As Pyrococcus PFD shares high sequence identity to human PFD and the PFD-homolog protein found in human brains, these results suggest that PFD may be involved in the formation of toxic s
Trang 1by the molecular chaperone prefoldin
Masafumi Sakono1,*, Tamotsu Zako1, Hiroshi Ueda2, Masafumi Yohda3and Mizuo Maeda1
1 Bioengineering Laboratory, RIKEN Institute, Saitama, Japan
2 Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Japan
3 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan
The neuropathology of Alzheimer’s disease (AD) is
characterized by loss of synapses and neurons in the
brain and the accumulation of senile plaques and
neurofibrillary tangles [1] The 39–43 amino acid Ab
peptides represent the principal components of
plaques, and are cleaved by secretases from parental
amyloid precursor protein localized to the plasma
membrane Synthetic Ab peptides have been shown to
spontaneously aggregate into b-sheet-rich fibrils
resem-bling those found in plaques These insoluble fibrillar forms were thought to cause neurotoxicity through oxidative stress both in vivo and in vitro However, the relevance of these plaques to AD pathogenesis remains unclear and is even questionable as there is no clear correlation between the number of amyloid plaque and the severity of dementia [2–5]
It has recently been suggested that soluble Ab species cause AD as the levels of these species correlate
Keywords
Alzheimer’s disease; amyloid b; molecular
chaperone; prefoldin; soluble 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
M Maeda, Bioengineering Laboratory,
RIKEN Institute, 2-1 Hirosawa, Wako,
Saitama 351 0198, Japan
Fax: +81 48 462 4658
Tel: +81 48 467 9312
E-mail: mizuo@riken.jp
*Present address
PRESTO, Japan Science and Technology
Agency, Saitama, Japan
(Received 16 June 2008, revised
5 September 2008, accepted 3 October
2008)
doi:10.1111/j.1742-4658.2008.06727.x
Alzheimer’s disease (AD) is a neurological disorder characterized by the presence of amyloid b (Ab) peptide fibrils and oligomers in the brain It has been suggested that soluble Ab oligomers, rather than Ab fibrils, contribute to neurodegeneration and dementia due to their higher level of toxicity Recent studies have shown that Ab is also generated intracellu-larly, where it can subsequently accumulate The observed inhibition of cytosolic proteasome by Ab suggests that Ab is located within the cytosolic compartment To date, although several proteins have been identified that are involved in the formation of soluble Ab oligomers, none of these have been shown to induce in vitro formation of the high-molecular-mass (> 50 kDa) oligomers found in AD brains Here, we examine the effects
of the jellyfish-shaped molecular chaperone prefoldin (PFD) on Ab(1–42) peptide aggregation in vitro PFD is thought to play a general role in
de novo protein folding in archaea, and in the biogenesis of actin, tubulin and possibly other proteins in the cytosol of eukaryotes We found that recombinant Pyrococcus PFD produced high-molecular-mass (50–250 kDa) soluble Ab oligomers, as opposed to Ab fibrils We also demonstrated that the soluble Ab oligomers were more toxic than Ab fibrils, and were capable
of inducing apoptosis As Pyrococcus PFD shares high sequence identity to human PFD and the PFD-homolog protein found in human brains, these results suggest that PFD may be involved in the formation of toxic soluble
Ab oligomers in the cytosolic compartment in vivo
Abbreviations
Ab, amyloid b; AD, Alzheimer’s disease; ADDL, Ab-derived diffusible ligand; HFIP, 1,1,1,3,3,3-hexa-fluoro-2-propanol; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PFD, prefoldin; PI, propidium iodide; PVDF, poly(vinylidene difluoride);
TEM, transmission electron microscopy; ThT, thioflavin T; TUNEL, terminal deoxynucletidyl transferase-mediated biotin-dUTP nick
end labeling.
