Surfactant properties of Alzheimer's A beta peptides and the mechanism of amyloid aggregation.

The major proteinaceous component of amyloid deposits associated with Alzheimer's disease is a self-assembling 40-42-residue peptide, known as A beta, which is generated by the proteolytic processing of the amyloid precursor protein (Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K., and Muller-Hill, B. (1987) Nature 325, 733-736; Haass, C., Hung, A. Y., Schlossmacher, M. G., Oltersdorf, T., Teplow, D. B., and Selkoe, D. J. (1993) Ann. N. Y. Acad. Sci. 695, 109-116; Estus, S., Golde, T. E., and Younkin S. G. (1992) Ann. N. Y. Acad. Sci. 674, 138-148) and is implicated as one of the causal factors in the pathology of the disease. A beta is now shown to display properties commonly associated with surfactants or detergents, which form micelles in solution. Increasing concentrations of A beta lower the surface tension of water up to a critical concentration, above which little further decrease in surface tension is observed. At concentrations above this critical concentration, increasing amounts of non-covalent aggregates of A beta are observed. A beta aggregation is also correlated with the formation of a hydrophobic environment that excludes water. The extent of this hydrophobic environment, as reflected by the partitioning of hydrophobic fluorescent probes, is much higher for the longer A beta 1-42 isoform, which may be more intimately associated with Alzheimer's disease pathology than A beta 1-40. Although the solution structure of A beta is not yet known, these results suggest that the structure of A beta has the same type of axial amphipathic organization as detergent molecules and that the same principles that govern micelle formation may also apply to A beta aggregation and amyloid fibril self-assembly.

the formation of a hydrophobic environment that excludes water. The extent of this hydrophobic environment, as reflected by the partitioning of hydrophobic fluorescent probes, is much higher for the longerApl-42 isoform, which may be more intimately associated with Alzheimer's disease pathology than Apl-40. Although the solution structure of AP is not yet known, these results suggest that the structure of AP has the same type of axial amphipathic organization as detergent molecules and that the same principles that govern micelle formation may also apply to AP aggregation and amyloid fibril self-assembly.
Investigations of the biological effects of AS on central nervous system neurons in vitro have provided data that support the idea that amyloid may contribute to neuronal degeneration. * This work was supported by the American Health Assistance Foundation and by National Institutes of Health Grants NS31230 (to C. G.) and AG00538. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The major form of AP associated with senile plaque amyloid deposits in Alzheimer's disease brain is Apl-42, while the major form associated with cerebrovascular amyloid deposits is Apl-40 (5, 6). Recent reports that indicate that Apl-42 production and deposition are enhanced in inherited, early onset forms of the disease suggest that Apl-42 is more intimately associated with AD pathology (7,8). Synthetic AP analogs of 1-36 residues or longer are toxic to cultured neuronal cells (9)(10)(11), and the toxicity is correlated with the increased aggregation state of the peptide (10,11).
These data and results from immunocytochemical studies suggesting that amyloid deposition may be a critical event in AD pathogenesis (4) have heightened interest in the structure of Ap a n d the mechanism of amyloid assembly. Electron microscopy has revealed that senile plaque amyloid deposits typically contain &IO-nm fibers (reviewed in Ref. 12). Rapid-freezing deep-etched images suggest that the amyloid fibril is hollow in the center and the wall of the fiber is composed of a helical arrangement of subunits with 5 s u b u n i t s h r n (13). Spectroscopic and x-ray diffraction studies indicate that amyloid fibril assembly is correlated with the adoption of a p sheet structure (14-17) and revealed an unusual cis peptide bond conformation between adjacent glycine residues in the carboxyl terminus of Ap (18). Biochemical studies of amyloid assembly in vitro have established many of the conditions that promote amyloid assembly, such as low pH (16,17,19), high concentrations of peptide (15,191, long incubation times (15,191, and salt concentration (15). Studies comparing the assembly properties of truncated forms ofAp and the effect of amino acid substitutions on fibril assembly have provided some information about the relationships between the structure of AP and its assembly properties (19, 20) and have implicated a nucleation step as a critical rate-limiting event in self-assembly (21).
