Báo cáo khoa học: Amyloid oligomers: formation and toxicity of Ab oligomers ppt

MINIREVIEW
Amyloid oligomers: formation and toxicity of Ab oligomers
Masafumi Sakono
1,2
and Tamotsu Zako
1
1 Bioengineering Laboratory, RIKEN Institute, Wako, Saitama, Japan
2 PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
Introduction
Alzheimer’s disease (AD) is an age-related, progressive
degenerative disorder characterized by the loss of
synapses and neurons from the brain, and by the accu-
mulation of extracellular protein-containing deposits
(referred to as ‘senile plaques’) and neurofibrillary
tangles [1]. Amyloid b-peptide (Ab; 39–43 amino acids
in length) is the principal component of plaques. Ab is
produced by the proteolytic cleavage of the parental
amyloid precursor protein (APP) that localizes to the
plasma membrane, trans-Golgi network, endoplasmic
reticulum (ER) and endosomal, lysosomal and mito-
chondrial membranes. Synthetic Ab spontaneously
aggregates into b-sheet-rich fibrils, resembling those
in plaques. As insoluble fibrillar aggregates are neuro-
toxic in vivo and in vitro, it has long been hypothesized
that fibrils cause neurodegeneration in AD [2].
However, debate over this ‘amyloid cascade hypothe-
sis’ remains contentious.
The number of senile plaques in a particular region
of the AD brain correlates poorly with the local extent
of neuron death or synaptic loss, or with cognitive
impairment [3]. However, recent studies show a robust

correlation between the soluble Ab oligomer levels and
the extent of synaptic loss and severity of cognitive
impairment [4–9]. The term ‘soluble’ refers to any form
of Ab that is soluble in aqueous buffer and remains in
solution after high-speed centrifugation, indicating that
it is not insoluble fibrillar Ab. Assemblies ranging from
dimers to 24-mers, or even those of higher molecular
weight (MW), have been reported as Ab oligomers
[5,10,11]. Soluble Ab oligomers are reportedly more
cytotoxic than fibrillar Ab aggregates in general, and
Keywords
Alzheimer’s disease; amyloid b; formation and
toxicity mechanism; intracellular and extracellular
oligomers; soluble amyloid oligomers
Correspondence
T. Zako, Bioengineering Laboratory, RIKEN
Institute, 2-1 Hirosawa, Wako, Saitama,
351-0198 Japan
Fax: +81 48 462 4658
Tel: +81 48 467 9312
E-mail:
(Received 4 September 2009, revised 11
December 2009, accepted 6 January 2010)
doi:10.1111/j.1742-4658.2010.07568.x
Alzheimer’s disease (AD) is an age-related, progressive degenerative dis-
order that is characterized by synapse and neuron loss in the brain and the
accumulation of protein-containing deposits (referred to as ‘senile plaques’)
and neurofibrillary tangles. Insoluble amyloid b-peptide (Ab) fibrillar
aggregates found in extracellular plaques have long been thought to cause
the neurodegenerative cascades of AD. However, accumulating evidence

suggests that prefibrillar soluble Ab oligomers induce AD-related synaptic
dysfunction. The size of Ab oligomers is distributed over a wide molecular
weight range (from < 10 kDa to > 100 kDa), with structural polymor-
phism in Ab oligomers of similar sizes. Recent studies have demonstrated
that Ab can accumulate in living cells, as well as in extracellular spaces.
This review summarizes current research on Ab oligomers, focusing on
their structures and toxicity mechanism. We also discuss possible formation
mechanisms of intracellular and extracellular Ab oligomers.
Abbreviations
AD, Alzheimer’s disease; ADDL, Ab-derived diffusible ligand; APP, amyloid precursor protein; Ab, amyloid-b peptide; ER, endoplasmic
reticulum; FCS, fluorescence correlation spectroscopy; HD, Huntington’s disease; LTP, long-term potentiation; MW, molecular weight;
NGF, nerve growth factor; NMDAR, N-methyl-
D-aspartate (NMDA)-type glutamate receptor; PD, Parkinson’s disease; polyQ, polyglutamine;
PrP
C,
cellular prion protein.
1348 FEBS Journal 277 (2010) 1348–1358 ª 2010 The Authors Journal compilation ª 2010 FEBS
inhibit many critical neuronal activities, including
long-term potentiation (LTP), a classic model for syn-
aptic plasticity and memory loss in vivo and in culture
[12–15]. These studies strongly support the idea that
soluble Ab oligomers are the causative agents of AD;
however, the biological and structural characteristics
of Ab oligomers and their formation mechanism
remain unclear.
Structure and size of soluble Ab
oligomers
Many types of natural and synthetic Ab oligomers of
different sizes and shapes have been reported, which
accounts for their biological and structural diversity

