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Keywords:

  • Alzheimer’s disease;
  • amyloid β;
  • molecular chaperone;
  • prefoldin;
  • soluble oligomers

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Alzheimer’s disease (AD) is a neurological disorder characterized by the presence of amyloid β (Aβ) peptide fibrils and oligomers in the brain. It has been suggested that soluble Aβ oligomers, rather than Aβ fibrils, contribute to neurodegeneration and dementia due to their higher level of toxicity. Recent studies have shown that Aβ is also generated intracellularly, where it can subsequently accumulate. The observed inhibition of cytosolic proteasome by Aβ suggests that Aβ is located within the cytosolic compartment. To date, although several proteins have been identified that are involved in the formation of soluble Aβ 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 Aβ(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 Aβ oligomers, as opposed to Aβ fibrils. We also demonstrated that the soluble Aβ oligomers were more toxic than Aβ 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 Aβ oligomers in the cytosolic compartment in vivo.

Abbreviations

amyloid β

AD

Alzheimer’s disease

ADDL

Aβ-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

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 Aβ peptides represent the principal components of plaques, and are cleaved by secretases from parental amyloid precursor protein localized to the plasma membrane. Synthetic Aβ peptides have been shown to spontaneously aggregate into β-sheet-rich fibrils resembling 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 Aβ species cause AD as the levels of these species correlate well with the extent of synaptic loss and severity of cognitive impairment [3–9]. The higher cytotoxicity of soluble Aβ species compared with Aβ fibrillar aggregates supports a casual relationship between the presence of soluble Aβ species and AD. It has been demonstrated that soluble Aβ 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 Aβ generation and oligomerization occur intracellularly [13–19]. While intracellular accumulation of Aβ occurs in the mitochondria, ER and Golgi, it is predominant in multivesicular bodies and lysosomes [13]. The presence of intracellular Aβ 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 Aβ levels [23]. As the proteasome is primarily located within the cytosol, these findings strongly support the notion that Aβ 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 proteins Hsp20, Hsp70 and Hsp90, prevent Aβ aggregation [18,26–28]. Several molecular chaperones are also known to be involved in the formation of toxic Aβ species. Aβ oligomers with low molecular mass (< 30 kDa) have been shown to form in vitro during incubation of Aβ and the molecular chaperone apolipoprotein J, which has been found in AD brains [10,29]. However, Aβ 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 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 β-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 Aβ 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 Aβ oligomers in the cytosol.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Fibrillation of Aβ peptide in the presence of PFD

In an effort to investigate the effects of PFD on fibrillation of Aβ(1–42) peptide, the major factor responsible for AD [1], Aβ fibrillation was examined by monitoring levels of the fluorescent dye thioflavin T (ThT) [43]. As shown in Fig. 1A, ThT fluorescence of the Aβ sample incubated at 50 °C in the absence of PFD increased after a lag phase of about 1 h, and reached a plateau within 5 h. Examination by transmission electron microscopy (TEM) confirmed the formation of amyloid fibrils (Fig. 2A). By contrast, when Aβ was incubated with an equimolar amount of PFD at 50 °C, the increase in ThT fluorescence was inhibited (Fig. 1A). This result suggests that Aβ fibrillation is inhibited by PFD. About a one-third molar ratio of PFD to Aβ was sufficient to inhibit Aβ 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).

image

Figure 1.  Aβ aggregation monitored by ThT fluorescence. (A) Time course of Aβ aggregation monitored by ThT fluorescence. Aβ 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 ΔThT fluorescence. The inset is an enlargement of the curves from 0–10 h. (B) ThT fluorescence of Aβ samples incubated with various PFD concentrations. A 30 μm aliquot of Aβ was incubated with PFD (0, 1, 3, 5, 10, 15, 25 and 30 μm) at 50 °C for 24 h, and added to ThT solution. The x axis indicates the ratio of PFD concentration to Aβ concentration.

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image

Figure 2.  Morphology of Aβ aggregates formed in the presence of PFD. (A) Aβ fibrils formed in the absence of PFD. Scale bar = 100 nm. (B) Aβ particles and protofibrils formed in the presence of PFD. Arrows indicate Aβ particles shown in (C). Scale bar = 100 nm. (C) Examples of the Aβ particles shown in (B). Scale bar = 50 nm.

