Nichols Et Al-2005-Microscopy Research and Technique

Amyloid-b Aggregates Formed at PolarNonpolar InterfacesDiffer From Amyloid-b Protobrils Produced inAqueous BuffersMICHAEL R. NICHOLS,1 MELISSA A. MOSS,1 DANA KIM REED,1 JAN H. HOH,2

AND TERRONE L. ROSENBERRY1*1Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, Florida 322242Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

KEY WORDS Alzheimers disease; peptide b-structure; brils; stability; hexauoroisopropanol

ABSTRACT The deposition of aggregated amyloid-b (Ab) peptides in the brain as senileplaques is a pathological hallmark of Alzheimers disease (AD). Several lines of evidence indicate thatbrillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important inthe etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that asingle polypeptide can fold into multiple amyloid conformations. Here we review our recent reportsthat Ab(1-40) in vitro can form soluble aggregates with predominant b-structures that differ instability and morphology. One class of aggregates involved soluble Ab protobrils, prepared by vig-orous overnight agitation of monomeric Ab(1-40) in low ionic strength buffers. These aggregateswere quite stable and disaggregated to only a limited extent on dilution. A second class of solubleAb aggregates was generated at polarnonpolar interfaces. Aggregation in a two-phase system ofbuffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexauoroisopro-panol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than10 minutes. Like Ab protobrils, these interfacial aggregates showed increased thioavin T uores-cence and were rich in b-structure by circular dichroism. However, electron microscopy and atomicforce microscopy revealed very different morphologies. The HFIP aggregates formed initial globu-lar clusters that progressed over several days to soluble brous aggregates. When diluted out ofHFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes.However, their stability increased as they progressed to bers. It is important to determinewhether similar interfacial Ab aggregates are produced in vivo. Microsc. Res. Tech. 67:164174,2005. ' 2005 Wiley-Liss, Inc.VVC 2004 Wiley-Liss, Inc.

INTRODUCTION

The brains of patients with Alzheimers disease (AD)contain large numbers of amyloid deposits in the formof senile plaques (Cohen and Calkins, 1959). The amy-loid core of the plaques consists of interwoven brils,each 79 nm in diameter (Terry et al., 1964), that canbe visualized by light microscopy after staining withCongo Red or thioavin S (Terry, 1985). The brils arecomposed of 40- and 42-residue peptides (Glenner andWong, 1984; Miller et al., 1993), denoted Ab(1-40) andAb(1-42). These peptides are produced by cleavage ofcellular amyloid precursor protein (APP) by two pro-teases called b- and g-secretase (reviewed in Selkoe,2001). As originally suggested by the amyloid cascadehypothesis (Hardy and Higgins, 1992), it appears likelythat Ab aggregates are important in the etiology of AD.The most striking evidence supporting this hypothesiscomes from the identication of numerous mutationslinked to early-onset familial AD (FAD) (Selkoe andPodlisny, 2002). These mutations are located withinthe APP gene or the genes for presenilins 1 and 2,which play an integral role in g-secretase activity. AllFAD mutations reported thus far increase either thelevel of the more amyloidogenic Ab(1-42) peptide

(reviewed in Selkoe and Podlisny, 2002) or the propen-sity of a mutated Ab peptide to form amyloid aggre-gates (Nilsberth et al., 2001).The amyloid hypothesis for the etiology of AD is also

supported by early results from therapeutic strategiesthat reduce the amount of aggregated Ab in the brain(Lansbury, 1997). An extremely promising example is avaccine comprised of Ab(1-42) (Schenk et al., 1999).While a clinical trial with this vaccine was haltedbecause of adverse side effects in 5% of individuals(Schenk, 2002), the antibodies generated by patients in

*Correspondence to: T.L. Rosenberry, Department of Neuroscience, MayoClinic College of Medicine, 4500 San Pablo Road, Jacksonville, FL 32224.E-mail: [email protected]

Contract grant sponsor: American Heart Association, Florida/Puerto RicoAfliate (to M.R.N. and M.A.M.)

Received 21 January 2005; accepted in revised form 21 March 2005

DOI 10.1002/jemt.20189

Published online inWiley InterScience (www.interscience.wiley.com).

Abbreviations: AD, Alzheimers Disease; AFM, atomic force microscopy; APP,amyloid precursor protein; CD, circular dichroism; DLS, dynamic light scattering;EM, electron microscopy; FAD, early-onset familial AD; HFIP, hexauoroisopropa-nol; MALS, multi-angle light scattering; SEC, size exclusion chromatography.

VVC 2005 WILEY-LISS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 67:164174 (2005)

this trial appeared to be effective. Twenty patients gen-erated antibodies against Ab, as determined by tissueamyloid plaque immunoreactivity assay. Thesepatients showed signicantly slower rates of decline ofcognitive functions and activities of daily living thanthose observed with patients who did not produce suchantibodies (Hock et al., 2003).Early evidence suggested that brillar deposits of Ab

initiate a cascade of events that result in neuronal celldeath and lead to the cognitive decline characteristic ofAD (Yankner, 1996). However, concerns were raisedbecause the number of amyloid deposits detected byneuropathological analysis of postmortem brains doesnot correlate well with the degree of cognitive impair-ment experienced by AD patients prior to death (Hardyand Selkoe, 2002). A number of investigators nowpropose that soluble aggregates of Ab (also calledoligomers or protobrils), rather than monomers orinsoluble amyloid brils, may be responsible for synap-tic dysfunction in the brains of AD patients and ADanimal models (Hardy and Selkoe, 2002; Hartley et al.,1999; Klein et al., 2001; Westerman et al., 2002;Kawarabayashi et al., 2004). This proposal is sup-ported by observations that soluble aggregates gener-ated in vitro from synthetic Ab(1-40) and (1-42)induced toxicity in cultured cells (Hartley et al., 1999;Lambert et al., 1998), that soluble Ab aggregates pro-duced in cell culture markedly inhibited hippocampallong-term potentiation in rats in vivo (Walsh et al.,2002), and that transgenic mice expressing human Abshow functional decits that precede extracellular dep-osition of brillar Ab (Hsia et al., 1999; Westermanet al., 2002).A mechanistic understanding of the brillogenesis

