Biochemistry 2006, 45, 15157-15167
15157
Solution State Characterization of Amyloid β-Derived Diffusible Ligands Robert W. Hepler,*,‡ Karen M. Grimm,‡,§ Deborah D. Nahas,‡ Robert Breese,| Elizabeth Chen Dodson,| Paul Acton,| Paul M. Keller,‡ Mark Yeager,‡ Hui Wang,‡ Paul Shughrue,| Gene Kinney,| and Joseph G. Joyce‡ Department of Vaccine & Biologics Research and Department of Alzheimer’s Research, Merck Research Laboratories, West Point, PennsylVania 19486 ReceiVed September 5, 2006; ReVised Manuscript ReceiVed October 30, 2006
A growing body of evidence suggests that soluble oligomeric forms of the amyloid β peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer’s disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide’s strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide’s interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.
ABSTRACT:
The amyloid β peptide (Aβ) is produced by proteolytic cleavage of amyloid precursor protein (APP) and is found in vivo primarily as a 39-43-residue peptide. Accumulation of Aβ, particularly the 42-amino acid form [Aβ(1-42)], leads to the formation of large, insoluble deposits (plaques) of aggregated peptide in the brain of patients with Alzheimer’s disease (AD) (1). It is this plaque deposition that is a hallmark in the post mortem analysis of the AD brain. The “amyloid cascade hypothesis” postulates that a time-dependent deposition of amyloid plaques in the brain is responsible for the neurodegeneration seen in AD (2). This hypothesis has been revised in recent years to account for the observation that early neurodegenerative symptoms in AD patients do not necessarily correlate with the onset of plaque deposition. A mounting body of evidence suggests that soluble oligomeric forms of Aβ known as amyloid-derived diffusible ligands (ADDLs) may constitute the acutely toxic peptide species (3-9). ADDLs have a demonstrated toxicity on neuronal cells in vitro (10) and can induce cognitive deficits when administered in vivo (11, 12). Long-term potentiation in hippocampal brain slices is negatively affected by preincubation of the slices with Aβ oligomers (13-15). The * To whom correspondence should be addressed: Department of Vaccine & Biologics Research, WP16-101, Merck Research Laboratories, West Point, PA 19486. Telephone: (215) 652-4786. Fax: (215) 652-7320. E-mail:
[email protected]. ‡ Department of Vaccine & Biologics Research. § Current address: Acologix, Inc., 3960 Point Eden Way, Hayward, CA 94545. | Department of Alzheimer’s Research.
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by cultured cells was shown to be affected by preincubation of the cells with oligomeric Aβ; however, the validity of this assay has been questioned (16). It is postulated that the physiological effects of ADDLs may be related to their ability to form ion channels in cell membranes, resulting in membrane depolarization (17). Aβ concentrations in vivo are in the low nanomolar range. This fact, coupled with its propensity to associate with other proteins, has precluded the isolation of large quantities of purified peptide from natural sources. The preparation of ADDLs was first reported by Lambert et al. (3), who incubated Aβ(1-42) with clusterin in the presence of components of cell culture media. Subsequently, Stine et al. (18) described a preparation method utilizing peptide alone in which synthetic Aβ(1-42) was first dissolved in hexafluoro2-propanol (HFIP) to eliminate any preexisting secondary structure that could act as a “seed” for aggregation. The HFIP was removed by evaporation, and the dried peptide film was then redissolved in dimethyl sulfoxide (DMSO) and diluted into cell culture medium followed by incubation at 2-8 °C to induce oligomer formation. More recent studies have reported that the HFIP-solubilized peptide may be directly diluted in water followed by evaporation of residual solvent and mechanical agitation to induce oligomer formation (19). Characterization of ADDL preparations has often utilized denaturing polyacrylamide gel electrophoresis (SDSPAGE), while atomic force microscopy (AFM) and, to a lesser degree, electron microscopy have been successfully used to visualize oligomeric assemblies and distinguish these
10.1021/bi061850f CCC: $33.50 © 2006 American Chemical Society Published on Web 12/06/2006
