Báo cáo khoa học: Amyloid oligomers: dynamics and toxicity in the cytosol and nucleus ppt

The aggregadysfunc-tion-prone nature of the mutant proteins suggests that misfolded proteins dis-turb neuronal cell functions via unnecessary interac-tions with normal proteins.. Moreove

Amyloid oligomers: dynamics and toxicity in the cytosol and nucleus

Akira Kitamura1and Hiroshi Kubota2

1 Department of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Kita-ku, Sapporo, Japan

2 Department of Life Science, Faculty of Engineering and Resource Science, Akita University, Akita, Japan

Introduction

The accumulation of misfolded proteins in the cytosol

and nucleus causes neurodegenerative disease [1–3]

For example, proteins harboring expanded

polygluta-mine (polyQ) tracts cause polyQ diseases, which

include Huntington’s disease and several

spinocerebel-lar ataxias [4,5], and mutations in superoxide

dismu-tase 1 (SOD1) lead to familial amyotrophic lateral

sclerosis (ALS) [6,7] In these diseases, inclusions of

the mutant proteins are found in the neuronal cells of

patients and the accumulation of misfolded proteins is

considered to be a primary cause of neuronal dysfunc-tion and death The aggregadysfunc-tion-prone nature of the mutant proteins suggests that misfolded proteins dis-turb neuronal cell functions via unnecessary interac-tions with normal proteins However, the mechanism

by which mutant proteins exert their cytotoxicity is lar-gely unknown Although these diseases have a late onset, as symptoms appear in adulthood, the molecu-lar mechanisms underlying the age-dependent onset are poorly understood Moreover, little is known about

Keywords

live cell imaging; misfolded protein;

molecular chaperone; neurodegenerative

disease; neuronal cell death; oligomer;

protein aggregation; protein degradation;

protein interaction; spectroscopic analysis

Correspondence

H Kubota, Department of Life Science,

Faculty of Engineering and Resource

Science, Akita University, 1-1

Tegatagakuen-cho, Akita 010-8502, Japan

Fax: +81 18 75 3053

Tel: +81 18 75 3053

E-mail: hkubota@ipc.akita-u.ac.jp

(Received 4 September 2009, revised 29

November 2009, accepted 1 December

2010)

doi:10.1111/j.1742-4658.2010.07570.x

The accumulation of misfolded proteins in the cytosol and nucleus of neuronal cells leads to neurodegenerative disorders Polyglutamine diseases are caused by polyglutamine-expanded proteins, whereas mutations in superoxide dismutase 1 lead to amyotrophic lateral sclerosis These struc-turally unstable mutant species perturb essential interactions between nor-mal proteins and tend to aggregate because of the presence of exposed hydrophobic surfaces Accumulating evidence suggests that soluble species, including misfolded monomers and oligomers, are more toxic than large insoluble aggregates or inclusions Spectroscopic analysis, including fluores-cence recovery after photobleaching and fluoresfluores-cence loss in photobleach-ing, in living cells revealed that protein aggregates of misfolded proteins are dynamic structures that interact with other proteins, such as molecular chaperones Fluorescence correlation spectroscopy analysis detected soluble oligomers⁄ aggregates of misfolded proteins in cell extracts Fluorescence resonance energy transfer analysis supported the notion that soluble oligo-mers⁄ aggregates are formed before the formation of inclusions in vivo Here, we reviewed the characteristics of oligomers and aggregates of misfolded proteins, with a particular focus on those revealed by spectro-scopic analysis, and discussed how these oligomers may be toxic to cells

Abbreviations

ALS, amyotrophic lateral sclerosis; AR, androgen receptor; CCT, chaperonin containing t-complex polypeptide 1; CFP, cyan fluorescent protein; FCCS, fluorescence cross-correlation spectroscopy; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; HDAC6, histone deacetylase 6; HSP, heat shock protein; polyQ, polyglutamine; RFP, red fluorescent protein; SCA, spinocerebellar ataxia; SOD1, superoxide dismutase 1; YFP, yellow fluorescent protein.