process would be very useful in identifying individualsteps as therapeutic targets (Teplow, 1998). Since thisunderstanding is difcult to attain in the complex envi-ronment of living cells, it is fortunate that syntheticAb(1-40) and Ab(1-42) form both soluble aggregatesand amyloid brils in vitro that share many featureswith the amyloid in AD plaques as well as the amyloidbrils formed by other proteins in a number of dis-eases. These brils contain peptide segments that alignto form b-sheets with extended peptide strands perpen-dicular to and interstrand H-bonds parallel to the brilaxis (Sunde et al., 1997; Serpell et al., 2000). Thiscross-b structure may underlie common properties,including the binding of dyes like thioavin T andCongo Red and of antibodies that react with a commonepitope in several amyloidogenic proteins (ONuallainand Wetzel, 2002; Kayed et al., 2003). However, recentreports also indicate a fascinating degree of amyloidpolymorphism at the molecular level. This feature wasrst noted with mammalian and yeast prion proteinswhen it was shown that a single polypeptide can mis-fold into multiple amyloid conformations (Chien et al.,2004). Specically, the yeast Sup35p prion protein wasaggregated at different temperatures into amyloid con-formations that could be distinguished by thermalstability and EPR spectroscopy, and infection of yeastwith these different conformations led to differentpropagating yeast [PSI] strains (Tanaka et al., 2004;King and Diaz-Avalos, 2004). Ab brils also show

molecular diversity. Solid-state NMR measurementsrevealed that Ab(1-40), Ab(1-42), and Ab(10-35) brilscontained in-register, parallel b-sheets (Tycko, 2003;Burkoth et al., 2000), while brils formed by theshorter peptides Ab(16-22), Ab(34-42), and Ab(11-25)adopted anti-parallel b-strand alignments (Balbachet al., 2000; Lansbury et al., 1995; Petkova et al.,2004). Very recently, two types of amyloid brils wereformed by Ab(1-40) following aggregation under mildlyagitated or quiescent conditions, and chemical shiftand line-width data from solid-state NMR for 33 of the40 residues indicated different underlying structures(Tycko, 2004; Petkova et al., 2005).The emergence of amyloid polymorphism under-

scores the need for measures that would distinguishamyloid aggregates formed under different conditions.The cellular environment provides a variety of inter-faces that are likely to dictate the formation of particu-lar amyloid structures, and it is important to developmeasures that eventually can be applied to in situ Abaggregates in tissue samples. In this review we incor-porate images obtained by EM and AFM in three of ourrecent reports (Nichols et al., 2002, 2005a,b) to enhanceour characterization of soluble Ab(1-40) aggregatesgenerated either in a homogeneous buffer or in two-phase systems at a polarnonpolar interface. Whilethese aggregates all showed enhanced uorescencewith thioavin T and enrichment in b-structure by CD,they differed strikingly in their stabilities on dilutionand their morphological features.

MATERIALS ANDMETHODSPreparation of Monomeric Ab(1-40)

Ab(1-40) peptide was obtained from QCB (Hopkin-ton, MA), rPeptide (Athens, GA), or the protein andpeptide core facility at the Mayo Clinic (Rochester,MN). Samples were routinely dissolved in water andany preformed aggregates were removed from stocksolutions by size exclusion chromatography (SEC) on a1 30 cm Superdex 75 HR 10/30 column (AmershamBiosciences, Piscataway, NJ) (see Teplow, 1998). A sin-gle elution peak was observed at a partially includedvolume (Nichols et al., 2002), and translational diffu-sion measurements by NMR (Tseng et al., 1999) as wellas direct Mw determination by static multiangle lightscattering (MALS) (Nichols et al., 2005a) indicated thatthis peak corresponds to monomeric Ab(1-40) (4.34.5 kDa).

Isolation of Ab(1-40) Protobrils

Monomeric Ab(1-40) was aggregated with vigorousagitation in Tris-EDTA buffers (550 mM Tris-HCl,5 mM EDTA at pH 8.0) containing 0150 mM NaCl at238C. Aggregation was monitored with thioavin T(Nichols et al., 2002), a uorophore that shows greatlyenhanced uorescence on binding to amyloid brils(LeVine, 1993) as well as protobrils (Walsh et al.,1999). Samples were microfuged for 10 minutes in atabletop centrifuge (Beckman Coulter, Fullerton, CA)at 18,000g. The supernatant was fractionated by SECon Superdex 75, and Ab eluting in the void volume wasdened as the protobril fraction (Nichols et al., 2002).Superdex columns were routinely pretreated with a

165AMYLOID-b AGGREGATION AT NONPOLAR INTERFACES

bolus of bovine serum albumin (50 mg) in running buf-fer to block nonspecic binding of Ab protobrils to theresin and occasionally were washed with 1 N NaOH.

Light-Scattering Measurements

Particle hydrodynamic radii (RH) were measured bydynamic light scattering (DLS) at room temperaturewith a DynaPro MSX instrument (Wyatt Technology,Santa Barbara, CA). Total light scattering intensity ata 908 angle in kilocounts/sec (kcts) was collected usinga 5-second acquisition time (Nichols et al., 2002).Weight average molecular masses (Mw) were estimatedby MALS with a DAWN EOS instrument (Wyatt Tech-nology, Santa Barbara, CA) in-line with a Superdex 75SEC column (Nichols et al., 2002). This SEC-MALSprocedure is attractive because, in contrast to DLS,Mwestimates can be made without assumptions of molecu-lar or aggregate shape.

Circular Dichroism (CD)

Spectra were obtained on an Aviv Model 215 CircularDichroism Spectrometer with a 0.1 cm pathlengthquartz cuvette (Hellma, Mullheim, Germany). Buffercontrol spectra were averaged and subtracted from theaverage of triplicate scans of each Ab sample spectra,and each resulting point ([y]obs, deg) was converted tomean residue ellipticity ([y], deg cm2 dmol1) (Nicholset al., 2005a).

Electron Microscopy (EM)

Samples of Ab aggregates were applied to 200-meshformvar-coated copper grids (Ernest F. Fullam,Latham, NY) and incubated for 1015 minutes at roomtemperature. The sample was then wicked off with lenspaper, washed briey by placing the grid face-down ona wash droplet, and stained by transferring the gridface-down to a droplet of 2% uranyl acetate (Polysci-ences, Warrington, PA) for 510 minutes before wick-ing off the solution and air drying. With samples gener-ated in 2% HFIP, all wash and treatment solutionsapplied to the grids also contained 2% HFIP. Gridswere visualized in a Philips EM208S transmission elec-tron microscope.