and for the complexity of AD pathology (reviewed in
[4,5,9–11]). SDS-stable dimers and trimers have been
found in the soluble fractions of human brain and
amyloid plaque extracts, which suggests that these
low-MW Ab oligomers could be the fundamental
building blocks of larger oligomers or insoluble amy-
loid fibrils [16–18]. Ab oligomers of similar sizes have
also been secreted from cultured cells and have been
shown to inhibit LTP in vitro [14]. The high toxicity of
low-MW Ab oligomers is also supported by in vitro
studies showing that Ab dimers are threefold more
toxic than monomers, and that Ab tetramers are
13-fold more toxic [19].
Recently, Lesne et al. [13] demonstrated that the level
of SDS-stable Ab nonamers and dodecamers (referred
to as Ab*56) correlated with memory deficits in an APP
transgenic Tg2576 mice model. Purified dodecamers
also induced a significant fall-off in the spatial memory
performance of wild-type rats. These results suggest
that nonamers and dodecamers are associated with
deleterious effects on cognition. However, it is unlikely
that these oligomers alone cause brain dysfunction. For
example, young Tg2576 mice showed decreased
dendritic spine density in the dentate gyrus, impaired
LTP and impaired contextual fear conditioning, all at
an age before the first dodecamer was detected [13,20].
Another recent report also showed that the Ab*56
levels are not correlated with memory deficits in a
certain transgenic mice model [21]. These results sug-
gest that Ab*56 is not the only key determinant of

memory impairment. These oligomers could be classified
as low-MW (< 50 kDa) oligomers. However, natural
Ab oligomers with a wide-ranging MW distribution
(from < 10 kDa to > 100 kDa) have been found in
the AD brain [22], suggesting that Ab oligomers of
various sizes are associated with the disease.
There are also many reports of toxic oligomers from
synthetic Ab. Synthetic Ab forms fibrillar aggregates
that have properties similar to those found in AD
plaques in the brain. In vitro studies using synthetic
Ab are useful to complement efforts to determine the
disease mechanism. Snyder et al. [23] detected the for-
mation of soluble Ab assemblies, rather than fibrils,
using an analytical ultracentrifugation technique, and
Lambert et al. [12] reported the formation of small Ab
globular oligomers (5 nm in diameter) in Hams-F12
medium, which were referred to as Ab-derived
diffusible ligands (ADDLs). Importantly, ADDLs
strongly bound to the dendritic arbors of cultured
neurons, caused neuronal cell death and blocked LTP.
The finding of ADDL in soluble brain extracts from
the human AD brain using ADDL-specific antibody
supports the idea that the existence of ADDLs in the
human AD brain causes disease [24].
The formation of annular Ab oligomers, with an outer
diameter of 8–12 nm and an inner diameter of 2.0–
2.5 nm (150–250 kDa), has also been reported [25,26].
As these annular Ab oligomers could be preferentially
formed from mutant Ab (such as those carrying the
Arctic mutation), and because the amyloid ‘pore’ resem-

