Growth, Structural features and cytotoxicity of amyloid oligomers: implications in alzheimer's disease and other diseases with amyloid deposits

Structural

features

and

cytotoxicity

of

amyloid

oligomers:

Implications

in

Alzheimer’s

disease

and

other

diseases

with

amyloid

deposits

Massimo

Stefani

*

DepartmentofBiochemicalSciencesandResearchCentreontheMolecularBasisofNeurodegeneration,UniversityofFlorence,V.leMorgagni50,50134Florence,Italy

Contents

1. Introduction... 000

2. Proteinfoldingandaggregationarecompetingprocesses... 000

3. Proteinfolding/misfoldingonsurfaces... 000

4. Theonsetofproteinaggregation ... 000

5. Generalstructuralfeaturesofamyloidoligomers... 000

6. Severalnewlyintroducedtechniquescanbeusedtostudythestructureofamyloidoligomers... 000

7. Amyloidfibrilscanbetoxiceitherdirectlyorasasourceoftoxicspeciesintissue... 000

8. Biologicalsurfacesareprimarysitesofamyloidinteractionandtoxicity... 000

9. Oligomer/fibrilpolymorphismunderliesdifferentamyloidinteractionwiththecellmembraneandtoxicity... 000

10. Concludingremarks ... 000

ARTICLE INFO

Articlehistory:

Received21October2011

Receivedinrevisedform8March2012 Accepted9March2012 Availableonlinexxx Keywords: Amyloid Amyloiddiseases Amyloidoligomers Amyloidcytotoxicity Proteinaggregation ABSTRACT

Amyloiddiseasesdisplaythepresence,intargetedtissuesandorgans,offibrillardepositsofspecific

peptidesorproteins.Increasingeffortsarepresentlyspentininvestigatingthestructuralfeaturesand

thestructure–toxicityrelationofthesolubleoligomericprecursorsarisinginthepathoffibrillizationas

wellastheimportanceofsurfacesastriggersofproteinmisfoldingandaggregationandaspossible

responsibleforamyloidpolymorphism.Presently,itisrecognizedthattheunstable,heterogeneous

pre-fibrillar aggregates are the main responsible for amyloid toxicity. Conversely, mature fibrils are

consideredstable,harmlessreservoirsoftoxicspecies,althoughdirectfibriltoxicityhasbeenreported.

Recentstudiesshowthatmaturefibrilsgrownatvariousconditionscandisplaydifferentstructural

features,stabilitiesandtendencytodisassemblewithleakoftoxicoligomers.Fibrilpolymorphismcan

resultfromproteinaggregationatdifferingconditionspopulatingmisfoldedmonomersandoligomers

withdistinctconformationalcharacteristics.Recentresearchhasstartedtounraveloligomerstructural

andbiophysicalfeaturesandtheirrelationtocytotoxicity.Increasinginformationsupportsthenotion

thatoligomer–membraneinteraction,disruptionofmembraneintegrityandcellimpairmentresults

frombotholigomerandmembranebiophysicalfeatures;accordingly,theformationoftheoligomer–

membranecomplex,oftenthefirststepofamyloidtoxicity,canbetheresultoftheinterplayofthese

events.This viewcanhelpexplainingthevariablevulnerabilityofdifferentcelltypestothesame

amyloidsandthelackofrelationbetweenamyloidloadandseverityofclinicalsymptoms;italsostresses

theimportance,forcell/tissueimpairment,ofthepresenceoffibrilsconformersofreducedstabilityasa

possiblesourceofoligomersresultingfromleakagepossiblyfavoredbytheinteractionwithsuitable

macromolecular/lipidsurfacesorbyotherenvironmentalconditions.

ß2012ElsevierLtd.Allrightsreserved.

Abbreviations:Ab,amyloidbeta;AD,Alzheimer’sdisease;FRET,fluorescenceresonanceenergytransfer;CD,circulardichroism;DLS,dynamiclightscattering;NMR,nuclear magneticresonance;MALDI-TOF,matrix-assistedlaserdesorption/ionization-time-offlight;NUP,nativelyunfolded/unstructuredprotein;PS,phosphatidylserine;ER, endoplasmicreticulum;APP,Amyloidbetaprecursorprotein;GM1,gangliosideM1;EM,electronmicroscopy;AFM,atomicforcemicroscopy;ESI-MS,electronspray ionization-massspectrometry;SAXS,small-angleX-rayscattering;RAGE,receptorforadvancedglycationend-products;NMDA,N-methyl-D-asparticacid;AMPA, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoicacid.

*Tel.:+390554598307;fax:+390554598905. E-mailaddress:stefani@scibio.unifi.it.

URL:http://www.unifi.it/unifi/scibio/pers/stefani.html

ContentslistsavailableatSciVerseScienceDirect

Progress

in

Neurobiology

j our na l h ome p a ge : w ww . e l se v i e r. co m/ l oc a te / p ne ur obi o

0301-0082/$–seefrontmatterß2012ElsevierLtd.Allrightsreserved. doi:10.1016/j.pneurobio.2012.03.002

Acknowledgements... 000

References... 000

1. Introduction

Amyloid diseases are a number of familial or, more often, sporadicconditionscharacterizedbythepresence,intheaffected tissues/organs of extracellular or intracellular proteinaceous deposits,genericallyknownasamyloid,whosecoreconstituents arefibrillarpolymersofoneoutofanumberofpeptides/proteins, each characteristic of a given disease [reviewed in Stefani and Dobson, 2003; Chiti and Dobson, 2006; Sipe et al., 2010]. The amyloidhistorycanbedatedbackto1838,whenthetermamyloid wasfirstintroducedbyMatthiasSchleiden,aGermanbotanist,to describeanormalamylaceousconstituentofplants.Subsequently, in 1854,the term wasused, albeit incorrectly, by theGerman physician Rudolph Wirchow to describe ‘‘cerebral corpora amylacea’’ in abnormal human brains staining blue by iodine and turning into violet by addition of sulfuric acid and thus consideredtobeidenticaltoplantstarch(Kyle,2001;Puchtlerand Sweat,1965).Afewyearslater(1859)FriedreichandKekule´ found that amyloidcontained nitrogenand was madeup of proteins ratherthanofcarbohydrates(FriedreichandKekule,1859).Atthe beginning of the 20th century, based on optical microscope observation,scientistsbelievedthatamyloidwasanamorphous material. Thefirst clues on the existenceof ordered structural organizationwithanisotropicopticalpropertieswithinamyloid depositscamein1927,whenDivryandFlorkinbypolarizedoptical microscopy found orange or apple-green birefringence upon treatmentofamyloidwithCongored(Divry andFlorkin,1927). After Divry and Florkin, congophilia and green birefringence became the signature used to recognize amyloid, whereas the fibrillarnatureof thedeposits wasfirstrecognized in1959 by ultrastructuralstudiesbyCalkinsandCohen(CohenandCalkins, 1959).Presently,itisknownthatthebasiccomponentofamyloid isafibrillarnetworkmadeupofpolymericunbranchedfibrilswith a beta sheet-richcore (the cross-beta structure)(Serpell et al., 2000)arising fromorderedpolymerization of specificproteins/ peptidesbasicallyunderconditionsfavoringareductionoftheir thermodynamic stability resulting in misfolding [reviewed in

StefaniandDobson,2003;ChitiandDobson,2006].

Amongthe27humanamyloiddiseasespresentlyidentifiedby theInternational Nomenclature Committee(Sipe et al.,2010), Alzheimer’sdisease(AD)isbyfarthemostimportantforitslarge occurrenceintheagedpopulation.Therefore,itisnotsurprising that,afteralargebodyofworkdoneonsystemicamyloidoses,the subsequentidentificationofA

b

asthemaincomponentoftheAD plaqueshasdrivenmuchoftheresearchonamyloidinthepast twentyyears. Thatpolymericfibrilsof A

b

, thepeptidearising fromtheproteolyticcleavageofthemembraneamyloidprecursor protein(APP),werethemaincomponentoftheplaquesfoundin thebrainparenchymaofADpeoplewasfirststatedin1984by WongandGlenner(GlennerandWong,1984)andsubsequently confirmedin1985byColinMastersincollaborationwithKonrad Bayreuther.Mastersidentifiedtheaminoacidcompositionand N-terminalsequence of theA

b

peptides and found thatboth correspondedto those of the main protein component of the amyloid deposits found in the brains of people affected by congophilicangiopathy,ADorDownsyndrome(Masterset al., 1985).Inthesameyearitwasalsoshownthattheprionprotein, previouslyreportedtopossesspropertiesofinfectious proteina-ceousmaterial,assembledintopolymericfibrilsdepositedinthe amyloid plaques found in scrapie affected brains (DeArmond etal.,1985).

