Báo cáo khoa học: Formation of highly toxic soluble amyloid beta oligomers by the molecular chaperone prefoldin pptx

Formation of highly toxic soluble amyloid beta oligomers by the molecular chaperone prefoldin Masafumi Sakono1,*, Tamotsu Zako1, Hiroshi Ueda2, Masafumi Yohda3 and Mizuo Maeda1 Bioengineering Laboratory, RIKEN Institute, Saitama, Japan Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Japan Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan 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 September 2008, accepted October 2008) 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 intracellularly, 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 doi:10.1111/j.1742-4658.2008.06727.x 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 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 5982 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS M Sakono et al well with the extent of synaptic loss and severity of cognitive impairment [3–9] The higher cytotoxicity of soluble Ab species compared with Ab fibrillar aggregates supports a casual relationship between the presence of soluble Ab species and AD It has been demonstrated that soluble Ab oligomers inhibit many critical neuronal activities, including long-term potentiation – 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 presence 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 nonnative 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 proteins Hsp20, Hsp70 and Hsp90, prevent Ab aggregation [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 apolipoprotein 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 biogenesis 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 Formation of amyloid beta oligomers by prefoldin 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 demonstrate that archaeal PFD from P horikoshii OT3 produces soluble and toxic high-molecular-mass Ab oligomers in vitro with a broad molecular mass distribution (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 fibrillation of Ab(1–42) peptide, the major factor responsible 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 50 °C in the absence of PFD increased after a lag phase of about h, and reached a plateau within h Examination by transmission electron microscopy (TEM) confirmed the formation of amyloid fibrils (Fig 2A) By contrast, when Ab was incubated with an equimolar amount of PFD at 50 °C, the increase in ThT fluorescence was inhibited (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 analysis by gel electrophoresis 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-molecularmass Ab oligomers with a broad range of molecular FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5983 Formation of amyloid beta oligomers by prefoldin M Sakono et al 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 determined 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-molecular-mass soluble Ab oligomers with a size distribution similar to that found in AD brains [30] A 40 ΔThT fluorescence intensity (A.U.) ΔThT fluorescence intensity (A.U.) 50 30 ThT fluorescence intensity (A.U.) 40 30 20 10 0 10 Incubation time (h) 20 10 + PFD – PFD 0 B 50 10 20 30 40 Incubation time (h) 50 60 50 40 30 20 10 0 0.2 0.4 0.6 [PFD]/[Aβ] 0.8 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 recognized by A11 antibody (Fig 5) Weak A11 immunoreactivity of the Ab ⁄ PFD sample was observed, but this might be due weak immunoreactivity with PFD rather than Ab, as shown in Fig This result suggests 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 Fig Ab aggregation monitored by ThT fluorescence (A) Time course of Ab aggregation monitored by ThT fluorescence Ab samples incubated in the presence (+PFD, closed circles) or absence ()PFD, open circles) of PFD were withdrawn at various time intervals and added to ThT solution Changes in the ThT fluorescence at 482 nm from the incubation time (0 h) are shown as DThT fluorescence 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 lM aliquot of Ab was incubated with PFD (0, 1, 3, 5, 10, 15, 25 and 30 lM) at 50 °C for 24 h, and added to ThT solution The x axis indicates the ratio of PFD concentration to Ab concentration mass (50–250 kDa) were observed Similar results were obtained when Ab was incubated with a lower concentration (1:10 ratio) of PFD at lower temperatures (37 and 42 °C) (data not shown) Ab oligomers formed in 5984 Dot-blot assay 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 molecular 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- FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS M Sakono et al Formation of amyloid beta oligomers by prefoldin A B 100 nm 100 nm C 50 nm Fig 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 ined the cytotoxicity of soluble Ab oligomers produced by the addition of PFD Ab aggregates of various concentrations 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-diphenyltetrazolium bromide (MTT) (Fig 6A) Addition of up to lm of Ab fibrils formed in the absence of PFD did not induce any major changes in cell viability FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5985 Formation of amyloid beta oligomers by prefoldin + PFD – PFD Incubation time (h) 48 M Sakono et al 1:Aβ/PFD 48 Antibody 250 anti-Aβ anti-PFD 2:Aβ monomer alone 3:PFD alone anti-Aβ anti-PFD 669 150 440 100 232 75 140 50 37 66 25 20 15 10 m (kDa) m (kDa) Fig SDS–PAGE analysis of Ab aggregates Samples incubated for or 48 h with PFD (+PFD) or without PFD ()PFD) were separated by SDS–PAGE (10–20% gels), probed using a mouse monoclonal Ab antibody (6E10), and visualized by chemiluminescence However, PC12 cell death was observed upon addition of lm Ab fibrils formed in the absence of PFD By contrast, addition of only 0.05 lm soluble Ab oligomers 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 observations 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 deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling (TUNEL) (Fig 7A) By contrast, only lowlevel 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 5986 Fig 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 lL of 50 lM sample mixture The same amount of Ab monomer alone and PFD alone were used as control samples An HMW native marker kit (GE Healthcare) 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 result is consistent with the results of the MTT assay, indicating a higher toxicity of PFD-induced Ab oligomers compared with Ab fibrils (Fig 6A) We also examined caspase-3 activation in PC12 cells PC cells exposed to 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 h of incubation in PFD-induced Ab oligomer samples, but was barely detected even after 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 formation 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 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS M Sakono et al Formation of amyloid beta oligomers by prefoldin Aβ/PFD + PFD – PFD A 100 Cell viability (%) 80 Aβ fibril A11-positive Aβ oligomer 60 40 20 0.01 0.05 0.1 0.2 0.5 Aβ concentration (μM) PFD Antibody A11 6E10 Fig Dot-blot assay of soluble Ab oligomers formed in the presence of PFD Samples of Ab oligomers formed in the presence 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 report our novel findings that the molecular chaperone PFD induces in vitro formation of soluble Ab oligomers 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 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 oligomerization also occurs intracellularly [13–19] Takahashi et al reported the existence of intracellular soluble Ab oligomers in Tg2576 transgenic mice [17], and Walsh et al showed that soluble oligomers are preferentially produced 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 shows a Cell viability (%) B 120 100 80 60 40 20 0.1 0.2 0.5 PFD concentration (μM) Fig 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 hypothetical model relating to the PFD-induced formation 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 substrate proteins was shown to be in dynamic equilibrium [36] This might also account for the fact that PFD has not been identified as one of the proteins that FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5987 Formation of amyloid beta oligomers by prefoldin Tunel PI A M Sakono et al + PFD – PFD – PFD + PFD B h 9 Procaspase-3 Activated caspase-3 β-actin Fig Apoptosis assays (A) DNA cleavage analyzed by the TUNEL assay PC12 cells were incubated with soluble Ab oligomers 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 labeling 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, or 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 bind to Ab peptides, as determined by co-immunoprecipitation 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 structural 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 synthesized or denatured proteins, and subsequently escort these to chaperonins for further assembly or final folding 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 ⁄ (the a-subunit) and PFD1 ⁄ ⁄ ⁄ (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, similar 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- PFD Inhibit/Slow down oligomerization LMW oligomer Aβ monomer 5988 HMW oligomer Aβ soluble oligomer Aβ protofibril Aβ fibril Fig Schematic model of the formation of soluble Ab oligomers in the presence of PFD FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS M Sakono et al tively; Fig S1) More interestingly, hydrophobic residues 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 various archaeal PFDs, are important for interaction and stabilization of a non-native substrate [35] Thus it is plausible that human PFD also utilizes these hydrophobic residues in the coiled coils to interact with its substrate 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 Pyrococcus PFD used in this study that might be important for their interaction with chaperonin [37] Thus, it is plausible that eukaryotic PFD could induce formation of Ab oligomers, as shown for archaeal PFD in this study This is speculative however, and further experiments using eukaryotic PFD are required to clarify possible involvement of PFD in AD pathology Experimental procedures Formation of amyloid beta oligomers by prefoldin 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 Lyophilized Ab(1–42) peptide (2 mgỈmL)1) was dissolved in HFIP, dried using a spin-vacuum system, and stored at )80 °C HFIP-treated peptide was dissolved to mm in distilled water with vortexing and sonication, immediately diluted to 50 lm in NaCl ⁄ Pi with or without 50 lm PFD, and then incubated at 50 °C for 48 h 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 NaCl ⁄ Pi at 50 °C Aliquots (2 lL) of the sample were withdrawn from the incubation mixture at various time intervals (0–48 h), and then added to 238 lL of 50 mm glycine–NaOH (pH 8.5) buffer containing lm ThT 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 concentrations (0, 1, 3, 5, 10, 15, 25 and 30 lm) in NaCl ⁄ Pi at 50 °C for 24 h Aliquots (2 lL) of the sample were added to 238 lL of lm ThT solution Each sample was excited at 445 nm (band width nm), and the emission