Atomic Force Microscopy (AFM)

Samples were applied to freshly cleaved mica thathad been modied with 30-aminopropyl-triethoxy-silane (APTES) and incubated for 15 minutes (Nicholset al., 2002, 2005a). The residual sample liquid wasaspirated off and the disk was then rinsed gently withwater and blown dry with compressed air. With aggre-gates generated in HFIP, the rinse included 2% HFIP.A Nanoscope III controller with a Multimode AFM(Digital Instruments, Santa Barbara, CA) was used forimaging by ambient tapping mode. Images wereobtained in either amplitude mode or height mode,where increasing brightness indicates greater dampingof cantilever oscillation (Harper et al., 1999) or increas-ing feature height, respectively. Height mode imageswere attened prior to measurement of particleheight distributions with NanoScope (R) III software(v. 5.13r5, Digital Instruments).

RESULTS AND DISCUSSIONFormation and Growth of Ab(1-40) Protobrils in

Aqueous Buffers

The term protobril was introduced to describesoluble Ab aggregates prepared in vitro and isolated bySEC. These aggregates exhibited Stokes radii of 1050 nm as measured by DLS (Walsh et al., 1997),showed enhanced uorescence with thioavin T, andgave a CD spectrum enriched in b-structure (Walshet al., 1999). Other groups have generated similar solu-ble Ab aggregates, sometimes called oligomers, but themorphology of the aggregates has varied somewhat.Initial EM and AFM reports showed mixtures ofroughly spherical globules and short, curly bers withlengths of 10200 nm (Harper et al., 1997, 1999; Walshet al., 1997), but other groups have observed similarglobules together with short rods, also with lengths of10200 nm (Goldsbury et al., 2000; Huang et al., 2000;Dahlgren et al., 2002). These morphological differencesprobably arise from variations in pH, ionic strength,cosolvent, and sample preparation. We prepared Ab(1-40) protobrils by aggregation of monomeric Ab at pH8.0 (Nichols et al., 2002). The reaction was monitoredby thioavin T uorescence and proceeded with adelay, or lag time, that is well known for amyloidogenicproteins (Jarrett and Lansbury, 1993) (see Fig. 2A).Samples were periodically microcentrifuged for 10minutes at 18,000g and the soluble aggregates thatremained in the supernatant were designated proto-brils and the sedimented aggregates as brils. Theyield of protobrils depended on the ionic strength,increasing from less than 20% of the total thioavin Tuorescence in 150 mM NaCl to more than 80% at orbelow 40 mM NaCl. Our protobrils isolated by SECshared several features of protobrils and soluble Aboligomers reported by others. DLS measurements indi-cated average hydrodynamic radii RH of 3070 nm, andAFM revealed small aggregates with a globularappearance, rods (with lengths of 20400 nm), andglobular aggregates with rods emanating from them(Fig. 1A). Protobril heights obtained from our AFMimages were about 3 nm, consistent with thosereported previously for Ab(1-40) protobrils (Harperet al., 1997, 1999). Mw values determined by SEC-MALS for several protobril preparations ranged from732 103 kDa (Nichols et al., 2002). These protobrilsthus are quite large, containing at least 1,500 mono-mers. Although DLS and MALS provide very sensitivemeasures of aggregation, we have not been able todetect the smaller Ab(1-40) aggregates reported byothers (Lashuel et al., 2003). Smaller aggregationintermediates under our conditions thus must occur atvery low steady-state levels.Since mature Ab brils are longer and have a larger

diameter than Ab protobrils, several possible mecha-nisms of protobril growth may exist. Studies of Abbril formation have examined Ab monomer aggrega-tion (Jarrett et al., 1993) and Ab bril extension bymonomer deposition (Naiki and Nakakuki, 1996; Esleret al., 1996, 2000). However, few reports have focusedon protobril growth in solution. We distinguished twogrowth processes, protobril extension by monomerdeposition, a process we termed elongation, and directprotobrilprotobril association (Nichols et al., 2002).

166 M.R. NICHOLS ET AL.

Elongation occurred immediately upon addition ofpuried Ab monomer to puried protobrils and couldbe conveniently followed by increases in thioavin Tuorescence or RH. This growth occurred at protobrilends. Both AFM and EM indicated a higher proportionof aggregates corresponding to rods with extendedlengths of several mm after elongation. Fractionation ofthe elongation reaction by SEC yielded a subpopulationof shorter rods that could be eluted from the column(Fig. 1B), and MALS analyses of this fraction showedconsistent decreases in mass per unit length (MPL)after the protobrils were elongated (Nichols et al.,2002). Protobril growth also occurred in the absenceof added Ab monomer, as observed previously by DLS(Walsh et al., 1999) and AFM (Harper et al., 1999). Thisgrowth involved association of the initial protobrilsand was very sensitive to ionic strength. It occurred ata very slow rate, if at all, in the absence of NaCl. Addi-tion of 150 mM NaCl to an aliquot of isolated proto-brils more than doubled the average RH over a 2-hourperiod with little change in thioavin T uorescence(Nichols et al., 2002). Values of MPL from SEC-MALSfor these associated protobrils were about 3 timeslarger than those for the elongated protobrils, andAFM showed that association involved clustering ofthe initial protobrils in a relatively disordered arraywith no extension of tendrils (Fig. 1C).

Interfacial Ab Aggregation

The concept that Ab brillogenesis is a nucleation-dependent polymerization process (Jarrett et al., 1993;Walsh et al., 1997) has provided a useful framework forin vitro Ab aggregation studies. Nucleus formation isconsidered rate-limiting in this process, but this rate(as measured by the delay or lag time to the maximalrate of aggregation) is highly variable even in homoge-neous aqueous solutions and is sensitive to many con-ditions including pH, ionic strength, temperature, andagitation (Wood et al., 1996; Harper et al., 1999; Nich-ols et al., 2002). Interfaces including phospholipidmembranes may play important roles in promoting

nucleation in vivo, and anionic phospholipid vesicles(Terzi et al., 1997) as well as vesicles containing GM1ganglioside (Choo-Smith et al., 1997; Choo-Smith andSurewicz, 1997) promoted Ab binding and b-sheet con-tent in vitro. Ab interactions with these vesiclesappeared restricted to the lipid polar head groups(Terzi et al., 1997; Matsuzaki and Horikiri, 1999; Yipand McLaurin, 2001). Other reports have indicatedthat amphiphilic interactions may be important andsuggested that micellar-like Ab structures may play arole in nucleation (Lomakin et al., 1996; Tjernberget al., 1999; Yong et al., 2001). Amphiphilic moleculestend to accumulate at interfaces between water and airor nonpolar liquids (Pratt and Pohorille, 2002), and anAb monolayer at an air/water interface did undergo arapid and substantial increase in interfacial b-sheetcontent compared to that of a similar Ab incubation inbulk solution (Schladitz et al., 1999).We have identied two systems in which amphiphilic