bles bacterial cytolytic b-barrel pore-forming toxins, it
has been suggested that these doughnut-like oligomers
could be responsible for the Ab-associated cytotoxicity
[26]. The largest globular assemblies are amylospheroids
[27], which are highly neurotoxic, off-pathway, sphe-
roidal structures with diameters of 10–15 nm.
Although A
b oligomer structures at atomic resolu-
tion are unclear, studies using conformation-dependent
antibodies suggest that structural variants could exist
among even morphologically similar Ab oligomers.
A difference in antibody-binding properties indicates a
difference in epitope exposure. For example, Glabe
et al. used two antibodies – A11 and OC – which are
specific for oligomers and fibrils, respectively. They
proposed two distinct types of oligomers: prefibrillar
oligomers that are A11-positive and OC-negative, and
fibrillar oligomers that are A11-negative and OC-posi-
tive [10]. As prefibrillar oligomers are not recognized
by the fibril-specific antibodies, and are considered to
be transient intermediates in the fibril-formation pro-
cess, a conformational change is necessary for them to
become fibrils. It should be noted that the oligomer-
specific A11 antibody also recognizes soluble oligomers
from various proteins, such as those from a-synuclein,
islet amyloid polypeptide, polyglutamine (PolyQ), lyso-
zyme, human insulin and prion peptide (106–126) [28].
These findings suggest that various proteins may form
prefibrillar oligomers that share a common structure
regardless of their amino acid sequence [8,28]. How-

ever, because the fibrillar oligomers are recognized by
the fibril-specific antibody, but not by A11, they at
M. Sakono and T. Zako Formation of toxic Ab oligomers
FEBS Journal 277 (2010) 1348–1358 ª 2010 The Authors Journal compilation ª 2010 FEBS 1349
least possess the structural characteristics of fibrils.
Thus, it is plausible that the fibrillar oligomer might
represent fibril nuclei to which the monomers can
attach before elongation [10]. Ab oligomers formed at
a low pH, but not those formed at a neutral pH, are
recognized by the 6E10 antibody [29]. These results
strongly suggest the existence of a structural polymor-
phism of A b oligomers.
There have been several other attempts to examine
Ab oligomer structures to elucidate the mechanism of
formation of Ab oligomers. Studies using atomic force
microscopy and scanning tunneling microscopy showed
that the structures of dimers, tetramers and other low-
MW Ab oligomers were consistent with the model of
the Ab monomers as b-hairpins [30,31]. These low-
MW Ab oligomers are relatively compact, being
1–3 nm in height and 5–10 nm in width ⁄ length, and
could be the fundamental building blocks of larger
oligomers and protofibrils.
Bernstein et al. [18] developed a new method, called
electrospray-ionization ion-mobility mass spectrometry,
to obtain oligomer size distributions and the qualita-
tive structure of each oligomer. Electrospray ionization
allows a fixed population of different Ab oligomer
states in solution to be isolated from one another, and
their size and shape could be determined using ion-

mobility spectrometry. By analyzing the cross-
sectional area of each oligomer obtained by ion mobil-
ity, the structure of the Ab
42
tetramer is theoretically
assumed to take an open ‘V’ form, which is neither
linear nor square. A planar hexagon form was
assumed for the Ab
42
hexamer. It is interesting to note
that a stacked hexamer paranuclei structure, rather
than side-by-side planar hexagons, was suggested for
the Ab
42
dodecamer, (Ab*56). These authors also
showed that oligomer size distribution was very
different between Ab
42
and Ab
40
, consistent with
previous studies, indicating that their oligomerization
pathways are different [25]. Although the oligomer
structure in the gas phase may not be completely
identical to that in solution, the information obtained
using this novel technique probably reflects the
characteristics of Ab oligomers, at least in part, and
may be useful for understanding the physical aspects
of Ab oligomers.
Further attempts to characterize Ab oligomers at a