was recorded at 482 nm on a spectrofluorometer (FP-6500; Jasco, Tokyo, Japan) The fluorescence intensity of lm ThT solution was used for background subtraction Materials TEM 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 Escherichia coli BL21 (DE3), and purified as previously 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 purchased from BioSource (Camarillo, CA, USA) Rat polyclonal 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, Kanagawa, Japan) Horseradish peroxidase-conjugated anti-rabbit The sample incubated at 50 °C for 48 h in the presence or 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 transmission electron microscope (JEOL, Tokyo, Japan) Analysis of Ab aggregates by SDS–PAGE/western blotting The sample mixture (5 lL) was diluted with lL SDS loading buffer containing 10% b-mercaptoethanol and then FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5989 Formation of amyloid beta oligomers by prefoldin M Sakono et al denatured at 98 °C for Following separation by SDS–PAGE using 10–20% Tris–glycine gels for 60 and a constant current of 20 mA, proteins were transferred onto poly(vinylidene difluoride) (PVDF) membranes (Millipore, Billerica, MA, USA) for h using a constant current of 140 mA For immunoblotting, the blot was blocked overnight in blocking reagent (Roche, Switzerland) at °C After washing away unbound material using NaCl ⁄ Tris containing 0.05% Tween-20 (0.05% NaCl ⁄ Tris-T), the membrane was incubated with mouse monoclonal Ab antibody (6E10, : 2000) for 40 at 37 °C, followed by secondary horseradish peroxidase-conjugated anti-mouse IgG (1 : 2000) Proteins were visualized using the ECL Plus blotting detection system (Amersham Biosciences) according to the manufacturer’s instructions Analysis of Ab aggregates by native PAGE/western blotting The sample mixture (5 lL) was diluted with 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 transfer to PVDF membrane, blots were probed using mouse monoclonal Ab antibody (6E10, : 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 molecular mass marker Dot-blot assay The dot-blot assay was performed as previously described [7] Peptide samples (30 lm) were incubated with or without 30 lm PFD in NaCl ⁄ Pi at 50 °C The sample mixture (3 lL) was spotted onto nitrocellulose membrane (0.22 lm; Whatman, Kent, UK) After blocking with 10% skim milk and 0.01% NaCl ⁄ Tris-T for h at room temperature, the membrane was incubated with rabbit polyclonal antibody to the oligomer (A11, : 500) or with mouse monoclonal Ab antibody (6E10, : 2000) for h at room temperature, followed by incubation with secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (each : 2000) for 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 incubated in NaCl ⁄ Pi at 25 °C for days as described previously [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) 5990 Rat PC12 cells (American Type Culture Collection, Manassas, VA, USA) were plated on poly-d-lysine-coated dishes in RPMI-1640 medium containing 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, 100 mL)1 penicillin, and 100 lgỈmL)1 streptomycin in humidified 5% CO2 incubators at 37 °C The medium was replaced every days PC12 cells (5 · 103 per well) were plated in 96-well plates 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 sample diluted in NaCl ⁄ Pi at various concentrations, which were taken from the 50 lm Ab samples incubated with or without PFD at 50 °C for 48 h As a control, the culture medium was replaced with the same volume of PFD samples in NaCl ⁄ Pi at various concentrations instead of Ab samples The cultures were incubated for 24 h, and then 10 lL of mgỈmL)1 MTT solution was added to each well and incubated for a further 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 chambers (4 · 105 cellsỈmL)1), and lm Ab aggregate was added to the culture medium Cultures were incubated for 24 h, fixed with 4% paraformaldehyde in NaCl ⁄ Pi for 25 at °C, permeabilized using 0.2% Triton X-100 in NaCl ⁄ Pi for at room temperature, and then incubated with propidium iodide (PI) and fluorescent-labeled 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 (excitation = 520–550 nm, emission > 580 nm) filter cubes Detection of activated caspase-3 PC12 cells exposed to Ab samples incubated with or without PFD for predefined times (3, or h) were lysed in RIPA buffer comprising 50 mm Tris ⁄ HCl (pH 7.2), 150 mm NaCl, 1% Triton X-100, 0.05% SDS, mm EDTA and mm MgCl2 Protein concentrations were determined 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 membrane, probed using mouse monoclonal b-actin antibody (1 : 2000) or rabbit polyclonal caspase-3 antibody (1 : 2000), and then detected as described above FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS M Sakono et al Acknowledgements We thank Dr Takao Yoshida, Japan Agency for Marine-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.) 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supplementary material is available: Fig S1 Sequence alignment of Pyrococcus prefoldin subunits and human prefoldin subunits This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5993 ... 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5983 Formation of amyloid beta oligomers by prefoldin M Sakono et al the presence of PFD were also separated by native PAGE and then subjected... 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... Examples of the Ab particles shown in (B) Scale bar = 50 nm ined the cytotoxicity of soluble Ab oligomers produced by the addition of PFD Ab aggregates of various concentrations were added to the