interfacial interactions appear to promote Ab aggrega-tion into structures rich in b-structure. The rstinvolves addition of monomeric Ab(1-40) to the upperaqueous phase of a buffer/chloroform two-phase system(Nichols et al., 2005b). Ab aggregates formed withinhours, in contrast to the formation of Ab protobrilsfrom the same 50100 mM concentrations of Ab mono-mers in dilute aqueous buffer, a process that typicallyinvolves a lag time of days in a quiescent one-phasesystem. The liquid/liquid interface was necessary forthis effect, as buffer saturated with chloroform failed toshow any acceleration of aggregation. The second sys-tem consists of dilute HFIP in buffer. Our study of thissystem was prompted by a report that 14% (v/v) HFIPstrongly accelerated amyloid ber formation by isletamyloid polypeptide (IAPP) (Padrick and Miranker,2002). This is less clearly a two-phase system, but wepresent evidence below to conrm a second phase com-posed of HFIP microdroplets. Rates of Ab aggregationin aqueous buffer alone and in dilute HFIP are com-pared in Figure 2. The formation of protobrils frommonomeric Ab(1-40) during vigorous agitation in dilute

Fig. 1. Protobril growth arising from elongation by added mono-mer or direct protobril association as monitored by AFM. Ab(1-40)protobrils were isolated by SEC in 5 mM Tris-EDTA and diluted to1 mM (in Ab monomer units) in 50 mM Tris-EDTA. The dilution forthe elongation reaction included 30 mM Ab(1-40) monomer and after1 hour the mixture was fractionated by SEC to isolate a subpopula-tion of elongated protobrils. The dilution for the association reaction

included 150 mM NaCl, but low recoveries precluded further fractio-nation by SEC. Aliquots (100 ml) from each of the three samples wereapplied to mica stubs and analyzed by AFM. Initial (A), elongated(B), and associated (C) protobrils are shown as 2.5 2.5 mm images(cropped from 5 5 mm elds). Images are presented in amplitudemode. Slightly different images from the same 5 5 mm elds werepreviously presented in height mode (Nichols et al., 2002).


buffer showed a lag time of about 6 hours in Figure 2A.Increasing the NaCl concentration to 150 mM furtherdecreased the lag time to less than 4 hours but resultedin a greater yield of brils than of protobrils (Woodet al., 1996; Harper et al., 1999; Nichols et al., 2002), asnoted above. In contrast, over a narrow range of HFIPconcentrations from 14%, aggregation of Ab(1-40)without agitation occurred in minutes and the usual

lag time for Ab aggregation was abolished (Fig. 2B).The aggregation did not require a particular mixingsequence, as similar rates were observed when stockAb in 50% HFIP was diluted or a small volume of neatHFIP was added to diluted Ab. HFIP at higher concen-trations is widely known as an effective solvent for Abpeptides (Zagorski et al., 1999), and we observed thatAb(1-40) protobrils solubilized at HFIP concentra-tions above 20% (v/v) were converted to monomeric Abas determined by SEC. CD spectra were compared todocument these differences. In agreement with pre-vious reports (Terzi et al., 1997; LeVine, 2002; Walshet al., 1999), SEC-puried Ab(1-40) monomer waslargely random coil in buffer alone (a single minimumat 197 nm) and a-helical in 30% HFIP (double minimaat 208 and 222 nm), while isolated protobrils showedpredominantly b-structure (a minimum at 216 nm anda maximum at 195 nm) (Fig. 3A). The CD spectrum ofAb(1-40) aggregates in the aqueous phase of the chloro-form/buffer system also showed a single minimum at216 nm, indicating considerable b-structure (Fig. 3B).Addition of monomeric Ab(1-40) to fresh 2% HFIP gavea spectrum consistent with signicant b-structure,while addition of the same Ab to 2% HFIP that hadbeen centrifuged to reduce the number of apparentHFIP microdroplets (see below) gave a smaller shift at216 nm in the corresponding CD spectrum (Fig. 3C).Inclusion of the random coil spectrum for control mono-meric Ab(1-40) in Figure 3C revealed an isodichroicpoint at 209 nm for these three solutions, indicating atwo-component mixture with a rapid equilibriumbetween the random coil and b-structures. Therefore,these CD spectral analyses demonstrated clear differ-ences in Ab structure at high and low concentrations ofHFIP.

Stabilities of Ab Aggregates

The stability of soluble Ab aggregates has receivedmuch less attention than their assembly pathways.Virtually the only quantitative estimate of an in vitroprotobril disaggregation rate involved Ab(1-40) proto-brils trace-labeled with 125I-Ab(1-40). The dialyzedprotobrils released only about 30% of their radioactiv-ity, mostly over the rst 2 days of dialysis (Walsh et al.,1999). We measured the stability of our Ab(1-40) proto-bril preparations by diluting Ab(1-40) aggregationreactions that were optimized for Ab protobril forma-tion as outlined above. Large dilutions were necessaryto minimize further monomer deposition and revealdisaggregation, indicated by a progressive decrease inthioavin T uorescence (Fig. 4A). The uorescencedecrease occurred in at least two kinetic phases, withinitial disaggregation followed by a much slower secon-dary phase. Less than half of the initial protobrils dis-aggregated over a 16-hour period, an observation quitecompatible with the extent of disaggregation reportedfor dialyzed 125I-Ab(1-40) protobrils (Walsh et al.,1999). Continued incubation of the aggregation reac-tions revealed a progressive stabilization of the Ab pro-tobrils, as demonstrated by decreased disaggregationrate constants (Nichols et al., 2005a) and increasedpercentages of stable aggregates after dilution at lon-ger incubation times. In Figure 4A, no disaggregationcould be detected after 5 days of incubation. Ab(1-40)protobrils isolated after SEC also were completely

Fig. 2. Aggregation of Ab(1-40) in one- and two-phase systems asmonitored by thioavin T uorescence (F). A: (Nichols et al., 2002)Monomeric Ab(1-40) (70 mM) in 50 mM Tris-EDTA and 40 mM (D) or150 mM (O) NaCl was shaken continuously at 600 rpm. Aliquots(10 ml) were diluted into 200 ml of 5 mM thioavin T in buffer at theindicated times. B: (Nichols et al., 2005a) Indicated concentrations ofHFIP (% v/v) were included in quiescent solutions containing mono-meric Ab(1-40) (20 mM) and 5 mM thioavin T in 30 mM Tris-HCl (pH8.0), and uorescence was recorded continuously. A control with buf-fer and 5 mM thioavin T in the absence of Ab(1-40) gave a uores-cence value of 1. Addition of 2% HFIP increased the value to 3.


