Synaptotoxicity of Alzheimer beta amyloid can be explained by its membrane perforating property.
ABSTRACT The mechanisms that induce Alzheimer's disease (AD) are largely unknown thereby deterring the development of disease-modifying therapies. One working hypothesis of AD is that Abeta excess disrupts membranes causing pore formation leading to alterations in ionic homeostasis. However, it is largely unknown if this also occurs in native brain neuronal membranes. Here we show that similar to other pore forming toxins, Abeta induces perforation of neuronal membranes causing an increase in membrane conductance, intracellular calcium and ethidium bromide influx. These data reveal that the target of Abeta is not another membrane protein, but that Abeta itself is the cellular target thereby explaining the failure of current therapies to interfere with the course of AD. We propose that this novel effect of Abeta could be useful for the discovery of anti AD drugs capable of blocking these "Abeta perforates". In addition, we demonstrate that peptides that block Abeta neurotoxicity also slow or prevent the membrane-perforating action of Abeta.
[show abstract] [hide abstract]
ABSTRACT: In its earliest clinical phase, Alzheimer's disease characteristically produces a remarkably pure impairment of memory. Mounting evidence suggests that this syndrome begins with subtle alterations of hippocampal synaptic efficacy prior to frank neuronal degeneration, and that the synaptic dysfunction is caused by diffusible oligomeric assemblies of the amyloid beta protein.Science 11/2002; 298(5594):789-91. · 31.20 Impact Factor
Article: Intranasal NAP administration reduces accumulation of amyloid peptide and tau hyperphosphorylation in a transgenic mouse model of Alzheimer's disease at early pathological stage.[show abstract] [hide abstract]
ABSTRACT: Accumulation of beta-amyloid (Abeta) peptide and hyperphosphorylation of tau in the brain are pathological hallmarks of Alzheimer's disease (AD). Agents altering these pathological events might modify clinical disease progression. NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is an octapeptide that has shown neuroprotective effects in various in vitro and in vivo neurodegenerative models. Previous studies showed that NAP protected against Abeta-induced neurotoxicity, inhibited Abeta aggregation, and, by binding to tubulin, prevented disruption of microtubules. In this study, we investigated the effect of NAP on Abeta and tau pathology using a transgenic mouse model that recapitulates both aspects of AD. We administered NAP intranasally (0.5 microg/mouse per day, daily from Monday through Friday) for 3 mo, starting from 9 mo of age, which is a prepathological stage in these mice. NAP treatment significantly lowered levels of Abeta 1-40 and 1-42 in brain. In addition, NAP significantly reduced levels of hyperphosphorylated tau. Of particular interest, hyperphosphorylation at the threonine 231 site was reduced; phosphorylation at this site influences microtubule binding. Our results indicate that NAP treatment of transgenic mice initiated at an early stage reduced both Abeta and tau pathology, suggesting that NAP might be a potential therapeutic agent for AD.Journal of Molecular Neuroscience 02/2007; 31(2):165-70. · 2.50 Impact Factor
Article: Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.[show abstract] [hide abstract]
ABSTRACT: The distinct protein aggregates that are found in Alzheimer's, Parkinson's, Huntington's and prion diseases seem to cause these disorders. Small intermediates - soluble oligomers - in the aggregation process can confer synaptic dysfunction, whereas large, insoluble deposits might function as reservoirs of the bioactive oligomers. These emerging concepts are exemplified by Alzheimer's disease, in which amyloid beta-protein oligomers adversely affect synaptic structure and plasticity. Findings in other neurodegenerative diseases indicate that a broadly similar process of neuronal dysfunction is induced by diffusible oligomers of misfolded proteins.Nature Reviews Molecular Cell Biology 03/2007; 8(2):101-12. · 39.12 Impact Factor
Synaptotoxicity of Alzheimer Beta Amyloid Can Be
Explained by Its Membrane Perforating Property
Fernando J. Sepulveda1., Jorge Parodi1., Robert W. Peoples3, Carlos Opazo2, Luis G. Aguayo1,4*
1Laboratory of Neurophysiology, Department of Physiology, University of Concepcio ´n, Concepcio ´n, Chile, 2Laboratory of Neurobiometals, Department of Physiology,
University of Concepcio ´n, Concepcio ´n, Chile, 3Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin, United States of America, 4Centro de
Investigacio ´n Avanzada en Educacio ´n, University of Concepcio ´n, Concepcio ´n, Chile
The mechanisms that induce Alzheimer’s disease (AD) are largely unknown thereby deterring the development of disease-
modifying therapies. One working hypothesis of AD is that Ab excess disrupts membranes causing pore formation leading
to alterations in ionic homeostasis. However, it is largely unknown if this also occurs in native brain neuronal membranes.
Here we show that similar to other pore forming toxins, Ab induces perforation of neuronal membranes causing an increase
in membrane conductance, intracellular calcium and ethidium bromide influx. These data reveal that the target of Ab is not
another membrane protein, but that Ab itself is the cellular target thereby explaining the failure of current therapies to
interfere with the course of AD. We propose that this novel effect of Ab could be useful for the discovery of anti AD drugs
capable of blocking these ‘‘Ab perforates’’. In addition, we demonstrate that peptides that block Ab neurotoxicity also slow
or prevent the membrane-perforating action of Ab.