how the mutant proteins specifically damage particular

neuronal cells

Recent progress in spectroscopic imaging analysis,

using proteins tagged with green fluorescent protein

(GFP) and related cyan, yellow and red fluorescent

proteins (e.g CFP, YFP, RFP respectively) allowed us

to trace the tagged proteins in living cells [8] Mutant

proteins that cause polyQ disease and ALS have been

tagged with these fluorescent proteins and analyzed by

fluorescence microscopy-based spectroscopic analysis,

as well as by conventional biochemical experiments The

spectroscopic techniques used for living cells include

flu-orescence recovery after photobleaching (FRAP),

fluo-rescence loss in photobleaching (FLIP) and

fluorescence⁄ Fo¨rster resonance energy transfer (FRET)

(Fig 1) These techniques reveal real-time movements

and interactions of misfolded proteins in living cells

The recent application of fluorescence correlation

spectroscopy (FCS), which is a microscopy-based

technique used for the analysis of fluorescent molecules

at the single-molecule sensitivity [9,10], to misfolded

mutant proteins succeeded in detecting their soluble

oligomers⁄ aggregates in cell extracts Together with

evidence from other cell biological and biochemical

analyses, we discussed the role of soluble oligomers of

toxic species in protein-misfolding diseases, including

polyQ disease and ALS

Soluble oligomers of misfolded

proteins as potentially toxic species

PolyQ-expanded proteins and ALS-linked mutant

SOD1 are structurally unstable [5,7] These proteins

thus tend to aggregate and interact with other proteins via exposed hydrophobic surfaces, leading to the pertur-bation of cellular activities (Fig 2) The hydrophobic surfaces of misfolded proteins can be masked by molec-ular chaperones, and the aggregation of misfolded pro-teins is inhibited by chaperones through this activity [11,12] However, the concentration of chaperones is limited in living cells, and these chaperones are required for the folding of newly synthesized normal proteins Thus, the overloading of cellular chaperoning capacity

by misfolded mutant proteins results in increased mis-folding of normal proteins and further enhancement of co-aggregation In this state, the degradation of mis-folded proteins is diminished by their insolubility, and cellular functions are severely damaged by a negative chain reaction This situation can be explained by escape from (or collapse of) the protein homeostasis net-work [13,14], and the accumulating incapacitation of protein homeostasis may explain, in part, the late onset

of neurodegenerative disorders associated with protein misfolding It should be noted that chaperone functions and substrate proteins differ among chaperones, to a certain extent, and their expression levels vary according

to cell type These differences may affect the protein species whose functions are inhibited and the cell types that are damaged under disease conditions

The decrease in the amount of functional proteins (e.g transcription factors) as a result of becoming trapped in aggregates may explain the toxicity of mis-folded proteins However, a number of studies suggest that sequestration of misfolded proteins into inclusions

is protective [15] The total amount of exposed hydro-phobic surfaces in misfolded proteins is much greater

Detection of conformational change

Dynamics of correctly folded monomer

Detection of small oligomer

Detection of soluble aggregate

or amyloid fibril

FRAP/FLIP

FCS

FCCS

FRET

Unsuitable Good Not applicable Not applicable

Unsuitable Not sensitive*

Less sensitive****

Good****

Unsuitable Less sensitive**

Good****

Less sensitive***

Unsuitable Good Good****

Less quantitative

Dynamics of inclusion body

Good Not applicable Not applicable Less quantitative

Fig 1 Suitability of spectroscopic methods for the analysis of protein aggregation Asterisks indicate the following: *, FCS can be applied only when the size of molecule is greatly altered by conformational change; **, FCS can detect most oligomers but this method is less sen-sitive for the detection of very small oliomers such as dimers or trimers; ***, FRET detects dimers and larger oligomers ⁄ aggregates as com-plexes but cannot determine their sizes; and ****, these methods have not been used in in vivo studies for the indicated purposes despite the availability FCCS, fluorescence cross-correlation spectroscopy; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching.