stable. However, chemical modication of the freeamino groups in Ab allowed a wider range of stabilitymeasurements. The somewhat less stable protobrilsprepared from reductively radiomethylated Ab(1-40)showed progressive stabilization on dilution of isolatedprotobrils as well as aggregation reactions (Nicholset al., 2005a).A striking feature of the interfacial Ab aggregates

generated both in chloroform/buffer and in dilute HFIPwas their instability on dilution. Ab aggregatesremoved from the aqueous phase after a 1-day incuba-tion in the two-phase system disaggregated rapidly fol-lowing dilution into buffer containing thioavin T,declining to background in less than 10 minutes (Fig.4B, left). Furthermore, simply removing an aliquot of

the aqueous phase from the interface was sufcient toinduce essentially complete disaggregation (Fig. 4B,right). The HFIP aggregates also were initially veryunstable. Fifteen-fold dilution of the aggregates formedafter 30 minutes in 2% HFIP into buffer with thioavinT resulted in a uorescence decrease too fast to meas-ure. The speed of the disaggregation resulted from lossof the HFIP subphase, because maintenance of 2%HFIP in the dilution buffer resulted in a much sloweruorescence decrease (Nichols et al., 2005a). Asobserved in Figure 4A with Ab protobrils, continuedincubation of Ab(1-40) in dilute HFIP resulted in amarked stabilization of the aggregates without anincrease in the overall thioavin T binding (Fig. 4C).Disaggregation rate constants on dilution into buffer

Fig. 4. Stabilities of Ab protobrils and interfacial aggregates asmonitored by continuous thioavin T uorescence (F) or dynamiclight scattering intensity (LS). SEC-puried monomeric Ab(1-40) wasaggregated as described below and incubated at 258C without furthermanipulation. At the indicated times samples were diluted to initiatedisaggregation. A: (Nichols et al., 2005a) Ab (100 mM) was aggregatedwith agitation for 22 hours in 5 mM Tris-EDTA and aliquots werediluted 40-fold into 5 mM Tris-EDTA buffer containing 5 mM thioa-vin T. B: (Nichols et al., 2005b) Ab (30 mM) in 50 mM Tris-HCl (pH

8.0) was incubated over chloroform for 30 hours and aliquots wereremoved from the aqueous phase and either diluted 15-fold into thesame buffer containing 5 mM thioavin T (left) or analyzed withoutdilution (right). C: (Nichols et al., 2005a) Ab (20 mM) was aggregatedfor 30 minutes without agitation in a solution containing 2% HFIPand 5 mM thioavin T in 5 mM Tris-EDTA and aliquots were diluted15-fold into the same thioavin T and buffer without HFIP. The9 hours disaggregation curve from A (PF) is reproduced to allow com-parison with Ab(1-40) protobrils.

Fig. 3. CD spectra of Ab monomers and aggregates. A: (Nicholset al., 2005a) CD spectra for monomeric Ab(1-40) (30 mM) in 5 mMTris-HCl (pH 8.0) (solid) or after addition of 30% HFIP (dots). Ab(1-40) protobrils were prepared as in Figure 2A and isolated by SEC(dashed). B: (Nichols et al., 2005b) Ab(1-40) (50 mM) in 5 mM Tris-HCl(pH 8.0) was incubated over chloroform for 24 hours and a CD spec-

trum was recorded for an aliquot of the aqueous phase (dashed). Acontrol spectrum of stock monomeric Ab(1-40) (solid) was also recorded.C: (Nichols et al., 2005a) CD spectra for monomeric Ab(1-40) (30 mM)in 5 mM Tris-HCl (solid) and after aggregation for 24 hours with 2%HFIP. The HFIP was added either neat (dots) or from 4% HFIP/waterafter a 10-minute centrifugation at 18,000g (dashes).


without HFIP were decreased and, after nearly 10days, approached the rate constant obtained for disag-gregation of Ab protobrils formed in the absence ofHFIP (Fig. 4C). The HFIP-induced aggregatesremained soluble despite this stabilization, as indi-cated by almost complete retention of thioavin T uo-rescence in the supernatant following centrifugation ofthe dilutions at 18,000g for 10 minutes.

Morphologies of Interfacial Ab Aggregates

We applied AFM and EM imaging to compare themorphologies of Ab aggregates formed in these interfa-cial systems with those of Ab protobrils. For compari-son with the two-phase aggregates, protobrils wererst elongated by brief deposition of Abmonomers. Thesubset of shorter elongated protobrils was isolated bySEC and examined by AFM (Fig. 5A,D). They consistedof rod-like laments with an average height of 3.1 nm,often emanating from globular cores that were typicalof the protobrils prior to elongation (see Fig. 1A,B).AFM images of the two-phase aggregates (Fig. 5B,E;C,F) revealed structures that we denote exible bers,with lengths of much greater than 1 mm, as well asnumerous species that appeared globular. The bersappeared to be composed of a collection of the globularspecies aligned roughly along a longitudinal axis. Themeasured heights for both bers and globules in Figure5E were similar (45 nm). Distinctions between theelongated protobrils in Figure 5A,D and the two-phase aggregate sample in Figure 5B,E and C,F are

clear: the two-phase aggregates included no rod-like l-aments, and the elongated protobrils showed nochain-like alignment of globules. Furthermore, Figure5F showed discontinuities within central sections ofseveral bers that appeared to reect disaggregation ofindividual globular components of the bers afteradsorption of the diluted sample and washing of theAFM disk. Such rapid disaggregation is consistent withthe ber instability in the aqueous phase shown inFigure 4B. One limitation of the two-phase chloroform/buffer system in our analysis of interfacial Ab aggrega-tion was the continued growth of the aggregates. Maxi-mum aggregate levels were obtained after 1-4 days,and this peak was followed by a rapid decline in aggre-gate concentration in the aqueous phase and formationof a cloudy white precipitate hovering at and just abovethe aqueous/chloroform interface (Nichols et al.,2005b). This precipitation prevented us from examin-ing long-term changes in the stability of the aggre-gates. In contrast, HFIP-induced Ab aggregatesremained soluble for several days, allowing the pro-gressive stabilization of the aggregates to be discerned(Fig. 4C).EM analyses, conducted following negative staining

of samples with uranyl acetate, initially were useful inclarifying the disperse state of dilute HFIP itself. Con-trol samples of freshly diluted 2% HFIP showed no fea-tures in EM images, but addition of bovine serum albu-min to the diluted HFIP resulted in the appearance ofcircular features (Fig. 6A). The number of these fea-