Citation: Sepulveda FJ, Parodi J, Peoples RW, Opazo C, Aguayo LG (2010) Synaptotoxicity of Alzheimer Beta Amyloid Can Be Explained by Its Membrane
Perforating Property. PLoS ONE 5(7): e11820. doi:10.1371/journal.pone.0011820
Editor: Howard E. Gendelman, University of Nebraska, United States of America
Received March 17, 2010; Accepted June 24, 2010; Published July 27, 2010
Copyright: ? 2010 Sepulveda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by FONDECYT grant 1060368, FONDECYT grant 1100502, Ring of Research PBCT ACT-04, CIE-05 (L.G.A. and C.O.). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: A patent application is pending and L.G.A, C.O. and J.P. might opt to receive royalties from licenses that the University of Concepcio ´n
negotiates. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
. These authors contributed equally to this work.
Alzheimer’s disease (AD) is a progressive and irreversible
neurodegenerative brain disorder that leads to major debilitating
cognitive deficits in the elderly. It is now believed that the
cellular and molecular alterations that cause brain dysfunctions
are slow in onset and that it probably takes several years to
develop the full blown disease . Surprisingly, the cellular and
molecular mechanisms that induce AD are largely unknown,
deterring the development of effective modifying or symptomatic
therapies. Thus, attempts to alleviate and stop AD symptoms are
actually based on compensating synaptic deficits and blocking
intracellular signaling cascades . However, the results are
minor because at clinical stages the AD brain is already too
It is accepted that the toxic effects of Ab, one etiological agent in
AD, depend on dimer formation and subsequent oligomerization
which include diverse structural forms . Thus, blocking
dimerization reduces aggregation and the ensuing peptide toxicity
. The working hypothesis of AD is that excess of Ab either i)
binds to membrane receptors affecting their functions , ii)
interferes with signaling cascades [6–8] or iii) directly disrupts
neuronal membranes causing pore formation leading to alterations
in ionic homeostasis . Although the latter is an attractive
hypothesis because it could explain several effects of Ab in brain
neurons, it is largely unknown if this can also occur in native brain
neuronal membranes. Additionally, the existence of this mem-
brane phenomenon will reveal that the target of Ab is not another
membrane protein, but that Ab itself is the cellular target and
explain the failure to interfere with the course of AD. In agreement
with this idea, atomic force microscopy (AFM) in lipid environ-
ments and molecular dynamic analysis have shown the presence of
molecular entities with inner diameters in the 1.5–2.6 nm range
[10,11] which were similar to those generated by other peptidergic
molecules known to form pores in cell membranes, such as amylin
and a-synuclein .
For many years it has been recognized that several peptides
with differing structures such as gramicidin, amphotericin and
a-latrotoxin can alter membrane permeability after inducing
pore formation [13,14]. Additionally, it is known that antifungal
antibiotics are toxic because they can attach to the cell wall and
steadily disrupt permeability. Electrophysiologists have utilized
gramicidin and amphotericin for more than 20 years to
perforate cell membranes and record whole cell ionic currents
with the patch clamp technique [13,15,16]. In the patch
perforated mode, the membrane is disrupted thereby making
holes that allow the continuous flow of ionic currents under the
patch pipette. Here, we report that Ab has a rapid and potent
perforating property in neuronal membranes. We postulate that these
perforations increase intracellular calcium leading to synaptic
transmission failure . Based on this membrane property,
similar to gramicidin and amphotericin, we have defined Ab as
a perforating toxic agent, rather than a classical pore-forming
PLoS ONE | www.plosone.org1July 2010 | Volume 5 | Issue 7 | e11820
Ab perforates hippocampal neuron membranes
The cell attached mode of the patch clamp technique allows for
stable measurements at the single molecule level. Thus, activation
of most single voltage or ligand activated channel proteins can be
adequately time resolved with open kinetics and complex cellular
regulations [16,18]. Alternatively, small peptides can produce
minute disturbances in membrane stability causing a low
resistance pathway under the membrane patch, known as a
‘‘perforated recording configuration’’ . In this study, cell-
attached recordings in hippocampal neurons were stable using a
control solution in the patch pipette (i.e. .30 min). For example,
the application of a 5 mV voltage pulse induced a stable, fast
current arising from a partly compensated electrode capacitance
(Fig. 1A). This current was markedly altered when 500 nM of pre-
aggregated Ab (see methods) was added into the patch pipette
solution and allowed to diffuse to the underlying membrane
(Fig. 1B). For example, the traces in figure 1B clearly show that the
amplitude and time course of the capacitive current increased with
time and this was similar to that induced by gramicidin (Fig. 1C).
Fig. 1D obtained with a fluorescent form of Ab and with Western
blot analysis shows that the peptide was able to associate with
neuronal membranes at times when it was producing membrane
perforation (i.e. 15–30 min). The data also show that the time it
took to form the perforated configuration by Ab was dependent on
the peptide concentration (Fig. 1E). For example, it took nearly
40 min to establish perforated recordings with 1 nM Ab and less
than 10 min with micromolar concentrations. Ab (2.2 mg/ml,
500 nM) was more potent and rapid than gramicidin in forming
the perforated configuration. However, the perforated configura-
tion formation with gramicidin (100 mg/ml) can take more than
30 min . Interestingly, Ab1–42 produced similar effects in
membrane charge and input resistance as those of Ab1–40(Fig.
S1B,C). Furthermore, the Ab-dependent actions were demon-
strated by their blockade with the Ab WO2 antibody that
recognizes residues 1 to 5 of Ab (Fig. S1B,C).
