Amyloid-β Membrane Binding and Permeabilization are Distinct Processes Influenced Separately by Membrane Charge and Fluidity

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  Amyloid-β Membrane Binding and Permeabilization are Distinct Processes Influenced Separately by Membrane Charge and Fluidity
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  Amyloid- β  Membrane Binding and Permeabilization areDistinct Processes Influenced Separately by MembraneCharge and Fluidity Pamela T. Wong 1 , Joseph A. Schauerte 1,2 , Kathleen C. Wisser 2 ,Hao Ding 2 , Edgar L. Lee 1 , Duncan G. Steel 2 and Ari Gafni 1,2 , * 1 Department of BiologicalChemistry, University of  Michigan, 930 N. University, Ann Arbor, MI 48109, USA 2 Biophysics Research Division,University of Michigan,930 N. University, Ann Arbor, MI 48109, USAReceived 15 August 2008;received in revised form25 November 2008;accepted 30 November 2008 Available online16 December 2008The40and42residueamyloid- β (A β )peptidesaremajorcomponentsoftheproteinaceous plaques prevalent in the Alzheimer's disease-afflicted brainand have been shown to have an important role in instigating neuronaldegeneration. Whereas it was previously thought that A β  becomes cyto-toxic upon forming large fibrillar aggregates, recent studies suggest thatsoluble intermediate-sized oligomeric species cause cell death throughmembrane permeabilization. The present study examines the interactions between A β 40 and lipid membranes using liposomes as a model system todetermine how changes in membrane composition influence the conversionof A β  into these toxic species. A β 40 membrane binding was monitoredusing fluorescence-based assays with a tryptophan-substituted peptide(A β 40 [Y10W]). We extend previous observations that A β 40 interacts pre-ferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of A β 40 to anionic, but notneutral, membranes involves insertion of the peptide into the bilayer, aswell as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel,liquid crystalline, and liquid ordered phases induce these conformationalchanges equally,membrane permeabilizationisreduceddramaticallyas thefluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that thelatter is also highly dependent on the fluidity and phase of the membrane.We conclude that binding and pore formation are two distinct steps. Thedifferences in A β  behavior induced by membrane composition may havesignificant implications on the development and progression of AD asneuronal membrane composition is altered with age. © 2008 Elsevier Ltd. All rights reserved. Edited by S. Radford  Keywords:  amyloid- β ; membrane; charge; fluidity; permeabilization *Corresponding author.  Department of Biological Chemistry, University of Michigan, 930 N. University, Ann Arbor, MI48109, USA. E-mail address: arigafni@umich.edu.Abbreviations used: A β , amyloid- β ; A β 40 [Y10W], A β 40with aTyr to Trp substitution at position 10; AD,Alzheimer'sdisease; AFM, atomic force microscopy; AMP, antimicrobial peptide; APP, amyloid precursor protein; CF,5(6)-carboxyfluorescein; Dns-DPPE,  N  -(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine); DPPC, 1,2-dipalmitoyl- sn -glycero-3-phosphocholine; DPPC/PG, liposomes with equimolarDPPC and DPPG; DPPG, 1,2-dipalmitoyl- sn -glycero-3-[phospho- rac -(1-glycerol)]; DPPS, 1,2-dipalmitoyl- sn -glycero-3-[phospho- L -serine]; FRET, fluorescence resonance energy transfer; L:P, lipid to peptide molar ratio; LC, liquid-crystalline;LO,liquid-ordered;LUV,largeunilamellarvesicle;NaP i ,sodiumphosphate;PFT,pore-formingtoxin;POPC,1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine; POPC/PG, liposomes with equimolar POPC and POPG; POPG, 1-palmitoyl-2-oleoyl- sn -glycero-3-[phospho- rac -(1-glycerol)]; RT, room temperature; SUV, small unilamellar vesicle; WT, wild-type. doi:10.1016/j.jmb.2008.11.060  J. Mol. Biol.  (2009)  386 , 81 – 96  Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.  Introduction Several age-related diseases are characterized byprotein misfolding and aggregation. Alzheimer'sdisease (AD) is acommon form of progressive seniledementia caused by a loss of neurons in the cerebralcortex and hippocampal region of the brain.Pathologically, AD is characterized by the presenceof neurofibrillar tangles and extracellular senileplaques in the affected regions of the brain. Theseplaques are insoluble proteinaceous deposits com-posed primarily of aggregates of fibrils of the 40 and42 residue amyloid- β  peptides (A β 40 and A β 42). Alarge body of work has implicated A β  as a keyfactor in instigating neurodegeneration, althoughthe mechanism by which this occurs is still notentirely understood. 