Trang 2well with the extent of synaptic loss and severity of
cognitive impairment [3–9] The higher cytotoxicity of
soluble Ab species compared with Ab fibrillar
aggre-gates supports a casual relationship between the
pres-ence of soluble Ab species and AD It has been
demonstrated that soluble Ab oligomers inhibit many
critical neuronal activities, including long-term
potenti-ation – a classic model for synaptic plasticity and
memory loss in vivo and in culture [10–12]
Numerous experiments have demonstrated that Ab
generation and oligomerization occur intracellularly
[13–19] While intracellular accumulation of Ab occurs
in the mitochondria, ER and Golgi, it is predominant
in multivesicular bodies and lysosomes [13] The
pres-ence of intracellular Ab within multivesicular bodies
has been shown to be linked to cytosolic proteasome
inhibition [20–22] Furthermore, it has been shown
that proteasome inhibition, both in vivo and in vitro,
leads to higher Ab levels [23] As the proteasome is
primarily located within the cytosol, these findings
strongly support the notion that Ab is also located
within the cytosolic compartment
Molecular chaperones are proteins that selectively
recognize and bind to exposed hydrophobic surfaces of
non-native proteins, subsequently preventing protein
aggregation and facilitating correct folding of
non-native proteins in vivo [24] Molecular chaperones are
also involved in many important aspects of protein
homeostasis, degradation and subcellular trafficking
[25] Consistent with this activity, it has been shown
that molecular chaperones, including heat-shock
pro-teins Hsp20, Hsp70 and Hsp90, prevent Ab
aggrega-tion [18,26–28] Several molecular chaperones are also
known to be involved in the formation of toxic Ab
species Ab oligomers with low molecular mass
(< 30 kDa) have been shown to form in vitro during
incubation of Ab and the molecular chaperone
apoli-poprotein J, which has been found in AD brains
[10,29] However, Ab oligomers with a wide molecular
mass distribution (< 10 to > 100 kDa) are found in
the AD brain [30], suggesting that other factors are
involved in their formation
Prefoldin (PFD) is a molecular chaperone that has
been proposed to play a general role in de novo protein
folding in archaea, and is known to assist in the
bio-genesis of actins, tubulins and possibly other proteins
in the cytosol of eukaryotes [24] Eukaryotic PFD is
likely to bind to substrate proteins that exist in an
unfolded state, and transfer these to the cytosolic
chaperonin-containing TCP-1 (CCT) for functional
folding [31–33] Archaeal PFDs from Methanobacterium
thermoautotrophicum and Pyrococcus horikoshii OT3
have also been shown to stabilize non-native proteins
and denatured actins prior to chaperonin-dependent folding in vitro [34–38] Eukaryotic and archaeal PFDs possess a similar jellyfish-like structure consisting of a double b-barrel assembly with six long and protruding coiled coils [39,40] Biochemical and structural studies have indicated that these ‘tentacles’ bind to substrate proteins [34,35,40] In the current study, we demon-strate that archaeal PFD from P horikoshii OT3 produces soluble and toxic high-molecular-mass Ab oligomers in vitro with a broad molecular mass distri-bution (50–250 kDa) as found in AD brains [30] As it has been shown that eukaryotic PFD is homologous
to archaeal PFD [33,41] and is expressed in the human brain [42], our results suggest a possible involvement
of PFD in the formation of toxic Ab oligomers in the cytosol
Results
Fibrillation of Ab peptide in the presence of PFD
In an effort to investigate the effects of PFD on fibril-lation of Ab(1–42) peptide, the major factor responsi-ble for AD [1], Ab fibrillation was examined by monitoring levels of the fluorescent dye thioflavin T (ThT) [43] As shown in Fig 1A, ThT fluorescence of the Ab sample incubated at 50C in the absence of PFD increased after a lag phase of about 1 h, and reached a plateau within 5 h Examination by trans-mission electron microscopy (TEM) confirmed the for-mation of amyloid fibrils (Fig 2A) By contrast, when