single molecule level have been performed. Dukes
et al. [32] and Ding et al. [33] recently reported
oligomer size determination with single molecule
spectroscopy using fluorescently labeled Ab.By
directly counting the photobleaching steps in the
fluorescence of each oligomer on a cover-glass sur-
face, the number of monomer molecules in individual
oligomers could be determined, enabling the determi-
nation of more precise oligomer size distributions.
For example, an Ab
40
sample incubated at a neutral
pH was shown to be a mixture of monomers,
dimers, trimers and tetramers, and the presence of
zinc ion in the sample buffer increased the number
of tetramers [33]. Although application of this
method is limited to small oligomers, the single mol-
ecule approach overcomes the limitations of resolu-
tion and sample heterogeneity.
Analyses of the size of the Ab oligomer in solution
at the single molecule level have also been performed
using fluorescence correlation spectroscopy (FCS),
which detects the fluorescence of dye-labeled molecules
in a very small confocal volume excited by a sharply
focused laser beam [34]; FCS enables estimation of the
size distribution of an oligomeric species in solution
over a wide range of sizes (from monomers to large
soluble particles) with a good time resolution ( 1
min). From the fluorescence intensity fluctuations, one
can calculate the number of molecules in the confocal

volume and their diffusion times (corresponding to
size). For example, the oligomer size distribution of
the incubated Ab
40
sample showed a peak ranging
from 50 to 120 nm, indicating the formation of large
oligomers [34]. It should be noted that a low concen-
tration (nm) of dye-labeled protein is required for
single molecule detection using FCS.
Orte et al. [35] used a two-color single-molecule
fluorescence technique (‘two-color coincidence detec-
tion’) to characterize oligomer formation of the SH3
domain of phosphatidylinositol 3-kinase, which is
known to form small granular toxic aggregates. In this
technique, fluorescence bursts from single oligomer
particles made from protein monomers, each labeled
with one of two fluorescent dyes that emit light at dif-
ferent wavelengths, are observed using optics similar
to that of FCS. The coincident detection of both emit-
ted wavelengths with dual excitation indicates the
presence of oligomers consisting of more than one
molecule. The size and population of oligomers can be
determined from the fluorescence intensity and the
frequency of such coincident bursts, respectively.
Oligomer stability at low concentrations can be exam-
ined from changes in the monomer in solution, which
can be evaluated from the frequency of noncoincident
monomer bursts. Experimental data suggest that the
stability of the SH3 domain of the phosphatidylinosi-
tol 3-kinase oligomer changes from unstable oligomer

to stable oligomers that show no monomer dissocia-
tion [35]. It would be interesting to apply this method
to examine the time-course of the stability of Ab
oligomers.
Formation of toxic Ab oligomers M. Sakono and T. Zako
1350 FEBS Journal 277 (2010) 1348–1358 ª 2010 The Authors Journal compilation ª 2010 FEBS
Although these in vitro studies provide insight into
how Ab monomers assemble into oligomeric com-
plexes, further characterizations, by such as visualiza-
tion of Ab oligomer at the molecular level in living
cells and animal models, may be required to elucidate
the mechanism of formation of Ab oligomers.
Possible mechanism of soluble
oligomer formation and toxicity
The mechanism of formation of soluble Ab oligomer
in vivo remains unclear. Glabe et al. [10] proposed that
multiple Ab oligomer conformations were produced
via different pathways, indicating the complexity of
the oligomer formation mechanism. The mechanisms
of formation may also differ for extracellular and
intracellular oligomers. In this section, we discuss pos-
sible formation mechanisms of extracellular and intra-
cellular Ab oligomers, and also discuss how these Ab
oligomers can cause cell death or neuronal impairment
(Figs 1 and 2).
Extracellular soluble Ab oligomer formation and
its toxicity
A recent study by Yamamoto et al. [36] showed the
formation of toxic Ab oligomers in the presence of
GM1 ganglioside. This Ab oligomer was spherical,