in the monomeric and oligomeric states than in the

inclusion state under conditions where the number of

misfolded proteins per cell is identical Although

misfolded monomers can be trapped and refolded by

molecular chaperones and degraded by the ubiquitin–

proteasome system [16], oligomers are more resistant

to refolding by chaperones and to degradation by the

proteasome Thus, soluble oligomers are considered as

highly toxic to cells Inhibition of oligomer formation

is probably useful to protect cells against the toxicity

of misfolded proteins

Sequestration of soluble oligomers⁄ aggregates into

inclusions or aggresomes by microtubule-dependent

transport is considered to play a role in the removal of

the potentially toxic soluble species from the cytosol

[17] Indeed, cells harboring polyQ-expanded

Hunting-tin inclusions were reported to be more resistant to the

toxicity of misfolded proteins than cells exhibiting

dif-fusible patterns [18] However, an opposing effect was

reported using an ALS-linked mutant SOD1 [19],

sug-gesting that the cell-protection activity exerted by

inclusion⁄ aggresome formation can be affected by dif-ferences in protein characters and other factors (e.g expression level, time course and cell type) Structural differences between inclusions⁄ aggresomes may also affect the cell-protection activity; polyQ-expanded Huntingtin proteins are tightly associated and immo-bile in the inclusion [20], whereas mutant SOD1 pro-teins are loosely packed in the aggresome and partly exchangeable with cytosolic SOD1 proteins [19] In a Drosophila model of spinobulbar muscular atrophy, which is a neurodegenerative disease caused by expan-sion of a polyglutamine repeat in the androgen recep-tor (AR), histone deacetylase 6 (HDAC6) was shown

to play an essential role in preventing polyglutamine toxicity [21] HDAC6 is a microtuble-associated pro-tein that interacts with polyubiquitinated misfolded proteins and dynein motors [22] Through these inter-actions, HDAC6 mediates inclusion⁄ aggresome forma-tion of misfolded proteins in a microtubule-dependent manner In this sequestration system, however, the details of transported species (e.g monomer, oligomer

Non-toxic conformation

Toxic monomer

Toxic small oligomer

Degradation

by proteasome

Degradation resistant

Soluble aggregate

Inclusion

Degradation

by autophagy

Chaperone Newly-synthesized polypeptide

Aberrant interaction with other proteins

Conformational change and further binding

Inactivation of other proteins

A

B

C

Depletion of chaperones

by oligomers

Increased misfolding

by impaired chaperoning activity

Further stimulation of aggregation

Fig 2 Possible mechanisms of oligomer

toxicity in neurodegenerative disease.

(A) Small oligomers are hardly degraded

by the proteasome and autophagy.

(B) Oligomers inhibit protein functions by

aberrant interaction and co-aggregation.

(C) Depletion of molecular chaperones by

oligomers leads to further stimulation of

co-aggregation.

or soluble aggregate) remain unknown Thus, these

observations are not contradictory with the notion that

soluble oligomers are highly toxic to cells

The autophagy–lysosome pathway plays a role in

the clearance of misfolded protein aggregates [23,24]

and this pathway is a candidate system for the removal

of soluble oligomers⁄ aggregates For example, beclin 1,

an essential component of the autophagy system, is

required for the effective removal of polyQ aggregates

[25] In the study by Pandey et al [21] the amount of

polyQ-expanded AR aggregates were significantly

decreased by the over-expression of HDAC6, but

increased by the knockdown of autophagy

com-ponents Interestingly, in the fly that expresses

polyQ-expanded AR, the rescue of eye degeneration by

HDAC6 was dependent on autophagic activity A

sim-ilar role of HDAC6 was shown in mammalian cells

[26], and an essential role of autophagy in preventing

neurodegeneration was reported using knockout mice

[27] These observations suggest a link between the

microtubule-dependent formation of aggresome and

the autophagy-dependent clearance of misfolded

pro-teins Recently, the ubiquitin-binding protein p62 (also

known as sequestosome 1) was shown to stimulate the

aggregation of ubiquitinated proteins and to interact

with LC3, an essential component of autophagy [28]