Ab was incubated with an equimolar amount of PFD
at 50C, the increase in ThT fluorescence was inhib-ited (Fig 1A) This result suggests that Ab fibrillation
is inhibited by PFD About a one-third molar ratio of PFD to Ab was sufficient to inhibit Ab fibrillation (Fig 1B) TEM observations showed that no mature amyloid fibrils were formed after 48 h incubation in the presence of PFD (Fig 2B) Intriguingly, small particles and protofibrils were observed in samples incubated with PFD TEM photographs show that the size of most particles was within 100 nm and that the particles vary in shape (Fig 2C) These structures were not observed in control samples containing only PFD (data not shown)
Incubated Ab samples were then subjected to analy-sis by gel electrophoreanaly-sis Samples were separated by SDS–PAGE and probed with a mouse monoclonal Ab antibody (6E10) (Fig 3) Most Ab aggregates that formed in the absence of PFD were insoluble, and no soluble oligomers were observed On the other hand, when Ab was incubated with PFD, high-molecular-mass Ab oligomers with a broad range of molecular
Trang 3mass (50–250 kDa) were observed Similar results were
obtained when Ab was incubated with a lower
concen-tration (1:10 ratio) of PFD at lower temperatures (37
and 42C) (data not shown) Ab oligomers formed in
the presence of PFD were also separated by native PAGE and then subjected to western blot analysis using Ab antibody As shown in Fig 4, Ab oligomers with a broad range of molecular mass were also detected using Ab antibody, which indicates that the
Ab oligomers were in a soluble form The molecular mass of Ab oligomers was greater than that deter-mined by SDS–PAGE, possibly due to binding of PFD molecules to Ab oligomers (as described below) These results suggest that PFD inhibits Ab peptide fibrillation and induces the formation of high-molecu-lar-mass soluble Ab oligomers with a size distribution similar to that found in AD brains [30]
Dot-blot assay
In order to examine structural characteristics of the soluble Ab oligomers formed in the presence of PFD, binding to A11 antibody was examined A11 antibody recognizes prefibrillar Ab oligomers and protofibrils and does not react with Ab monomer or fibrils [7,44,45] A11-positive Ab oligomers were prepared as previously described [44,45] Interestingly, soluble Ab oligomers formed in the presence of PFD were not rec-ognized by A11 antibody (Fig 5) Weak A11 immuno-reactivity of the Ab⁄ PFD sample was observed, but this might be due weak immunoreactivity with PFD rather than Ab, as shown in Fig 5 This result sug-gests that the Ab oligomer conformation is different from that of A11-positive Ab oligomers This is consistent with recent results that suggest multiple Ab intermediate conformations [44,45]
Interaction between Ab oligomers and PFD
In an effort to elucidate the molecular mechanism of soluble Ab oligomer formation, binding of PFD with
Ab oligomers formed in the presence of PFD was analyzed by native PAGE⁄ western blot analysis using
Ab antibody and PFD antibody As shown in Fig 4, PFD that was bound to Ab oligomers of higher molec-ular mass was detected using PFD antibody This result indicates that Ab oligomers are formed as a complex with PFD The higher molecular mass of Ab oligomers than that shown by SDS–PAGE also supports formation of a complex between PFD and
Ab oligomers
Toxicity of Ab oligomers Soluble Ab oligomers are highly cytotoxic and are found in AD brains, and are therefore considered to
be the causative agents of the disease [3–6] We
exam-+ PFD – PFD
Incubation time (h) 0
10
20
30
40
50
A
B
Incubation time (h) 0
10 20 30 40 50
0 2 4 6 8 10
0
10
20
30
40
50
60
[PFD]/[A β]
Fig 1 Ab aggregation monitored by ThT fluorescence (A) Time
course of Ab aggregation monitored by ThT fluorescence Ab
sam-ples incubated in the presence (+PFD, closed circles) or absence
( )PFD, open circles) of PFD were withdrawn at various time
inter-vals and added to ThT solution Changes in the ThT fluorescence at
482 nm from the incubation time (0 h) are shown as DThT
fluore-scence The inset is an enlargement of the curves from 0–10 h.