The analysis of the charge transferred during the capacitative
response showed that the effect of Ab was similar to those of
gramicidin and amphotericin, two peptides commonly used to
perforate neuron membranes (Fig. 1F). On the other hand, the
effect of Ab1–40was not produced by the reverse Ab peptide,
supporting the idea that Ab aggregation leads to membrane
damage. Finally, the membrane currents induced by Ab were very
similar to those induced by positive pressure in the whole cell
configuration, suggesting that they can transfer significant charge,
equivalent to that induced by positive pressure, which is believed
to completely burst the membrane under the patch pipette .
Figure 1. Ab peptide induced formation of membrane perforation in hippocampal neurons. A, currents induced by a 5 mV depolarizing
pulse recorded with control solution at two times in cell-attached mode. B, effect of application of 500 nM (2.2 mg/ml) Ab via the patch pipette on
capacitative membrane current. C, effect of gramicidin (100 mg/ml) on membrane current. D, confocal image shows a neuron stained for 15 minutes
with 500 nM fluorescent Ab. Western blot shows the time dependent association of low molecular weight oligomers with hippocampal cell
membranes. E, effect of increasing Ab concentrations on the time to establish the perforated configuration. F, effects of Ab gramicidin, and
amphotericin on the transferred membrane charge induced by 5 mV depolarization. Each point (mean 6 SEM) was measured in a least 6 different
hippocampal neurons. * denotes a P,0.05 (n=6–7 neurons).
Perforations Induced by Ab
PLoS ONE | www.plosone.org2July 2010 | Volume 5 | Issue 7 | e11820
Ab displayed a ‘‘gramicidin-like’’ behavior in neuronal
Peptides that perforate cell membranes can form pathways
which are more or less selective to cations or anions [15,16].
Gramicidin and amphotericin, for example, are used to record
GABAA and glutamatergic whole-cell currents, respectively,
because while the former generates mainly cationic pores in the
membrane, the latter is somewhat more selective for anions.
Consistent with a time dependent membrane perforation process,
the application of extracellular GABA or glutamate only 30 s after
GV seal formation was unable to induce detectable membrane
currents. This demonstrates the existence of a high resistance
pathway between the membrane and the patch pipette containing
500 nM Ab. After 15 minutes of Ab application to the patch
membrane, on the other hand, extracellular applications of both
neurotransmitters induced membrane currents, demonstrating the
formation of pathways in the membrane capable of conducting
ionic currents through the Ab-containing pipette (Fig. 2A, lower
traces). Additionally, the data show that Ab induced perforated
patches in a time-dependent manner making it possible to detect
synaptic currents arising from synapses distant to the recording
patch electrode (Fig. 2B). These results overwhelmingly show that
Ab is acting in a similar fashion to other pore forming peptides (i.e.
gramicidin, amphotericin) well known to perforate neuronal
membranes. Additionally, these novel results are appealing
because they show that Ab resembles other well known potent
cytotoxic compounds, providing a novel molecular mechanism for
neuronal toxicity of the Ab peptide.
We next studied the current-voltage (I–V) relationships  to
compare the ionic selectivity properties of perforates produced by
Ab (20 min of application) with those of gramicidin and
amphotericin, known to form cationic and anionic selective pores,
respectively. The application of GABA, the agonist for the GABAA
Cl2current present in hippocampal neurons, showed that Ab
behaved like gramicidin, but not like amphotericin (Fig. 2C). For
example, the Cl2current recorded with Ab in the pipette reversed
direction near the expected equilibrium potential for Cl2in the
perforated mode . Amphotericin, on the other hand, which
dissipates the Cl2gradient, reversed the GABAAcurrent at 0 mV.
The data also shows that the AMPA current reversed close to
0 mV with the three perforating peptides (Fig. 2D), which is near
the expected value for a non-selective cationic channel.
Ab action on conductance is not mediated by a classical
Some biophysical studies have indicated that Ab can increase
intracellular calcium and membrane conductance in artificial lipid
bilayers and clonal cell lines [22–24], but demonstration of actual
channel or pore formation in native brain membranes has been
inadequately resolved. In our experiments, it was clear that Ab was
able to inducean increaseinmembrane noise beforeestablishing the
perforated configuration in hippocampal neurons. However, the
noisy nature of the neuronal membrane related to activation of
endogenouschannelsprecluded us fromstudying the pore properties
in more detail. To circumvent this, we recorded from HEK 293 cells
before the formation of a perforated configuration. Recordings done
Figure 2. Ab peptide produced cationic perforates comparable to gramicidin. A, currents were recorded using a cell-attached configuration
at the beginning (30 s) and after 15 min of Ab application via the patch pipette. AMPA and GABA (50 mM) applied to the extracellular membrane
induced membrane currents after formation of membrane perforation. B, gradual appearance of synaptically mediated membrane currents after
establishing the cell-attached conformation. C, GABA-induced anionic current-voltage relationships obtained during perforated mode with either
gramicidin, amphotericin-B or Ab. D, AMPA induced cationic current using gramicidin, amphotericin -B or Ab. Each point (mean 6 SEM) was
measured in a least 5 different hippocampal neurons.