Previousdescriptionsofsystemicamyloidosesdidnotneedto stateanamyloidhypothesistorelatecell/tissuedegenerationto thepresenceofamyloiddeposits;infact,therewasnodoubtthata relationshipdidexistbetweenamyloiddepositsanddiseaseand thatamyloidfibrilsor,insomecases,non-fibrillaraggregates,were thepathologicalentities.Atvariancewithsystemicamyloidoses, the link between neurodegeneration and presence of amyloid plaquesinADbrainwasfirstproposedin1990,whenitwasshown thataggregatedA

b

wasneurotoxicinvitro(Yankneretal.,1990). AtthesametimeseminaldiscoveriesintheADgeneticspavethe way to the formulation of the ‘‘amyloid cascade hypothesis’’, statingthatA

b

peptidesoriginatingfromtheproteolyticcleavage of their precursor protein (APP) are the main culprits of AD symptomsatthemolecularlevel(Selkoe,1991).Rapidly,yetwith diffuse resistances in AD scientists, the ‘‘amyloid cascade hypothesis’’replacedpreviousproposalssuchasthe‘‘cholinergic hypothesis’’;yet,thelatterhasrecentlybeenre-proposed,though inamodifiedform(Craigetal.,2011).

Since1990,thedevelopmentoftransgenicADanimalmodels hasdramaticallyadvancedourknowledgeofseveralaspectsofthe ADpathophysiologyandpathogenicmechanisms,eventhougha completelysatisfactorymousemodelofthepathologyisnotyet available.After1990,huntingtinand

a

-synucleinwereidentified astheproteinspolymerizedinthefibrilsdepositedinthebrainsof peopleaffectedbyHuntington’sandParkinson’sdisease, respec-tively(Mayersetal.,1993;Spillantinietal.,1998),eventhoughthe plaques deposited in the latter did not display the histologic characteristics of those found in the previously described amyloidoses. At thesame time, datasuggesting a basicshared neurotoxicmechanism of amyloidsstarted toappear (Schubert etal.,1995).

Untiltheendof1990s thedataavailableandthegeneticsof amyloiddiseases supported a quite general consensusthat the basictoxicspeciesinamyloidplaqueswereamyloidfibrils,even thoughnomechanisticdatasupportingfibrilcytotoxicityhadbeen clearlyreported.Therefore,itappearedlikelythatthepathogenic featuresunderlyingADandotherneurodegenerativeandsystemic amyloidoses weretheresultof thepresenceof extracellular or intracellular deposits of amyloid type. Besides providing a theoretical frame to understand the molecular basis of these diseases,suchascenariostimulatedtheexplorationoftherapeutic approachestoamyloidosesthatweremainlyfocusedathindering thegrowthanddepositionofamyloidfibrils.However,attheend ofthe1990stheattentionshiftedtothecytotoxicityofamyloid fibril precursors, notably amyloid oligomers and protofibrils (Lambertetal.,1998);dataalsostartedtoappearindicatingthat neuronalsynapseswerethemaintargetofcytotoxicity(Walshand Selkoe,2004)andthattheimpairmentofthelong-term potentia-tion was the main effect resulting fromthe oligomer–synapse interaction(Walshetal.,2002).Atthesametime,onthebasisof experimental evidence, the ‘‘generic hypothesis’’ of amyloid formationwasproposed(Dobsonand Karplus,1999). Thelatter statesthattheabilitytoassembleintoorderedcross-

b

structures cannotbeconsidered anunusualbehavior exhibitedbya small sub-set of peptides and proteins with special sequence or structural properties, but as a property inherent to the basic structure of polypeptide chains.Soon after,in 2002,data were reportedsuggestingforthefirsttimethatcytotoxicityisageneric propertyofamyloidoligomers(Bucciantinietal.,2002)associated witha shared ‘‘toxic’’ fold (Kayed et al., 2003). This newview shifted thetargetof pharmacologicalresearchaimedatfinding

molecules useful to combat cell/tissue impairment in amyloid diseases from counteracting fibril growth to hindering the appearanceofamyloidoligomers.

Atthepresent,thepivotalrole of amyloidoligomersaskey players of amyloid cytotoxicity is widely recognized and the amyloidhypothesishasbeenextendedtootheramyloiddiseases; presentlythedetrimentalroleofamyloidoligomersandother pre-fibrillarentitiesissupportedbyalargenumberofdataonmanyin vivo and in vitro amyloidogenic proteins indicating a direct cytotoxic effect of amyloid aggregates (Selkoe, 2003). The cytotoxicityofpre-fibrillaramyloidassemblieshasbeenconfirmed for allproteins and peptidesassociated withamyloiddiseases, includingA

b

peptides,

a

synuclein,amylin,beta2-microglobulin, transthyretinandothers(Walshetal.,2002;Clearyetal.,2005; Bhatia et al., 2000; Conlon et al., 2001; Conway et al., 2000; Nilsberthetal.,2001;Sousaetal.,2001;Poirieretal.,2002;Chung et al., 2003a). Pre-fibrillar assemblies of many proteins and peptides not associated with disease have been found to be cytotoxicaswell(DobsonandKarplus,1999;Sirangeloetal.,2004; Anderluhetal.,2005).

The growing awareness that the early oligomeric species transientlyarisinginthefibrillizationpathofproteins/peptidesare themost potent toxins to cells stems from a large number of studies on A

b

aggregation both in vitro and in vivo and A

b

aggregateneurotoxicity[reviewedinWalshandSelkoe,2004].The idea that A

b

oligomers appear inside neuronal cells and are responsibleforneuronalimpairmentwasfirstproposedin1998by theWilliamKleingroup(Lambertetal.,1998).However,oneofthe firstevidencesthatA

b

oligomersarereallyproducedincultured cellsandimpairneuronalcellphysiologyandviabilitycameonlyin 2002,whentheDennisSelkoegroupshowedthatA

b

oligomersare generatedandsecretedsoonafterpeptideappearanceinmodel cultured cells overexpressing APP containing the AD mutation V717F.Theauthorsalsoshowedthatmicroinjectingintoratbrains thecellmediumcontainingtheseoligomers,butnotA

b

monomers orfibrils,potentlyinhibitedhippocampallong-termpotentiation, oneof thesignsof thediseasein animalmodels (Walsh etal., 2002).Sincethen,manystudieshaveconfirmedtheSelkoeresults eventuallyleadingtoassess(i)thenatureofthoseA

b

oligomersas variously sized spherical dimer–hexamer to 24mer aggregates (ADDLs, amylospheroids) (Cleary et al., 2005; Townsend et al., 2006; Chromy et al., 2003; Hoshi et al., 2003); (ii) their phosphorylation state (Kumar and Walter, 2011); (iii) their presenceinsideADbrains(Gongetal.,2003;Lesne´ etal.,2006; Gouras et al., 2000);(iv) their formation within neuronal cells (Walshetal.,2000;Billingsetal.,2005;Muckeetal.,2000);(v) theirabilitytoimpairmemoryandcognitivefunction(Gongetal., 2003; Lesne´ et al., 2006; Gouras et al., 2000); and (vi) their involvementinphysicaldegenerationofsynapses(Muckeetal., 2000). Similar results have also been reported for amyloid oligomersgrown frommany other peptides/proteins[reviewed inCaugheyandLansbury,2003]providingsolidsupporttotheidea thattoxicityisageneralfeatureofthosespecies.

Thisreviewwillfocussomeaspectsofproteinmisfoldingand aggregationaswellasofamyloidoligomergrowth,structureand cytotoxicity. It will also stress the importance of biological surfaces,notably macromoleculesand phospholipid bilayers,as keyplayersofproteinmisfoldingandaggregation.Theimportance oftheinterplaybetweenmembraneandoligomer physicochemi-calfeaturesmodulatingamyloidcytotoxicityinthemembrane– oligomercomplexwillalsobetakenintoconsideration.

2. Proteinfoldingandaggregationarecompetingprocesses Since 1998, when it was first reported that two proteins unrelatedtoanyamyloidpathologywereabletoaggregateinvitro

into fibrillar assemblies of amyloid type (Gujiarro et al., 1998; Litvinovichetal.,1998),ithasincreasinglybeenrecognizedthat thetendencytoaggregate intoamyloidassemblies ofproteins/ peptidesisageneralpropertyofthepeptidebackbone(Stefaniand Dobson,2003;Chiti andDobson,2006;Bucciantini etal.,2002; Fa¨ndrichet al.,2001). Sucha propertyarises fromtheintrinsic tendency of polypeptide chains toself-organize into polymeric assemblies stabilizedbyinter-molecularhydrogen bonds estab-lished between the peptide bonds in parallel or anti-parallel polypeptide stretches in the beta-strand conformation. The resulting ordered arrangement, known as cross-beta structure, characterizestheamyloidfold[reviewedinSerpelletal.,2000]. The key role of the main-chain contacts does not reduce the importance,foramyloidfibrilgrowth,ofthesidechainsequence, that determinestheenvironmental conditionsunderwhich the polypeptide chain can aggregate. This view considers natural proteinsasagroupofaminoacidpolymersselectedbyevolution so as their amino acid sequences are optimized to disfavor aggregationwhilst favoring foldinginto compact,yet notrigid, states;theseresultmainlyfromthetertiaryinteractionsamong thesidechainsthatshieldnotonlythehydrophobiccorebutalso the peptide backbone. Conversely, protein aggregation into amyloid polymers, which are mainly stabilized by secondary interactions,canbeconsideredtheresultoftheemergence,under non-naturalconditions,oftheintrinsicprimordialtendencyofthe peptide backbonetogivesecondaryintermolecular interactions (reviewedinStefaniandDobson,2003;ChitiandDobson,2006). Such a paradigm considers peptides and proteins as polymers endowedwiththeintrinsicpropertytoself-organizeintohigher orderpolymericaggregates;it alsosuggeststhatproteinfolding andproteinaggregationmustbedistinctbutcompetingprocesses andthattheenvironmentalconditionsdictatewhichoneisfavored for a given polypeptide chain (Fig. 1). Accordingly, extensive studieshavebeencarriedoutinvitrotoinvestigatethetransition between natively folded statesand soluble aggregate-precursor states, and between the latter and mature amyloid fibrils (Wisemanetal.,2005).