(B) ThT fluorescence of Ab samples incubated with various PFD
concentrations A 30 l M aliquot of Ab was incubated with PFD
(0, 1, 3, 5, 10, 15, 25 and 30 l M ) at 50 C for 24 h, and added to
ThT solution The x axis indicates the ratio of PFD concentration to
Ab concentration.
Trang 4ined the cytotoxicity of soluble Ab oligomers produced
by the addition of PFD Ab aggregates of various
con-centrations were added to the culture medium of rat
pheochromocytoma PC12 cells, and cell viability was
assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (Fig 6A) Addition of up
to 1 lm of Ab fibrils formed in the absence of PFD did not induce any major changes in cell viability
50 nm
100 nm A
C
B
100 nm
Fig 2 Morphology of Ab aggregates formed in the presence of PFD (A) Ab fibrils formed in the absence of PFD Scale bar = 100 nm (B)
Ab particles and protofibrils formed in the presence of PFD Arrows indicate Ab particles shown in (C) Scale bar = 100 nm (C) Examples of the Ab particles shown in (B) Scale bar = 50 nm.
Trang 5However, PC12 cell death was observed upon addition
of 5 lm Ab fibrils formed in the absence of PFD By
contrast, addition of only 0.05 lm soluble Ab
oligo-mers formed in the presence of PFD markedly induced
PC12 cell death The observed level of cytotoxicity was
similar to that of Ab-derived diffusible ligands
(ADDL) [46] Control samples containing only PFD in
the same concentration range showed no detectable
cell toxicity (Fig 6B) Taken together with our
obser-vations concerning molecular size, these results support
our hypothesis that PFD mediates formation of Ab
oligomers similar to those found in AD brains
Apoptosis assay of cell death induced by
Ab aggregates
Ab peptides have been shown to induce apoptosis [47]
In an effort to determine whether this is also true of
soluble Ab oligomers produced in the presence of
PFD, we examined DNA fragmentation and activation
of the caspase cascade DNA fragmentation in PC12
cells incubated with PFD-induced Ab oligomers was
observed by green fluorescence using terminal
deoxy-nucleotidyl transferase-mediated biotin-dUTP nick end
labeling (TUNEL) (Fig 7A) By contrast, only
low-level green fluorescence was detected in PC12 cells
incubated with Ab fibrils formed in the absence of
PFD and in control cells incubated with NaCl⁄ Pi This
result is consistent with the results of the MTT assay, indicating a higher toxicity of PFD-induced Ab oligo-mers compared with Ab fibrils (Fig 6A)
We also examined caspase-3 activation in PC12 cells
PC cells exposed to 1 lm Ab samples incubated in the presence or absence of PFD were lysed and then subjected to western blotting analysis using caspase-3 antibody and a control b-actin antibody (Fig 7B) Activated caspase-3 was detected within 3 h of incuba-tion in PFD-induced Ab oligomer samples, but was barely detected even after 9 h of incubation in samples
of Ab fibrils formed in the absence of PFD Therefore,
we conclude that soluble Ab oligomers formed in the presence of PFD induce PC12 cell death via apoptotic pathways
Discussion Several molecular chaperones are involved in the for-mation of the low-molecular-mass (< 30 kDa) soluble
Ab oligomers or protofibrils that have been indicated
as the causative agents of AD [10,29,48] Here we
10
m (kDa)
– PFD Incubation time (h)
+ PFD
20
37
150
250
100
75
50
25
15
Fig 3 SDS–PAGE analysis of Ab aggregates Samples incubated
for 0 or 48 h with PFD (+PFD) or without PFD ( )PFD) were
sepa-rated by SDS–PAGE (10–20% gels), probed using a mouse
mono-clonal Ab antibody (6E10), and visualized by chemiluminescence.
alone
2:A β monomer alone
Antibody
669
440
232
140
66
m (kDa)
Fig 4 Native PAGE analysis of soluble Ab oligomers formed in the presence of PFD Ab aggregates formed in the presence of PFD (Ab ⁄ PFD) were analyzed by native PAGE, and probed using a mouse monoclonal Ab antibody (6E10) or a mouse polyclonal PFD antibody as indicated PFD bound to Ab oligomers is indicated by
an arrow The samples used comprised 5 lL of 50 l M sample mix-ture The same amount of Ab monomer alone and PFD alone were used as control samples An HMW native marker kit (GE Health-care) comprising thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and albumin (66 kDa) was used as a molecular mass marker.