Perforations Induced by Ab
PLoS ONE | www.plosone.org3 July 2010 | Volume 5 | Issue 7 | e11820
in more than 40 cells did not show membrane events reminiscent of
typical singlechannel behavior,inthesenseofhaving wellstructured
open and closing behavior, in the presence of Ab. Therefore, the
noisy nature of the microscopic current events produced by Ab did
not allow for a good discrimination betweenconformationalstates or
to construct open and shut distributions (Fig. 3A). Nevertheless, plots
of all-point current distributions from different patches showed
multiple levels of peak conductance (20062, 260640, 360660,
440620 and 680660 pS) supporting the occurrence of multiple
membrane disruptions by Ab (Fig. 3B) more than formation of a
single unitary channel. In parallel experiments, we found that
fluorescent Ab was able to associate to cell membranes giving a
morphological correlation to the membrane-perforating actions of
the peptide (Fig. 3C). Furthermore, in some patches, Ab produced a
large transient increase in membrane current (1000–2000 pS) which
we interpreted as spontaneous breakage-resealing of the membrane
produced by Ab that sometimes resulted in a whole cell
configuration (Fig. 3F–H). Interestingly, Ab was able to bind widely
to HEK 293 cells and also caused the generation of a perforated
configuration in these cells, as indicated by parallel monitoring of
membrane capacitance (Fig. S3 and Fig. 3C). These data, therefore,
indicate that Ab affects the membrane inducing a range of current
responses which are different from those of membrane channels,
which have well defined conductance and time distributions due to
their gating properties [25,26]. For instance, the analysis of single
of comparing a typical ion channel having a single channel
conductance of 9263 pS with Ab activity (Fig. 3D,E). The previous
experiments showed small and large microscopic membrane current
events induced by Ab. While the smaller Ab perforations might
exhibit a degree of ion selectivity (Fig. 2C), it is likely that the large
onesmight allowthe entryof othermoleculesintothe cell,whichcan
be examined using fluorescent probes loaded in the pipette.
The amyloid pore allows entry of a large molecule into
The data in figure 4 show combined patch clamp-imaging
recordings using patch pipettes filled with ethidium bromide in the
presence and absence of Ab (Fig. 4A,C). From this data, it is evident
that a large (M.W. 394.3, ,1.3 nm van der Waals diameter, PDB
ID: 2ZOZ) organic molecule can enter the neuron in parallel with
the process of electrical membrane perforation (Fig. 4A, D). In the
absence of Ab in the pipette (Fig. 4C), or with Na7, an Ab-pore
blocking peptide  (Fig. 4B,E), ethidium bromide was unable to
enter into the cell. The size of this fluorescent molecule allows us to
place a diameter of at least 1.5 nm for the large perforation induced
by Ab, which agrees with previousAFM data [10,11]. Clearly, these
large perforations might cause enormous homeostatic consequences
for neuronal functions.
The amyloid perforates can be inhibited by small
One of the main issues related to the toxicity of Ab in brain
neurons is the identification of potential targets for the development
of pharmaceutics capable of blocking its effects. In line with the idea
that pore formation is relevant to Ab toxic actions, it was reported
that the increase in Ca2+influx and lipid bilayer conductance was
Figure 3. Ab induced a non channel-like increase in microscopic membrane conductance. A, current traces show high sensitivity patch
recordings obtained in the presence of 500 nM Ab. The red line represents zero current. B, graph shows an all-point histogram obtained from the
recordings in A. C, confocal micrograph shows the peripheral association of fluorescent Ab to HEK cells. D current trace showing typical single
channel behavior from a cell expressing alpha 1 human glycine receptors. Unlike Ab the current expansion shows clear transitions between closed
and open states. E, All point histogram fitted to a single conductance of 92 pS. F–H, traces show either partial or full membrane perforation in the
presence of Ab.
Perforations Induced by Ab
PLoS ONE | www.plosone.org4 July 2010 | Volume 5 | Issue 7 | e11820
blocked by two small peptides . Interestingly, these peptides also
inhibited Ab-induced cell death . Furthermore, the increase in
charge transfer and entry of ethidium bromide into the cell was well
inhibited by this peptide (Fig. 4B,E). In addition, we found that the
Na7 peptide produced a blockade of Ab effects on membrane
resistance (1/G) in hippocampal neurons in a concentration-
dependent fashion (Fig. 5A). In experiments using hippocampal
neurons loaded with fluo-4, Ab produced a reversible increase in
intracellular calcium, demonstrating the diffusible nature of Ab.
by other blockers of ligand-gated or voltage-dependent calcium
channels, suggesting that this effect was mainly mediated by Ab-
induced membrane perforation.
Consistent with the critical role of calcium in synaptic
transmission and in agreement with recently published data ,
we found that 500 nM of Ab enhanced the release of synaptic
vesicles from hippocampal neurons. This synaptic facilitation was
blocked by the presence of the Na7 peptide suggesting the
participation of Ab perforation in this phenomenon (Fig. 5C).
Na7 and Na4a, another structurally related peptide (Fig. S1A), but
not the inactive analogs Na13 and Na15 , also antagonized the
delayed synaptotoxic effects of Ab on synapsin I and SV2, two
vesicular proteins (Fig. 5D), and in addition altered membrane
charge and resistance (Fig. S1B,C). In conclusion, the data indicate
that the perforating effects of Ab are associated to microscopic
forms of Ab (Fig S2B). This data might be important for future
pharmacological applications in terms of the neurotoxic activity of
Ab. Furthermore, these results strongly suggest that Ab perforations
areinvolvedinsynapticdysfunctionmediated byAb oligomers.