The p62 protein is required for the prevention of

pol-yQ toxicity [29] and interacts with ALS-linked mutant

SOD1 [30] Thus, p62-bound misfolded proteins in a

soluble state (e.g soluble aggregates) may be removed

by an autophagy-mediated degradation system

How-ever, the autophagy-mediated pathway is probably

inefficient for the removal of small oligomers (e.g

trimer, tetramer, etc.), even though proteins such as

p62 assist specific recognition, because this system uses

bulk sequestration of cytosolic regions via the

forma-tion of double membranes Further investigaforma-tions are

required to understand what size of aggregated species

is effectively removed by the autophagy-mediated

deg-radation system in a selective manner in living cells

Two types of neurodegenerative

diseases caused by misfolded proteins

in the cytosol and nucleus

PolyQ diseases

Expansion of the polyQ tract in at least nine proteins

causes neurodegenerative disorders [4,5,31,32] PolyQ

expansion in the Huntingtin protein causes

Hunting-ton’s disease, and polyQ-expanded ataxin-1 leads to

spinocerebellar ataxia type 1 (SCA1; also known as

olivopontocerebellar atrophy type 1) PolyQ expansion

in ataxin-3 is the cause of the most common dominant ataxia, spinocerebellar ataxia type 3 (SCA3, also known as Machado–Joseph disease) SCA2, SCA6, SCA7 and SCA17 are also caused by polyQ expansion, and expansion of the polyQ repeat in the AR is responsible for spinal and bulbar muscular atrophy The expanded polyQ tract is encoded by a CAG repeat

in the causative genes, and polyQ diseases are inherited dominantly PolyQ repeats contain approximately 10–30 glutamine residues in healthy individuals, whereas they are expanded to more than 40 repeats in patients

In the polyQ diseases, inclusions containing polyQ-expanded proteins are found in the nucleus and⁄ or cytoplasm of neuronal cells in the brain of patients The function of neuronal cells is progressively dis-turbed, leading to cell death PolyQ-expanded proteins aggregate easily in vivo and in vitro, and these aggre-gates are very difficult to dissolve, even in the presence

of strong detergents, such as SDS [33] PolyQ-expanded proteins trap normal functional proteins in the aggregation process, which suggests a possible mechanism of toxicity PolyQ aggregates have been shown to be rich in b-sheets [34], and Nagai et al [35] demonstrated that b-sheet-containing monomers and oligomers produced in vitro are toxic to cultured neu-ronal cells when introduced by microinjection

A recent study of the conformation and toxicity of polyQ-expanded Huntingtin indicated that fragile amy-loid, which is rich in exposed and flexible regions, is significantly more toxic than rigid amyloid, which comprises buried and fixed regions [36] As the former

is considered to break into oligomers and to be accessible to other proteins more easily than the latter, these observations are consistent with the notion that oligomers with specific conformations are toxic to cells

Molecular chaperones, including heat shock protein (HSP)70, cognate of HSP70 (HSC70), chaperonin con-taining t-complex polypeptide 1 (CCT) (also called TRiC), HSP40 (DnaJ) and small HSPs (e.g HSP27 and crystalins), play crucial roles in the protection of cells against the toxicity of polyQ-expanded proteins [11,12] For example, HSP70, which is a cytosolic molecular chaperone that interacts with the hydropho-bic surfaces of denatured and misfolded proteins, pre-vents aggregation of polyQ-expanded proteins and inhibits their toxicity [37–39] The cytosolic chaperonin CCT is a molecular chaperone that prevents b-sheet aggregation by recognizing hydrophobic b-strands [40]; this chaperone prevents aggregate formation and toxic-ity of polyQ-expanded proteins [41–44] CCT weakly recognizes monomeric and oligomeric forms (2–5 mers)

of Huntingin-Q53, but not fibril forms of the protein

[43] Depletion of CCT activity by RNA interference

in polyQ-expressing cells results in increased amounts

of soluble aggregates and cell death [42], which

sug-gests that CCT interferes with polyQ aggregation by

trapping monomers or small oligomers, thus inhibiting

their toxicity These observations support the notion

that b-sheet-rich oligomers of polyQ-expanded proteins

are toxic to cells and that inhibition of oligomer

for-mation is an effective strategy for the inhibition of

polyQ toxicity

Familial ALS caused by mutant SOD1

ALS is a neurodegenerative disorder characterized by

the progressive loss of motor neurons Although 90%

of ALS cases are sporadic, the remaining 10% are

caused by genetic mutations that are inherited

domi-nantly Dominant inheritance of familial ALS suggests

a toxic gain of function, similarly to other

protein-misfolding diseases, such as the polyQ diseases

However, the molecular mechanisms of ALS toxicity

are largely unknown Moreover, little is known about

how the mutant gene products specifically damage and

kill motor neurons Several causative genes have been

identified for familial ALS, including SOD1, TDP-43

and FUS⁄ TLS [45] Mutations in SOD1 are the most

common cause of familial ALS, and SOD1 mutants

aggregate in the cytosol of neuronal cells [6,46,47]