Trang 6report our novel findings that the molecular chaperone
PFD induces in vitro formation of soluble Ab
oligo-mers with a high molecular mass (50–250 kDa) similar
to that found in AD brains Soluble Ab oligomers
formed in the presence of PFD were more toxic
compared with Ab fibrils, and exhibited similar
toxicities as ADDL via apoptotic cell-death pathways
(Figs 6 and 7) These data suggest that PFD might
also participate in the in vivo formation of highly toxic
Ab oligomers that lead to AD development
Recently, it has been reported that Ab
oligomeriza-tion also occurs intracellularly [13–19] Takahashi et al
reported the existence of intracellular soluble Ab
oligo-mers in Tg2576 transgenic mice [17], and Walsh et al
showed that soluble oligomers are preferentially
pro-duced intracellularly rather than extracellularly [16]
More importantly, inhibition of cytosolic proteasomes
by Ab implies that Ab is located within the cytosolic
compartment [13,20–23] It has been shown that a
PFD-like gene is expressed in the human brain [42]
These observations support the notion that PFD
participates in the formation of Ab oligomers within
the cytosolic compartment
In an effort to elucidate the mechanism pertaining
to the PFD-induced formation of high-molecular-mass
soluble Ab oligomers, we examined their interaction
with PFD As shown in Fig 4, bound PFD was
detected in soluble Ab oligomers Figure 8 shows a
hypothetical model relating to the PFD-induced for-mation of soluble Ab oligomers In this model, PFD inhibits or slows the oligomerization of Ab peptides by binding to the peptides in their oligomeric state Figure 1B suggests that the number of PFD molecules binding to one Ab oligomer molecule is at the most one-third the number of Ab molecules in one oligomer, which suggests that their interaction is non-specific Binding of PFD to protofibrils is indirectly supported
by TEM observations indicating that no Ab fibrils were formed in the presence of PFD (Fig 2) It is plausible that soluble Ab oligomers with a wide range
of molecular mass are produced due to repeated PFD binding and release, as the binding of PFD to sub-strate proteins was shown to be in dynamic equilib-rium [36] This might also account for the fact that PFD has not been identified as one of the proteins that
A
B
A β concentration (μ M )
+ PFD – PFD
0 20 40 60 80 100
0.01 0.05 0.1 0.2 0.5 1 5
PFD concentration (μM )
0 20 40 60 80 100 120
Fig 6 Cytotoxicity assays of soluble Ab oligomers formed in the presence of PFD against PC12 cells using the MTT method (A) Ab aggregates formed with PFD (+PFD, black bars) or without PFD ( )PFD, white bars) were incubated with cells at the indicated monomer concentrations (B) PFD was incubated with cells at the indicated concentrations.
A β/PFD
PFD
A β fibril
A11-positive
A β oligomer
Fig 5 Dot-blot assay of soluble Ab oligomers formed in the
pres-ence of PFD Samples of Ab oligomers formed in the prespres-ence of
PFD (Ab⁄ PFD), Ab fibrils formed in the absence of PFD (Ab fibril),
A11-positive Ab oligomers and PFD alone were prepared Aliquots
were spotted onto nitrocellulose membranes and probed with A11
and 6E10 antibodies.