15158 Biochemistry, Vol. 45, No. 51, 2006 from monomer and fibrils. Clusterin-derived ADDLs examined by AFM were globular in nature; however, peptide prepared in the absence of clusterin tended to be more fibrillar in nature (3). The globules were shown to have a diameter of approximately 3.8-4.7 nm, which was theorized to correspond to a protein of 17000-42000 Da. Further support for the low-mass oligomer structure of ADDLs was provided by nondenaturing electrophoresis where bands were observed at predicted masses of 17 000 and 27 000 Da. Size exclusion chromatography (SEC), analytical ultracentrifugation (AU), and fluorescent dye binding have also been utilized in characterizing the components of this complex system, although to a much more limited degree. In 2003, Chromy et al. (6) characterized ADDL preparations using AFM, nondenaturing electrophoresis, and SEC to separate ADDLs into two distinct peaks. They found the preparation to be composed of two species, an early-eluting high-mass component and a more retained low-mass component. They further showed that while the higher-mass peak exhibited punctate binding to primary neurons, the lower-mass peak (estimated to be approximately 13 000 Da) did not bind. In this study, we sought to provide a definitive profile of the solution state of various forms of the Aβ peptide and to resolve the discrepancies between gel-based and solutionbased methods of analysis. Toward this end, we utilized SEC coupled with multiangle laser light scattering detection (SEC/ MALLS) and AU to characterize the solution behavior of monomeric peptide, ADDLs, and fibrils. Importantly, we provide definitive evidence using MALLS that previous reports identifying the low-molecular mass component as being composed of low-n oligomers are in error, that this species actually represents monomeric peptide, and that the high-molecular mass components are a polydisperse mixture of oligomeric species with molecular masses significantly higher than those previously proposed. We show definitively that intermediate-sized oligomers, postulated by gel-based analyses to be the major component of ADDL preparations, actually represent an insignificant mass of the total peptide in solution and are likely detergent-induced or detergentstabilized artifacts. EXPERIMENTAL PROCEDURES Peptides and Reagents. Aβ(1-42) and Aβ(1-40) peptides along with their N-terminal biotinylated analogues were purchased from American Peptide Co. (Sunnyvale, CA) at purity levels of >95%. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and anhydrous DMSO were purchased from SigmaAldrich (St. Louis, MO). Polyacrylamide gels, buffers, SYPRO-Ruby protein stain, and electrophoresis equipment were purchased from Invitrogen (Carlsbad, CA). ADDLs were prepared using slight modifications of previously described methods (20). Fibrils were prepared as described previously (18) by dissolving 0.5 mg of Aβ(142) peptide in 1 mL of 10 mM hydrochloric acid and incubating the mixture at 37 °C for 16-18 h. Alternatively, peptide was dissolved to a concentration of 5 mM in DMSO, diluted to 0.5 mg/mL in phosphate-buffered saline, and incubated at 37 °C for 16-18 h. Aβ monomer was prepared by dissolving 0.5 mg of HFIP-treated Aβ(1-40) or Aβ(142) in 1.0 mL of 25 mM sodium tetraborate (pH 8.5). These preparations were either used immediately [Aβ(1-42)] or
Current Topics flash-frozen and stored at -70 °C [Aβ(1-40)]. Biotinylated ADDLs (bADDLs) and monomer were prepared by the same protocols using N-terminally biotinylated peptides. Chromatography. Size exclusion chromatography was performed on an Agilent (Wilmington, DE) 1100 series HPLC system equipped with a diode array detector, a Wyatt Technology (Santa Barbara, CA) Optilab DSP interferometric refractometer, and a Wyatt DAWN EOS multiangle laser light scattering (MALLS) detector. Wyatt Astra software (version 4.90.07) was used to analyze all light scattering data. The refractive index increment for monomeric Aβ(1-42) was determined empirically using flow injection analysis of HFIP-treated peptide. Chromatographic separations were performed in 25 mM sodium phosphate (pH 7.3) at a flow rate of 0.5 mL/min on a 10 mm × 30 cm Superdex 75 column (Amersham Biosciences, Piscataway, NJ) preceded by a 5 mL guard column composed of prep grade Superdex 75. The column was run at room temperature, while the refractometer and light scattering cells were kept at 35 °C. Large-scale ADDL preparations were separated on a highresolution 1.6 cm × 60 cm Sephacryl S-200 column (Amersham Biosciences) run in 25 mM sodium phosphate (pH 7.3) at a flow rate of 0.5 mL/min. Five-minute fractions were collected on ice, and the peaks were pooled on the basis of the absorbance at 205 nm. Analytical Ultracentrifugation. Sedimentation velocity experiments were performed at 4, 20, and 30 °C on a Beckman (Fullerton, CA) XLI analytical ultracentrifuge using an An-60 Ti rotor and double-sector cells. Rotor speeds ranged from 20 000 to 50 000 rpm, and the sample absorbance was monitored at 280 nm for samples prepared in DMSO/F12 or at 215 nm for fractionated SEC peaks in phosphate buffer. Time derivative analysis was performed using the Beckman XLI data analysis software and DC/DT+ software (21, 22). Sedimentation