and not molecules of the sizes observed in the
present study. Our results are, therefore, consistent
with a major disruption of mitochondrial membrane
integrity, caused by large-scale but transient mitochondrial membrane lesions, such as those recently
reported.60 Such an incomplete release, as a result of
damage of a subpopulation of brain mitochondria,
may also be due to heterogeneity of brain
mitochondria61,62 and prevention of more extensive
damage through binding of the monomeric protein
present in the oligomer preparations, similar to the
effect of monomeric α-synuclein in suppressing
prefibrillar permeabilization of phospholipid
vesicles.56 Our own preliminary results indicate
that mitochondrial heterogeneity may play an
important role in the present investigation, since
greater extents of enzyme release were observed in
liver mitochondria, known to be less heterogeneous
in nature62 (data not shown). Therefore, heterogeneity in susceptibility to disruption, as opposed to
transient disruption, may also account for the
Membrane disruption without a size-selective
pore formation is not without precedent. As an
example, incomplete and transient permeabilization
of large unilamellar vesicles loaded with lowmolecular-weight and high-molecular-weight dextrans induced by rabbit neutrophil defensins may be
mentioned.63 If our hypothetical mechanism is
correct, the amount of enzyme released would be
limited by the diffusion rate of the molecules,63
which is inversely proportional to their size. It is
therefore possible that the lack of release of GDH in
the present study (see Fig. 3a) is due to its very large
size (336 kDa).64 Consequently, our finding here
demonstrates that oligomeric HEWL species can
cause mitochondrial membrane disruption in a
nonspecific detergent-like manner, perhaps through
the formation of transient lesions.
We believe that several findings presented in this
communication are especially noteworthy. First,
contrary to our own expectations before embarking
on the study, membrane permeabilization was
very excessive, allowing for release of large proportions of mitochondrial enzymes. Second, the
defects are apparently quite large but probably
limited in size, allowing for release of proteins as
large as 102 kDa (CS) but not 336 kDa (GDH).
Third, release of the matrix enzymes appears to
require appreciably greater concentrations of the
oligomeric structures, suggesting that the inner
Membrane Integrity and Amyloid Cytotoxicity
membrane is less susceptible to damage. This may
be due to greater rigidity of the inner membrane,
differences in lipid/protein compositions or the
fact that inner-membrane damage requires the
initial disruption of the outer membrane. Fourth,
release of the enzymes is never complete, presumably being mainly due to the transient nonequilibrium nature of the defects and/or mitochondrial
heterogeneity.61,62 Fifth, as a technical limitation of
the present study, the minimum time at which
release of enzymes from mitochondria could be
determined was 3 min, at which most of the
release was observed. However, release could be
very fast, probably being initiated immediately
after interaction. Sixth, since hydrophobicity and
flexibility of lysozyme oligomers were not affected
by the presence of calcium (data not shown), it is
reasonable to assume that membrane stabilization
is the main protective event. Formation of smaller
defects could be tested upon oligomer treatment in
the presence of calcium by looking for release of
small molecules rather than the large protein
structures examined in the present study. Finally,
interaction of the oligomeric structures with
mitochondrial membranes may cause subtle
changes in lipid/protein interactions, causing
alteration in some important functional properties
of many membrane proteins. Such fundamentally
important membrane perturbation mechanisms
may explain some of the reasons for the occurrence of a large array of events describing
mitochondrial dysfunction and the prominent
role of mitochondria in the pathogenesis of
neurodegenerative disorders.
Concluding Remarks
The present communication describes a model
study for the mechanism of prefibrillar cytotoxicity
at the membrane level, demonstrating how the
oligomeric and not the fibrillar structures formed in
the course of HEWL fibrillation may have the
capacity to interact with mitochondria. Hydrophobicity and flexibility of the amphiphilic oligomeric
structures appear to provide them with the capacity
to interact with the membranes,38,49 causing destabilization and subsequent permeabilization, by a
nonspecific detergent-like mechanism. Such damaging behavior may cause large defects in the
membranes, with subsequent release of mitochondrial enzymes. To a limited extent, release of the
proteins may occur irrespective of their size. The
limitation is presumably controlled by the size of the
lesions transiently occurring upon membrane destabilization. It is suggested that the mitochondrion,
with its two membranes of well-defined protein and
phospholipid compositions, biophysical properties
and compartmental diversity, may provide an
extremely useful model for the study of the
mechanism of cytotoxicity at the membrane level.