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Incubated Aβ samples were then subjected to analysis by gel electrophoresis. Samples were separated by SDS–PAGE and probed with a mouse monoclonal Aβ antibody (6E10) (Fig. 3). Most Aβ aggregates that formed in the absence of PFD were insoluble, and no soluble oligomers were observed. On the other hand, when Aβ was incubated with PFD, high-molecular-mass Aβ oligomers with a broad range of molecular mass (50–250 kDa) were observed. Similar results were obtained when Aβ was incubated with a lower concentration (1:10 ratio) of PFD at lower temperatures (37 and 42 °C) (data not shown). Aβ oligomers formed in the presence of PFD were also separated by native PAGE and then subjected to western blot analysis using Aβ antibody. As shown in Fig. 4, Aβ oligomers with a broad range of molecular mass were also detected using Aβ antibody, which indicates that the Aβ oligomers were in a soluble form. The molecular mass of Aβ oligomers was greater than that determined by SDS–PAGE, possibly due to binding of PFD molecules to Aβ oligomers (as described below). These results suggest that PFD inhibits Aβ peptide fibrillation and induces the formation of high-molecular-mass soluble Aβ oligomers with a size distribution similar to that found in AD brains [30].

image

Figure 3.  SDS–PAGE analysis of Aβ aggregates. Samples incubated for 0 or 48 h with PFD (+PFD) or without PFD (−PFD) were separated by SDS–PAGE (10–20% gels), probed using a mouse monoclonal Aβ antibody (6E10), and visualized by chemiluminescence.

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image

Figure 4.  Native PAGE analysis of soluble Aβ oligomers formed in the presence of PFD. Aβ aggregates formed in the presence of PFD (Aβ/PFD) were analyzed by native PAGE, and probed using a mouse monoclonal Aβ antibody (6E10) or a mouse polyclonal PFD antibody as indicated. PFD bound to Aβ oligomers is indicated by an arrow. The samples used comprised 5 μL of 50 μm sample mixture. The same amount of Aβ 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.

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Dot-blot assay

In order to examine structural characteristics of the soluble Aβ oligomers formed in the presence of PFD, binding to A11 antibody was examined. A11 antibody recognizes prefibrillar Aβ oligomers and protofibrils and does not react with Aβ monomer or fibrils [7,44,45]. A11-positive Aβ oligomers were prepared as previously described [44,45]. Interestingly, soluble Aβ oligomers formed in the presence of PFD were not recognized by A11 antibody (Fig. 5). Weak A11 immunoreactivity of the Aβ/PFD sample was observed, but this might be due weak immunoreactivity with PFD rather than Aβ, as shown in Fig. 5. This result suggests that the Aβ oligomer conformation is different from that of A11-positive Aβ oligomers. This is consistent with recent results that suggest multiple Aβ intermediate conformations [44,45].

image

Figure 5.  Dot-blot assay of soluble Aβ oligomers formed in the presence of PFD. Samples of Aβ oligomers formed in the presence of PFD (Aβ/PFD), Aβ fibrils formed in the absence of PFD (Aβ fibril), A11-positive Aβ oligomers and PFD alone were prepared. Aliquots were spotted onto nitrocellulose membranes and probed with A11 and 6E10 antibodies.

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Interaction between Aβ oligomers and PFD

In an effort to elucidate the molecular mechanism of soluble Aβ oligomer formation, binding of PFD with Aβ oligomers formed in the presence of PFD was analyzed by native PAGE/western blot analysis using Aβ antibody and PFD antibody. As shown in Fig. 4, PFD that was bound to Aβ oligomers of higher molecular mass was detected using PFD antibody. This result indicates that Aβ oligomers are formed as a complex with PFD. The higher molecular mass of Aβ oligomers than that shown by SDS–PAGE also supports formation of a complex between PFD and Aβ oligomers.