More than 100 ALS-linked mutations have been

iden-tified in SOD1 and these mutant proteins are

structur-ally unstable [7,48] Because of structural instability,

the SOD1 mutants are thought to expose hydrophobic

surfaces more easily than the wild-type protein Thus,

these proteins tend to aggregate and potentially exert

their toxicity via aberrant interactions with other

normal proteins

Recently, Wang et al [49] used a mouse model of

ALS-linked mutant SOD1 (G85R) to show that soluble

oligomers of mutant SOD1 are detectable biochemically

in spinal cord extracts before the onset of visible motor

neuron dysfunction Similar oligomers were also

detected biochemically in Caenorhabditis elegans

expressing the G85R mutant [50] In the mouse model,

insoluble aggregates were detected at the onset of

symp-toms, which suggests that soluble oligomers are further

aggregated into inclusions These observations suggest

that soluble oligomers of mutant SOD1 appear when

cellular chaperoning and other quality-control pathways

are overwhelmed by the accumulation of misfolded

proteins Although the molecular chaperone HSC70 was

associated with soluble species of mutant SOD1 at any

stage, HSP110, which is a nucleotide exchange factor of

HSP70⁄ HSC70, was associated with the mutant protein after the initiation of motor neuron dysfunction The structure and toxicity of soluble oligomers may differ according to the stage of disease progression

Extracellular oligomers have been suggested to be a pathogenic factor of neurodegenerative diseases, including Alzheimer’s disease and prion diseases [51– 53] For example, amyloid-b peptide (Ab) oligomers induce synaptic disfunction, probably by interfering with receptor-dependent signaling pathways via bind-ing to synaptic plasma membranes In the case of the ALS-linked mutant, SOD1, Urushitani et al [54] indi-cated (using cultured cells) that these mutants are secreted from neuronal cells through a chromogranin-mediated pathway and that extracellular mutant SOD1 triggers microgliosis and neuronal cell death In a mouse model of ALS, a conditional knockout of mutant SOD1 in astrocytes revealed that these cells affect the disease progression, but not the onset, of ALS in a noncell autonomous manner [55] In this report, extracellular mutant SOD1 was suggested as a candidate for the mediator These observations suggest that extracellular mutant SOD1 may play an addi-tional role in the pathogenesis of mutant SOD1-medi-ated ALS As in vitro studies for mutant SOD1 indicate that post-translational events (including metal binding and disulfide formation) affect oligomer and fibril formation [56,57], the aggregation state may be altered by extracellular environmental conditions Thus, like other neurodegenerative diseases, extracellu-lar oligomers of mutant SOD1 might act as a toxic species, although this possibility remains to be investigated

In vivo dynamics of misfolded proteins revealed by spectroscopic imaging analyses

FRAP and FLIP analyses of aggregates and interacting proteins

Time-lapse observation of fluorescently labeled mole-cules is often used to trace the movement of cellular structures However, this method cannot analyze the mobility of molecules distributed uniformly and is unsuitable for the determination of molecular-exchange rates from one structure to another FRAP is a method that measures the mobility of rapidly moving fluores-cent molecules in a living cell [8] Molecules labeled with

a fluorescent protein (i.e GFP and related proteins) are bleached in a region of interest for a short time-period and the subsequent movement of fluorescent molecules from the unbleached area is quantitatively analyzed by

the recovery of fluorescence intensity This method is

useful for the quantitative analysis of the mobility of

aggregation-prone proteins in a living cell (Fig 1) In

FLIP analysis, fluorescently labeled molecules are

con-tinuously bleached in a region and the fluorescence of

unbleached areas is measured FLIP can be used for the

analysis of molecular transfers between two or more

regions, regardless of the speed of movement, even if

this method is less quantitative than FRAP Thus,

FRAP is very useful for determining the locoregional

mobility of proteins, whereas FLIP can

comprehen-sively analyze protein trafficking

The mobility of polyQ-expanded proteins in

inclu-sions has been analyzed by FRAP and FLIP FRAP

analysis of polyQ-expanded ataxin-3 tagged with GFP

(GFP-ataxin-3-Q82) revealed that polyQ-expanded

ataxin-3 is immobile in the nuclear inclusion [58] In

addition, FLIP analysis indicated that polyQ-expanded

ataxin-3 is unable to shuttle between the inclusions and

the nucleoplasm These results demonstrate that the

inclusion body formed by polyQ-expanded ataxin-3 is a

structure that is immobilized in the nucleus By contrast,

FRAP analysis of GFP-ataxin-1-Q84 demonstrated that

ataxin-1 is mobile in nucleoplasmic inclusions [59]

