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2008, 112, 16249–16252 Published on Web 09/13/2008
How Does Shear Affect Aβ Fibrillogenesis? Paul Hamilton-Brown, Innocent Bekard, William A. Ducker, and Dave E. Dunstan* Department of Chemical and Biomolecular Engineering and Particulate Fluids Processing Centre, UniVersity of Melbourne, Victoria 3010, Australia ReceiVed: June 15, 2008; ReVised Manuscript ReceiVed: July 15, 2008
The fibrillogenesis of Aβ1-40 proceeds via three main stages: (i) formation of aggregates from monomers, (ii) linear association of these aggregates to form “beaded” protofibrils, and (iii) fusion and structural reorganization of protofibrils into mature fibrils. We have studied the effect of shear on the rate of each of these steps through a combination of fluorescence, atomic force microscopy, and circular dichroism experiments. We find that shear increases the rate of the first two stages (aggregation and protofibril formation) and inhibits the third. Our hypothesis is that in the first stage shear mechanically perturbs the peptide from its native state inducing aggregation via hydrophobic interactions; in the second stage, shear enhances the linear alignment of aggregates due to minimization of drag in the shear flow field; in the third stage, exposure to constant and uniform shear inhibits the formation of mature fibrils. The partial unfolding of the native state of a protein can often lead it to aggregate along a nucleation-dependent pathway into well-defined fibrillar structures that are associated with disease states.1,2 For example, brain amyloid plaques composed of structured fibrillar forms of the amyloid-β protein (Aβ) are pathologic markers of Alzheimer’s disease (AD).3-5 Accumulating evidence suggests that AD is a vascular disorder associated with deterioration in vascular microcirculation in the brain.6,7 These changes in circulation may be accompanied by changes in shear stresses which can destabilize protein structure, and therefore could be a factor in the fibrilization of Aβ. Therefore, it is interesting to study the effect of shear on the development of amyloid fibrils from unstructured Aβ. Recently, we demonstrated that shearing dramatically accelerates the rate of amyloid formation from Aβ1-40 solutions.8 Thioflavin T (ThT) fluorescence increases rapidly when Aβ1-40/ ThT solutions are stirred with a magnetic stirrer bar (heterogeneous shear) or exposed to Couette flow (uniform shear). Atomic force microscopy (AFM) images indicate that fibrillogenesis proceeds via the formation of aggregates, the growth of “beaded” protofibrils, and then the fusing of protofibrils. Circular dichroism (CD) spectroscopy shows a concomitant increase in helical content followed by a transition to a β-sheet. We have also shown that ThT fluorescence is principally a determinant of the mature fibril content of the solution. Our observations of shear-induced acceleration of amyloid formation raise the following question: How does shear increase the rate of amyloid formation? The consensus of existing research is that the first step in amyloid formation is the partial unfolding of the protein from the native state.9,10 Our experiments8 show that shear-induced fibrillogenesis of Aβ1-40 occurs in the following stages: (i) monomers f aggregates, (ii) aggregates f protofibrils, (iii) protofibrils f mature fibrils; the same stages occur in the absence of shear.11-14 An increase in the overall rate requires an increase in the rate-determining step. Shear may accelerate only one stage; if so, which one? Here, 10.1021/jp805257n CCC: $40.75
TABLE 1: Descriptions of the Different Shearing Protocols protocol
experimental procedure
a
Sheared at 37 °C for ∼3 h. Stopped shearing when I < I0. Incubated at 37 °C for a further 24 h. Sheared at 37 °C for ∼4 h. Stopped shearing when I ) I0. Incubated at 37 °C for a further 17 h. Incubated at 37 °C for 27 h, without shearing. Sheared (at 37 °C) for a further 21 h.