Although some of the key structural features and biochemical properties of AP have been identified, the mechanism of amyloid fibril assembly is not clear. The sequence of Ap is amphipathic; the amino-terminal residues 1-28 ofAP are polar, derived from the extracellular domain, and residues 2 9 4 2 a r e hydrophobic, corresponding to the transmembrane domain of APP (1). We have investigated how this amphipathic organization affects the biochemical properties of AS isoforms. These results indicate that the naturally occurring AP isoforms display a number of properties associated with surfactants and suggest that the same principles that govern micelle formation may apply to amyloid self-assembly.
EXPERIMENTAL PROCEDURES Peptides-All AP analogs were synthesized by fluoren-g-ylmethoxycarbonyl chemistry using a continuous flow semiautomatic instrument, purified by reverse-phase high performance liquid chromatography, and the expected structure was verified by sequencing and electrospray mass spectrometry as described previously (19). Estimates of the fraction of expected product range from 85 to 90%, and no single failure product represents more than 1% of the total, which is typical of synthetic peptides of this size (19).
Surface Tension-Surface tension measurements were carried out using a Wilhelmy plate apparatus consisting of a 1-cm platinum plate suspended by a Cahn 2000 RG Electrobalance connected to a Perkin-Elmer R lOOA chart recorder. Solutions were measured in a Pyrex 9-well crystallization dish. The plate and dish were cleaned with SDS and then flamed to pyrolyze detergent residue and any dirt or dust particles. The force required to break the surface tension was recorded six times for each sample. The variation between individual measure-

Surfactant Properties of Amyloid
AP Peptides ments of the same sample was k0.2 dynes/cm. The variation between different samples was on the order of 21 dynes/cm. The surface tension was calculated from the chart recorder values using a calibration series of 10 standard solutions of known surface tension. Gel Filtration-Gel filtration analysis was performed with a Pharmacia Superdex 75 HR 10/30 column using a Pharmacia Biotech Inc. 500 fast protein liquid chromatography system, consisting of a Controller LCC-500 Plus, Pump P-500, Optical Unit UV-1, Control Unit UV-1, FRAC-100, and a REC 2. Lyophilized samples of Apl-40 and Apl-42 peptides were dissolved in 0.1 M Tris, pH 7.4, 0.02% sodium azide (column buffer) a t a concentration of 1 mg/ml and incubated at room temperature for 24 h. Samples were subsequently centrifuged at 14,000 rpm for 3 min, and a 200-pl aliquot of the supernatant was loaded onto a Pharmacia Superdex 75 HR 10/30 column and eluted a t a flow rate of 1 mumin. The peptide was detected by UV absorbance at 280 nm. The column was calibrated with the following standards: thyroglobulin, bovine serum albumin, ovalbumin, soybean trypsin inhibitor, ribonucletides with molecular weights of 2560, 1657, 1191, 1104, and 722, ase A, cytochrome c, aprotinin, insulin, insulin p chain, synthetic peptyrosine, and glucose.
SPEX Fluorolog-2 model F112A with a SPEX DMlB spectroscopy lab-Fluorescence-Fluorescence measurements were carried out using a oratory coordinator system. Experiments were carried out essentially as described (22). Stock solutions of Ap were prepared by dissolving lyophilized peptide in 88% formic acid immediately before use (a total time of less than 30 min). No evidence of peptide formylation was observed over this time period based on matrix-assisted laser desorption ionization mass spectroscopic measurements. Five pl of Ap stock solution were added to 95 pl of 0.1 M Tris, pH 7.4, 5 p 1,6-diphenyl-1,3,5-hexatriene (DPH) (Sigma), and fluorescent measurements of the samples were taken after incubation for 30 min in the dark. The excitation wavelength was 358 nm, and the emission wavelength was 430 nm.