with a diameter of 10–20 nm and a molecular mass of
200–300 kDa, and therefore much larger than ADDL.
Furthermore, Ab monomers produced extracellularly
can interact with GM1, and an Ab complex with GM1
has been found in AD brain [37]. These observations
support the idea that extracellular soluble Ab oligo-
mers could be formed by GM1. The Ab oligomer–
GM1 complex is not recognized using a seed-specific
mAb, suggesting that the GM1-induced Ab oligomer
is formed via a pathway distinct from that of fibril
Fig. 1. Formation and toxicity mechanisms of extracellular Ab oligomers. Ab is released extracellularly as a product of proteolytically cleaved,
plasma membrane-localized amyloid precursor protein (APP). Extracellular Ab oligomers can be formed in the presence of GM1 ganglioside
on the cell membrane. GM1 induces Ab oligomer-induced neuronal cell death mediated by nerve growth factor (NGF) receptors. Toxic non-
fibrillar Ab is also produced in the presence of aB-crystallin and ApoJ. A cellular prion protein (PrP
C
) acts as an Ab oligomer receptor with
nanomolar affinity, and mediates synaptic dysfunction. Furthermore, the membrane pore is formed by Ab oligomers. The pores allow abnor-
mal flow of ions, such as Ca
2+
, which causes cellular dysfunction. Binding of Ab oligomers to the NMDA-type glutamate receptor (NMDAR)
also causes abnormal calcium homeostasis, leading to increased oxidative stress and synapse loss. Binding of Ab oligomers to the Frizzled
(Fz) receptor can inhibit Wnt signaling, leading to cell dysfunctions such as tau phosphorylation and neurofibrillary tangles. Moreover, Ab
oligomer can induce insulin receptor loss from the neuronal surface and impaired kinase activity related to long-term potentiation.
M. Sakono and T. Zako Formation of toxic Ab oligomers
FEBS Journal 277 (2010) 1348–1358 ª 2010 The Authors Journal compilation ª 2010 FEBS 1351
formation [36]. Furthermore, nonfibrillar Ab can be
produced in the presence of aB-crystallin [38] and clus-
terin (also known as Apo J) [39], suggesting that extra-
cellular Ab oligomers could be formed by various bio-
components such as proteins and gangliosides (Fig. 1).

The GM1-induced Ab oligomer induces neuronal
cell death mediated by nerve growth factor (NGF)
receptors, suggesting that binding of the Ab oligomer
to the NGF receptor is important for the toxicity
mechanism [36] (Fig. 1). Potent alternation of NGF-
mediated signaling by ADDL supports this concept
[40]. Moreover, previous studies suggested that apopto-
tic cell death occurs through the interaction of Ab with
low-affinity NGF receptor [pan neurotrophin receptor
(p75NTR)] and the activation of downstream signaling
molecules, such as c-Jun N-terminal kinase (reviewed
in ref. [41]). However, it has also been demonstrated
that p75NTR promotes neuronal survival and differen-
tiation, indicating that p75NTR might have diverse
functions in both cell death and cell survival [42].
Consistent with this notion, there are also conflicting
reports showing that p75NTR is protective against
Ab toxicity [43,44]. These results imply that the
NGF-mediated toxicity mechanism is complicated.
Other reports on neuronal receptor-mediated toxicity
mechanisms (reviewed in ref. [9]) have shown that
ADDL binding to an N-methyl-d-aspartate (NMDA)-
type glutamate receptor (NMDAR) causes abnormal
calcium homeostasis, leading to increased oxidative
stress and synapse loss [45,46]. ADDL can also induce
the loss of insulin receptors from the neuronal surface
[47,48] and impair LTP-associated kinase activity [49].
However, such insulin receptor impairment is inhibited
by extracellular insulin, suggesting that insulin plays
an important role in oligomer-induced cell death.