b c
we determine which stages of Aβ fibrillogenesis are affected by shear. By selectively shearing Aβ1-40 solutions at particular times, we discover that shear accelerates the first two stagessformation of aggregates and protofibrilssand inhibits the thirdsformation of mature fibrils. To determine the effect of shear at each stage of fibrillogenesis, we performed a series of experiments where shearing was stopped, or started, at discrete points in the three-stage pathway (protocols a-c). The protocols are described in Table 1. The kinetics of fibrilization was determined from the fluorescence of ThT, the morphology of species in solution were imaged using AFM, and the secondary structure was determined using CD spectroscopy, as described earlier.8 Aβ1-40 (Keck Laboratories) solutions were prepared by direct dissolution of solid protein in 10 mM phosphate buffer (Sigma) and 50 µM aqueous thioflavin T (ThT) (Sigma) to yield a final protein concentration of 0.2 mg/mL (pH 7). The fluorescence was measured in situ in the cuvette cell, whereas both the CD spectroscopy and the AFM were done after an aliquot was removed from the cell. The AFM samples were imaged after the solution was dried onto mica. The fluorescence curves for various Aβ1-40 solutions sheared at particular times are shown in Figure 1. The fibrilization kinetics for a continuously sheared control sample at 37 °C (0) is consistent with results from our previous study; we observe the typical Aβ fibrilization sigmoid.11,14 This consists of a flat “lag phase” where AFM images show aggregates and a small 2008 American Chemical Society
16250 J. Phys. Chem. B, Vol. 112, No. 51, 2008
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Figure 1. ThT fluorescence for sheared Aβ (0.2 mg/mL, pH 7)/ThT solutions at 37 °C. The time scale has been offset so that the time is the same at I ) I0 for all experiments. Shearing was stopped at different time periods (fluorescence intensities) reflecting varying degrees of protofibril formation. (0) Continuously sheared control; (-) protocol a, shearing was stopped when I < I0 (short arrow at t ≈ 3 h); ([) protocol b, shearing was stopped when I ) I0 (long arrow at t ≈ 4 h); (+) protocol c, preheated for 27 h at 37 °C; (×) protocol c, preheated and then sheared.
number of protofibrils, followed by the so-called “elongation phase” (I/I0 > 1) where there is an increasing number of protofibrils and mature fibrils, and finally a plateau in fluorescence when the sample contains mainly mature fibrils. A solution incubated at 37 °C over the same time period without shearing, or any other form of agitation, exhibits no increase in fluorescence intensity. Our previous study showed that such a sample does not form fibrils even after three days at 37 °C. Thus, continuous shearing accelerates the formation of fibrils. We also note that, in all experiments, the fluorescence intensity initially decreases (I/I0 < 1) slightly during the lag phase. This may be due to a wavelength shift in the emission. In protocol a, shearing commences and is stopped in the lag phase (I/I0 < 1; t ∼3 h), i.e., when the sample contains mainly aggregates (Figure 2a1). When shearing is stopped during the lag phase, the rate of fluorescence increases slowly (Figure 1, protocol a), that is, much more slowly than if shearing were to be continued but more rapidly than if the solution were never sheared. Therefore, shearing in the lag phase does increase the rate of amyloid formation, but this is not the whole picture. The CD spectrum on cessation of shearing shows mainly random coil structures, while the sample at 27 h has a broad minimum denoting a combination of helices and β-sheets (Figure 3a). Images on cessation of shearing show mainly aggregates and few fibrils, whereas there are many fibrils and few aggregates at 27 h. In contrast, the continuously sheared sample at 27 h consists of mature fibrils, the fluorescence intensity has long plateaued, and the CD spectrum shows mainly β-sheets. Thus, shearing until partway through the lag phase accelerates the formation of aggregates and fibrils, but the progression of Aβ to the mature fibrils is much slower than that for a continuously sheared sample. In protocol b, we stopped shearing only after the fluorescence had begun to rise (I/I0 ) 1; t ∼ 4 h) (Figure 2b1). At the time that we stop shearing, CD data (Figure 3b) show that the R-helix content has increased, and from our earlier AFM work,8 we know that there are fibrils in solution at this stage. Even after we cease shearing, fluorescence continues to increase rapidly.
Figure 2. AFM images of amyloid fibril development in Aβ solutions exposed to different shear protocols. Protocol a: Shearing stopped at initial formation of protofibrils (a1), then incubated, unsheared, at 37 °C for a further 24 h (a2). Protocol b: Solution sheared until many protofibrils were formed (b1), then incubated, unsheared, at 37 °C for a further 17 h (b2). Protocol c: Aβ solution heated at 37 °C for 27 h until aggregates form (c1). Preaggregated solution from (c1) subsequently sheared for 20 h (c2). Scale bars represent 1 µm.