RESULTS AND DISCUSSION
A classical property of surfactants is the ability to lower the surface tension of water due largely to the ordering of the amphiphile at the air-water interface with the hydrophobic moiety oriented away from the aqueous phase. Like detergents, the A0 peptides found in amyloid deposits lower the surface tension of water (Fig. 1). Increasing concentrations of AP analogs of 1-36 residues or longer progressively lower the surface tension of water up to a concentration of approximately 25 p~ (approximately 100 pg/ml). At higher concentrations there is little additional decrease in surface tension. The concentration above which little additional change in surface tension occurs defines the critical micelle concentration for detergents and is indicative of the fact that the amounts of detergent above this value contribute largely to micelle formation. All of the surfaceactive AP analogs display approximately the same critical concentration value of 25 p~. AP analogs of 30 residues or less cause little or no change in surface tension over the same concentrations, indicating that the hydrophobic carboxyl-terminal domain, which is derived from the transmembrane region of APP, is necessary for the detergent-like behavior of AP. A variety of control proteins, such as ovalbumin (not shown), apoferritin, and alcohol dehydrogenase (23), show a much more gradual decrease in surface tension as a function of concentration, which is typified by the behavior of Apl-28 (Fig. 1). Although the details of the solution structure of AP are not clear, its activity in lowering the surface tension of water indicates that the folded structure of AP is linearly amphipathic in the sense that one end is polar and the other end is non-polar, like the structures of surfactants or detergents.
The behavior of Ap in lowering the surface tension of water suggests that Ap may form micelle-like aggregates at concentrations above the critical concentration defined by the surface tension measurements described above. A@ aggregates have been demonstrated by a variety of methods, but one of the earliest reports of aggregate formation came from SDS-PAGE analysis of isolated amyloid deposits and synthetic A@ analogs The surface tension was measured using a Wilhelmy plate apparatus and calibrated using a standard curve as described under "Experimental Procedures." (1,15,19). Our previous studies demonstrated that Apl-39 and shorter analogs do not form SDS-stable aggregates. The concentration dependence of SDS-resistant aggregate formation by AP analogs is shown in Fig. 2. At concentrations below approximately 100 pg/ml, a single band with the expected apparent molecular mass of 4.0 kDa is observed. At concentrations above 100 pg/ml, increasing amounts of aggregates migrating with an apparent mass of 16 kDa are observed for peptides of 41 residues or longer. As previously noted, this band may correspond to a tetramer ofAp (19). These results indicate that the formation of AP aggregates, like micelle formation, is dependent on concentrations above the critical concentration defined by the surface tension measurements. Although Apl-40 does not form SDS-resistant aggregates, higher molecular weight aggregates are readily detected by gel filtration (Fig. 3). Apl-40 and Apl-42 were dissolved in 0.1 M Tris, pH 7.4, at a concentration of 0.22 mM, incubated at 20 "C for 24 h, and then centrifuged to remove any fibrillar assembled peptide (19). Both peptides elute as two well resolved, symmetrical peaks. The column was calibrated with 14 different protein and peptide standards in the mass range of 669 kDa to 722 Da. The first peak elutes at approximately the void volume of the column, which may represent aggregates greater than approximately 100 kDa. The second peak elutes at a position that corresponds to a molecular mass of 8-9 kDa, suggesting that it may represent a dimeric species as described previously (15,24). The void volume peak is the major species observed for Apl-42, while the dimer peak is much larger for Apl-40. The relative amounts of peptide that elute in the void volume peak and the apparent dimer peak depend on the peptide concentration. At concentrations of Ap below the critical concentration (25 p~) and as low as 10"O M, the dimer peak predominates (data not shown). The apparent aggregation number observed for Ap analogs by gel filtration is much higher than that ob- weight. This suggests that some of the peptide contact surfaces in the amyloid fibril are more sensitive to detergent.