That protein folding and aggregation can be competing processes is suggested by several theoreticaland experimental evidences.Forexample,thehighintra/extracellularconcentration of macromolecules, averaging 300–400mg/ml inside the cell, appearsoneoftheenvironmentalconditionsaffectingthechoice betweenfoldingandaggregation.Thisfeature,oftenreferredtoas macromolecularcrowding,affectsthermodynamicallythe confor-mationalstatesofproteins(Ellis,2001).Averyhigh macromolec-ular concentration meansthat thevolumefreelyavailable toa macromoleculeisonlyafractionofthetotalvolumewhereitis dissolved; therefore, theresulting excluded volume effects can favor thermodynamically, though to a different extent for any givenprotein,compactstructures,includingbothnativelyfolded andaggregatedstates.Onthisaspect,ithasbeencalculatedthatan increaseinmacromolecularcrowdingfrom30%to33%(intermsof thevolumeofagivenspaceoccupiedbymacromolecules)could resultinariseofthemolecularbindingaffinitiesbyasmuchasan orderofmagnitude(Ellis,2001).Theexcludedvolumeeffectscan also favor compact states resulting from aggregate nucleation whenproteinsandpeptidesareunabletoundergoefficientfold into monomeric compact states. However, it must also be consideredthattheincreasedviscosityofthemediumcanlower the diffusion-limited growthrate of the aggregationnuclei by reducingthetranslationalmovementsofthemacromolecules.The generalvalidityoftheseconsiderationsmustalsotakeintoaccount thattheystemfromstudiescarriedoutinvitromainlywith non-physiologicalcrowdingagents(PEGs,dextransandothers);these agentsdonotestablishinteractionswithproteinssimilartothose occurring with the crowding molecules found inside the cell

(nucleicacidsandproteins,includingmolecularchaperones)orin theextracellularmilieu(glycosaminoglycansandproteins, includ-ingmolecularchaperones).

Besidesmacromolecularcrowding,anumberofphysicochemical parametersofthepolypeptidechainaffectsimilarlybothprotein foldingandaggregation;theseincludeasignificantpropensityto gainsecondarystructure,alow netchargeanda relativelyhigh contentofhydrophobicresidues(Chitietal.,2003).Theimportance ofthesepropertiesforproteinfoldingandaggregationissupported bythestructuraladaptationsfoundinthenativelyunfoldedproteins (NUPs)(Uversky,2002).Itisalsoconfirmedbythedataindicating that mutations neutral to the thermodynamic stability but increasingthemeanhydrophobicityorthepropensitytogenerate betastructureorreducingthenetchargeofanyprotein/peptidecan favorkineticallyitsaggregationfromtheunfoldedstatesindynamic equilibriumwiththefoldedstructure(Chitietal.,2003).Thecaseof nativelyunfoldedproteinsrisesinterestingconsiderationsonthe relationbetweenstabilityandfunctioninproteinevolution.Protein thermodynamicstabilitycannotbeconsideredanabsolutegoalof evolution independently from the environmental conditions (temperature, ionic strength, pH) at which a protein folds and performsitsbiologicalfunction.Theexampleofnativelyunfolded proteinsandoftheproteinsofthehyperthermophilesclearlyshow thateachnaturalpolypeptidechainisaproductofevolutionwhich displaysspecificthermodynamicfeatures;thesemustensureproper stabilityofthenativelyfoldedproteinanditsfoldingintermediates makingitpossibletobeeasilyunfolded forcorrectdegradation. Moreover,properstabilityalsoresultsinoptimalflexibilityofthe proteinallowingit toperformitsbiologicalfunction atthe best underphysiologicalconditions.

Overall,thefindingsshowingthatproteinfoldingandprotein aggregationdependonthesamephysicochemicalfeaturesofthe polypeptidechainandofthemediumwhereitisdissolved,confirm theideathatbothpathwaysareinclosecompetition;itresultsthat anypolypeptidechaincanundergoeitherprocessdependingonits

structuralandphysicochemicalfeaturesandthemedium condi-tions. These considerations apply more correctly to protein domainsthantowholeproteins;infact,itappearsthatinproteins containing potentially amyloidogenic domains, evolution has embedded those domains in larger intramolecular contexts hinderingtheirtendencytoaggregate.However,theaggregation propensity appears when the domain is removed from its ‘‘chaperoned’’ context, is mutated or cleaved. Actually, recent datasuggestthatnaturalproteinsareengineeredbyevolutionso that,attheconditionswheretheyaresynthesizedandperform theirfunctions,foldingisfavoredoveraggregation,eventhough thelatterwouldbethermodynamicallypreferred;thiscanhappen inseveralways,includingthepresenceofhighkineticbarriersto unfoldingandtheevolutionofeukaryoticgenomesencodingboth proteinswithhighmolecularweights,thatlimittheirpropensity togrowintopolymersofamyloidtype(Uversky,2002)andalarge numberofmolecularchaperones.

Theviewthatproteinfoldingandaggregationarein competi-tion to each other considers both as distinct yet not mutually excludingprocessesrelyingonamoregeneralenergylandscape includingconformationalstates notdirectlyinvolvedin protein folding,yetpotentiallyavailabletoapolypeptidechain(Jahnand Redford,2008)(Fig.1,modifiedfromJahnandRedford,2008).The twosidesoftheproteinenergylandscapehighlightthe competi-tionbetweenintramolecular(folding)andintermolecular (aggre-gation)interactions,whichincreasesconsiderablytheroughness ofthewholelandscape.TheschemedepictedinFig.1showsthe complexity of the overall energy landscape of protein folding/ aggregation, which includes some of the main conformational statesapolypeptidechaincanreachduringitsself-organization eventuallyculminatingwiththeappearanceof thermodynamical-lyfavoredcompactmonomeric(folded)orpolymeric(aggregated) states (Jahn and Redford, 2008). In a living environment (cell, tissue), either stable final state may be even more favored thermodynamically by the above mentioned macromolecular Fig.1.Theenergylandscapeoftheconformationalstatesavailabletoapolypeptidechaininthefoldingandaggregationsections.Foldingandordered(amyloid)aggregation areprocessesrelyingonsimilarphysicochemicalparametersandhenceincompetitiontoeachother.

crowding, whose excluded volume effects favor stabilizing interactions offunctional, orpure thermodynamic, significance. Even though there is a substantial lack of information on the energy barriers a folded or a partially unfolded protein must overcometogainaccesstotheconformationalspacesallowingitto re-organize into aggregation nuclei, it is believed that these structuraltransitionscanbefavored,amongothers,bysurfaces. This is a key issue, considering that in the intracellular environment an extremely large surface area is provided by macromolecules and phospholipid bilayers. Actually, either biologicalorsyntheticsurfaces,besidesfavoring,inspecialcases, protein folding (molecular chaperones), can also be potent enhancersofproteinmisfoldingandacceleratorsofaggregation. 3. Proteinfolding/misfoldingonsurfaces

Mostofthepresentknowledgeonproteinfoldingarisesfroma multi-disciplinary approach including the use of a variety of simulationandexperimentalmeasurementscarriedouton wild-typeandside-directedmutantsoftheinvestigatedproteins.The mostwidelyusedexperimentaltoolsincludespectroscopic(CD, DLS,NMR),fluorimetric(fluorescencespectroscopy,FRET), diffrac-tion(smallangleX-raydiffractionscattering),massspectroscopy (MALDI-TOF coupled to limited proteolysis) and mutational (protein engineering) techniques as well as theoretical and in silicostudies(moleculardynamicssimulationsandmodel build-ing).Theexperimentalresultsprovidedbythesetechniquesareof immensevalue,however,ingeneral,theyhavebeenobtainedin testtubeexperimentswhereaverysimpleenvironmentispresent (usuallyabufferwithadefinedpHandionicstrengthcontaining someco-solventordenaturingagent).Theseconditionsarevery different from those found in the intracellular medium where proteinfoldingtakesplace;infact,theylackthepresenceofother factors potentially affecting protein folding, misfolding and aggregationsuchasspecificproteinligands,molecularchaperones andthemacromolecularcrowdingmentionedabove,withitsvery largesurface areaincludingtheoverall macromolecularsurface andthatprovidedbythecellmembranes(Ellis,2001).