Fig. 5. Comparison of Ab aggregates induced at the chloroform/buffer interface with those of Ab protobrils by AFM (Nichols et al.,2005b). Images are presented in both amplitude mode (AC) andheight mode (DF). A,D: Ab(1-40) protobrils were prepared by vigo-rous continued vortexing of monomeric Ab (180 mM) in 5 mM Tris-EDTA overnight at 258C. The protobrils were elongated by incuba-tion with monomeric Ab (30 mM) for 0.5 hours and isolated a secondtime by SEC. A 100-ml sample (1 mM in Ab monomer units) was

applied to the mica surface. B,C,E,F: Ab aggregates were induced by24-hour incubation in buffer containing 50 mM Ab(1-40) in 50 mMTris-HCl (pH 8.0) over chloroform. A 20-ml aliquot of the aqueousphase was diluted 5-fold with 50 mM Tris-HCl (pH 8.0) directly on themica surface. Images are 2.5 2.5 mm (A,B,D,E; insets are the samescale) or 10 10 mm (C,F) and were taken from the same sample disk.Arrows in F indicate areas of ber discontinuity.


tures was greatly reduced when the 2% HFIP solutionswere centrifuged at 18,000g for 10 minutes before andafter addition of albumin (data not shown), indicatingthat the albumin had slightly adsorbed to sphericalHFIP microdroplets. This conclusion was supported byobservations that centrifugation of 24% HFIP stocksremoved particles responsible for 90% of the DLSintensity from the supernatant and decreased the rateand extent of aggregation when Ab was introduced(Nichols et al., 2005a; Fig. 3C). It is also consistentwith a report of hydrated HFIP oligomers in diluteHFIP solutions (Yoshida et al., 2003).To determine whether the stabilization of Ab aggre-

gates formed in dilute HFIP was accompanied by mor-phological changes, we examined the aggregation reac-tions by EM and AFM. A solution of Ab(1-40) was pre-pared in freshly diluted 2% HFIP and aliquots wereremoved at several time points, centrifuged at 18,000gfor 10 minutes, and examined by EM. After 1 hour ofincubation, clustered globular structures were the pre-dominant negatively stained feature (Fig. 6B). Someindividual globular structures were also observed (Fig.6B inset). The diameter of the globules extended over arange of 20200 nm and punctate deposits could beseen on several of the larger globules. The globules pre-sumably represented HFIP microdroplets upon whichAb had deposited and begun to aggregate. However,based on their uorescence with thioavin T, these Ab

deposits differed from the albumin-coated microdrop-lets by remaining soluble after centrifugation at18,000g. Two days later the clustered globular struc-tures were incorporated into a mesh or lattice of ber-like elements (Fig. 6C), and after 9 days more distinctbers were apparent (Fig. 6D). These included longbers from which many short bers branched and, veryrarely, structures resembling initial microdropletson which bers had formed (Fig. 6D, inset). After 23days (Fig. 6E) the HFIP-induced bers appeared simi-lar to control brils produced by elongation of Ab(1-40)protobrils (Fig. 6F), but numerous short branchesremained that were not found on the control brils.The bers at this time still remained soluble, as theyfailed to sediment during 18,000g centrifugation.AFM images supported and extended the features of

HFIP-induced Ab aggregates observed by EM. Thesame mixture of clustered globular structures and indi-vidual globules was observed 1 hour after adding Ab(1-40) to dilute HFIP (Fig. 7A and inset). The heights ofthe clustered structures typically ranged from 1015 nm but in some cases extended to over 50 nm. After23 days, bers with heights of 45 nm had appearedand numerous very short rods with heights of 2.54 nm had emerged that were barely evident in the 1-hour sample (Fig. 7B). These short rods were less clearin the EM images, perhaps because of differences inretention on the EM grid and AFM mica surfaces or

Fig. 6. Electron micrographs of HFIP-induced Ab(1-40) aggre-gates reveal progressive changes in morphology (Nichols et al.,2005a). Bovine serum albumin (0.25 mg/ml) (A) or SEC-puriedmonomeric Ab(1-40) (40 mM) (BE) were incubated in 5 mM thioavinT, 5 mM Tris-HCl (pH 8.0), and 2% HFIP (neat) at room temperature.After 0.1 hours a 10-ml sample of the albumin solution was removedand at the indicated times aliquots of the Ab(1-40) solution were cen-trifuged at 18,000g for 10 minutes and supernatant samples (10 ml)

were removed. These samples were processed for negative stainingand EM. F: SEC-puried monomeric Ab(1-40) (100 mM) in 5 mM Tris-EDTA was incubated with agitation for 22 hours. An aliquot was cen-trifuged (18,000g for 10 minutes) and diluted 10-fold and the proto-brils in the supernatant were elongated by addition of monomericAb(1-40) (40 mM) for 15 minutes. A 10-ml sample was removed for neg-ative staining and EM as in panels AE. Images are shown relative toa calibration bar of 200 nm.


because they showed little negative staining. A 9-dayimage showed a ber-like structure with severalbranching arms (Fig. 7B, inset), consistent with thebranched bers observed by EM.Interfaces between aqueous and nonpolar phases

promote Ab aggregation by concentrating amphiphilicAb monomers at the interface in a conformation thatfavors formation of a nucleus. Polymerization thenoccurs by addition of monomer from the aqueous phaseand/or from a monomer pool already associated at theinterface. The process by which the initial Ab aggre-gates formed on HFIP microdroplets convert to bersmay involve additional steps. Because of the initialinstability of these HFIP-induced aggregates, thegrowth of bers may reect monomer dissociation fromthe globular clusters and redeposition on more stablenascent bers. However, the rare cases of relatively

well-formed bers on spherical structures that resem-ble microdroplets (Fig. 6D, inset) suggest that somereorganization of the initial b-structured aggregatescan occur directly on the microdroplet surface.