Magdesian et al. [50] showed that Ab oligomers bind-
ing to the Frizzled (Fz) receptor, an acceptor of Wnt
protein, inhibited Wnt signaling, leading to cellular
dysfunction. Wnt signaling, which promotes progenitor
cell proliferation and directs cells into a neuronal
Fig. 2. Formation and toxicity mechanisms of intracellular Ab oligomers. Ab can be localized intracellularly by the uptake of extracellular Ab
or by the cleavage of APP in endosomes generated from the ER or the Golgi apparatus. Extracellular Ab is internalized through various
receptors and transporters, such as formyl peptide receptor-like protein 1 (FPRL1) or scavenger receptor for advanced glycation end-products
(RAGE). These receptor–Ab complexes are internalized into early endosomes. Most Ab in the endosome is degraded by the endosome ⁄ lyso-
some system. However, Ab in the lysosome can leak into the cytosol by destabilization of the lysosome membrane. Although cytosolic Ab
can be degraded by the proteasomal degradation system, inhibition of the proteasome function by Ab oligomers causes cell death. Suppres-
sion of protein aggregation by interactions with various cellular proteins, such as prefoldin (PFD) or other molecular chaperones, may cause
the formation of Ab oligomers.
Formation of toxic Ab oligomers M. Sakono and T. Zako
1352 FEBS Journal 277 (2010) 1348–1358 ª 2010 The Authors Journal compilation ª 2010 FEBS
phenotype during brain development, inactivated
glycogen synthase kinase-3b (GSK-3b) and increased
b-catenin levels. Inhibition of Wnt signaling by Ab
oligomers causes tau phosphorylation and neurofibril-
lary tangles, which suggests a Wnt ⁄ b-catenin toxicity
pathway [50].
A recent report by Nimmrich et al. [51] showed that
Ab oligomers can also impair presynaptic P ⁄ Q-type
calcium currents, which are related to neurotransmis-
sion and synaptic plasticity in the brain, at both gluta-
matergic and gamma-amino butyric acid (GABA)-ergic
synapses. This impairment is specific for Ab oligomers,
but not for Ab monomer or fibrils. Although the
detailed mechanism of this impairment remains
unclear, the interaction of Ab oligomers with synaptic

proteins or channels may cause modification of the
P ⁄ Q current. By contrast, another study showed that
the cell membrane could be destabilized by the Ab oli-
gomer [52]. The membrane pores formed by the Ab
oligomer would allow the abnormal flow of ions, such
as Ca
2+
, suggesting another plausible mechanism for
Ab oligomer toxicity [53,54]. Recent observations by
Lauren et al. [55] indicate that cellular prion protein
(PrP
C
) can act as an Ab oligomer receptor with a
nanomolar affinity, mediating synaptic dysfunction.
Although misfolded prion protein (PrP
Sc
) is thought to
cause prion disease, the interaction between the Ab oli-
gomer and the prion does not require the infectious
PrP
Sc
conformation. This interaction may disrupt the
interaction between PrP
C
and a co-receptor, such as
NMDAR, impairing the neuron signal-transduction
pathways. This discovery by Lauren et al. also suggests
that AD is linked with other neurodegenerative
diseases.
Recently, interactions between Ab and a-synuclein

in vivo and in vitro have recently been observed [56,57].
Alpha-synuclein is an aggregation-prone protein that
causes Parkinson’s disease (PD), and interactions
between a-synuclein and Ab therefore indicate that
AD and PD could be related. Ab also promotes a-syn-
uclein aggregation and toxicity. These results suggest
that the AD and PD pathologies could overlap. Inter-
estingly, interactions between Ab and a-synuclein
induce the formation of hybrid pore-like oligomers
[58]. Ab-treated cells expressing a-synuclein display
increased current amplitudes and calcium influx,
consistent with the formation of cation channels. It is
therefore assumed that the hybrid pore-like oligomers
may alter neuronal activity and cause neurodegenera-
tion. These observations support the idea that
there are various Ab oligomer-formation pathways,
and that cell death might occur via multiple pathways
(Fig. 1).
Intracellular Ab
Although Ab was first identified as a component of
extracellular amyloid plaques, ample evidence has
demonstrated that Ab is also generated intracellularly
(reviewed in ref. [6]). Besides the plasma membrane,
APP localizes to the trans-Golgi network, to the ER
and to the endosomal, lysosomal and mitochondrial
membranes. Ab is produced by the sequential cleavage
of APP by b-secretase (also known as BACE) and
c-secretase in endosomes as well as at the plasma
membrane [59]. Ab is also produced intracellularly
within the ER and the trans-Golgi network system