Although suggestions have been made here on
possible mechanistic features of oligomeric cytotoxicity, a clear definition of the events leading to
membrane damage and cell death would avidly
require more detailed structural information on the
protofibrillar oligomers and the processes involved,
leading to membrane permeabilization. Such
endeavors would undoubtedly prove to be of
major physiological significance.
Materials and Methods
HEWL (EC, ThT, acetyl coenzyme A and 7fluorobenz-2-oxa-1,3-diazole-4-sulfonamide were purchased from Sigma (St. Louis, MO, USA). D-Luciferin
was obtained from Synchem Corp. ANS was purchased
from Fluka. All other chemicals were obtained from Merck
(Darmstadt, Germany) and were reagent grade.
Preparation of HEWL oligomers and fibrillar
Protein solutions (1 mM) were prepared in 50 mM
glycine buffer (pH 2.2) (pH adjusted with HCl), and
aliquots were incubated at 57 °C for 5 days, as oligomer
preparations. The oligomer concentrations are given in
terms of the monomer protein concentration throughout
the article. Mature HEWL fibrils were prepared by
incubating the stock protein solution (1 mM) at 57 °C
up to 15 days. Fibril concentration was estimated by
centrifugation (40 min, 21,000g) and determination of
the protein concentration in the supernatant using an
extinction coefficient (ɛ1 mg/ml) of 2.63 at 280 nm,65
subtracting it from the original (1 mM) protein
ThT assay
All fluorescence experiments were carried out on a
Cary Eclipse VARIAN fluorescence spectrophotometer.
For monitoring the growth of HEWL fibrils, we
performed ThT fluorescence assays in a mixture of
20 μM protein solutions and 25 μM ThT, with excitation
fixed at 440 nm and emission at 482 nm. The excitation
and emission slit widths were set at 5 nm and 10 nm,
ANS binding assay
Emission spectra of ANS were recorded between
400 nm and 600 nm, using an excitation wavelength of
350 nm. Aliquots of the incubated mixtures were diluted
(final concentration of 5 μM) in glycine buffer (50 mM,
pH 2.2) containing 100 μM ANS. Excitation and emission
slit widths were both set at 5 nm.
Fluorescence quenching
Fluorescence quenching was analyzed according to the
Stern–Volmer relationship (F0/F = 1 + Ksv[Q]), where F0 is
the fluorescence in the absence of quencher, F is the
fluorescence at molar quencher (acrylamide) concentration [Q] and Ksv is the Stern–Volmer constant obtained
from the slope of a plot of F0/F versus [Q].66 The excitation
wavelength was 280 nm, and acrylamide concentration
ranged from 0 M to 0.2 M.
CD measurement
CD spectra were recorded using an AVIV 215 spectropolarimeter (Aviv Associates, Lakewood, NJ, USA) and a
0. 05-mm-path cell. Aliquots were taken after regular time
intervals and diluted (final concentration of 20 μM) in
glycine buffer (50 mM, pH 2.2), and spectra were recorded
in the 190- to 260 -nm range.
Oligomer characterization
Size-exclusion chromatography
For SEC, protein samples were centrifuged at 21,000g
for 30 min to remove any insoluble aggregates. Twenty
microliters of the supernatant was then applied onto a
Sephadex S2000 HR (Amersham Pharmacia) gel-filtration
column equilibrated with glycine buffer (50 mM, pH 2.2),
using a flow rate of 0.8 ml/min while monitoring UV
absorbance at 280 nm. Relative amounts of prefibrillar
oligomers were determined by calculating the area under
the void peaks using LC Solution Software (Version 1.22
SP1; Shimadzu). The column was calibrated with several
molecular weight standards.