Interestingly, there are two types of ataxin-1 inclusions:

one undergoes fast and complete exchange with a

nucle-oplasmic pool and the other exhibits slow exchange

rates The slowly exchanging inclusions contain high

levels of ubiquitin and low levels of proteasome, which

suggests a role that is distinct from that of the rapidly

exchanging inclusions Inverse FRAP analysis of

ataxin-1 indicated that wild-type ataxin-ataxin-1 shuttles between the

nucleus and the cytosol, whereas polyQ-expanded

ataxin-1 is not exported from the nucleus [60] These

observations suggest that the ataxin-1 accumulated in

the nucleus becomes a species that is unable to pass

through nuclear pores FRAP was also used to analyze

the dynamics of ALS-linked mutant SOD1 in cytosolic

inclusions [19] Mutant SOD1 shuttles dynamically

between the inclusion body and the cytosol in neuronal

cells, which suggests that the inclusion body of mutant

SOD1 is not an immobile structure By contrast, polyQ

and polyQ-expanded Huntingtin formed immobile

inclusions in the cytosol and in the nucleus [20] Thus,

there are at least two types of inclusions – mobile

inclu-sions and immobile incluinclu-sions – which is consistent with

a recent study proposing two distinct inclusion-like

compartments for protein quality control [61]

FRAP and FLIP are also useful for analyzing the

transient association of other proteins with the

inclu-sions FRAP analysis of HSP70–YFP in

Huntingtin-150Q–CFP inclusions revealed that HSP70 is mobile

within the inclusion [20] As the movement of HSP70 is

significantly slower in the inclusion than in the cytosol, HSP70 appears to interact transiently with aggregated mutant proteins in the inclusion These observations indicate that HSP70 localized in inclusions is not co-aggregated in inclusions and thus may play a role in the modulation of the potentially toxic hydrophobic sur-faces of polyQ aggregates Interaction of HSP70 with the ALS-linked mutant SOD1 was analyzed using FLIP [19] By continuous photobleaching of YFP–SOD1 in a small cytosolic region, the fluorescence intensity of the nonbleached area was decreased more slowly in aggre-gate-containing cells than in aggregate-free cells These observations suggest a dynamic interaction between HSP70 and mobile inclusions of mutant SOD1 in living cells HSP70 might shuttle with misfolded mutant SOD1 between the inclusions and the cytosol

FCS analysis of oligomers and soluble aggregates

The mobility or exchange rate of aggregate-prone proteins in inclusions has been estimated using FRAP,

as described above However, this method is unsuitable for determining the diffusion coefficients of rapidly moving molecules (or particles), because the diffusion rates are faster than the image capture rate on the detector By contrast, FCS is appropriate for this pur-pose [8–10] For FCS analysis, a very small fluorescence detection volume (the so-called confocal volume) is cre-ated using optics similar to that of a confocal micro-scope In the FCS optics, fluorescent molecules are excited by a diffracted narrow laser beam and detected

in a pinhole aperture-regulated thin layer When fluo-rescent molecules pass through the confocal volume, fluorescence fluctuation is detected using a highly sensi-tive photodetector The fluctuation is analyzed as a cal-culated autocorrelation function, which provides the residence time of diffusing molecules in the confocal volume As diffusion coefficients correlate with the fric-tion between the molecule and the solvent, the molecu-lar mass of the molecules can be calculated by assuming the molecular shape (e.g sphere or rod) FCS analysis allows determination of the concentration of fluorescent molecules and of fluorescence intensity per molecule (or counts per particle) thus, the distribution

of differently sized oligomeric species can be estimated

As FCS analysis can be performed in living cells as well

as in solution [10,62], this technique is becoming a pow-erful tool for the quantitative analysis of protein com-plexes, including soluble oligomers⁄ aggregates of misfolded proteins, as described below

