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

Amyloid oligomers: formation and toxicity of Ab oligomers Masafumi Sakono1,2and Tamotsu Zako1 1 Bioengineering Laboratory, RIKEN Institute, Wako, Saitama, Japan 2 PRESTO, Japan Science a

Amyloid oligomers: formation and toxicity of Ab oligomers Masafumi Sakono1,2and Tamotsu Zako1

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: zako@riken.jp

(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.

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 Ab oligomer structures at atomic resolu-tion are unclear, studies using conformaresolu-tion-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

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 Ab 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 Ab42 tetramer is theoretically

assumed to take an open ‘V’ form, which is neither

linear nor square A planar hexagon form was

assumed for the Ab42hexamer It is interesting to note

that a stacked hexamer paranuclei structure, rather

than side-by-side planar hexagons, was suggested for

the Ab42 dodecamer, (Ab*56) These authors also

showed that oligomer size distribution was very

different between Ab42 and Ab40, 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 Ab40 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 Ab40 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

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 vivoremains 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.

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 lysolyso-some can leak into the cytosol by destabilization of the lysolyso-some 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.

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 Ca2+, suggesting another plausible mechanism for

Ab oligomer toxicity [53,54] Recent observations by

Lauren et al [55] indicate that cellular prion protein

(PrPC) can act as an Ab oligomer receptor with a

nanomolar affinity, mediating synaptic dysfunction

Although misfolded prion protein (PrPSc) is thought to

cause prion disease, the interaction between the Ab

oli-gomer and the prion does not require the infectious

PrPSc conformation This interaction may disrupt the

interaction between PrPC 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 vivoand 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 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

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 Ab 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 celluMolecu-lar

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 Ab 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

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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