Surfaces can favor reversible unfolding/refolding of specific proteins,forexamplewhentheyphysiologicallytranslocateacross a membrane (Bychkova et al., 1998); in other cases, suitable surfaces or environmentsprovided by specificmacromolecules foundinsideacellcanfavorfoldingofanewlysynthesizedorofa misfoldedprotein.Thisisthecaseofthelargeandheterogeneous family of the molecular chaperones, including the prokaryotic GroES/GroELandDnaK/DnaJsystemsaswellasmanyeukaryotic assemblies,bothintracellular(theHsp70/Hsp40system,otherHsp proteins and the crystallins) and extracellular (clusterin,

a

2-macroglobulin,haptoglobin).Thesemolecularchaperonesprovide asuitableenvironment,inmostcasesasurface,whereaprotein can fold rapidly and efficiently still exploiting exclusively the informationencodedinitsaminoacidsequence(Hartland Hayer-Hartl,2002).Inmostcases,themolecularchaperonesareableto specificallyrecognizeexposedhighlyaggregation-pronesegments avoiding their inappropriate interactions with other cellular componentsorwithsimilarsegments exposedonotherprotein moleculesthat couldpossibly result in aggregatenucleation.A recentsurveyintheproteindatabankhasshownthatthesehighly aggregation-prone segments are most often capped by basic residues. Besides hindering the mutual interaction of these stretchesthatcouldenhancethegenerationofaggregationnuclei, this also favors recognition of these stretches by molecular chaperones, thusfeaturing co-evolution of thelatter (Rousseau etal.,2006a).

Anotherexampleofsurfacesfavoringproteinfoldingconcerns thelargefamilyof theNUPs(see above),which includesmany

proteins involved in key functions such as gene transcription, controlofcellcycleandsignaltransduction(Romeroetal.,1998). ThemolecularfeaturesofNUPs,includingalowmean hydropho-bicityandahighnetcharge,besidesallowingthesemoleculesto remainunfoldedintheabsenceofbindingpartners,arealsoableto reducetheirintrinsictendencytoaggregateinthehighlycrowded intracellularmilieu(Minton,1994).Here,theunstructuredstateof most NUPs favors their binding to the molecular chaperones during their short living time before they interact with their specifictargetsoraredegraded(Uversky,2002). Actually,many NUPs are ‘‘short lived’’ proteins and adopt specific three-dimensionalstructuresonlyupon interactionwiththeirspecific targetsthatarethoughttoprovidethemasurfacesuitabletoallow theirfoldinginsome‘‘assisted’’manner(Dedmonetal.,2002).Itis also possible that the target protein provides some structural informationneededforthespecificNUPtoreachitscorrectfoldin theprotein-proteincomplex,inparticularchargedand hydropho-bicresiduescomplementingitsstructuraldeficiencies.Finally,the unfoldedstateofNUPsfavorstheirrapidintracellularturnoverby thecellularclearancemechanisms(WrightandDyson,1999).The latterfeaturecouldbeanadvantageforcertaincellularfunctions, providingafurtherlevelofcontroltoenablethecelltorespond rapidly,effectivelyandreversiblytoperturbationsofthecellular environment.

Thehugesurfaceextensionintheintra/extracellular environ-mentcanalsoinfluenceprofoundlythetendencyofaproteinto lose its native fold and to gain alternative conformations (misfolding)favoringaggregatenucleation.Forinstance,peptides and proteins can interact with, and be actively recruited by, biological surfaces thus modifying their conformational states. Indeed,intheadsorbedstate,theinteractionofproteinresidues withsurface-exposedhydrophobicorchargedgroupscaninduce localormoreextensiveunfoldingpopulatingnon-native, aggre-gation-proneconformations(Bokvistetal.,2004). Infact,under these conditions many protein molecules will loosen their structureandexternalizenormallyburiednon-polargroupsand thepeptidebackbonethatwillinteractwiththesurface-exposed hydrophobicclustersinaprocessthatcanbedifferentfromthat occurringinbulksolutionandenergeticallyfavored(Sethuraman andBelfort,2005).Suchaviewleadstoproposethat,similarlyto catalyzedreactions,surfacescanfavoramyloidaggregate nucle-ationandgrowthbyamechanismsubstantiallydifferentfromthat occurring in bulk solution (Zhu et al., 2002). Finally, besides populatingaggregation-proneconformerspossiblydifferentfrom those arising in solution, surfaces can actively recruit them increasingconsiderablytheirlocalconcentration;thiseffectcan remarkablyspeedaggregatenucleation(SethuramanandBelfort, 2005; Zhu et al., 2002) and induce structural alterations of membraneintegrity(Yipetal.,2001;Poratetal.,2003;Knightand Miranker,2004).

These considerations account for the increasing interest in investigatingthephysicochemicalfeaturesofproteininteraction withnatural or artificialsurfacesand therelationbetween the latterandproteinaggregation.Indeed,manystudieson heteroge-neousinterfacescarriedoutmainlyusinginorganicorsynthetic surfaceshaveshown thatbasicphysicochemical featuresofthe surface such as net electrostatic charge, hydrophilicity or hydrophobicity affect the conformational properties of the interacting peptide/protein (Necula et al., 2002; Hoyer et al., 2004; Arce et al., 2011). These studies have raised increasing interestontheroleperformedinproteinaggregationbybiological surfaces,notablymembranes,eveninrelationtotheirstructure andlipidcomposition.Inadditiontomembranes,othersurfacesin cellortissuecanalsofavorproteinaggregation.Forexample,ithas beenreportedthatbeta2-microglobulinbindstothecollagentriple helix and that binding affinity fluctuations could influencethe

concentrationofboththewild-typeandtheN-truncatedformsof the protein in the proximity of collagen fibers and hence its propensityto aggregate (Giorgetti et al., 2005). More recently, amyloidfibrilsofbeta2-microglobulinsproutingfromthesurface ofcollagenfibershavebeenimaged(Relinietal.,2006)providing mechanisticsupporttotheproposed explanationof the tissue-specificityofdialysis-relatedamyloidosis(Homma,1998). Other examples of biological surfaces favoring protein fibrillization includedifferentanionic macromoleculessuch as glycosamino-glycans(Suketal.,2006;Monsellieretal.,2010)andnucleicacids (NandiandNicole,2004;Chernyetal.,2004).Thesemolecules,by providingatrueinterfacetoproteinmoleculesactasmodifiersof the protein surface properties affecting protein stability and structure.

Membraneenvironmentisalsooffundamentalimportancefor the regulation of membrane protein degradation upon co-localizationofmembraneproteasesandtheirsubstrates; accord-ingly,inseveralcasesmembranescontributetoamyloid fibrillo-genesis as the primary sources of the aggregating peptide monomers.Examplesinclude theAbetapeptidesresultingfrom APP processing, the ABri/ADan peptides resulting from BRI processingandthepeptidesmedinandgelsolinarisingfromthe partialproteolysisoflactadherinandwild-typegelsolin, respec-tively(Pengetal.,2005;Huffetal.,2003).Onthisaspect,thestudy ofproteincompartmentalizationin discrete orderedmembrane domainssuchascaveolaeandlipidraftscanprovidefurtherclues tothe regulatoryeffects of membrane dynamics to membrane proteinturnoverbyproteolysis(RushworthandHooper,2010).In addition,thehydrophobicinterioroftheplasmamembranefavors structural changes in proteins and peptides and increases the strengthof thesecondary interactions,often leadingtoprotein aggregation,asithasbeenshownfortheprionprotein,amylinand Abetapeptides(Bokvistetal.,2004;Yipetal.,2001;Kazlauskaite etal.,2003a;Engeletal.,2006;Lu¨hrsetal.,2006).Finally,lipid membranescan enhance proteinaggregation also favoring the growthofsupramolecularlipidproteincomplexes,asithasbeen reported for the islet amyloid polypeptide (Domanov and Kinnunen,2008).

The ability of lipid membranesto unfold monomersand to nucleateandrecruitamyloidoligomerssuggeststhatmembrane lipidcompositionisofrelevancefortheseeffects.Indeed,several reports highlight the key role of anionic surfaces and anionic phospholipid-rich membranes as triggers of protein/peptide fibrillization(Neculaetal.,2002).Negativelychargedmembrane surfaceshavealsobeenproposedaspotentinductorsofbeta-sheet structures by acting as conformational catalysts for amyloids (Jayakumaretal.,2004;Zhaoetal.,2004).Furthermore,ithasbeen shownthatphosphatidylserine(PS)-containingliposomesinduce amyloidaggregationofavarietyofproteinsinvitro(Engeletal., 2006)andthatannexin5protectsagainstA

b

-peptidecytotoxicity bycompetingatacommonPS-richsite(Leeetal.,2002).Finally,it hasbeenproposedthatmembranesrichinanionicphospholipids couldinteractwithamyloidaggregatespossiblybyrecognizinga sharedfold (Zhaoet al.,2004). Thesefindings alsosuggest that amyloid aggregate toxicity can be different depending on the aggregatecellular localization. In fact, PS is almost exclusively foundinthecytosolicleafletoftheplasmamembraneinallcells other than apoptotic, cancer and vascular endothelial cells in tumors,whereitmovestotheouterleaflet(Utsugietal.,1991;Ran andThorpe, 2002);this can beof importance,consideringthat recent data suggest that A

b

peptide aggregates display their cytotoxicpropertiesmainlyintheintracellularlocation(Kaminski Schierleetal.,2011).