Trang 7bind to Ab peptides, as determined by co-immunopre-cipitation studies [49,50] It should be noted that PFD does not facilitate or catalyze oligomer formation in this model This is supported by our observation of a ThT fluorescence time lag, which was not shortened by the addition of PFD (Fig 1A) Further studies are necessary to determine the precise mechanism of PFD-mediated oligomer formation
Archaeal PFD shares many biochemical and struc-tural characteristics with eukaryotic PFD [32–41,51] Both archaeal and eukaryotic PFDs share a jellyfish-like structure [39,40], and can bind and stabilize newly syn-thesized or denatured proteins, and subsequently escort these to chaperonins for further assembly or final fold-ing into active conformations In addition, archaeal PFDs are homologous to eukaryotic PFDs [33,41] Six distinct subunits of eukaryotic PFD can be grouped into two separate classes corresponding to the archaeal ones, represented by PFD3⁄ 5 (the a-subunit) and PFD1⁄ 2 ⁄ 4 ⁄ 6 (the b-subunit) The results of secondary structure prediction for human PFD showed that each human PFD subunit contains central b-hairpin(s) flanked N- and C-terminally by coiled-coil helices, simi-lar to Pyrococcus PFD (Fig S1) The coiled-coil helices within each Pyrococcus PFD subunit assemble in an antiparallel orientation [51] The result of primary sequence alignment also showed that Pyrococcus PFD shares high sequence identity to human PFD (59 and 62% similarity for the a-subunit to PFD3 and PFD5, respectively, and 62, 53, 58 and 68% similarity for the b-subunit to PFD1, PFD2, PFD4 and PFD6,
respec-Activated caspase-3
β-actin
+ PFD
A
B
– PFD
Tunel PI
Procaspase-3
Fig 7 Apoptosis assays (A) DNA cleavage analyzed by the
TUNEL assay PC12 cells were incubated with soluble Ab
oligo-mers formed in the presence of PFD (+PFD) or Ab fibrils formed
without PFD ( )PFD), and TUNEL-positive green fluorescence was
observed when DNA was cleaved into fragments (right) PI
label-ing indicates DNA in whole-cell nuclei (PI, left) (B) Time course of
caspase-3 activation PC12 cells were exposed to Ab aggregates
formed in the presence (+PFD) or absence ( )PFD) of PFD for 3, 6
or 9 h Equal amounts of proteins were separated on SDS–PAGE
using 10–20% gradient gels and probed using rabbit polyclonal
caspase-3 antibody or mouse monoclonal b-actin antibody as a
control.
PFD
LMW oligomer HMW oligomer
Inhibit/Slow down oligomerization
Fig 8 Schematic model of the formation of soluble Ab oligomers in the presence of PFD.
Trang 8tively; Fig S1) More interestingly, hydrophobic
resi-dues located at the first (a) and fourth (d) positions of
the heptad repeat (abcdefg) of the coiled-coil helices are
well conserved in both PFDs It has been shown that
the partially buried hydrophobic residues in these a⁄ d
positions, which are conserved in the coiled coils of
vari-ous archaeal PFDs, are important for interaction and
stabilization of a non-native substrate [35] Thus it is
plausible that human PFD also utilizes these
hydropho-bic residues in the coiled coils to interact with its
sub-strate The b-subunit of Pyrococcus PFD also shares
high sequence identity to the PFD-like protein (57%
similarity) found in the human brain [42] It should be
noted that the isoelectric point of Ab(1–42) peptide
(5.24) calculated from the amino acid sequence is similar
to that of known substrates of eukaryotic PFD, namely
b-actin (5.18) and b-tubulin (4.64), suggesting that Ab
peptide is a potential substrate for eukaryotic PFD This
idea is supported by the fact that there are hydrophilic
residues at the tips of the eukaryotic PFD tentacles that
appear to be important for interaction with substrate
proteins [40,52,53] Although archaeal PFDs have been
considered to bind a wide range of substrates through a