Dot blot analysis
Dot immunoblot analysis was carried out to investigate
reactivity of the anti-oligomeric A11 antibody (Chemicon)
against HEWL oligomers. Aliquots (2 μl) from protein
samples incubated for 0 day, 5 days and 15 days, for
monomer, prefibrillar oligomer and fibrils, were spotted
onto nitrocellulose membranes and air dried for 30 min.
Membranes were blocked for 1 h with 10% nonfat dry
milk in Tris-buffered saline–Tween 20 [20 mM Tris
(pH 7.4), 150 mM NaCl and 0.05% Tween 20] and were
incubated with A11 antibody (1:1000 dilution) overnight
at 4 °C. Blots were then incubated for 1 h with horseradishperoxidase-conjugated anti-rabbit IgG secondary antibody (IMGENEX) at a 1:2000 dilution, and dots were
visualized with the enhanced chemiluminescent light plus
chemiluminescence kit (Amersham Biosciences).
Dynamic light scattering
DLS experiments were carried out using a zeta potential
and particle size analyzer (Brookhaven Instrument,
Holtsville, NY 11742-1896, USA). Aliquots of incubated
solutions at a concentration of ∼ 33 μM were filtered
through a 0. 2-μm syringe filter before measurements. A
laser of 657 nm with a fixed detector angle of 90° was used,
and DLS experiments were performed at least in triplicate.
Membrane Integrity and Amyloid Cytotoxicity
Transmission electron microscopy
Five microliters of 10× diluted samples was adsorbed
onto copper 400 mesh grid, previously covered by carboncoated film. After 2 min, a drop of 1% uranyl acetate was
added, and after a few seconds, the samples were
observed by a CEM 902A Zeiss microscope. For fibril
preparations, a 20× dilution was used.
Preparation of rat brain mitochondria
Mitochondria from the brains of male rats (250–300 g)
were removed, washed and homogenized in isolation
buffer [10 mM Tris–HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.32 M sucrose]. Homogenates were centrifuged at 3000g for 10 min, and
mitochondrial fraction from the resulting supernatant
was isolated according to Kabir and Wilson67 and was
stored at 15 mg/ml in isolation buffer in liquid nitrogen.
Mitochondrial membrane integrity was confirmed by
determination of specific activities of marker enzymes:
MDH, GDH, rotenone-insensitive NADH cytochrome c
reductase, cytochrome c oxidase, CS and AK, as described
previously.68 Protein concentration was measured by the
method of Lowry et al.69
Incubation of isolated mitochondria with HEWL
monomers and its fibrillation products
Aliquots of solutions containing HEWL monomer,
oligomers and fibrils or glycine buffer as control were
added to 200 μl of mitochondrial homogenate (1 mg/ml),
followed by incubation for 30 min at 30 °C. The incubation
time of 30 min was used to allow sufficient time for release
of the enzymes, based on the data obtained for MDH.
Triton X-100 (at a final concentration of 0.1%) was used as
a positive control for maximum release. At the end of the
incubation period, mitochondrial suspensions were centrifuged at 21,000g for 15 min, and resulting supernatants
were used for release of mitochondrial enzymes. To
correct for possible binding of the matrix enzymes to
mitochondrial membranes, we treated final pellets with
50 mM KCl for MDH and CS70 and 500 mM NH4Cl for
GDH.71 Data are expressed as a fraction of the maximum
effect (Triton X-100): (measured signal − blank signal)/
(maximum signal − blank signal). Since Triton X-100
interferes with the luciferase luminescence assay, AK
activities are expressed relative to control.
Mitochondrial enzyme release assays
Bioluminometric assay of MAO activity
MAO activity was measured using a bioluminescent
assay kit (Promega). The two-step bioluminescence assay
was performed exactly according to the manufacturer's
instructions. In the MAO reaction (step 1), 20 μl of
mitochondrial supernatant was incubated with the substrate for 3 h at 25 °C in the reaction buffer containing
100 mM Hepes (pH 7.5) and 5% glycerol. In the luciferase
detection reaction (step 2), 40 μl of a luciferin detection
reagent was added to the MAO reaction, and after a
20-min incubation period, the luminescent signal was
Membrane Integrity and Amyloid Cytotoxicity
measured as relative light unit on a Berthold Detection
System luminometer.