Ab Aggregate Polymorphism

Since the interfacial Ab aggregates in this report dif-fer from Ab protobrils in morphology and stability,their molecular structures must differ at least in subtleways. One measure of this conformational specicity isthe efciency with which these aggregates acted asnuclei or seeds for elongation by monomeric Ab(ONuallain et al., 2004). HFIP-induced Ab aggregatesthat had stabilized for 3 days and Ab(1-40) protobrilcontrols were diluted to equivalent concentrations(1 mM) and incubated with 60 mM Ab(1-40). The netelongation rate of the protobrils as measured by thio-avin T uorescence was almost 30 times faster thanthat of the HFIP-induced Ab aggregates (Nichols et al.,2005a). The difference in elongation rates was not dueto a difference in aggregate and protobril sizes, as theRH values measured by DLS were similar. We con-cluded that the seeding efciency of the HFIP-inducedaggregates was much lower than that of protobrils, atleast in aqueous buffers without HFIP.Further studies are required to determine the molec-

ular interactions in these aggregates, but models ofamyloid brils suggest ways in which differences couldarise. In one structural model for Ab(1-40) based onsolid-state NMR data, a cross-b unit is composed of twob-strand segments involving residues 12-24 and 30-40separated by a segment with a bend angle of 1808,allowing interpeptide hydrogen bonding and separateparallel b-sheet formation from both b-strands (Tycko,2003). In contrast, low-angle X-ray diffraction analysisof Ab(11-25) brils indicated an antiparallel alignmentof fully extended b-strands (Sikorski et al., 2003). Inthese brils the b-sheets appeared to stack by slippingrelative to each other by the length of two amino acidunits (Sikorski et al., 2003), and the registry of thehydrogen bonding appeared to vary with pH (Tycko,2003). It is possible then that Ab(1-40) protobrils andHFIP-induced aggregates differ by their b-strandalignments or the registry of their b-sheets or hydrogenbonding, and solid-state NMR measurements will berequired to resolve this issue.It will also be of interest to determine whether

agents or conditions that favor rapid interfacial forma-tion of unstable Ab aggregates occur in vivo. Analogs ofbiological membranes, namely, GM1 gangliosidemicelles and articial lipid rafts that contain GM1(Kakio et al., 2002), as well as lipoprotein particles(Chan et al., 1996) have been reported to bind Ab pep-tides in a saturable manner and to convert the peptideconformations to b-structures in the complexes. Thestability of these aggregates has not been investigated.A recent study has also found that inhaled anestheticspromote Ab aggregation (Eckenhoff et al., 2004). Thehaloalkane halothane and the haloether isourane,like HFIP, are highly uorinated, and both increasedthe rates of Ab aggregation as measured by thioavinT uorescence. Furthermore, an EM image of the Abaggregates induced by halothane at concentrations

Fig. 7. AFM images of HFIP-induced Ab(1-40) aggregates alsoshow changes in morphology with time (Nichols et al., 2005a). Sam-ples were taken at the indicated times from the same incubated solu-tion of Ab(1-40) in 2% HFIP described in Figure 6 and diluted 25-foldwith 2% HFIP. Aliquots (100 ml) were applied directly to the mica sur-face. Images, presented in amplitude mode, are 2.5 2.5 mm, andinsets are on this same scale.


achieved in routine clinical anesthesia (

Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, LiosatosM, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C,Finch CE, Drafft GA, Klein WL. 1998. Diffusible, nonbrillarligands derived from Ab1-42 are potent central nervous system neu-rotoxins. Proc Natl Acad Sci U S A 95:64486453.

Lansbury Jr PT. 1997. Inhibition of amyloid formation: a strategy todelay the onset of Alzheimers disease. Curr Opin Chem Biol 1:260267.

Lansbury Jr PT, Costa PR, Grifths JM, Simon EJ, Auger M, Halver-son KJ, Kocisko DA, Hendsch ZS, Ashburn TT, Spencer RG, TidorB, Grifn RG. 1995. Structural model for the b-amyloid bril basedon interstrand alignment of an antiparallel-sheet comprising a C-terminal peptide. Nat Struct Biol 2:990998.

Lashuel HA, Hartley DM, Petre BM, Wall JS, Simon MN, Walz T,Lansbury Jr PT. 2003. Mixtures of wild-type and a pathogenic(E22G) form of Ab40 in vitro accumulate protobrils, includingamyloid pores. J Mol Biol 332:795808.

LeVine H. 1993. Thioavine T interaction with synthetic Alzheimersdisease b-amyloid peptides: detection of amyloid aggregation in sol-ution. Protein Sci 2:404410.

LeVine 3rd H. 2002. 4,40-Dianilino-1,10-binaphthyl-5,50-disulfonate:report on non-b-sheet conformers of Alzheimers peptide b(1-40).Arch Biochem Biophys 404:106115.

Lomakin A, Chung DS, Benedek GB, Kirschner DA, Teplow DB.1996. On the nucleation and growth of amyloid b-protein brils:detection of nuclei and quantication of rate constants. Proc NatlAcad Sci U S A 93:11251129.

Matsuzaki K, Horikiri C. 1999. Interactions of amyloid b-peptide(1-40) with ganglioside-containing membranes. Biochemistry 38:41374142.

Miller DL, Papayannopoulos IA, Styles J, Bobin SA, Lin YY, BiemannK, Iqbal K. 1993. Peptide compositions of the cerebrovascular andsenile plaque core amyloid deposits of Alzheimers disease. ArchBiochem Biophys 301:4152.

Naiki H, Nakakuki K. 1996. First-order kinetic model of Alzheimersb-amyloid bril extension in vitro. Lab Invest 74:374383.

Nichols MR, Moss MA, Reed DK, Lin W-L, Mukhopadhyay R, HohJH, Rosenberry TL. 2002. Growth of b-amyloid(1-40) protobrils bymonomer elongation and lateral association. Characterization ofdistinct products by light scattering and atomic force microscopy.Biochemistry 41:61156127.

Nichols MR, Moss MA, Reed DK, Cratic-McDaniel S, Hoh JH, Rosen-berry TL. 2005a. Amyloid-b protobrils differ from amyloid-b aggre-gates induced in dilute hexauoroisopropanol in stability and mor-phology. J Biol Chem 280:24712480.

Nichols MR, Moss MA, Reed DK, Hoh JH, Rosenberry TL. 2005b.Rapid assembly of amyloid-b peptide at a liquid/liquid interface pro-duces unstable b-sheet bers. Biochemistry 44:165173.

Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axel-man K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG,Naslund J, Lannfelt L. 2001. The Arctic APP mutation (E693G)causes Alzheimers disease by enhanced Ab protobril formation.Nat Neurosci 4:887893.