set of hydrophobic residues located at the tips of the
tentacles [34,35,39,52], it has also been shown that there
are basic residues in the distal regions of the tentacles of
PyrococcusPFD used in this study that might be
impor-tant for their interaction with chaperonin [37] Thus, it
is plausible that eukaryotic PFD could induce
forma-tion of Ab oligomers, as shown for archaeal PFD in this
study This is speculative however, and further
experi-ments using eukaryotic PFD are required to clarify
possible involvement of PFD in AD pathology
Experimental procedures
Materials
Ab(1–42), ThT, 1,1,1,3,3,3-hexa-fluoro-2-propanol (HFIP)
and RPMI-1640 medium were purchased from Sigma (St
Louis, MO, USA) P horikoshii PFD was expressed in
described [38] Rabbit polyclonal caspase-3 antibody was
purchased from Calbiochem (San Diego, CA, USA) Mouse
monoclonal b-actin antibody and mouse monoclonal Ab
antibody (6E10) were purchased from Abcam (Cambridge,
UK) A11 anti-oligomer rabbit polyclonal antibody was
pur-chased from BioSource (Camarillo, CA, USA) Rat
poly-clonal antibody to Thermoccocus PFD, which is highly
similar to P horikoshii PFD [54], was a kind gift from
T Yoshida (Extremobiosphere Research Center, Japan
Agency for Marine-Earth Science and Technology,
Kana-gawa, Japan) Horseradish peroxidase-conjugated anti-rabbit
IgG and horseradish peroxidase-conjugated anti-mouse IgG were purchased from R&D systems (Minneapolis, MN, USA) Enhanced chemiluminescence and western blotting detection systems were purchased obtained from Amersham Biosciences (Chalfont St Giles, UK) The cell proliferation kit (MTT) and the DeadEnd fluorometric TUNEL system were purchased from Roche (Indianapolis, IN, USA) and Promega (Madison, WI, USA), respectively
Preparation of Ab aggregates
HFIP, dried using a spin-vacuum system, and stored at )80 C HFIP-treated peptide was dissolved to 1 mm in distilled water with vortexing and sonication, immediately
ThT fluorescence assay
Ab fibrillation was assessed by the ThT assay as described previously [43] For the time-course assay, 30 lm peptide sample was incubated with or without 30 lm PFD in
withdrawn from the incubation mixture at various time intervals (0–48 h), and then added to 238 lL of 50 mm
Changes in the ThT fluorescence from the incubation time (0 h) are shown as DThT fluorescence Peptide samples (30 lm) were also incubated with PFD of various
to 238 lL of 5 lm ThT solution Each sample was excited
at 445 nm (band width 3 nm), and the emission was recorded at 482 nm on a spectrofluorometer (FP-6500; Jasco, Tokyo, Japan) The fluorescence intensity of 5 lm ThT solution was used for background subtraction
TEM
absence of PFD was diluted 10-fold with distilled water and placed on a carbon-coated copper grid and allowed to adsorb Excess sample was removed from the grid using filter paper, and the grid was air-dried prior to negative staining with uranyl acetate Excess stain was then removed from the grid by air drying Samples were observed with an excitation voltage of 100 kV using a JEM-1011 transmis-sion electron microscope (JEOL, Tokyo, Japan)
Analysis of Ab aggregates by SDS–PAGE/western blotting
The sample mixture (5 lL) was diluted with 5 lL SDS loading buffer containing 10% b-mercaptoethanol and then
Trang 9denatured at 98C for 3 min Following separation by
SDS–PAGE using 10–20% Tris–glycine gels for 60 min and
a constant current of 20 mA, proteins were transferred onto
poly(vinylidene difluoride) (PVDF) membranes (Millipore,
Billerica, MA, USA) for 2 h using a constant current of
140 mA For immunoblotting, the blot was blocked
membrane was incubated with mouse monoclonal Ab
sec-ondary horseradish peroxidase-conjugated anti-mouse IgG
(1 : 2000) Proteins were visualized using the ECL Plus
blotting detection system (Amersham Biosciences)
accord-ing to the manufacturer’s instructions