The presence of soluble oligomers (or soluble aggre-gates) has been demonstrated by FCS analysis using

extracts of cultured cells expressing long polyQ repeats

or polyQ-expanded Huntingtin tagged with GFP or

YFP [42,63] The amount of soluble oligomers⁄

aggre-gates was significantly increased by the RNA

interfer-ence-mediated knockdown of the cytosolic chaperonin

CCT, which suggests that CCT prevents oligomer

for-mation of polyQ-expanded proteins in an early step of

aggregation, under normal conditions [42] In this

study, records of count rate indicated that bright

parti-cles of Q82–GFP and Huntingtin-Q143–YFP passed

through the confocal volume in the CCT-depleted cell

extract In another study using FCS, fluorescence

intensity per particle increased for Q45–GFP and

Q81–GFP in a time-dependent manner [63]

Further-more, a polyQ-binding polypeptide (QBP1)

signifi-cantly inhibited the increase of fluorescence intensity

per particle for Q45–GFP These observations suggest

that the soluble oligomers⁄ aggregates detected by FCS

contain multiple polyQ-expanded proteins, which are

probably homo-oligomeric, at least in part

Fluorescence cross-correlation spectroscopy (FCCS)

detects the direct interaction between two fluorescent

molecules at a near single-molecule sensitivity [10,64]

For FCCS measurements, two molecules are labeled

with different fluorophores that are distant in

wave-length, and a solution of these labeled molecules is

analyzed using FCS equipment An interaction

between the denatured proteins and small HSPs was

determined using FCCS in vitro [65] Although this

study was carried out in vitro, FCCS can be performed

in living cells or in cell lysates Thus, this method has

the potential to analyze the interaction between

aggre-gation-prone protein and binding protein (e.g

chaper-ones) in living cells

FRET measurements to analyze molecular

interactions in aggregates

FRET provides a useful tool with which to detect

interactions between proteins labeled with a fluorescent

tag FRET analysis is a method that measures energy

transfer from a donor fluorophore to a nearby acceptor

chromophore FRET efficiency is highly dependent on

the distance between the donor and the acceptor

Sev-eral methods have been used to detect FRET signals,

including spectral scanning, ratio imaging, the recovery

of donor fluorescence after acceptor photobleaching

and fluorescence lifetime measurement of the donor

Molecular interactions between neurodegenerative

disease-associated proteins in aggregates have been

ana-lyzed by FRET using the ratio imaging method For

example, polyQ-expanded proteins show strong FRET

in the inclusions when they are tagged with enhanced

(E)CFP as a donor and EYFP as an acceptor [20,66] PolyQ-expanded proteins (Q82–CFP) co-aggregate with normal-length polyQ (Q19–YFP), as detected by FRET [20] Because FRAP analysis in Q82–FLAG-based inclusions indicates that the exchange rate of Q19–GFP

is faster than that of Q82–GFP, Q19 interacts weakly with Q82 Although TATA-box binding protein, which contains a short polyQ tract, is also sequestered in the Q82 inclusion, this protein exhibited more rapid exchange than Q19–GFP Thus, protein mobility in inclusions appears to depend on the external polypep-tide sequence as well as on the length of the polyQ repeat In this report, the FRET efficiency was variable among cells, which suggests the presence of cell-to-cell heterogeneity in the molecular interactions within Q82–CFP⁄ Q19–YFP aggregates As FRET analysis via fluorescence recovery of the donor after acceptor photo-bleaching for Q40 in C elegans also indicates the pres-ence of heterogeneity in living neurons [67], the molecular interactions of polyQ proteins may be affected

by unknown cellular conditions FRET was also used to screen for inhibitors of polyQ aggregation in cultured cells, which indicates that this method has high-through-put potential [68] Recently, Takahashi et al [69] reported that soluble FRET-positive species of polyQ-expanded proteins were detected before inclusion-body formation, and FRET signals were significantly decreased by inhibitors of aggregation These observa-tions suggest that soluble oligomers⁄ aggregates of pol-yQ-expanded proteins are formed before the formation

of inclusion bodies, which is consistent with the results

of FCS analysis described above [42,63] and with studies reporting 4-50 nm particles of immunopurified polyQ-expansion proteins by atomic force microscopy [70,71] Fluorescence lifetime measurement of a donor fluorophore is available for quantitative FRET analy-sis [72] Fluorescence lifetime imaging microscopy can determine the distribution of fluorescence lifetime and