1 – 4 A β 40 and A β 42 are produced by cleavage of thetransmembrane protein, amyloid precursor protein by several secretases. 5 The A β  portion of APP isreleased extracellularly as a soluble peptide, and isfound in the plasma and cerebrospinal fluid (CSF)under both normal and disease states. Developmentof AD cannot be explained by over-expression, sincetotal levels of A β  found in the CSF are not con-sistently clearly higher in the diseased brain. 6,7 While certain point mutations in APP found ininherited forms of early-onset AD lead to signifi-cantly increased levels of A β  production, reports onA β  levels in late-onset  “ sporadic ”  AD brain areinconsistent, and do not show a clear elevation inA β . 6,8,9 A β 40 is presumably cleaved as a monomerfrom APP, and forms a stable dimeric or trimericspecies. 10 – 12 It subsequently oligomerizes to a highdegree, eventually forming large insoluble amyloidfibrils, which are characterized by a unique highlyordered cross β -sheet structure. In addition to fibrilsand monomers, lower-order oligomers and interme-diate order oligomers or protofibrils ( N 20-mer) can be formed, and are present in neuronal tissue takenfrom AD patients. 13 – 17 Whereas historically it wasassumed that A β  becomes cytotoxic when it formslarge insoluble fibrillar aggregates in the brain,recent studies have found little correlation betweenamyloid plaque load and the clinical severity of thedisease. 18,19 Furthermore, neuronal abnormalitiesand cognitive deficits begin to appear well beforeamyloid plaques are detected in transgenic animalsover-expressing APP. 20 – 23 Moreover, recent studiesprovide evidence suggesting that the soluble inter-mediate-sized oligomeric species of A β  effectivelycause cell death, potentially through membranepermeabilization. Indeed, a number of studies havedemonstrated that neurotoxicity is influenced by theinteraction of A β  with membrane lipids. 24 – 30 Sig-nificant increases in calcium influx into cells, 31,32 increased conductivity in membrane bilayers trea-ted with A β , 25,33 – 36 and AFM images revealingannular A β  structures bound to lipid membranes, 35 have provided evidence supporting a model inwhich A β  forms channels or pores in the plasmamembrane, thereby permitting rapid influx of cations such as calcium into the cell. Calcium influxmay subsequently trigger cell death directly or indi-rectly by stimulating apoptosis signaling path-ways. 37 However, what specific factors promoteconversion of this normally expressed peptide into aneurotoxic entity remains elusive.An intriguing aspect of AD is that amyloidplaques are found only in specific regions of the brain, and are toxic to only certain types of neurons(cerebral cortex and hippocampal cells in particular)and not to other cells in the body. 38,39 This points toan important role of the local environment, inparticular the plasma membrane, in promoting A β toxicity. Therefore, changes in the plasma mem- brane composition may be crucial in the develop-ment and progression of AD.A number of studies have shown that A β  caninteract with synthetic lipids  in vitro , 35,40 – 42 and thatthe presence of A β  induces changes in permeabilityand conductivity across lipid bilayers. 25,33 – 36 In thepresent study, the specificity of A β  membrane binding, and how peptide binding leads to per-meabilization, was further examined using lipo-somes as model membranes. A β 40 membraneinteractions were characterized using the fluores-cence of a tryptophan-substituted peptide (A β 40[Y10W]). The effects of lipid composition, inparticular the membrane charge and fluidity, onA β 40 binding, conformation, oligomerization state,and permeabilization activity were determined.Building on previous studies showing that A β interacts preferentially with anionic, as opposed toneutral, membranes, 40 – 45 we present evidence forthe presence of at least two kinds of A β 40 inter-actions with the membrane, which are highlydependent on the charge of the lipid. Significantly,we demonstrate that bilayer phase and fluidity asdetermined by phospholipid acyl chain saturationand cholesterol content, while not affecting A β  binding, are critical in determining the permeabi-lization activity of the peptide.A β  shares several characteristics with pore-forming toxins (PFT) such as melittin,  α -hemolysin,aerolysin and antimicrobial peptides. 