The above mentioned data couldexplain the selective anti-tumorspecificityofeitherendostatin(Zhaoetal.,2004,2005)and thefoldingvariantsof

a

-lactalbumin(Svanborgetal.,2003)and

apoptin(Zhangetal.,2003).Itmustalsoberemindedthatamyloid aggregatesarefoundinawidevarietyofmalignancies( Kazlaus-kaiteetal.,2003a);thelatterfeaturehasledtosuggestthatthe presence of PS at the surface of cancer cells and vascular endothelial cells in tumors could favor the formation of toxic aggregates by these anti-tumor proteins. Such idea is further supported by the observation that a number of antimicrobial peptides known to target phosphatidylglycerol in microbial membranesarealsoabletokillcancercells(HancockandScott, 2000).Finally, recentresearchreports

b

-amyloid-induced mod-ificationsofthestructureandpermeabilityofthemitochondrial, lysosomal and ER membranes (Kaminski Schierle et al., 2011; Ferreiroetal.,2004,2006;Aleardietal.,2005;Caspersenetal., 2005),althoughasubstantiallackofinformationstillexistsonthe effectiveoccurrenceoftheinteractionofamyloidaggregateswith cellorganellesandonthemolecularfeaturesofsuchinteraction. An increasing number of data highlight the importance of membrane lipid composition in modulating either protein aggregation at the membrane level and aggregate interaction withcellmembranesalsoforwhatthecontentofcholesteroland gangliosidesin concerned. Itis known that APP andsecretases preferentially localize into ganglioside and cholesterol-rich membrane microdomains(lipid rafts)(Lee etal., 1998;Ehahalt et al., 2003; Kakio et al., 2003). Several lines of evidence also supporttheideathatthepathologicalconversionofthecellular (PrPc_{)tothescrapie(PrP}sc_{)formoftheprionproteinoccursatthe}

lipidraft level(Prusineretal.,1998; Harris,1999;Kazlauskaite etal.,2003b;Sarnataroetal.,2004)andthatthePrPcconformation is stabilized upon association with lipid rafts in the secretory pathway(Sarnataroetal.,2004).Ithasthereforebeenproposed thataggregationofsolubleA

b

peptidesandtheprionproteincan beraft-associatedprocesses (Ehahalt et al.,2003)and that any alteration of cholesterol homeostasis can be a shared primary causeofseveralneurodegenerativediseases(Kakioetal.,2003).

TheinteractionofA

b

peptideswiththecellsurface,particularly at sites rich in membrane cholesterol, has beenconsidered an important requirement for neurotoxicity (Kakio et al., 2002; KoudinovandKoudinova,2005).Ontheotherhand,reducedlevels ofcholesterolarefoundinbrainsfromADpatients(Masonetal., 1992)andalossofcholesterolinthebrainleadsto neurodegen-eration(ArispeandDoh,2002).Inthisregarditisinterestingto notethatrecentstudieshaveshownthattheexpressionof seladin-1,adesmosterolreductaseinvolved,amongothers,incholesterol synthesis,appearstobedownregulatedinADbrains(Benvenuti et al., 2006) even though many data indicate that increasing cholesterolinneuronalmembranesresultsinenhanced produc-tionofA

b

peptides(reviewedinStefaniandLiguri,2009).Recent reportsalsoshowthatlossofcholesterolinneuronalmembranes enhances amyloid peptide generation (Abad-Rodriguez et al., 2005) and that cell interaction with pre-fibrillar aggregates supplemented to the culture mediaand aggregate cytotoxicity are reduced upon enriching in cholesterol the cell membrane (ArispeandDoh,2002;Cecchietal.,2005).Finally,theimportance of membrane cholesterol in cell membrane resistance against disruption by A

b

peptides has recently been reported by a molecular dynamics simulation study (Qui et al., 2011). The relationbetweencholesterolcontent,A

b

aggregategenerationand cytotoxicityrequiresmoreextensiveresearch;however,thedata presentlyavailablesupporttheideathat,ingeneral,thepresence ofcholesterolinthecellmembranecanmodulateconformational changesofspecificprotein/peptidesandthatahighermembrane rigidityfollowingincreasedcholesterolcontentcanbeprotective againstaggregate interactionwithcell membranesandreduces theircytotoxicityarisingfrommembraneintegrityperturbation. GM1,themostabundantraftganglioside,particularlyenriched inneuronal cellmembranes,isthemaincarrierofthenegative

chargeoccurringontheouterleafletoftheplasmamembraneof mostcells.Thecontentofgangliosides,particularlyGM1andGM2 isincreasedindetergent-resistantmembranes(DRMs,considered tocorrespondfunctionallytothemembranerafts)purifiedfrom neuronal membranes of AD people, further supporting their importanceinA

b

pathology (Molander-Melin etal., 2005).The latterisconfirmedbyalargebodyofevidencethatGM1andother gangliosides, together with their negatively chargedsialic acid moietiesandtheir lipidenvironment,arekey sitesnotonlyfor nucleationof A

b

peptide aggregates (Yuyama and Yanagisawa, 2010)but also for oligomer interaction at the cell membrane, possibly upon ganglioside clustering (Fujita et al., 2007). In particular, several studies have indicated that the preferential binding to GM1 of monomeric and oligomeric A

b

stimulates peptidefibrillization(WakabayashiandMatsuzaki,2007; Matsu-zaki etal., 2010;Malchiodi-Albedi etal., 2010; Mclaurinetal., 1998).Finally,recentevidenceindicatesthatA

b

depositionstarts atpresynapticterminalsintheADbrainandthatthelevelsofGM1 are significantly increased in amyloid-positive synaptosomes obtained from AD brains, suggesting that age-dependent GM1 clusteringatthepresynapticterminalsofneuritescanbeakeystep forA

b

depositioninAD(Yamamotoetal.,2008).Similareffects havebeenreportedforotherpeptidessuchasamylin( Wakabaya-shiandMatsuzaki,2009)andsalmoncalcitonin(sCTO)(Diociaiuti etal.,2006)suggestingthepossibility thattheseeffects canbe somehow generalized. The importance of the negative charge carriedbygangliosidesisconfirmedbythedataindicatingthat treatmentwithneuraminidase,whichremovesfromgangliosides thesialic acidmoiety withits negativecharge, inhibits amylin neurotoxicity and its associated biochemical modifications in exposedcells(WakabayashiandMatsuzaki,2009).

Takentogether,thedataontheeffectofmembranecholesterol andgangliosideson protein/peptidemisfoldingand aggregation andoligomerinteractionwiththecellmembranefurthersupport theimportanceoflipidraftsaskeysiteswheretheseprocesses occur. They also provide a possible rationale of the acute vulnerabilityofneuronalcellstoamyloidsconsideringthelarge surfaceareaofthesecellsandtheenrichmentinmembraneraftsof theirplasmamembranes.

4. Theonsetofproteinaggregation

Thegenerationof aggregationnuclei isconsidered the rate-limitingstepintheonsetofproteinaggregation,accountingforthe delaytimesof polymerappearancethatarerecordedininvitro protein aggregation experiments. However, at variance with proteinfolding,wheresignificantknowledgehasbeen accumulat-ed in the last decade, presently much less is known on the conformational states available to an aggregating polypeptide chainandontheatomiclevelstructuralfeaturesoftheoligomeric assembliesarisingintheearlystepsofaggregation.Actually,some of the energy minima in the aggregation side of the energy landscape(Fig.1)are expectedtobepoorlydefineddue tothe broadheterogeneityofunstable,highlydynamicearlyoligomeric statesendowedwithcomparablefreeenergies.Onthecontrary, theenergyminimaofthemuchmorestructurallydefined,stable higher order species (protofibrils, protofilaments and mature fibrils)can be muchmore easily identifiable. For example, the stabilities,andhencetheenergyminima,ofmatureamyloidfibrils, and their structural variants grown at different conditions (Petkovaetal.,2005)areexpectedtobemorepronouncedthan thoseoftheirnativelyfoldedmonomersconsideringthereduced molecular dynamics and the consistency of the ordered core structureofthefibrils.Thenucleation-dependentpolymerization mechanismoffibrilgrowth,whosephysicalbasisapproachesthat oftheorderedassemblyoccurringincrystalgrowth,alsosupports

fibril stabilityand represents a key difference between protein foldingandaggregation.

Aproteincanbeshiftedfromthefoldingtotheaggregation side of its energy landscape by enhancing the factors that promotemoreconsistentlyaggregationoverfolding(Fig.1).For example,proteinstabilitycanbereduceduponmodificationof theenvironmentalconditions(temperature,pH,medium polari-ty, ionic strength) or the structural features of the specific protein/peptide (mutations, truncations, chemical modifica-tions),orevenbymerelyincreasingitsconcentration.Thelatter canresultinaggregatenucleationwhentheconcentrationofthe nucleationprecursorsexceedsa criticalthreshold(reviewedin

ChitiandDobson,2006).Inmostcases,proteinaggregationcan be started in the presence of mildly destabilizing medium conditions;theseincludeamildshiftofthetemperatureorthe pHorthepresenceofmoderateamountsofdenaturingagentsor of co-solvents such as trifluoroethanol. The latter modify the dielectricconstantofthemediumthusincreasingthestabilityof the secondary contacts while reducing that of the tertiary interactionsinthefoldedprotein(ColonandKelly,1992;Chiti etal.,1999;McParlandetal.,2000).Undertheseconditions,the destabilizedproteinopensitscloselypackedfoldedstructureand populates partially unfolded states exposing hydrophobic patches and aggregation-prone regions normally buried into thehydrophobiccore,includingthepeptidebackbone,normally shieldedbythesidechains.Thesepartiallyunfoldedstructures can bear similaritiestothe folding intermediates(Chitiet al., 2003;Apetrietal.,2004;Uverskyetal.,2001)ortosomeofthe near-native conformationsindynamicequilibriumwithafully folded protein. The link between native state dynamics and fibrillaraggregationofa proteinhasbeenhighlightedbymass spectrometryexperimentsin thecaseoflysozyme. Ithasbeen shown that the relative instability of the partially folded precursors of the latteris the driving force allowingthem to re-organizeintostillpoorlystable,andoftenthermodynamically disfavored, transientaggregation nucleirich inbeta structure, establishedbetweenstretchesofpolypeptidechainsinthebeta strandconformation(Dumoulinetal.,2005).