Only recent studies have dealt with the action of Ab at
concentrations without overt neurotoxicity on synaptic properties
. Although controversy exists on whether Ab can up or down
Figure 4. Ab perforation causes entry of a small organic molecule in parallel with the increase in membrane conductance. A, the time
dependent increase in cellular fluorescence associated with entry of ethidium bromide in presence of Ab in the pipette. B, the effect of Ab was
blocked by the Na7 peptide. C, Ethidium bromide was unable to enter into the cell in the absence of Ab. D–E, effect of Ab on membrane current
transferred and fluorescence in the absence and presence of Na7. * denotes a P,0.05 (n=7–8 neurons).
Perforations Induced by Ab
PLoS ONE | www.plosone.org5July 2010 | Volume 5 | Issue 7 | e11820
regulate specific components of synaptic transmission, several
studies in rodent hippocampus showed that Ab alters pre and
postsynaptic components governing LTP, NMDA- and AMPA
neurotransmissions and calcium homeostasis. No definitive
mechanism is available to explain this variety of effects [30–32],
hindering the development of anti Ab therapies. On the other
hand, due to the urgency of generating disease-modifying
therapeutics to treat people suffering from AD, we believe that
the present data provide novel insights into innovative strategies to
interfere with the toxic processes likely initiated at the neuronal
membrane level . Future studies should decipher the
characteristics of Ab perforate formation in brain membranes.
For example, although pore forming peptides have been in use for
more than 40 years, most of their mechanisms for membrane
insertion, pore formation and membrane conductance initiation
have remained largely undetermined . For example, from lipid
bilayer studies, it was postulated that gramicidin required
simultaneous insertion of two monomers on opposite faces of the
lipid bilayer to perforate the membrane. However, this phenom-
enon might not occur in biological membranes. Our most recent
experiments have shown that gramicidin forms oligomeric
complex structures in aqueous solution and induces membrane
perforations, similar to Ab, rather than single channel currents in
native cell membranes (unpublished results). Nevertheless, because
Ab can internalize rapidly , it might break the membrane
inserting itself in both faces.
In agreement with the data in the present study, AFM and
molecular dynamic studies of Ab pores in bilayers support the
presence of diverse, small and large molecular entities that
possibly correspond to the functional perforation described in this
study. The Ab inner pore diameter appears to be much larger (at
least 2.6 nm) than ion selective channels, which have an
estimated diameter of 0.6 nm . Overall, studies with AFM,
molecular simulations and single channel conductances suggest a
high range of pore sizes , and provide additional support to
the idea that the phenomenon of insertion and conductance of
Ab are very complex. The proposal of a complex pore structure is
consistent with a recent study that proposed a model for the Ab
pore . Additionally, because these conducting Ab entities
appear to lack most regulatory mechanisms (i.e. post transduc-
tional modifications, inactivation, membrane anchoring, stable
pore size) important for channel gating, we believe that they do
not behave as classical ion channels to allow selective ion
permeation. Since these membrane disruptions are important for
neuronal toxicity, their blockade would be expected to inhibit
synaptotoxicity, neurodegeneration and subsequently AD pro-
Figure 5. The effects of Ab on membrane conductance and synaptotoxicity can be inhibited by small peptides. A, effect of Ab(N) on
membrane resistance in the absence and presence of Na7 (# 1mM, & 3 mM, % 7 mM, m 100 mM). B, effect of Ab application on intracellular calcium
increase and its inhibition by Na7, but not by other ion channel blockers. C, effects of Na7 and low calcium on Ab-induced destaining of FM1–43. The
insets show destaining of FM1–43 (arrows) at 20 min. D, time dependent reduction of synapsin and SV2 induced by Ab and its inhibition by Na4a and
Na7 (50 mM). Each point (mean 6 SEM) was measured in a least 5 different hippocampal neurons.
Perforations Induced by Ab
PLoS ONE | www.plosone.org6July 2010 | Volume 5 | Issue 7 | e11820
gression. Furthermore, this membrane permeabilization action of
Ab is in agreement with the vesicular depletion recently reported
Interestingly, the actions of Ab show strong similarities,
although to a lesser extent, to the effect of a-latrotoxin (LTX) on
neurotransmission . For example, after a strong enhancement
of synaptic transmission, LTX induced vesicle depletion and
diminution in miniature potentials by a pore forming mechanism,
having conductance and kinetic properties very similar to those of
pores formed by Ab in lipid bilayers .
In summary, our working model to explain the toxicity of Ab in
Alzheimer’s disease proposes the existence of diverse membrane
structures that can progress from a small, ion selective pore, to a
large membrane perforation (Fig. 6). All these Ab perforations are
capable of producing a wide range of toxic effects ranging from
synaptotoxicity to cell death.
Materials and Methods
All animals were handled in strict accordance with the Animal
Welfare Assurance (permit number 2008100A) and all animal
work was approved by the appropriate Ethics and Animal Care
and Use Committee of the University of Concepcion.
Hippocampal neurons were obtained from 18 day pregnant
mouse embryos (C57BL/J6) or Sprague-Dawley rat embryos as
previously described  in accordance with NIH recommenda-
tions. Human Embryonic Kidney 293 cells (HEK) were cultivated
in D-MEM (Dulbecco’s Modified Eagle Medium, Life Technol-
ogies, Inc. USA) supplemented with 10% fetal bovine serum (Life
Technologies Inc. USA.) and streptomycin-penicillin (200 units
each, Life Technologies Inc. USA). Cells were maintained with
5% CO2at 37uC. HEK 293 cells were kindly provided by Dr.