46,28 Like A β ,these peptides exist in an unstructured randomcoiled state in solution, and undergo conforma-tional changes in the presence of membranes. 47 – 49 These peptides are also capable of forming oligo-meric structures, and have been shown to alter thepermeability of their target membranes, 50,51 Thus,greater insight into the mechanism of A β  mem- brane interaction may be obtained by comparingits mechanism of action with that of AMPs andPFTs. Work with several AMPs (e.g., melittin,alamethicin, magainin, and protegrin) revealedthat in order to insert into a membrane and formchannels, a threshold ratio of lipid to peptide (L:P),the critical L:P, must be reached. 52 The critical L:Pvalue is specific for the peptide and depends on themembrane it interacts with. When the L:P is abovethe critical value, the AMPs remain in an inactive,non-pore-forming state, with the axis of the helicalpeptide parallel with the plane of the mem- brane. 49,52 On the other hand, below the critical 82  A β  Membrane Interactions Depend on Lipid Composition   L:P, the peptide undergoes a change in conforma-tion and orientation, allowing it to penetrate themembrane with the helical axis perpendicular tothe plane of the membrane, forming channels orpores. Here, we propose a similar mechanism forA β , and compare the critical L:P values for dif-ferent membranes. Membrane binding and per-meabilization are pivotal steps in A β  toxicity.Therefore, determining which factors promote orinhibit these processes is critical to understandingthe etiology of AD. Results Membrane binding affinity of low-orderoligomers of A β 40 increases with membrane netnegative charge A fluorescence resonance energy transfer (FRET)- based assay was used to determine the lipid bindingspecificity of A β . small unilamellar vesicles (SUVs)containing mixtures of DPPC and DPPG at differentratios and 5% (mol/mol) Dns-DPPE were made.DPPC is a neutral zwitterionic lipid, whereas DPPGis an anionic lipid with a net negative charge of 1.Incubation of 5  μ M A β 40[Y10W] with Dns-SUVsresulted in an increase in Dns and a decrease intryptophan emission upon excitation of the trypto-phan, indicative of FRET and association of A β 40with the membrane. Dissociation constants weredetermined by fitting the binding curves to a simple bimolecular association model (Eq. (1)), using theassumption that the binding affinity was indepen-dent of total peptide concentration as an estimation.A β 40 interacted very weakly with pure DPPCliposomes. However, the  K  D  of A β 40 lipid bindingdecreased dramatically with increasing relativeamounts of DPPG, and is saturable, reaching aminimum at 70:30 DPPC:DPPG ( K  D  38.8±7.0  μ M)and remains about constant thereafter with increas-ing amounts of DPPG (Table 1). These resultsconfirm what has been reported by other groupsusing different techniques that A β  interacts prefer-entially with anionic lipids. 40 – 45,53,54 The A β 40samplesusedintheseexperimentswerefreshly dissolved from a NaOH-disaggregated lyo-philate resulting in a soluble low molecular massspecies (monomer – tetramer) as seen by size-exclu-sion chromatography (see below) and glutaralde-hyde crosslinking (see below). To determine thedependence of membrane-binding affinity on theoligomerization state of A β 40, binding to DPPC/PGSUVs was measured at various time-points forpeptide incubated alone at 125  μ M at room tempe-rature (RT) over three days. The dissociation cons-tantsdidnotchangesignificantlyoverthethreedays(Fig. 1a). However, the final Dns fluorescencedecreasedoverthisperiodoftime(Fig.1 b),reflectinga decrease in the total amount of bound A β  (the Trpfluorescence of A β  did not decrease over time (datanot shown)). Fibril formation as assessed by ThT Fig. 1.  