Thekineticsofnucleationoftheaggregatesofagivenprotein/ peptidecandependontheproteinconcentrationandstructureas well as on the medium conditions affecting the height of the energeticbarriertobeovercomeforamyloidaggregatenucleation can occur; atvariance, the subsequentthermodynamically and kineticallyfavorednucleielongationproceedsuntilcompletionof fibril assembly. In some cases, instead of aggregation nuclei, sphericaloligomersandotherpre-fibrillarforms,includingcurvy protofibrils,canbeformed,apparentlyresultingfroma nucleation-independent path intheabsence ofanylag phase (Harper and Lansbury,1997;Modleretal.,2003;Gosaletal.,2005;Smithetal., 2006a). In most cases, it is not clear the difference between oligomersandaggregationnucleinorwhetheroligomersare on-pathwayproducts,growingbydirectbindingofmonomers,or off-pathwayentitiesrepresentingdead-endreversibleintermediates (Gosaletal.,2005;Bitanetal.,2003;Baskakovetal.,2002;Necula etal.,2007).Forexample,beta-2-microglobulinandotherproteins can exist in different aggregation states depending on protein structuralfeaturesandmediumconditions.Someofthesestates (oligomeric species and beaded protofibrils) are off-pathway products (Petkova et al., 2005; Gosal et al., 2005; Kad et al., 2003)arisingfromthepolymerizationofpartiallyfoldedspecies retaining significant amount of native structure and involving someofthenativebetastrands(McParlandetal.,2002);thelatter speciesaredifferentfromtheoligomersappearinginthepathof fibril formation, which involves extensive structural rearrange-ment until the stable cross-beta structure of amyloidfibrils is reached(Smithetal.,2006a).

Thepresenceofsignificantnativestructureinthe conforma-tional states of a protein/peptide undergoingthe first steps of aggregationisfurthersupportedbyrecentfindingsshowingthat, at least in some cases, a protein can aggregate by initially populatingmonomeric or oligomeric states where it maintains substantially its natively folded structure before undergoing structuralrearrangements intoamyloids(see later).In addition totheabovementionednativelyfoldedbeadedprotofibrilsof beta-2-microglobulin,otherproteinshave beenproposedtoundergo orderedfibrillarpolymerizationretainingtheirnativefoldatleast intheinitialsteps.Theseincludetheserpins(reviewedinLomas andCarrell,2002),whereadomainswappingmechanismhasbeen shown,transthyretin,forwhicha modelwithdirect stackingof natively folded monomeric subunits has been proposed (Serag etal.,2002),T7 endonucleaseI(Guoand Eisenberg,2006), and p13suc1(Rousseauetal.,2006b).Apartserpins,wherethewhole polymerizationprocessoccurswithoutgrossmodificationofthe proteinglobularstructure,inallthesecasesmarkedmodifications ofthesecondarystructureaccompanythefinalproductsofprotein polymerization. A similar mechanism could also underlie the generationofnative-likefibrilsbytheyeastprionUre2p(Bousset etal.,2002)andthefirststepoftheamyloidaggregationofthe acylphosphatasefromthehyperthermophileSulfolobussolfataricus (Plakoutsietal.,2005).

5. Generalstructuralfeaturesofamyloidoligomers

Due to the variable cytotoxicity of structurally different amyloid assemblies, the study of the amyloid structure is becomingamainfocusintheinvestigationofthemolecularbasis of amyloid diseases. As reported above, amyloid nucleation is considereda key eventintheonsetof proteinaggregationand oftenthe rate-limiting step of fibrilgrowth. However, reduced knowledgeiscurrentlyavailableonthestructuralandbiophysical featuresofpre-fibrillarassemblies,particularlyamyloidoligomers grownatdifferentconditions.

Manyinvitrostudiesperformedinthelastdecadehaveshown themorphologicalmodificationsoccurringinamyloidassemblies grown from different disease-related and disease-unrelated peptides and proteins undergoing fibrillization (Quintas et al., 2001; Relini et al., 2004), and a theoretical frame of these modificationshasbeenprovidedbymoleculardynamics simula-tionstudies(Cheonetal.,2007).Thesestudieshaveshownthat severalmore-or-lessdefinedstepsareinvolvedinamyloidfibril growth.EMandAFMimageshaveshownthat,inmanycases,atthe onsetoftheaggregationprocesstransient,unstable,roundishor tubular particles 2.5–5.0nm in diameter generally, but with exceptions, enriched in

b

-structure, often called ‘‘amorphous aggregates’’arepresent (Reliniet al.,2004; Cheonet al.,2007; Lashuel et al., 2002; Poirier et al., 2002). These species are characterized as protein/peptide oligomeric assemblies that frequentlyassociate to each other into bead-like chains, small annularrings(‘‘doughnuts’’or‘‘pores’’),orcurvyprotofibrils.The latteroftenappeartobeprecursorsofhighlyorganizedamyloids suchaslargeclosedrings,ribbons(Relinietal.,2004;Lashueletal., 2002;Poirieretal.,2002;Linetal.,2001)orlongerprotofilaments eventually generating maturefibrils [reviewed in Caughey and Lansbury,2003].Overall,thedataemergingfromtheseandother studiesdepictamyloidfibrilgrowthasahierarchicalprocesswith ‘‘deadend’’routesinwhichboththemonomerbasicstructureand theenvironmentalparameters concurtodetermine thegeneral conformationalpropertiesofthemisfolded/unfolded monomers initiallypresent.These propertiesdictate,toa large extent, the structuralmodificationsresultinginmonomerarrangementinto less or more compact oligomers with variable hydrophobic exposure and subsequently in oligomer re-organization into

increasingly more complex, ordered and stable

b

-sheet-rich assemblies.Thatsuchaprocesscanbephysicallyapproachedto crystal growth is shown by the data indicating that seeds of polymorphicmaturefibrilsortheirintermediatesgrownfromthe samepeptide/proteinoroffibrilsgrownfromdifferentmonomers oftenareabletospeedupthegrowthofoligomersandmature fibrils(Baldwinetal.,2011);furthermore,thesespecies,though sharing the general basic cross-beta structure of amyloids, maintainthesamespecificstructuralandmorphologicalfeatures oftheseedingassemblies.

Thepresenceofprefibrillaramyloidshasbeenshowninthe aggregationpathofmanyproteinsandpeptideseitherassociated (A

b

peptides,

a

-synuclein,superoxidedismutase,huntingtin,the androgenreceptor,amylin,transthyretin,serumamyloidA,and others)(Quintas etal., 2001;Cheon etal., 2007;Lashuel et al., 2002;Poirieretal.,2002)ornotassociated(Bucciantinietal.,2002, 2004;Sirangeloetal.,2004;Relinietal.,2004;Ceruetal.,2008) with amyloid disease. Although most of these assemblies are consideredintermediatesarisinginthepathoffibrillization,some ofthem,suchasthefrequentlyimagedsmallannularoligomers (‘‘doughnuts’’)mentionedabove,couldbe‘‘deadend’’productsof theprocessor,atanyrate,structurallyandfunctionallydistinct typesofamyloidoligomers(Kayed etal.,2009). Finally,soluble oligomers arising in the aggregation of several peptides and proteinshavealsobeenrepeatedlydetectedin,andinsomecases purified from,cultured cells and tissues where themonomeric precursorsareexpressed(Clearyetal.,2005;Lesne´ etal.,2006; Billingsetal.,2005;Koffieetal.,2009).Thesedatareinforcethe idea that amyloidoligomers are really formedin vivo and are directlyassociatedwithcell/tissueimpairment(Koffieetal.,2009). Theincreasingbodyofknowledgesupportingthecytotoxicity ofearlyaggregatesofpeptidesandproteinseitherassociatedor not associated with amyloid disease implies that amyloid cytotoxicityarisesfromsharedcharacteristicsofthe supramolec-ular structure of the aggregates rather than from any specific feature of the amino acid sequences of their parent soluble polypeptides(Bucciantinietal.,2004;Demuroetal.,2005).This concept appears in contrast with the properties of functional proteins, whose native structures and biological functions are specificallydeterminedbytheiraminoacidsequences.Itcanbe concludedthatneitherthestructurenorthefunctionalproperties (notablycytotoxicity)ofproteinconformationalstatesotherthan thenativeone are directlydetermined, as in thelatter,by the specific interactions among side-chains (reviewed in Chiti and Dobson,2006).