Olate (University of Concepcion) and have been previously
described in the lab .
Human Ab1–40 labeled with Rhodamine Green at its N-
terminus and unlabeled were purchased from Anaspec (CA, USA)
and Tocris (MO, USA), respectively. Ab1–40 was dissolved in
DMSO (10 mg/ml) and stored in aliquots at 220uC. For the
preparation of Ab aggregates (80 mM), aliquots of peptide stock
(250 mg in 25 ml of DMSO) were added to 700 ml of PBS (Gibco,
USA) and continuously agitated (200 RPM at 37uC) for
90 minutes and stored at 4uC. Ab1–40Rhodamine Green (Abs/
Em=502/527 nm) was dissolved in DMSO (4 mg/ml) and
immediately stored in aliquots at 220uC.
Patch pipettes having a resistance between 1 and 3 MV were
prepared from filament-containing borosilicate micropipettes.
Currents were measured with the whole-cell patch-clamp
technique at a holding potential of 260 mV using an Axopatch
200B (molecular devices, USA) amplifier as previously described
[37,38]. Perforated recordings were obtained as follows: the
perforating agent was added into the pipette solution and a 5 mV
pulse was used to monitor the formation of the perforation.
Gramicidin and amphotericin were used at 100 mg/ml. Short
applications of Ab, GABA (100 mM) and AMPA (100 mM) were
done via lateral motion of a multi-pipette array (approx. 200 mm
in diameter). Some experiments involved an external solution
without added calcium, Na7 (20–100 mM), Na4a (20 mM) or the
inactive peptides Na13 and Na15 (20 mM).
Standard Western blotting procedures were followed. Equal
amounts of protein were separated on 10% SDS-PAGE gels.
Protein bands were transferred onto nitrocellulose membranes,
blocked with 5% milk and incubated with a primary antibody
using the following concentrations: anti-Ab (NAB228, Santa Cruz
Biotechnology, CA, USA) 1:500, anti-Synapsin I (AB1543,
Chemicon, MA, USA) 1:1000, anti-SV2 (Developmental Studies
Hybridoma Bank, IA, USA) 1:200. Immunoreactive bands were
Figure 6. The scheme is a simplified model for association, micro and macro perforation induced by Ab in cellular membranes. A,
aggregation and binding (association) of Ab to the neuronal membrane B, smaller perforations are associated to a selective ion influx (gramicin-like
ion influx). C, larger perforations allow the entry of large molecules, which include EtBr (,1.3 nm). All these Ab effects are blocked by application of
anti- Ab antibody.
Perforations Induced by Ab
PLoS ONE | www.plosone.org7 July 2010 | Volume 5 | Issue 7 | e11820
visualized with ECL plus Western Blotting Detection System
(PerkinElmer, MA, USA).
Intracellular Calcium Imaging
Neurons were loaded with Fluo-4 AM (1 mM in pluronic acid/
DMSO, Molecular Probes, Eugene, OR, USA) for 30 min at
37uC. The neurons were then washed twice with external solution
and incubated for 30 min at 37uC. The cells were mounted in a
perfusion chamber that was placed on the stage of an inverted
fluorescent microscope (Eclipse TE, Nikon, USA). The cells were
briefly (200 ms) illuminated using a computer-controlled Lambda
10-2 filter wheel (Sutter Instruments, USA). Regions of interest
(ROI) were marked in a field having usually more than 10 cells.
Images were collected at 2–5 s intervals during a continuous 5-min
period. The imaging was carried out with a SensiCam camera
(PCO, Germany) using Axon Instruments Workbench 2.2
software. The calcium channel inhibitors used were conotoxin
(1 mM), agatoxin (1 mM), nifedipine (3 mM), CNQX (4 mM) and
D-AP5 (50 mM).
FM1-43 Loading and Unloading
Presynaptic vesicles were labeled by exposure to FM1-43
(15 mM, Molecular Probes, USA) during a high-K+depolarization
for 5 min and immediately washed, as previously described
[39,40]. Coverslips were mounted on a rapid switching flow
perfusion chamber with an inverted fluorescent microscope
(Eclipse TE, Nikon, USA) equipped with a 1006 objective (oil
immersion, NA 1.4). Depolarization-dependent destaining was
induced by bath perfusion with 30 mM K+
replacement of Na+).
Hippocampal neurons treated during 15 minutes with 500 nM
fluorescent Ab were fixed for 15 min with 4% paraformaldehyde
and permeabilized with 0.1% triton X-100 in PBS and incubated
with anti-MAP2 1:300 (Santa Cruz Biotechnology, CA, USA).
Secondary anti-rabbit IgG (Jackson ImmunoResearch Laborato-
ries, PA) conjugated with Cy3 was used at 1:500 for 2 hours.
Calculation of Ethidium Bromide Size
The van der Waals diameter of EtBr was measured with Swiss
PDBviewer using atomic coordinates for the crystal structure of
the ethidium-bound form of the multi-drug binding transcriptional
repressor CgmR (PDB ID: 2ZOZ).
Non-lineal analysis was performed using Origin (Microcal).
Membrane charge was analyzed by integrating the transient
capacitative current after subtracting the pipette capacitance. The
values are expressed as mean 6 SEM (standard error mean).