equilibrium experiments were performed on Aβ(1-42) ADDL preps at 476, 238, and 151 µg/mL using six-channel cells. Experiments were performed at 4 °C at speeds of 10 000, 15 000, 20 000, 30 000, and 34 000 rpm. For Aβ(1-40) peptide prepared following the ADDL protocol, measurements were taken at sample concentrations of 476 and 238 µg/mL at a rotor speed of 35 000 rpm. Samples were centrifuged for 20 h before equilibrium absorbance measurements were taken. Molecular masses were calculated using Beckman XLI data analysis software. Gel Electrophoresis. SDS-PAGE was performed using Novex (Invitrogen) 10 to 20% Tricine gels, along with a Novex sample, and running buffers. Atomic Force Microscopy. Atomic force microscopy was performed essentially as described previously (3, 23) using a MultiMode atomic force microscope (Digital Instruments/ Veeco Metrology, Santa Barbara, CA) controlled by a NanoScope IIIa with NanoScope Extender electronics and Q-Control (nanoAnalytics, Mu¨nster, Germany). NanoScope operating software version 4.31 was used to acquire the data images, and Nanoscope offline software was used to render the data and perform the particle and section analyses after zero-order flattening of the image background. The z-height of >50 globules from several different areas on the mica was measured. Particle analysis was performed by choosing a threshold height equivalent to 1/2 the average z-height of
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Biochemistry, Vol. 45, No. 51, 2006 15159
FIGURE 1: (A) Refractive index trace of a standard ADDL preparation with an overlay of the actual molecular mass as calculated by MALLS using an empirically determined dN/dC of 0.241. (B) SEC-MALLs comparison of Aβ(1-42) (black circles) and Aβ(1-40) (red triangles) prepared using the standard ADDL preparation method.
the globules. This measures globule diameter at fwhm (full width at half-maximum). Primary Neurons. Primary hippocampal cultures were prepared from in-house frozen isolated rat hippocampal cells or commercial frozen dissociated rat hippocampal cells (Cambrex, East Rutherford, NJ) that were thawed and plated according to the manufacturer’s protocol in 96-well plates (Corning Costar, Acton, MA) at a concentration of 20 000 cells per well. The cells were maintained in Gibco Neurobasal medium without L-glutamine, supplemented with B27 (Invitrogen) for a period of 2-3 weeks before being used for binding studies. Neuronal Binding of the High- and Low-Molecular Mass Fractions of ADDLs. Neuronal binding of the high- and lowmass components of a bADDL preparation was investigated as described below by incubating cells with the high- and low-mass peaks of a bADDL preparation that had been isolated by SEC and adjusted to a concentration of 5 µM to match the protein concentration of the unfractionated bADDL control. Primary hippocampal neurons (cultured for 14-21 days) were incubated with 5 µM bADDLs or fractionated bADDL components for 1 h at 37 °C, and then the cells were washed three or four times with warm culture media [Gibco Neurobasal medium without L-glutamine, supplemented with B27 (Invitrogen)] to remove unbound peptide or oligomers. The cells were then fixed with 4% paraformaldehyde diluted in PBS (Electron Microscopy Sciences, Fort Washington, PA) for 10 min at room temperature. This solution was then removed, and fresh fixative was added for an additional 10 min at room temperature. The cells were then permeabilized in 4% paraformaldehyde and 0.1% Triton X-100 (Sigma Aldrich) for 10 min, washed six times with PBS, and incubated for 1 h at 37 °C with blocking buffer [PBS with 10% bovine serum albumin (BSA), Sigma]. For the detection of bADDL binding, the cells were incubated overnight with a 1:1000 dilution of a polyclonal antiserum raised against tau (Sigma Aldrich). The following morning, the cells were washed three times with PBS and incubated for 1 h at room temperature with an Alexa 488-labeled antirabbit secondary and an Alexa 594-labeled streptavidin (1: 1000 and 1:500, respectively, in PBS with 1% BSA) (Invitrogen), washed five or six times in PBS, and then visualized with a fluorescence microscope. Cell nuclei were labeled with a 1:1000 dilution of DAPI (Invitrogen) following standard protocols. To detect bound bADDLs with