The rate is about 10-20% of the rate that occurred when we continued to shear right into the fluorescence plateau (Figure 1, curve [) and much greater than the rate if we stopped shearing when I/I0 < 1. After the fluorescence has reached a plateau (I/I0 ∼ 7), the sample consists of mainly mature fibrils (Figure 2b2) containing a high concentration of β-sheet structure. These data show that most of the direct modification of amyloid by shearing occurs before the sudden rise in fluorescence. The shearing encourages the formation of protofibrils, and the development beyond this stage (to mature fibrils) can proceed at a rapid pace without the need for further shearing. In comparison to protocol a, we see that the shearing must continue beyond I ) I0 or else the effect of shearing is greatly diminished. To further determine the effect of shear on the second stage of fibrillogenesis, we started shearing the Aβ solution only after a long lag period with no shearing (27 h at 37 °C) (Figure 1, protocol c). Normally, the formation of aggregates and protofibrils is accelerated when we shear solutions at 37 °C; however, only aggregates are formed within 24 h at 37 °C without shearing. For protocol c, we want to shear a solution which initially consists of just aggregates. We determined the point at which shearing was to start when AFM images showed that the sample consisted almost entirely of aggregates (Figure 2c1). The aggregates in this sample are indistinguishable from those produced by shearing at either 37 or 20 °C and have ap-
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Figure 4. Schematic depicting the effect of shear on the stages of Aβ fibrillogenesis.
Figure 3. CD spectra for Aβ solutions which were exposed to different shear protocols. Protocol a: Solutions heated at 37 °C for 27 h (corresponding to Figure 2a1), and preheated and subsequently sheared (Figure 2a2). Protocol b: Solution sheared until initial formation of protofibrils (corresponding to Figure 2b1), then incubated, unsheared, at 37 °C for a further 24 h (Figure 2b2). Protocol c: Solution sheared until many protofibrils were formed (Figure 2c1), then incubated without shearing, at 37 °C for a further 17 h (Figure 2c2).
proximately the same CD spectrum as at time zero (i.e., there is no increase in R-helix or β-sheet structures) (Figure 3c). As soon as we shear these species, the fluorescence intensity rises rapidly (Figure 1, curve ×), and mature fibrils (Figure 2c2) containing β-sheet structures (Figure 3c) are found in the sample. These results show that shear dramatically accelerates the conversion of aggregates to protofibrils. The aggregates can be formed by heating alone, but protocol a shows that their formation is enhanced by shear.8 In summary, shear dramatically enhances the rate of formation of protofibrils from aggregates, and does also enhance the rate of formation of aggregates. Note also that our CD data are in disagreement with previous studies which suggest that aggregates (nuclei) for fibril formation have unordered helical and intermolecular nonfibrillar β-structures;11,13,15 we only observe helical structures when the sample contains protofibrils. When we use Couette shear flow (as opposed to heterogeneous shear), we do not observe the formation of mature fibrils.
The fibrils observed resemble 3 nm high “beaded” protofibrils. We previously observed an identical result when comparing β-lactoglobulin solutions exposed to both types of shear.16 Furthermore, when solutions are sheared extensively, i.e., past the point at which mature fibrils have formed, we observe fibril breakage. The ensuing fibril fragments may act as “seeds” for the formation of subsequent fibrils incorporating Aβ monomers or aggregates.17,18 Hence, shear can indirectly contribute to an increased rate of fibril formation. Our findings on the effect of shear are summarized in Figure 4. The fibrillogenesis of Aβ1-40 proceeds via three principal stages: (i) unfolding and aggregation of monomers to form nuclei, (ii) linear association of these nuclei to form “beaded” protofibrils, and (iii) fusion and structural reorganization of protofibrils into mature fibrils. Shear accelerates the first two stages and inhibits the third. (i) Shearing increases the rate of Aβ aggregate formation. The rate can also be increased by raising the temperature: we find that the rate of aggregate formation is also higher at 37 °C than at 25 °C. A possible mechanism is that heating and/or shearing induce aggregation by thermally or mechanically perturbing the peptide from its “native state”. Some of these perturbed states may expose hitherto buried hydrophobic regions, hydrogen-bonding sites, or other groups that produce attractive forces between molecules and lead to aggregation of monomers.19,20 Furthermore, fibril formation occurs at a faster rate for solutions in the cuvette cell (as opposed to the Couette cell). In this instance, mixing facilitates the frequency of association of partially folded monomers resulting in their coalescence into aggregates, in agreement with molecular dynamics studies.21,22