is appropriate for cell-based assays Fluorescence life-time imaging microscopy analysis led to the detection

of FRET signals for an ubiquitination substrate pro-tein tagged with EGFP as a donor and ubiquitin tagged with REACh (a non-fluorescent variant of yellow fluorescent protein) as an acceptor in cultured cells, which indicates that this method can be used to analyze the distribution of ubiquitin conjugates of spe-cific proteins [73] The ubiquitination of ALS-linked SOD1 in cultured cells was analyzed using this system, which revealed that the distribution of fluorescence lifetime was different for the G85R and G93A mutants [74] These observations are consistent with the fact that mutant SOD1 is polyubiquitinated for rapid degradation by the proteasome [75,76] and that the

G85R mutant is more structurally unstable than the

G93A mutant [6,7] Interestingly, the FRET efficiency

of the polyubiquitinated mutant, SOD1–G85R, was

stronger in a region near the plasma membrane than

in inclusions, which suggests a role for the

juxtamem-brane region in protein quality control As

proteaso-mal function is important for eliminating the potential

toxicity of mutant proteins, such as SOD1, detailed

spatiotemporal examination of the polyubiquitination

of mutant SOD1 using FRET may be useful for

inves-tigating the details of the role of the

ubiquitin–protea-some system in neurodegenerative diseases in vivo By

contrast, polyubiquitinated SOD1–G93A emitted

strong FRET signals in perinuclear inclusions The

localization of the polyubiquitinated mutant SOD1

may be affected by the structure of the mutant

pro-teins, because the G93A mutant is more structurally

stable than the G85R mutant The FRET efficiency of

SOD1–ubiquitin conjugates in cultured cells was highly

correlated with that of SOD1–HSP70 complexes,

which suggests that HSP70 plays a role in the

poly-ubiquitination of mutant SOD1, perhaps by

maintain-ing mutant SOD1 in a ubiquitination-competent state

Although FRET signals indicate steady-state

molec-ular interactions, the combination of FRET analysis

with other spectroscopic methods, including FRAP

and FCS, provides important information on the

dynamic biophysical properties of protein aggregates

The optical system of FCS can be applied to

single-molecule FRET analysis This method was used to

analyze aggregate formation and protein interactions

of polyQ-expanded proteins in vitro [43] In this study,

the fluorescence intensity of FRET and non-FRET

sig-nals was measured at the single-molecule sensitivity

level using a dual-color system, and direct interaction

between polyQ-expanded Huntingtin (Huntingtin-Q53)

and the molecular chaperone CCT was detected using

single-molecule FRET Similar methods may be

appli-cable to the analysis of the molecular structure of

polyQ-expanded proteins or ALS-linked SOD1 in

oligomers and aggregates formed in living cells

Inter-molecular FRET analysis has been applied to yeast

Sup35 prion proteins and revealed that Sup35 exists as

a monomer at low concentrations in vitro and adopts a

compact state in yeast [77] Intermolecular FRET may

also be useful for the analysis of the aggregation of

polyQ-expanded proteins or ALS-linked SOD1 in vivo

Conclusions and perspectives

We reviewed the dynamics and toxicity of misfolded

proteins (including polyQ-expanded proteins and

ALS-linked SOD1) in living cells, with a particular focus on

soluble oligomers⁄ aggregates Accumulating evidence strongly suggests that soluble oligomers of misfolded proteins are toxic to cells However, the exact molecu-lar mechanisms that underlie this cytotoxicity remain unknown Real-time spatiotemporal observation of misfolded proteins in vivo is essential for the full understanding of these mechanisms, and spectroscopic analyses in living cells will greatly aid the detailed analysis of the processes involved in these diseases In addition to the techniques described in this review, single-molecule observations in living cells may be required to elucidate how misfolded proteins produce toxic oligomers and interact with other proteins The improvement of microscopic techniques will promote the understanding of the dynamics and toxicity of mis-folded proteins in living cells in the near future

Acknowledgements

AK was supported by a fellowship of the Japan Society for the Promotion of Science (JSPS) HK was supported by Grant-in-Aid for Scientific Research Programs from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japanese Society for the Promotion of Science

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