Analysis of Ab aggregates by native
PAGE/western blotting
The sample mixture (5 lL) was diluted with 5 lL native
PAGE sample buffer and then subjected to native PAGE
using a Tris–glycine 10–20% gradient precast gel (Wako,
Osaka, Japan) Samples containing Ab monomer alone or
PFD alone were used as control samples Following
trans-fer to PVDF membrane, blots were probed using mouse
monoclonal Ab antibody (6E10, 1 : 2000) or rat polyclonal
PFD antibody (1 : 2000) Bound antibodies were visualized
as described above An HMW native marker kit (GE
Healthcare, Chalfont St Giles UK) was used as the
mole-cular mass marker
Dot-blot assay
The dot-blot assay was performed as previously described
[7] Peptide samples (30 lm) were incubated with or without
was spotted onto nitrocellulose membrane (0.22 lm;
What-man, Kent, UK) After blocking with 10% skim milk and
mem-brane was incubated with rabbit polyclonal antibody to the
oligomer (A11, 1 : 500) or with mouse monoclonal Ab
anti-body (6E10, 1 : 2000) for 1 h at room temperature, followed
by incubation with secondary horseradish
peroxidase-conju-gated anti-rabbit or anti-mouse IgG (each 1 : 2000) for 1 h
at room temperature Proteins were visualized as described
above To prepare A11-positive Ab oligomers as a control
sample, 45 lm Ab(1–42) peptide samples diluted from
NaOH stock (2 mm Ab dissolved in 100 mm NaOH) were
previ-ously [44] Aliquots (2 lL) were spotted onto the membrane
Toxicity assay
Cell viability was determined by the MTT reduction assay
[55] according to the manufacturer’s instructions (Roche)
Manassas, VA, USA) were plated on poly-d-lysine-coated dishes in RPMI-1640 medium containing 10% heat-inacti-vated horse serum, 5% heat-inactiheat-inacti-vated fetal bovine serum,
replaced every 2 days
coated with poly-d-lysine, and covered with 100 lL culture medium Following plating, 20 lL medium was removed from each well, and replaced with the same volume of Ab
were taken from the 50 lm Ab samples incubated with or
medium was replaced with the same volume of PFD
samples The cultures were incubated for 24 h, and then
and incubated for a further 4 h Following incubation,
100 lL of 10% SDS in 0.01 m HCl was added to each well, and the cultures were incubated overnight The adsorption values at 550 nm were determined using a model 680 microplate reader (Bio-Rad, Hercules, CA, USA)
TUNEL assay
Apoptosis was detected by performing a TUNEL assay according to the manufacturer’s instructions (Promega) Briefly, PC12 cells were grown in poly-d-lysine-coated slide
added to the culture medium Cultures were incubated for
nucleotide in the presence of terminal deoxynucleotidyl transferase Cells were then examined using a fluorescence microscope (IX71; Olympus, Tokyo, Japan) Fluorescein and
PI were detected using U-MGFPHQ (excitation = 460–
480 nm, emission = 495–540 nm) and U-MWIG2 (excita-tion = 520–550 nm, emission > 580 nm) filter cubes
Detection of activated caspase-3
PC12 cells exposed to Ab samples incubated with or with-out PFD for predefined times (3, 6 or 9 h) were lysed in
150 mm NaCl, 1% Triton X-100, 0.05% SDS, 1 mm EDTA
by the Bradford assay using BSA as a standard Equal amounts of proteins were separated on a Tris–glycine 10– 20% gradient precast gel, transferred to a PVDF mem-brane, probed using mouse monoclonal b-actin antibody
(1 : 2000), and then detected as described above
Trang 10We thank Dr Takao Yoshida, Japan Agency for
Mar-ine-Earth Science and Technology (JAMSTEC) for
providing the antibody to Thermococcus PFD Funds
for this research were provided by RIKEN (M.S., T.Z
and M.M.) and the Ministry of Education, Science,
Sports, Culture and Technology of Japan (MEXT)
(T.Z., M.Y and M.M.) M.S is Special Postdoctoral
Researcher of RIKEN
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