In spiteof theincreasingnumber of techniquesused toget information on oligomer structural features and growth (see below), theexperimental information accumulated in the past yearsontheseissuesisstillremarkablypoor.Severalmodelshave beenproposedtodescribemechanisticallytheself-organization into oligomeric assemblies of misfolded monomeric peptides/ proteins. According to the widely accepted model known as ‘‘nucleatedconformationalconversion’’(Carullaetal.,2009;Petty andDecatur,2005),agroupofunstablemisfoldedmonomersin solution coalesce generating relatively disordered ‘‘molten’’ or ‘‘metastable’’ oligomerswhose constituting monomersundergo extensivestructuralreorganizationwhichtemplatesnew incom-ing misfolded monomers. Such a reorganizationand monomer recruitment results in increasingly structured oligomers and higherorderassemblieseventuallyculminatingwiththe appear-ance of mature fibrils (Cerda`-Costaet al., 2007). Although this model has been supported by experimental and theoretical observations (Plakoutsi et al., 2005; Serio et al., 2000; Bader etal.,2006),anindepthexperimentallybaseddescriptionatthe molecular level of the phenomenon, particularly of its earliest steps,remainslargelyelusive.

In most of the investigated systems, experimental and theoreticalobservationssupportageneric‘‘twostep’’mechanism ofoligomergrowththatappearshighlydependentonthedegreeof hydrophobicityandtheextentofexposedhydrophobicsurfaceof themisfoldedmonomers.Hydrophobicityfavors monomer coa-lescence into initially disordered oligomers, whose subsequent conformationalreorganizationgivesrisetoincreasingly ordered species that sequester more efficiently a progressively more compacthydrophobiccore (Plakoutsiet al.,2005; Cheon etal., 2007;Carullaetal.,2009;PettyandDecatur,2005;Cerda`-Costa etal.,2007;Serioetal.,2000;Baderetal.,2006;Nguyenetal., 2007).In caseofinsufficientmonomerhydrophobicity,thefirst coalescencestepcanbeskippedandthemonomerscanorganize directlyintoorderedoligomerswheresecondaryinteractionssuch ashydrogenbondspredominate(Cheonetal.,2007).Anexample ofthis‘‘onestep’’mechanismcanbeprovidedbyaggregationfrom nativelyfoldedproteins [Chiti andDobson, 2009], inwhich the degreeof exposure of hydrophobicresidues in theaggregating monomers is likely to be low. This view suggests that the intermolecular hydrophobic interactions, together with protein concentration and temperature, are among themajor determi-nantsof therate of thehydrophobiccollapse of themisfolded monomersintodifferenttypesofoligomers.Italsosupportsthe ideathatthesequence ofappearanceofdisordered–ordered,or only ordered, oligomers depends on the balance between the rapidly forming intermolecular hydrophobic interactions and theslower exchangeofthedirectionalhydrogen bondsintothe assemblingoligomers(Baderetal., 2006). Finally,it provides a theoreticalframetothewidelyacceptedideasthataggregationisa generic property of virtually every polypeptide chain and that amyloidsgrownfromstructurallydifferentpeptidesandproteins shareabasicstructuralmodel.

Theimportanceoftherelativecontributionsofthehydrophobic forces and the hydrogen bonds rationalizes the findings that different solution conditions favoring or disfavoring hydrogen bondingcanpromotethegrowthofdifferentaggregates(fibrillar ornon-fibrillar) withsimilar

b

-sheetcontent, yetwithdistinct morphological features (Calamai et al., 2005). Therefore, it provides a molecular basis to the polymorphism of amyloid oligomersgrownfromthesamepeptide/proteinunderdifferent solutionconditionsor in thepresenceof differingdestabilizing agents (temperature,pressure, urea, co-solvents,ionicstrength, and others). The importance of oligomer polymorphism is increasinglyrecognized,explainingseveralobservations,suchas the propagation of prion strain infectivity and other protein polymorphisms(Petkovaetal.,2005;Calamaietal.,2005;Chien etal.,2004;JonesandSurewicz,2005),thevariablecytotoxicityof amyloids grown from the same peptides/proteins at different conditions(Kayedetal.,2009;Chafekaretal.,2008;Deshpande etal.,2006;Iijimaetal.,2008),theappearanceofin-pathor off-pathintermediatesoffibrilgrowth(Poirieretal.,2002;Gosaletal., 2005; Iijimaet al., 2008; Dusa etal., 2006), and thestructural heterogeneityofamyloidfibrilsandtheirprecursorsgrownfrom thesame peptide/proteinunder differentenvironmental condi-tions(Gosaletal.,2005;Mayer-Luhemanetal.,2006;Goldsbury etal.,2005)(seePar.9).

6. Severalnewlyintroducedtechniquescanbeusedtostudy thestructureofamyloidoligomers

Aspointedoutabove,inspiteoftheconsiderableinformation currently available on the cytotoxicity of amyloid pre-fibrillar aggregates,theheterogeneity,remarkableinstabilityand intrinsi-callydisorderednatureofthesespeciesmakesitverydifficultto get solid data on their conformational features. An important contribution to unravel this point has came from the use of

conformationalantibodiesraisedagainstamyloidfibrilsandtheir precursorsthatareabletorecognizespecificstructuralfeaturesin different forms of these assemblies and hence to discriminate amongthem.In2002aseminalstudyreportedthegenerationofan antibodyrecognizingagenericconformationalepitopeinamyloid fibril,thushighlightingforthefirsttimethepresenceofashared conformationinfibrilsgrownfromdifferentpeptidesandproteins (O’NuallainandWetzel,2002).Thisknowledgewasimplemented andsubstantiatedbysubsequentreportsdescribingthegeneration of antibodies able to specifically recognize amyloid oligomers grownfromdifferingpeptidesandproteinsinsteadthanmature fibrils(Kayedetal.,2003)ortocross-reactwitheitheramyloid poresandwithporesformedbypore-formingproteins(Yoshiike etal.,2007).Anotherrecentreportdealswiththegenerationofan anti-A

b

amyloid fibril antibody that recognizes a shared, sequence-independent, conformational epitopepresent in amy-loidfibrilsgrownfromdifferentpeptides(Kayedetal.,2007).This antibody alsorecognizes soluble A

b

fibrillaroligomers but not apparentlysimilarprefibrillaroligomers,indicatingtheexistence of structurally distinct families of amyloid oligomers with potentially different cytotoxicities (Par. 9). These data suggest the existenceof at least two alternative aggregationnuclei for amyloidfibrilsgrownfromA

b

and,possibly,otherpeptides:one typeevolvingintomaturefibrilsdirectlybutonlyafterextensive structuralreorganization,andanothertype,possiblythetruefibril precursor,growingintoincreasinglysizedaggregatesbyaddition ofmonomersthatacquireanewstructurewhenincorporatedinto the oligomer. Finally, a recent study reports the generation of conformation-dependent monoclonal antibodies able to distin-guish between different subsets of replicating strains of con-formers in the same population of pre-fibrillar A

b

oligomers displayingdifferentsizedistributions.ThesedataindicatethatA

b

oligomerstructurevarieswithsizeandconfirmtheexistenceof structural polymorphisms in amyloid oligomers (Kayed et al., 2010)(Par.9).

In general, theuseof conformationalantibodiesrecognizing specificallyamyloidoligomers,pre-fibrillarassembliesormature fibrils hasprovided avaluable tooltodistinguishthesedistinct entities, clearly showing the existence of differences in their structuralfeatures(Glabe,2004;Mamikonyanetal.,2007;Kayed andGlabe,2006).Ithasalsoallowedtodefinitivelydemonstrate(i) thatthesameprotein/peptidecangenerateoligomericand pre-fibrillarassembliesapparentlyundistinguishable,yetwith differ-encesintheirfinestructuraldetails(Kayedetal.,2007;Kumarand Udgaonkar,2009),(ii)that,conversely,structuralsimilaritiesare presentincomparableassembliesgrownfromdifferentpeptides andproteins(Yoshiikeetal.,2007;Kayedetal.,2007;Glabe,2004); (iii)thatimmunologicaltoolssuchasmonoclonalandpolyclonal antibodies,nanobodies,orotherscanbeofkeyimportancetostudy amyloid polymorphism. Finally, it has provided knowledge showingthatthesameprotein/peptideattheonsetofaggregation cangenerateoligomericandpre-fibrillarassemblieswith distinct-lydifferentstructuralfeatures(Kayedetal.,2007,2010;Kumar and Udgaonkar, 2009);accordingly, monoclonal conformational antibodiesprovideatool toinvestigatethestructural modifica-tionsunderlyingthehierarchicalgrowthofamyloidfibrilsaswell asoligomerstructuralheterogeneity.

Besides the above described immunological tools, in silico molecular dynamics simulations (Berryman et al., 2011) and traditional highresolutionimaging techniquesarealsoof great valuetogetinformationonthestructuralfeaturesand polymor-phism of fibrilnuclei, oligomersand other amyloid assemblies (reviewedin Dobson,2003). Theseinclude electronand atomic forcemicroscopy(Dobson,2003),X-raycrystallographyandother recently introduced biophysical tools, such as single-molecule spectroscopic(notablyfluorescence)techniques(Orteetal.,2008;

CalamaiandPavone,2011;Kamibnski-Schierleetal.,2011),ESI/ MS(Smithet al.,2006a), SAXS in solution(Giehm etal., 2011; Oliveiraetal.,2009)andss-NMR((Mustataetal.,2009),reviewed inLangkildeandVestergaard,2009).Forexample,arecentSAXS study of

a

-synuclein fibrillation in solution has revealed the presence of a wreath-shaped oligomer made of around 16 monomerswitha centralpore.Theoligomerappearstobehave as an in-path fibril building block by a stacking process and displays intrinsic cytotoxicity most likely through membrane permeabilization(Oliveiraetal.,2009).