ONuallain B, Wetzel R. 2002. Conformational Abs recognizing ageneric amyloid bril epitope. Proc Natl Acad Sci U S A 99:14851490.

ONuallain B, Williams AD, Westermark P, Wetzel R. 2004. Seedingspecicity in amyloid growth induced by heterologous brils. J BiolChem 279:1749017499.

Padrick SB, Miranker AD. 2002. Islet amyloid: phase partitioningand secondary nucleation are central to the mechanism of brillo-genesis. Biochemistry 41:46944703.

Petkova AT, Buntkowsky G, Dyda F, Leapman RD, Yau WM, Tycko R.2004. Solid state NMR reveals a pH-dependent antiparallel b-sheetregistry in brils formed by a b-amyloid peptide. J Mol Biol335:247260.

Petkova AT, Leapman RD, Guo Z, Yau WM, Mattson MP, Tycko R.2005. Self-propagating, molecular-level polymorphism in Alz-heimers b-amyloid brils. Science 307:262265.

Pratt LR, Pohorille A. 2002. Hydrophobic effects and modeling of bio-physical aqueous solution interfaces. Chem Rev 102:26712692.

Schenk D. 2002. Opinion: amyloid-b immunotherapy for Alzheimersdisease: the end of the beginning. Nat Rev Neurosci 3:824828.

Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K,Jiping-Huang, Johnson-Wood K, Khan K, Kholodenko D, Lee M,Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vas-quez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D,

Seubert P. 1999. Immunization with amyloid-b attenuates Alz-heimer-disease-like pathology in the PDAPP mouse. Nature400:173177.

Schladitz C, Vieira EP, Hermel H, Mohwald H. 1999. Amyloid-b-sheetformation at the air-water interface. Biophys J 77:33053310.

Selkoe DJ. 2001. Alzheimers disease: genes, proteins, and therapy.Physiol Rev 81:741766.

Selkoe DJ, Podlisny MB. 2002. Deciphering the genetic basis of Alz-heimers disease. Annu Rev Genomics Hum Genet 3:6799.

Serpell LC, Sunde M, Benson MD, Tennent GA, Pepys MB, FraserPE. 2000. The protolament substructure of amyloid brils. J MolBiol 300:10331039.

Sikorski P, Atkins ED, Serpell LC. 2003. Structure and texture ofbrous crystals formed by Alzheimers Ab(11-25) peptide fragment.Structure (Camb) 11:915926.

Sunde M, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CC.1997. Common core structure of amyloid brils by synchrotron X-ray diffraction. J Mol Biol 273:729739.

Tanaka M, Chien P, Naber N, Cooke R, Weissman JS. 2004. Confor-mational variations in an infectious protein determine prion straindifferences. Nature 428:323328.

Teplow DB. 1998. Structural and kinetic features of amyloid b-proteinbrillogenesis. Amyloid: Int J Exp Clin Invest 5:121142.

Terry RD. 1985. Textbook of Neuropathology. Baltimore: Williams &Wilkins. p 824841.

Terry RD, Gonatas NK, Weiss M. 1964. Ultrastructural studies in Alz-heimers presenile dementia. Am J Pathol 44:269297.

Terzi E, Holzemann G, Seelig J. 1997. Interaction of Alzheimer b-amy-loid peptide(1-40) with lipid membranes. Biochemistry 36:1484514852.

Tjernberg LO, Pramanik A, Bjorling S, Thyberg P, Thyberg J, Nord-stedt C, Berndt KD, Terenius L, Rigler R. 1999. Amyloid b-peptidepolymerization studied using uorescence correlation spectroscopy.Chem Biol 6:5362.

Tseng BP, Esler WP, Clish CB, Stimson ER, Ghilardi JR, Vinters HV,Mantyh PW, Lee JP, Maggio JE. 1999. Deposition of monomeric, notoligomeric, Ab mediates growth of Alzheimers disease amyloidplaques in human brain preparations. Biochemistry 38:1042410431.

Tycko R. 2003. Insights into the amyloid folding problem from solid-state NMR. Biochemistry 42:31513159.

Tycko R. 2004. Progress towards a molecular-level structural under-standing of amyloid brils. Curr Opin Struct Biol 14:96103.

Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB. 1997.Amyloid b-protein brillogenesis: detection of a protobrillar inter-mediate. J Biol Chem 272:2236422372.

Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Loma-kin A, Benedek GB, Selkoe DJ, Teplow DB. 1999. Amyloid b-proteinbrillogenesis: structure and biological activity of protobrillarintermediates. J Biol Chem 274:2594525952.

Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS,Rowan MJ, Selkoe DJ. 2002. Naturally secreted oligomers of amy-loid b protein potently inhibit hippocampal long-term potentiationin vivo. Nature 416:535539.

Westerman MA, Cooper-Blacketer D, Mariash A, Kotilinek L, Kawar-abayashi T, Younkin LH, Carlson GA, Younkin SG, Ashe KH. 2002.The relationship between Ab and memory in the Tg2576 mousemodel of Alzheimers disease. J Neurosci 22:18581867.

Wood SJ, Maleeff B, Hart T, Wetzel R. 1996. Physical, morphologicaland functional differences between pH 5.8 and 7.4 aggregates of theAlzheimers amyloid peptide Ab. J Mol Biol 256:870877.

Yankner BA. 1996. New clues to Alzheimers disease: unraveling theroles of amyloid and tau. Nat Med 2:850852.

Yip CM, McLaurin J. 2001. Amyloid-b peptide assembly: a criticalstep in brillogenesis and membrane disruption. Biophys J80:13591371.

Yong W, Lomakin A, Kirkitadze MD, Teplow DB, Chen SH, BenedekGB. 2001. Structure determination of micelle-like intermediates inamyloid b-protein bril assembly by using small angle neutron scat-tering. Proc Natl Acad Sci U S A 99:150154.

Yoshida K, Yamaguchi T, Adachi T, Otomo T, Matsuo D, Takamuku T,Nishi N. 2003. Structure and dynamics of hexauoroisopropanol-water mixtures by x-ray diffraction, small-angle neutron scattering,NMR spectroscopy, and mass spectrometry. J Chem Phys 119:61326142.

Zagorski MG, Yang J, Shao H, Ma K, Zeng H, Hong A. 1999. Methodo-logical and chemical factors affecting amyloid beta peptide amyloi-dogenicity. Methods Enzymol 309:189204.


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