along the secretory pathway. Identification of the
intracellular protein, endoplasmic reticulum associated
binding protein (ERAB), which binds to Ab,
also strongly suggests the existence of intracellular Ab
[60].
In addition to Ab being produced intracellularly,
previously secreted Ab that forms the extracellular Ab
pool can be taken up by cells and internalized into
intracellular pools through various receptors and trans-
porters, such as the nicotinic acetylcholine receptor,
low-density lipoprotein receptor, formyl peptide recep-
tor-like protein 1, NMDAR and the scavenger recep-
tor for advanced glycation end-products [6] (Fig. 2).
These receptor-associated Ab complexes could be
internalized into endosomes. Recent findings also sup-
port the idea that Ab is present within the cytosolic
compartment. Intracellular accumulation of Ab in the
multivesicular body is linked to cytosolic proteasome
inhibition [61]. Furthermore, in vivo and in vitro pro-
teasome inhibition also leads to higher Ab levels
[62,63]. As the proteasome is primarily located within
the cytosol, these findings strongly suggest that Ab is
also located within the cytosolic compartment. Extra-
cellular Ab can enter the cytosolic compartment and
inhibit the proteasome activity of cultured neuronal
cells [62]. Clifford et al.
[64] showed that fluorescently
labeled Ab which is injected into the tail of mice with
a defective blood–brain barrier (which is common in
AD patients) accumulates in the perinuclear cytosol of

pyramidal neurons in the cerebral cortex. These obser-
vations strongly support the notion that neurons can
take up extracellular Ab in the cytosolic compartment.
The destabilization of intracellular membranes
may also contribute to the presence of cytosolic Ab.
A high proportion of autophagy-related vesicular
structures, which would suggest impaired maturation of
autophagosomes to lysosomes, has been found in the
AD brain, but not in the normal brain [65]. Although
most Ab formed in endosomes is normally degraded
within lysosomes, Ab can accumulate in lysosomes in
M. Sakono and T. Zako Formation of toxic Ab oligomers
FEBS Journal 277 (2010) 1348–1358 ª 2010 The Authors Journal compilation ª 2010 FEBS 1353
the AD brain. Ab within the lysosomal compartment
destabilizes its membrane [66], which would also lead to
the presence of Ab in the cytosolic compartment.
Intracellular soluble A b oligomer formation and
its toxicity
How intracellular Ab monomers assemble and form
soluble oligomers remains unclear. One possibility is
that the uptake of extracellularly-produced Ab oligo-
mers occurs via endocytic pathways or various recep-
tors and transporters, as described above (Fig. 2).
Another possibility is that the interaction of Ab with
intracellular proteins results in oligomer formation.
Recent observations by Yuyama et al., [67] showing
GM1 accumulation in early endosomes, support the
idea that intracellular GM1 could also induce Ab oli-
gomer formation. Recently, we found formation of
toxic high-MW (50–250 kDa) soluble Ab oligomers by

the cytosolic molecular chaperone protein, prefoldin,
in vitro [68]. In general, molecular chaperones stabilize
and mediate the folding of unfolded proteins. Molecu-
lar chaperones play essential roles in many cellular
processes, such as protein folding, targeting, transpor-
tation, degradation and signal transductions [69].
Prefoldin reportedly captures and delivers denatured
protein to another cytosolic chaperone, chaperonin
[70–73]. Our results also suggested that the interaction
between prefoldin and A b oligomers prevents further
aggregation and stabilizes the oligomer structure
(Fig. 2).
Molecular chaperones are potent suppressors of pro-
tein aggregation, leading to neurodegenerative disor-
ders such as AD, PD and Huntington’s disease (HD)
[74–76]. Various molecular chaperones are upregulated
in patients and co-localize with aggregated proteins in
plaques ⁄ inclusion bodies. These molecular chaperones
prevent aggregation in vivo and in vitro; for example,
the cytosolic chaperonin CCT can inhibit aggregation
of the polyglutamine (polyQ) expansion protein, which
causes HD in vivo and in vitro [77–79]. Reduced CCT
levels also enhance the aggregation and toxicity of pol-
yQ in neuronal cells, strongly supporting the idea that
molecular chaperones can be a defense against the
aggregation of misfolded protein. Importantly, how-
ever, our findings also suggest the possibility that the
suppression of protein aggregation may cause the for-
mation of toxic oligomeric species, which is consistent
with previous results showing that toxic nonfibrillar