Bioluminescence assay of AK activity
This was performed in the direction of ATP formation,
coupled to luciferin/luciferase reaction according to Wu et
al. with some modifications.72 Twenty microliters of
mitochondrial supernatant was added to 27 μl of reaction
mixture (50 mM Tris and 20 mM MgCl2, pH 7.4), followed
by 20 μl of 0.5 mM ADP. After 5 min of incubation at room
temperature, 3 μl of 20 mM D-luciferin and 5 μl of 20 μg/
ml luciferase were added and mixed, and the relative light
unit signal was recorded on a Berthold Detection System
MDH activity determination
For MDH fluorometric assay, 5 μl of mitochondrial
supernatant was added to 485 μl of reaction buffer [50 mM
potassium phosphate, 0.5 mM EDTA, 2.5 μM rotenone,
2 mM NaN3 and 15 mM malate (pH 7.5)]. Upon
incubation for a few seconds, 10 μl of 50 mM NAD+ was
added, and fluorescence was measured at 25 °C using an
excitation wavelength of 340 nm and an emission
wavelength of 465 nm. The excitation and emission slit
widths were set as 5 nm and 10 nm, respectively.
at a dilution of 1:1000 overnight at 4 °C and finally with
secondary goat anti-mouse IgG antibody conjugated with
horseradish peroxidase (Abbiotec™) at a dilution of 1:2500
for 1 h at room temperature. The membrane was
processed for cytochrome c detection using the chemiluminescent light plus chemiluminescence kit (Amersham
Aggregation measurement
Aggregation of mitochondria in the presence of oligomers was investigated by DLS experiments.74 Aliquots of
the HEWL oligomers (20 μM final concentration) were
added to mitochondrial homogenates and diluted with
filtrated mitochondrial extraction buffer to 1 mg/ml, and
scattered light was collected as described above for
oligomer characterization, for 5 min at 30-s intervals.
Statistical analysis
All experiments were performed at least three times
with triplicate assays. The results are presented as mean ±
standard deviation, and a Student's paired t-test was
utilized to calculate the statistical significance. The level of
statistical significance was set as p b 0.05 treated mitochondria compared to control: *, p b 0.05; **, p b 0.01.
GDH activity determination
For GDH fluorometric assay, 20 μl of mitochondrial
supernatant was added to 470 μl of reaction buffer
[100 mM potassium phosphate, 5.55 mM α-ketoglutarate,
55.5 mM NH4Cl and 0.2 mM EDTA (pH 7.8)]. Upon
incubation for a few seconds, 10 μl of 2 mM NADH was
added, and fluorescence was measured at 25 °C as
described above for MDH.
Fluorometric assay of CS activity
Mitochondrial CS activity was determined fluorometrically according to Hassett and Crockett with some
modifications.73 Briefly, 10 μl of mitochondrial supernatant was added to 145 μl of reaction buffer [0.44 mM
acetyl coenzyme A and 0.5 mM oxaloacetate in 40 mM
Hepes buffer (pH 7.5)]. After 45 min of incubation at
25 °C, 35 μl of 2 M KHCO3 was added, followed by
addition of 20 μl of 7-fluorobenz-2-oxa-1,3-diazole-4sulfonamide solution (500 μM), and the mixture was
incubated at 40 °C for 45 min. Excitation and emission
slit widths were set as 10 nm.
Cytochrome c release measurement
Mitochondrial homogenate (1 mg/ml) was incubated in
the presence of HEWL oligomer, HEWL fibril or glycine
buffer (control) for 30 min at 30 °C, followed by
centrifugation at 12,000g for 10 min. Aliquots of the
mitochondrial supernatant were subjected to 15% SDSPAGE and transferred to polyvinylidene difluoride
membrane for 1 h at 100 V. The membrane was blocked
by 5% nonfat dry milk and was probed with primary
monoclonal antibody to cytochrome c (BD Pharmingen™)
This work was supported by a grant from the
Iranian National Science Foundation (INSF). We
thank Dr. Saman Hosseinkhani from the Department of Biochemistry, Tarbiat Modares University
(Tehran, Iran) for his help with bioluminometric
assays and for providing luciferase.
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