B
Another important property of detergents is the formation of a hydrophobic environment that excludes water upon micelle assembly. This hydrophobic core can be detected by the partitioning. of hvdroDhobic dyes into this environment. which re-

Surfactant Properties of Amyloid AP Peptides
Our results demonstrate that the naturally occurring AP peptides display a number of properties commonly associated with surfactants. AP analogs greater than 33 residues lower the surface tension of water, and this effect displays a critical concentration point above which there is little additional change in surface tension. The fact that AP is surface active indicates that the solution structure of AP is axially amphipathic, where the amino-terminal domain is polar and the carboxyl-terminal domain is hydrophobic. Although this fact might seem trivial since the amino acid sequence is also amphipathic, this type of structural arrangement is unusual for proteins where most of the hydrophobic residues are buried within the interior of globular proteins in solution (25) . Endothelin 1 (26) and a-crystallin (22) also display surfactant-like properties and self-assemble to form higher molecular weight aggregates. Taken together, our results suggest that the same principles that govern the assembly of surfactants into micelles may also apply to the assembly of AP into amyloid fibrils. The assembly is stabilized by the "hydrophobic effect" (271, which minimizes the ordering of water molecules around the hydrophobic parts of the molecule. Although the hydrophobic effect may play an important role in amyloid self-assembly, this does not imply that AP self-assembly is necessarily as simple a process as micelle formation by common detergents. Unlike simple detergents, the polar domain ofAp is capable of stable self-association to form fibrils at acid pH, even in the absence of the stabilizing influence of the hydrophobic carboxyl terminus (16, 19).
Our current working model of amyloid fibril assembly postulates that the A@ peptide is organized within the amyloid fibril in the same fashion as detergent molecules are organized in a "tubular micelle" (also known as a hexagonal I type phase). The model predicts that the polar domain ofAp (approximately residues 1-28) forms the outer wall of the amyloid fibril, while the hydrophobic domain fills the center of the fiber. This model is consistent with the major structural features deduced from x-ray fiber diffraction investigations (14, 28). The "micellar" aggregates may represent short sections of the fibril, which may be intermediates in fibril assembly process, but this remains to be established.
The amphipathic structure and properties of AP may also have implications for the mechanisms of amyloid accumulation and neuronal degeneration. The most obvious possibility might be that A@ could act as a detergent to destabilize cellular membranes, but a substantial amount of evidence argues against a direct effect on cells by solubilizing membrane lipids (29, 30). We have previously found that when human fibroblasts are incubated in Apl-42 at concentrations above the critical concentration, the peptide is internalized by cells and accumulates in an aggregated form in lysosomes (31). This finding, coupled with the surfactant-like properties, suggests that AP may be a lysosomotropic detergent. This class of surfactant accumulates in lysosomes and ultimately results in cell death (32). The mechanism of cytotoxicity induced by lysosomotropic detergents involves a release of lysosomal contents into the cytoplasm followed by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death (33). Surprisingly, the effects of lysosomotropic detergents on lysosomal membrane permeability are not due to a direct solubilizing effect of the detergent on the lysosomal membrane (34). A number of laboratories have reported that AD is toxic to neurons (9-11,35,36) and that toxicity is correlated with the aggregation state of the peptide (10, 11). The LD,, values reported for AP toxicity are closely correlated with the critical concentration for AP aggregation defined by the surface tension measurements.
The amphipathic properties of AP are similar to the properties of another type of molecule that accumulates in aging and A D , lipofuscin (37). Lipofuscin from retinal pigmented epithelium has recently been shown to be a condensation product of retinaldelyde and ethanolamine and displays the properties of a self-assembling lysosomotropic detergent (37). The findings that AP is lysosomotropic and detergent-like may explain the increased accumulation of residual bodies in Alzheimer's disease and its association with a variety of lysosomal pathologies (38, 39).