Ab was produced in the presence of aB-crystallin [38]
and clusterin (also known as Apo J) [39]. These results
suggest that intracellular Ab oligomers could be pro-
duced by interaction with various cellular proteins,
including molecular chaperone proteins (Fig. 2).
The toxicity mechanism of intracellular Ab oligo-
mers also remains unclear. Microinjection of Ab or a
cDNA-expressing cytosolic Ab induces the cell death
of primary neurons and the simultaneous formation of
low-MW Ab oligomers [80]. Furthermore, intracellular
Ab accumulation is closely correlated with apoptotic
cell death via the P53-BAX pathway [81]. Recently,
Mousnier et al. [82] reported a possible prefoldin-medi-
ated proteasomal protein-degradation pathway. It is
therefore plausible that Ab oligomer–prefoldin com-
plexes could bind to proteasome, causing proteasome
dysfunction and subsequent cell death. This idea is
supported by interaction studies between Ab oligomers
and proteasome, which showed that the proteasomal
function was inhibited while interacting with Ab [63].
Impairment of proteasomal function by the Ab oligo-
mer also leads to age-related pathological accumula-
tion of Ab and tau protein [63]. Recent research has
shown that the dysfunction of autophagy, a lysosomal
pathway for degrading organelles and proteins, is
related to neurodegenerative diseases, including AD
and PD [65,76]. These observations support the idea
that the toxicity mechanism of intracellular oligomers
may be different from that of extracellular oligomers
(Fig. 2). However, more studies, particularly those

focused especially on the proteolysis system in AD
brains, are necessary to understand AD pathology in
relation to intracellular soluble Ab oligomers.
Concluding remarks
It has long been argued that insoluble Ab fibrillar
aggregates found in extracellular amyloid plaques initi-
ate the neurodegenerative cascades of AD. However,
recent emerging results indicate that prefibrillar soluble
Ab oligomers are the key intermediates in AD-related
synaptic dysfunction. Various amyloidogenic proteins
can form toxic soluble oligomers, suggesting that solu-
ble oligomers are the general key factors in various
diseases such as AD, PD, HD and other amyloidosis
[5,28,83]. Although much research effort is being direc-
ted towards characterizing oligomer states, their con-
formations and formation mechanisms remain unclear.
Recent evidence suggests that the size of Ab oligomers
is distributed in a wide MW range (from < 10 kDa to
> 100 kDa), and that there is structural polymor-
phism of Ab oligomers, even for those of a similar
size. The biochemical properties of these oligomers in
relation to disease pathology also seem to differ
depending on their sizes and structures.
Formation of toxic Ab oligomers M. Sakono and T. Zako
1354 FEBS Journal 277 (2010) 1348–1358 ª 2010 The Authors Journal compilation ª 2010 FEBS
Ab can form various distinct oligomeric states via
various pathways. The formation and toxicity mecha-
nisms of extracellular and intracellular Ab oligomers
can also be different from one another. Regardless of
the complexity of the oligomer-formation mechanism,

recent findings suggest that Ab oligomers can be
formed through interactions between Ab mono-
mers ⁄ oligomers and cellular proteins ⁄ biomolecules,
such as molecular chaperones and lipids. Prevention of
aggregation may cause the formation ⁄ stabilization of
oligomer states.
Acknowledgements
The authors are grateful for financial support from the
Japan Science Technology Agency (PRESTO Program,
MS), RIKEN (Nano-scale Science and Technology
Research, TZ) and the Japanese Society for the Pro-
motion of Science (TZ). We wish to thank Drs Karin
So
¨
rgjerd, Naofumi Terada (RIKEN) and Hiroshi Ku-
bota (Akita University) for helpful comments.
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