Time evolution of A β 40[Y10W] membrane binding ability and fibrilization. (a) A β 40[Y10W] mem- brane association was measured as an increase in Dnsfluorescence at 514 nm due to FRET from the tryptophanexcitedat280nm.A β 40[Y10W]wasdissolvedat125 μ Min10 mM NaPi pH 7.4 and incubated at 25 °C for 72 h.Aliquots (5 μ L) were titrated with Dns-SUVs composed of equimolar DPPC/DPPG. (b) WT A β 40 incubated at125  μ M as in a was assayed for fibril formation by ThT binding. ThT fluorescence emission intensity was mea-sured at 485 nm upon excitation at 440 nm. Endpoint Dnsemission from the titrations in a are plotted for compa-rison. The amount of bound A β decreased as fibrils beganto appear. (c) WT A β 40 (125  μ M) was allowed tooligomerize by incubation at RT. Aliquots were removedat timed intervals and crosslinked with 0.01% (v/v)glutaraldehyde. Reactions were quenched with glycine,and boiled in loading buffer before separation by SDS-PAGE. The gel was immunoblotted, and probed with amouse monoclonal  α -A β  antibody (6E10). 83 A β  Membrane Interactions Depend on Lipid Composition    binding begins to increase after 48 h (Fig. 1 b).Because the Y10W mutant does not bind ThT verywell (the ThTsignal is very low), wild type (WT) A β was used for measuring fibril formation rates.Furthermore,higher-order oligomerswere observed by chemical crosslinking after 48 h (Fig. 1c). TheY10W mutant and WT A β 40 have been shown tofibrilize similarly. 10,11 From these results, we con-clude that the species capable of binding to themembrane is present immediately after dissolution,anddiminishesovertimeduetoconversiontolargeroligomers or fibrils that no longer bind. A β 40 inserts into anionic membranes uponbinding as evidenced by a fluorescence shift While the FRET binding experiments demonstratetighter binding of A β 40 to anionic membranes, theydo not distinguish between surface binding andpenetration of the peptide into the bilayer. Sincetryptophan fluorescence emission upon partitioninginto a hydrophobic environment is blue shifted andfrequently enhanced in intensity relative to that inaqueous solution due to solvent dipole reorienta-tion, membrane insertion wasmeasured by ashiftinA β  tryptophan emission. A β 40 [Y10W] (5  μ M) wasallowed to bind to SUVs containing DPPC or POPC,or the mixtures DPPC/PG or POPC/PG. POPC andPOPG possess the same lipid head groups as DPPCand DPPG, respectively, but are in the liquid crys-talline (LC) phase at RT (due to an unsaturated acylchain), whereas DPPC and DPPG are in the gelphase. For each point in the titration, A β 40 wasincubated with the SUVs before measuring theemission spectrum of A β 40. The spectra were fit toGaussian curves as described in Materials andMethods to determine the fraction of A β  insertedas a function of lipid concentration. This was fit to asimple binding isotherm to yield apparent  K  D . The K  D  values obtained this way were similar to thosefound by FRET at the same concentration of A β .Binding of A β 40[Y10W] to DPPC/PG SUVsresulted in a 15 nm blue shift in the emission maxi-mum wavelength at only 70  μ M lipid, reflectingsignificantinsertionofthepeptideintothebilayer.Incontrast,bindingtoDPPCSUVsevenat170 μ Mlipidresultedin ablueshift of only3 nm (Fig.2a). POPC/PG showed the same magnitude shift as DPPC/PG for the same amount of lipid added, demon-strating that insertion is independent of membranefluidity (Fig. 2c). These results confirm what wasfound by FRET, that A β 40 binds preferentially tonegatively charged lipids, but still interacts weaklywith neutral lipids. Thus, A β 40 membrane inser-tion is highly dependent on the negative charge of the membrane.Apparent dissociation constants for membrane binding were determined for different concentra-tions of A β . The calculated  K  D  values were linearlydependent on the concentration of A β , at the con-centrations of A β  where fluorescence could bemeasured accurately. This dependence is indicativeof limiting surface area on the liposome membrane,resulting in a higher estimate of the  K  D  than theactual value at the higher concentrations of peptide.Thus, the calculated  K  D  value is only an apparent binding constant. Since the workable concentrationof peptide was limited by fluorescence detectionlimits, we can only conclude that the actual  K  D  is atleast as tight as the apparent  K  D  of 0.6 μ M calculatedat 100 nM peptide. Fig. 2.  Effect of lipid binding on tryptophan emissionspectra. A β 40 [Y10W] (2.5  μ M) was titrated with SUVscontaining DPPC (a) or an equimolar mixture of DPPC:DPPG(b).Peptidewasallowedtobindtotheliposomesasin the FRET experiments, and tryptophan fluorescenceemission spectra were measured upon excitation at280 nm. (c) Plot of the emission maxima wavelengths versus  lipid concentration for DPPC and DPPC/PG binding demonstrated a strong dependence on membranecharge for peptide insertion. 