Toxicity of Aβ oligomers

Soluble Aβ 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 examined the cytotoxicity of soluble Aβ oligomers produced by the addition of PFD. Aβ 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 1 μm of Aβ fibrils formed in the absence of PFD did not induce any major changes in cell viability. However, PC12 cell death was observed upon addition of 5 μm Aβ fibrils formed in the absence of PFD. By contrast, addition of only 0.05 μm soluble Aβ oligomers formed in the presence of PFD markedly induced PC12 cell death. The observed level of cytotoxicity was similar to that of Aβ-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 Aβ oligomers similar to those found in AD brains.

image

Figure 6.  Cytotoxicity assays of soluble Aβ oligomers formed in the presence of PFD against PC12 cells using the MTT method. (A) Aβ 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.

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Apoptosis assay of cell death induced by Aβ aggregates

Aβ peptides have been shown to induce apoptosis [47]. In an effort to determine whether this is also true of soluble Aβ 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 Aβ oligomers was observed by green fluorescence using terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling (TUNEL) (Fig. 7A). By contrast, only low-level green fluorescence was detected in PC12 cells incubated with Aβ 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 Aβ oligomers compared with Aβ fibrils (Fig. 6A).

image

Figure 7.  Apoptosis assays. (A) DNA cleavage analyzed by the TUNEL assay. PC12 cells were incubated with soluble Aβ oligomers formed in the presence of PFD (+PFD) or Aβ 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 Aβ 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 β-actin antibody as a control.

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We also examined caspase-3 activation in PC12 cells. PC cells exposed to 1 μm Aβ 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 β-actin antibody (Fig. 7B). Activated caspase-3 was detected within 3 h of incubation in PFD-induced Aβ oligomer samples, but was barely detected even after 9 h of incubation in samples of Aβ fibrils formed in the absence of PFD. Therefore, we conclude that soluble Aβ oligomers formed in the presence of PFD induce PC12 cell death via apoptotic pathways.

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Several molecular chaperones are involved in the formation of the low-molecular-mass (< 30 kDa) soluble Aβ oligomers or protofibrils that have been indicated as the causative agents of AD [10,29,48]. Here we report our novel findings that the molecular chaperone PFD induces in vitro formation of soluble Aβ oligomers with a high molecular mass (50–250 kDa) similar to that found in AD brains. Soluble Aβ oligomers formed in the presence of PFD were more toxic compared with Aβ 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 Aβ oligomers that lead to AD development.

Recently, it has been reported that Aβ oligomerization also occurs intracellularly [13–19]. Takahashi et al. reported the existence of intracellular soluble Aβ 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 Aβ implies that Aβ 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 Aβ oligomers within the cytosolic compartment.

In an effort to elucidate the mechanism pertaining to the PFD-induced formation of high-molecular-mass soluble Aβ oligomers, we examined their interaction with PFD. As shown in Fig. 4, bound PFD was detected in soluble Aβ oligomers. Figure 8 shows a hypothetical model relating to the PFD-induced formation of soluble Aβ oligomers. In this model, PFD inhibits or slows the oligomerization of Aβ peptides by binding to the peptides in their oligomeric state. Figure 1B suggests that the number of PFD molecules binding to one Aβ oligomer molecule is at the most one-third the number of Aβ 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 Aβ fibrils were formed in the presence of PFD (Fig. 2). It is plausible that soluble Aβ 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 bind to Aβ 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.

image

Figure 8.  Schematic model of the formation of soluble Aβ oligomers in the presence of PFD.

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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 PFD3/5 (the α-subunit) and PFD1/2/4/6 (the β-subunit). The results of secondary structure prediction for human PFD showed that each human PFD subunit contains central β-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 α-subunit to PFD3 and PFD5, respectively, and 62, 53, 58 and 68% similarity for the β-subunit to PFD1, PFD2, PFD4 and PFD6, respectively; 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 β-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 Aβ(1–42) peptide (5.24) calculated from the amino acid sequence is similar to that of known substrates of eukaryotic PFD, namely β-actin (5.18) and β-tubulin (4.64), suggesting that Aβ 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 Aβ 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

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

Aβ(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 β-actin antibody and mouse monoclonal Aβ 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 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 Aβ aggregates

Lyophilized Aβ(1–42) peptide (2 mg·mL−1) was dissolved in 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 diluted to 50 μm in NaCl/Pi with or without 50 μm PFD, and then incubated at 50 °C for 48 h.