16252 J. Phys. Chem. B, Vol. 112, No. 51, 2008 (ii) The rate of formation of “beaded” protofibrils, consisting of linearly aligned aggregates, is greatly accelerated in solutions that are exposed to shear. This alignment of aggregates in uniform shear stress fields has previously been observed for the von Willebrand factor (vWF).9 We hypothesize that the formation of linearly aligned aggregates (protofibrils) is due to minimization of drag in the flow field. Protofibrils also form over extended periods even in the absence of a flow field, so there must also be another force (e.g., attraction between R-helices) that drives aggregate alignment into fibrils. (iii) Shearing inhibits the net conversion of protofibrils to mature fibrils; we do not observe mature fibrils in solutions obtained from continuous and uniform shear (from a Couette cell). In contrast, heterogeneous shear (created by a magnetic stirrer at the base of the cuvette) provides a variety of shear stresses over time: high shear near the stirrer, and low at the top of the cuvette, away from the stirrer. Fusion and reorganization of protofibrils into β-sheet structures occurs in the low shear environment which does not exist in the constant shear flow of the Couette cell. Our results demonstrate that the formation of protofibrils is a rate-limiting step in Aβ fibrillogenesis: once these are formed, mature fibrils form rapidly. Acknowledgment. We thank Dr Kevin Barnham for the use of his CD spectrometer. Funding for this project was received from the Melbourne Research Grants System and CSL Ltd. W.A.D. is the recipient of an Australian Research Council Federation Fellowship (FF0348620). Note Added after ASAP Publication. This paper was published ASAP on September 13, 2008. Dave E. Dunstan was
Letters added as an author, and the Acknowledgment section was modified. The updated paper was reposted on November 21, 2008. References and Notes (1) Dobson, C. M. Nature 2003, 426, 884. (2) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329. (3) Harper, J. D.; Lieber, C. M.; Lansbury, P. T., Jr. Chem. Biol. 1997, 4, 951. (4) Walsh, D. M.; Lomakin, A.; Benedek, G. B.; Condron, M. M.; Teplow, D. B. J. Biol. Chem. 1997, 272, 22364–22372. (5) Sipe, J. D.; Cohen, A. S. J. Struct. Biol. 2000, 130, 88. (6) de la Torre, J. C. Neurol. Res. 2004, 26, 517. (7) Crawford, J. G. Med. Hypotheses 1996, 46, 367. (8) Hamilton-Brown, P.; Asimakis, P.; Ducker, W.; Dunstan, D. E. Soft Matter, submitted for publication, 2008. (9) Siediecki, C.; Lestini, B.; Kottke-Marchant, K.; Eppell, S.; Wilson, D.; Marchant, R. Blood 1996, 88, 2939. (10) Jarrett, J. T.; Lansbury, P. T., Jr. Biochemistry 1992, 31, 12345. (11) Benseny-Cases, N.; Cocera, M.; Cladera, J. Biochem. Biophys. Res. Commun. 2007, 361, 916. (12) Blackley, H. K. L.; Sanders, G. H. W.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Wilkinson, M. J. J. Mol. Biol. 2000, 298, 833. (13) Serpell, L. C. Biochim. Biophys. Acta 2000, 1502, 16. (14) Fezoui, Y.; Teplow, D. B. J. Biol. Chem. 2002, 277, 36948. (15) Chen, Y. R.; Huang, H. B.; Chyan, C. L.; Shiao, M. S.; Lin, T. H.; Chen, Y. C. J. Biochem. 2006, 139, 733. (16) Hamilton-Brown, P.; Asimakis, P.; Dunstan, D. E.; Ducker, W. Biomacromolecules, submitted for publication, 2008. (17) Goldsbury, C.; Frey, P.; Olivieri, V.; Aebi, U.; Muller, S. A. J. Mol. Biol. 2005, 352, 282. (18) Petkova, A. T.; Leapman, R. D.; Guo, Z.; Yau, W.-M.; Mattson, M. P.; Tycko, R. Science 2005, 307, 262. (19) Schladitz, C.; Vieira, E. P.; Hermel, H.; Mohwald, H. Biophys. J. 1999, 77, 3305. (20) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 6036. (21) Nguyen, H. D.; Hall, C. K. J. Biol. Chem. 2005, 280, 9074. (22) Smith, A. V.; Hall, C. K. J. Mol. Biol. 2001, 312, 1878.
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