84  A β  Membrane Interactions Depend on Lipid Composition   The tryptophan of A β 40 is protected fromdynamic quenching upon binding to anionicliposomes To confirm that the shift in tryptophan fluores-cence was due to A β 40 insertion, the ability of liposomes to provide protection to the tryptophanfrom the collisional quencher acrylamide wasmeasured. A β 40 [Y10W] was allowed to bind tovarious concentrations of SUVs, and the tryptophanemission was measured with increasing concentra-tions of acrylamide. The Stern-Volmer constants( K  SV ) were determined using Eq. (2). DPPC/PGdecreased the A β  K  SV  dramatically (97% reductionat 100  μ M lipid), compared to no change of   K  SV  forfree tryptophan in the presence of the sameconcentrations of DPPC/PG (Fig. 3a and b). Nochange was seen for RNase A in the presence of DPPC/PG SUVs (data not shown), indicating alarge decrease in the solvent-accessibility of thetryptophan residue in anionic membranes, provid-ing further evidence supporting insertion of A β 40into DPPC/PG. Minimal protection from quench-ing was observed for A β  in the presence of DPPCSUVs; however, this is trivial, since there is verylittle binding of the peptide to the neutral mem- branes, as reflected by the FRET and Trp shiftexperiments. Binding to anionic but not neutral liposomesinduces oligomerization of A β  and changes inits conformation Theeffectsofanionicandneutralliposomesontheconformation of A β 40 were studied using far-UVCDintherange190 – 260nm.Thespectrumoffreshlyprepared 25  μ M WT A β 40 revealed primarilyrandom coil structure, with a large negative peakat 200 nm (Fig. 4). The peptide was allowed to bindto the liposomes for 10 min before taking the mea-surements, and this initial spectrum did not changeover the time of the experiment ( ∼ 3 h). Addition of either gel phase DPPC SUVs or LC phase POPCSUVs to A β 40 did not produce any significantchange of the conformation of the peptide, even atconcentrations  N 500  μ M lipid (at which the loose binding to the neutral membrane seen by FRETandthe slight shift in tryptophan emission has reachedsaturation) as seen in Fig. 4a and c. In contrast,progressive addition of SUVs containing eitherDPPC/PG or POPC/PG induced a dramatic two-step transition from random coil to  β -sheet to  α -helical structures. The spectra were analyzed forsecondary structure composition.  β -Sheet structureis induced at intermediate L:P ratios (10:1 ∼ 20:1).The content of   β -sheet structure in peptide alone isless than 35%, but increases to 45% upon addition of 500  μ M DPPC/PG SUVs (L:P of 25:1) or 300  μ MPOPC/PGSUVs(L:Pof15:1)(Fig.5aandb).Furtherincreasing the levels of DPPC/PG or POPC/PGSUVs to high L:P ratios where A β  is presumablydilutedon theliposome, resulted ina decrease in thecontent of   β -sheet structure, and an increase in  α -helical structure from 5% with no SUVs to 18% withDPPC/PG SUVs and 35% with POPC/PG SUVs atL:P of 80:1 (Fig. 5a and b). In contrast, no change inany of these structural components was seen forDPPC or POPC SUVs. These results further distin-guish the interactions between A β 40 with neutraland anionic membranes. Notably, the inducedchanges in conformation are independent of thephase and fluidity of the membrane, dependingsolely on the identity of the lipid head groups.Increasing the ionic strength by including NaCl inthe buffer reduced the magnitude of the structuralchanges in the peptide however, the same randomcoil- β -sheet/ α -helix transitions were seen, support-ing the involvement of more than just electrostaticinteractions in A β  membrane association.Binding to the membrane increases the local con-centration of A β  relative to that in the solution,which may help promote oligomerization. To test Fig. 3.  Acrylamide quenching of A β 40 [Y10W] in thepresence of SUVs. (a) A β 40 [Y10W] (5  μ M) or (b) 5  μ MNATA was incubated with SUVs composed of a 1:1mixture of DPPC:DPPG and titrated with acrylamide. TheStern-Volmer plots are shown and the Stern-Volmerconstants ( K  SV ) were calculated using a linear fit of theplots. A sharp drop in the  K  SV  values for DPPC/DPPGwith respect to lipid concentration reflects protection of the tryptophan from quenching. No change of   K  SV  wasobserved for NATA in the presence of DPPC/PG. 85 A β  Membrane Interactions Depend on Lipid Composition 
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