Statistical differences were determined using Student’s t test or
ANOVA. The experiments were performed in triplicate.
small peptides. A, sequence of Ab and mini peptides used in this
study (NA7, NA4a, NA13 and NA15). B–C, shows the effect of Ab
(500 nM) and Ab plus mini peptides (20 mM) on the transferred
membrane charge and resistance, respectively. The bars are
means 6SEM. * denotes a P,0.05.
Found at: doi:10.1371/journal.pone.0011820.s001 (0.80 MB TIF)
Blockade of Ab induced membrane disruption by
offibril-like structures.A,the upperelectronmicrographshowsactive
structures labeled with 5 nm gold-particles. B, the current trace show
that these structures caused membrane perforations in rat hippo-
campal neurons. Lower panels show a more globular Ab structure
that was found to be inactive. Data is typical from 6 experiments.
Found at: doi:10.1371/journal.pone.0011820.s002 (2.18 MB TIF)
The confocal micrograph shows the peripherical association of
fluorescent Ab to HEK cells (30 min). B, capacitative membrane
currents were recorded using a cell-attached configuration at the
beginning (0 min) and after 30 min of Ab application via the patch
pipette. C, effects of Ab on the transferred membrane charge
induced by 5 mV depolarization pulse in HEK cells, hippocampal
and cortical neurons. Each point (mean 6 SEM) was measured in a
least 6 different cells.
Found at: doi:10.1371/journal.pone.0011820.s003 (1.58 MB TIF)
Ab induce membrane perforations in HEK293 cell. A,
We thank Lauren Aguayo for revising the paper and Claudia Lopez for
technical assistance. We thank Dr. Kogan from the University of Chile for
the gold-labeled peptide.
Conceived and designed the experiments: FJS JP CO LGA. Performed the
experiments: FJS JP RWP LGA. Analyzed the data: FJS JP. Wrote the
paper: FJS JP CO LGA.
1. Selkoe DJ (2002) Alzheimer’s disease is a synaptic failure. Science 298: 789–791.
2. Matsuoka Y, Gray AJ, Hirata-Fukae C, Minami SS, Waterhouse EG, et al. (2007)
Intranasal NAP administration reduces accumulation of amyloid peptide and tau
hyperphosphorylation in a transgenic mouse model of Alzheimer’s disease at early
pathological stage. (Translated from eng) J Mol Neurosci 31: 165–170.
3. Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration:
lessons from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol 8:
4. Soto C, Estrada L (2005) Amyloid inhibitors and beta-sheet breakers. Subcell
Biochem 38: 351–364.
5. Wang Q, Walsh DM, Rowan MJ, Selkoe DJ, Anwyl R (2004) Block of long-term
potentiation by naturally secreted and synthetic amyloid beta-peptide in
hippocampal slices is mediated via activation of the kinases c-Jun N-terminal
kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as
well as metabotropic glutamate receptor type 5. J Neurosci 24: 3370–3378.
6. Garrido JL, Godoy JA, Alvarez A, Bronfman M, Inestrosa NC (2002) Protein
kinase C inhibits amyloid beta peptide neurotoxicity by acting on members of
the Wnt pathway. Faseb J 16: 1982–1984.
7. Maccioni RB, Otth C, Concha II, Munoz JP (2001) The protein kinase Cdk5.
Structural aspects, roles in neurogenesis and involvement in Alzheimer’s
pathology. Eur J Biochem 268: 1518–1527.
8. Daniels WM, Hendricks J, Salie R, Taljaard JJ (2001) The role of the MAP-
kinase superfamily in beta-amyloid toxicity. Metab Brain Dis 16: 175–185.
9. Arispe N, Pollard HB, Rojas E (1994) The ability of amyloid beta-protein [A
beta P (1–40)] to form Ca2+ channels provides a mechanism for neuronal death
in Alzheimer’s disease. Ann N Y Acad Sci 747: 256–266.
10. Lin H, Bhatia R, Lal R (2001) Amyloid beta protein forms ion channels:
implications for Alzheimer’s disease pathophysiology. Faseb J 15: 2433–2444.
11. Jang H, Zheng J, Nussinov R (2007) Models of beta-amyloid ion channels in the
membrane suggest that channel formation in the bilayer is a dynamic process.
Biophys J 93(6): 1938–1949.
12. Quist A, Doudevski I, Lin H, Azimova R, Ng D, et al. (2005) Amyloid ion
channels: a common structural link for protein-misfolding disease. Proc Natl
Acad Sci U S A 102: 10427–10432.
13. Andersen OS, Koeppe RE, 2nd, Roux B (2005) Gramicidin channels. IEEE
Trans Nanobioscience 4: 10–20.
14. Orlova EV, Rahman MA, Gowen B, Volynski KE, Ashton AC, et al. (2000)
Structure of alpha-latrotoxin oligomers reveals that divalent cation-dependent
tetramers form membrane pores. Nat Struct Biol 7: 48–53.
15. Ebihara S, Shirato K, Harata N, Akaike N (1995) Gramicidin-perforated patch
recording: GABA response in mammalian neurones with intact intracellular
chloride. J Physiol 484: 77–86.
Perforations Induced by Ab
PLoS ONE | www.plosone.org8July 2010 | Volume 5 | Issue 7 | e11820
16. Tajima Y, Ono K, Akaike N (1996) Perforated patch-clamp recording in cardiac
myocytes using cation-selective ionophore gramicidin. Am J Physiol 271:
17. Parodi J, Sepulveda FJ, Roa J, Opazo C, Inestrosa NC, et al. (2010) b-amyloid
causes depletion of synaptic vesicles leading to neurotransmission failure. J Biol
Chemn 285: 2506–2514.
18. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-
clamp techniques for high-resolution current recording from cells and cell-free
membrane patches. Pflugers Arch 391: 85–100.
19. Le Foll F, Castel H, Soriani O, Vaudry H, Cazin L (1998) Gramicidin-
perforated patch revealed depolarizing effect of GABA in cultured frog
melanotrophs. J Physiol 507: 55–69.
20. Rhee JS, Ebihara S, Akaike N (1994) Gramicidin perforated patch-clamp
technique reveals glycine-gated outward chloride current in dissociated nucleus
solitarii neurons of the rat. J Neurophysiol 72: 1103–1108.
21. Hille B (2001) Ion channels of excitable membranes, (Sinauer Associates, Inc.)
pp xviii, 814.
22. Demuro A, Mina E, Kayed R, Milton SC, Parker I, et al. (2005) Calcium
dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism
of soluble amyloid oligomers. J Biol Chem 280: 17294–17300.
23. Alarcon JM, Brito JA, Hermosilla T, Atwater I, Mears D, et al. (2006) Ion
channel formation by Alzheimer’s disease amyloid beta-peptide (Abeta40) in
unilamellar liposomes is determined by anionic phospholipids. Peptides 27:
24. Kawahara M, Kuroda Y, Arispe N, Rojas E (2000) Alzheimer’s beta-amyloid,
human islet amylin, and prion protein fragment evoke intracellular free calcium
elevations by a common mechanism in a hypothalamic GnRH neuronal cell
line. J Biol Chem 275: 14077–14083.
25. Beato M, Groot-Kormelink PJ, Colquhoun D, Sivilotti LG (2004) The activation
mechanism of alpha1 homomeric glycine receptors. J Neurosci 24: 895–906.
26. Morales A, Nguyen QT, Miledi R (1994) Electrophysiological properties of
newborn and adult rat spinal cord glycine receptors expressed in Xenopus
oocytes. Proc Natl Acad Sci U S A 91: 3097–3101.
27. Arispe N (2004) Architecture of the Alzheimer’s A beta P ion channel pore.
J Membr Biol 197: 33–48.
28. Arispe N, Diaz JC, Simakova O (2007) Abeta ion channels. Prospects for
treating Alzheimer’s disease with Abeta channel blockers. Biochim Biophys Acta
29. Diaz JC, Linnehan J, Pollard H, Arispe N (2006) Histidines 13 and 14 in the
Abeta sequence are targets for inhibition of Alzheimer’s disease Abeta ion
channel and cytotoxicity. Biol Res 39: 447–460.
30. Glabe CG, Kayed R (2006) Common structure and toxic function of amyloid
oligomers implies a common mechanism of pathogenesis. Neurology 66:
31. Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, et al. (2005) Regulation of
NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8: 1051–1058.
32. Stephan A, Laroche S, Davis S (2001) Generation of aggregated beta-amyloid in
the rat hippocampus impairs synaptic transmission and plasticity and causes
memory deficits. J Neurosci 21: 5703–5714.
33. Kandimalla KK, Scott OG, Fulzele S, Davidson MW, Poduslo JF (2009)
Mechanism of neuronal versus endothelial cell uptake of Alzheimer’s disease
amyloid beta protein. PLoS One 4: e4627.
34. Kourie JI, Henry CL, Farrelly P (2001) Diversity of amyloid beta protein
fragment [1–40]-formed channels. Cell Mol Neurobiol 21: 255–284.
35. Ashton AC, Volynski KE, Lelianova VG, Orlova EV, Van Renterghem C, et al.
(2001) alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers
exocytosis of two pools of synaptic vesicles. J Biol Chem 276: 44695–44703.
36. Tapia JC, Mentis G, Navarrete R, Nualart F, Figueroa E, et al. (2001) Early
expression of glycine and GABAA receptors in developing spinal cord neurons:
effects on neurite outgrowth. Neuroscience 108: 493–506.
37. Sepu ´lveda FJ, Opazo C, Aguayo LG (2009) Alzheimer beta-amyloid blocks
epileptiform activity in hippocampal neurons. Mol Cell Neurosci 41: 420–428.
38. Pancetti F, Oyarce M, Aranda M, Parodi J, Aguayo LG, et al. (2004) S-
methylcysteine may be a causal factor in monohalomethane neurotoxicity.
Neurotoxicology 25: 817–823.
39. Ryan TA, Smith SJ, Reuter H (1996) The timing of synaptic vesicle endocytosis.
Proc Natl Acad Sci U S A 93: 5567–5571.
40. Ryan TA, Reuter H, Wendland B, Schweizer FE, Tsien RW, et al. (1993) The
kinetics of synaptic vesicle recycling measured at single presynaptic boutons.
Neuron 11: 713–724.
41. Yevenes GE, Peoples RW, Tapia JC, Parodi J, Soto X, et al. (2003) Modulation
of glycine-activated ion channel function by G protein subunits. Nat Neurosci 6:
42. Jang H, Arce FT, Capone R, Ramachandran S, Lal R, et al. (2010) Misfolded
amyloid ion channels present mobile beta-sheet subunits in contrast to
conventional ion channels. Biophys J 97: 3029–3037.
Perforations Induced by Ab
PLoS ONE | www.plosone.org9 July 2010 | Volume 5 | Issue 7 | e11820