monoclonal antibodies, cells were incubated overnight with an anti-ADDL monoclonal antibody (ACU-914, Acumen Pharmaceuticals, Inc., South San Francisco, CA), and in the morning, the immunoreactivity was detected with an Alexa 488-labeled anti-mouse secondary antibody. Bound bADDLs were visualized with an Alexa 594-labeled streptavidin, and the nuclei were stained with DAPI. RESULTS Biophysical Characterization of Amyloid Peptide Preparations Solution State Analysis. A primary objective of this study was to accurately characterize the solution state behavior of amyloid peptide formulations and determine how these data compared with previously reported analyses. To assess the size distribution of ADDLs, we employed SEC (a widely accepted method for size-based determinations), coupling it with laser light scattering detection, which allowed for precise determination of molecular masses. Figure 1A shows a representative refractive index trace of an ADDL preparation, which resolves into two distinct peaks. We marked the column with cytochrome c (Mw ) 12 500 Da) and bovine serum albumin (Mw ) 69 000 Da), and the elution position of these proteins is indicated above the chromatogram. When calibrated against these markers, the ADDL-related peaks have predicted molecular masses of 12000-15000 Da (tR ) 27 min) and 65000-80000 Da (tR ) 18 min). In contrast, the absolute mass determination made by multiangle laser light scattering indicates that the 27 min peak has an actual mass of approximately 4500 Da and thus represents monomeric peptide, while the 18 min peak is a polydisperse mixture of oligomers ranging in size from 150 000 Da at the trailing edge to nearly 1 000 000 Da at the leading edge of the peak. A very small amount of polydisperse material ranging in size from approximately 9000 to 150 000 Da can be detected between the two main components; however, no definitive peaks are observed in this region. Whole and fractionated ADDLs were also analyzed by AU, and these data are summarized in Table 1. ADDLs analyzed by sedimentation velocity runs at 4 °C were characterized by the presence of a high-molecular mass component which sedimented early in the run and a lowmolecular mass component which did not sediment under the same conditions. Time derivative analysis of the high-
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Table 1: Summary of AU and SEC-MALLS Data on the Molecular Masses of Various Amyloid β Preparations analytical ultracentrifugation velocity
a
equilibrium
SEC-MALLS
species
average S
estimated Mw (Da)
estimated Mw (Da)
estimated Mw (Da)
Aβ(1-42) ADDLs Aβ(1-40) ADDLs SEC high-molecular mass peak SEC low-molecular mass peak Aβ(1-42) fibrils
8.0 ND 6.7 0.55 31
175000 ND 223000 4500 >1000000
120000-540000 5400 ND ND ND
NAa 3500-12000 150000-1000000 4500 ND
Not applicable because SEC separates Aβ(1-42) ADDLs into two distinct peaks.
mass species revealed a unimodal distribution of oligomers that ranged in size from 4 to 12 S with an average sedimentation coefficient of 8 S, corresponding to an estimated molecular mass of 175 000 Da. Analyses performed at 20 and 30 °C showed a shift toward higher average S values, suggesting that aggregation was occurring as a function of increasing temperature (data not shown). We were able to achieve sedimentation of the lower-molecular mass component at higher rotor speeds; however, due to its small contribution to the total absorbance, it was difficult to obtain a reliable S value for this species. Equilibrium analyses were performed on ADDLs at three concentrations using multiple rotor speeds. A portion of the material in the ADDL preparations reached equilibrium during 10 000 and 15 000 rpm runs and yielded average masses of 120000-540000 Da. However, at higher speeds (20000-34000 rpm), very little of the remaining material reached an equilibrium state, suggesting that the high-mass material was completely sedimented early in the run while the lower-mass species remained in solution. Since the heterogeneous nature of an ADDL preparation rendered data interpretation and mass assignment by AU inconclusive, we scaled up the SEC fractionation protocol to obtain sufficient material for AU measurements to be made on the individual high- and low-molecular mass species. Since the stability of these fractions was tenuous, care was taken to tightly coordinate isolation with subsequent analysis. An additional benefit of this strategy was that formulation medium components that resulted in high background absorbances when analyzing whole ADDL preparations were removed by the fractionation procedure. The total peptide recovery through the scaled-up SEC separation was 70%, with approximately 10 and 90% recovered in the oligomer and monomer fractions, respectively. AU analysis of fractionated ADDLs confirmed the mass distributions observed using the MALLS data. Sedimentation velocity analysis performed on the oligomer fraction yielded an average sedimentation coefficient of 6.7 S with an estimated mass of 223 000 Da. The overall distribution was broad, ranging from approximately 3 to 11 S, again suggesting that a polydisperse population of oligomers is present in this peak. One significant observation from this analysis was the absence of any nonsedimenting material at S values of