ThT fluorescence assay

Aβ fibrillation was assessed by the ThT assay as described previously [43]. For the time-course assay, 30 μm peptide sample was incubated with or without 30 μm PFD in NaCl/Pi at 50 °C. Aliquots (2 μL) of the sample were withdrawn from the incubation mixture at various time intervals (0–48 h), and then added to 238 μL of 50 mm glycine–NaOH (pH 8.5) buffer containing 5 μm ThT. Changes in the ThT fluorescence from the incubation time (0 h) are shown as ΔThT fluorescence. Peptide samples (30 μm) were also incubated with PFD of various concentrations (0, 1, 3, 5, 10, 15, 25 and 30 μm) in NaCl/Pi at 50 °C for 24 h. Aliquots (2 μL) of the sample were added to 238 μL of 5 μm 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 μm ThT solution was used for background subtraction.

TEM

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 Aβ aggregates by SDS–PAGE/western blotting

The sample mixture (5 μL) was diluted with 5 μL SDS loading buffer containing 10%β-mercaptoethanol and then denatured at 98 °C 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 overnight in blocking reagent (Roche, Switzerland) at 4 °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 Aβ antibody (6E10, 1 : 2000) for 40 min 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 Aβ aggregates by native PAGE/western blotting

The sample mixture (5 μL) was diluted with 5 μL native PAGE sample buffer and then subjected to native PAGE using a Tris–glycine 10–20% gradient precast gel (Wako, Osaka, Japan). Samples containing Aβ monomer alone or PFD alone were used as control samples. Following transfer to PVDF membrane, blots were probed using mouse monoclonal Aβ 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 molecular mass marker.

Dot-blot assay

The dot-blot assay was performed as previously described [7]. Peptide samples (30 μm) were incubated with or without 30 μm PFD in NaCl/Pi at 50 °C. The sample mixture (3 μL) was spotted onto nitrocellulose membrane (0.22 μm; Whatman, Kent, UK). After blocking with 10% skim milk and 0.01% NaCl/Tris-T for 1 h at room temperature, the membrane was incubated with rabbit polyclonal antibody to the oligomer (A11, 1 : 500) or with mouse monoclonal Aβ antibody (6E10, 1 : 2000) for 1 h at room temperature, followed by incubation with secondary horseradish peroxidase-conjugated 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 Aβ oligomers as a control sample, 45 μm Aβ(1–42) peptide samples diluted from NaOH stock (2 mm Aβ dissolved in 100 mm NaOH) were incubated in NaCl/Pi at 25 °C for 7 days as described previously [44]. Aliquots (2 μL) were spotted onto the membrane.

Toxicity assay

Cell viability was determined by the MTT reduction assay [55] according to the manufacturer’s instructions (Roche). 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 U·mL−1 penicillin, and 100 μg·mL−1 streptomycin in humidified 5% CO2 incubators at 37 °C. The medium was replaced every 2 days.

PC12 cells (5 × 103 per well) were plated in 96-well plates coated with poly-d-lysine, and covered with 100 μL culture medium. Following plating, 20 μL medium was removed from each well, and replaced with the same volume of Aβ sample diluted in NaCl/Pi at various concentrations, which were taken from the 50 μm Aβ 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 Aβ samples. The cultures were incubated for 24 h, and then 10 μL of 5 mg·mL−1 MTT solution was added to each well and incubated for a further 4 h. Following incubation, 100 μL 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 cells·mL−1), and 1 μm Aβ aggregate was added to the culture medium. Cultures were incubated for 24 h, fixed with 4% paraformaldehyde in NaCl/Pi for 25 min at 4 °C, permeabilized using 0.2% Triton X-100 in NaCl/Pi for 5 min 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 Aβ samples incubated with or without PFD for predefined times (3, 6 or 9 h) were lysed in RIPA buffer comprising 50 mm Tris/HCl (pH 7.2), 150 mm NaCl, 1% Triton X-100, 0.05% SDS, 1 mm EDTA and 1 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 β-actin antibody (1 : 2000) or rabbit polyclonal caspase-3 antibody (1 : 2000), and then detected as described above.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

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.). M.S. is Special Postdoctoral Researcher of RIKEN.

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Fig. S1. Sequence alignment of Pyrococcus prefoldin subunits and human prefoldin subunits.

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