Overall, the data obtained by these immunological and biophysical techniques indicate that pre-fibrillar aggregates of different proteins and peptides share some basic structural features that are different from those displayed by the folded monomers or their fibrillar counterparts. These features can include the different exposure to theaqueous environment of hydrophobicpatchesnormallyburiedintothecompactstructure of the monomeric parent protein following its unfolding. For example,extensive investigationon alpha-synuclein hasshown that itstransiently formed oligomericspecies are rich in beta-sheet,exposehydrophobicclustersanddisplayapartiallyfolded structure(Uverskyetal.,2001;Dusaetal.,2006).Onthisaspect, chemicalmodificationsand,moregenerally,altered environmen-talconditionscanplayapivotalroleinaggregatenucleationand growth; in fact, they can not only promote/hinder protein misfolding and aggregation but also affect the conformational featuresoftheaggregationnucleithustargetingthemtoorganize intofibrilprecursorsandmaturefibrilswithdifferentstructures, stabilitiesandcytotoxicities(ChenandKokholyan,2005;Danzer et al., 2007; Bravo et al., 2008; Bolognesi et al., 2010). The conformationalfeaturesofamyloidoligomersbesidesexplaining their intrinsic instability also provide clues to explain their tendency,andthat ofotherunstable pre-fibrillaraggregates, to interactinappropriatelywithcellularcomponentsand, according-ly,theirtoxicpotential.

7. Amyloidfibrilscanbetoxiceitherdirectlyorasasourceof toxicspeciesintissue

Asreportedabove,presentlythemosttoxicspeciesamongst amyloidsareconsideredtheearlyoligomersandotherpre-fibrillar aggregates whereas amyloid fibrils are taken as by far less cytotoxicoreveninertreservoirsoftoxicspeciesand,assuch,with protectivesignificanceforthecell.However,insomecases,direct fibrilcytotoxicity(Gharibyanetal.,2007;Novitskayaetal.,2006; Bucciantini et al., 2012), or cytotoxicity associated with fibril assemblyandgrowthonlipidmembranes(Engeletal.,2008),has beenreported,supportingtheideathatanyoversimplificationof thisthemecanbemisleading.

Eventhoughtheamyloidcascadehypothesisremainsthefirst choicetoexplainADpathogenesis,itispossiblethattheeffective importanceofnon-fibrillarprecursorsofamyloidaskeyplayersof cell/tissueimpairmentinvivohasbeenoveremphasizedandthat thescenarioismuchmorecomplexinvivo.Actually,arecent re-evaluationoftheamyloidcascadehypothesisproposesthatthree key events are likely to occur in sequence in the aged brain developing sporadic AD: (i) an initiating injury (including a numberofdifferentnoxiousstimulifavoringA

b

productionand extracellular A

b

accumulation as amyloid deposits and toxic oligomers);(ii)theresultinginflammatoryresponsetriggeredby themicrogliaand(iii)thesubsequentfunctional changesofthe braincells(Herrup,2010).Thisnewviewintegratestheamyloid hypothesiscascade intoanamyloiddeposition cyclewhere the amyloid plaque pathology, stemming from extracellular A

b

accumulation, by reinforcing microglial activation is strictly correlatedwith,but distinctfrom,thefundamentalstepsofAD

pathogenesismentionedabove.ExtracellularA

b

accumulationcan alsodependonspecificneuronalfunctionalfeatures,asshownby recent dataindicating thatin APP transgenicmicetheregional vulnerability to A

b

deposition is modulated by the level of neuronalactivity(Beroetal.,2011).

Inadditiontotriggeranoxiousinflammatoryresponseintissue, amyloidfibrilscanaffectcellviabilityalsoasprovidersoftoxic species.Thatfibrillardepositscanleaktoxicspeciesinvitroandin vivo issupported by several recentinvestigations. Afirst study concernsthepositiveeffectofsulfatedpolysaccharidesonA

b

42 assemblyintohighlystable,thinandharmlessamyloidfibrilswith very reduced hydrophobic exposure and minimal tendency to spontaneouslydisassemble(Bravoetal.,2008).Theconclusionsof such study agree with the general idea that polysaccharides promotetheformationofinnocuousformsofamyloidoligomers andfibrilspossiblythroughtheformationofglycatedderivatives; thecross-linksinthelattercouldstabilizeoligomersandfibrilsby makingthemmorerigidandstableandhencelesspronetointeract with cell membranes (oligomers) or toleak oligomers (fibrils) (DeGroot,2004).AsecondstudyreportsthatA

b

fibrilsgrownin vitroaredestabilizedbytheinteractionwithvesiclesmadefrom biologically relevant lipids, including brain lipid extracts. The authors found that, under these conditions, the fibrils were disassembled into heterogeneous populations of neurotoxic oligomersandprotofibrils;theseappearedverysimilartothose precedingfibrilappearanceintheforwardaggregationprocessand were internalized by exposed neurons (Martins et al., 2008). Another investigation, carried out by an array tomography approach,imagedasharpreductionofdendriticspinedensityin neuronalcellssurroundedbyamyloidplaquesintissueslicestaken fromADtransgenicmice.Inthisstudy,theauthorshaveshown that such a reduction depends strictly on the gradient of A

b

oligomersirradiatingfromthefibrillarplaquesdepositedintissue, stronglysuggestingthatthelattercanbeasourceoftoxicspecies (Koffieetal.,2009).Theseandotherevidencesagreewiththeidea thatmaturefibrilscanbenotsostableasusuallybelievedandcan bedisassembledundersuitablemildconditions.Theyalsosupport thesuggestiontoconsiderwithcaretheinformationobtainedby chromatographic techniques, electrophoresis and AFM on the structure and molecular mass of the amyloids, particularly oligomers,presentinasample;infact,theAFM,chromatographic or electrophoresis support, or the detergent micelles could disassemble intosmaller oligomerslarge aggregates presentin the original sample (Hepler et al., 2006). Recent data on solubilizationofbeta2-microglobulinfibrilsintotoxicoligomers by a number of tetracyclines (Giorgetti et al., 2011) further reinforcetheideathatamyloidfibrils,underapanelofdifferent conditions,canbepartiallydisassembledor,atleast,canleaktoxic oligomers.Inotherwords,theeffectofsurfaces,previouslyknown to favor protein aggregation into oligomers and maturefibrils (Stefani,2007)insomecasescanresultintheopposite,inducing fibrildisassembly.

Inconclusion,thedataindicatingbothdirectandindirectfibril toxicity imply that amyloid plaques in tissue should not be considered always protective as recruiters of toxic assemblies arisingfrompeptide/proteinmisfolding;rather,theycouldalsobe potential sourcesof toxicity. Actually, recentdata suggest that fibrils of the same protein/peptide grown from structurally differentoligomersor depositedunderdifferingconditions,and hencewithvariablestructuresandstabilities,candisplaydifferent cytotoxicities possibly resultingfrom their differing abilities to leak toxic oligomers. These data concern monomer/oligomer recycling within amyloid fibrils both in vitro (Carulla et al., 2005)andinvivo(Shankaretal.,2009),thedifferentstabilitiesand biophysicalpropertiesoffibrilsgrownfromthesamemonomerat differentconditions(Smithetal.,2006b)andtheapparentleakage

oftoxicoligomersaroundfibrillardepositsinvivo(Koffieetal., 2009).Accordingly,spontaneousfibrildecompositionor fragmen-tationintissuecanbeseenasapossibleimportantdeterminantof amyloidcytotoxicity.

8. Biologicalsurfacesareprimarysitesofamyloidinteraction andtoxicity

Besidesrecruitingproteinmonomersfavoringtheirmisfolding andaggregation,surfaces,notablycellmembranes,canalsobind actively the unstable oligomeric assemblies preceding the appearance of mature amyloid fibrils. The importance of the relationbetweenmembranelipidcompositionandtheabilityof early amyloid aggregates to bind to, and to disassemble,lipid membraneshasbeenextensivelyinvestigated(Par.3).Aspointed outabove,manystudieshighlighttheimportantroleplayedby anionicsurfacesandbymembranescontaininganionic phospho-lipidsassitesnotonlyofprotein/peptidemisfolding,butalsoof oligomerinteraction;infact,clustersofnegativecharges,together with the strong electrostatic field therein, can be sites of preferentialinteractionwithpre-fibrillaraggregatesresultingin phospholipid bilayer or cell membrane destabilization (Arispe etal.,1993;Mirzabekovetal.,1996;Linetal.,1997;Kourie,1999; VollesandLansbury,2001;HirakuraandKagan,2001;Kourieand Henry,2002;Dingetal.,2002;Hirakuraetal.,2002).Theseeffects modifymembranepermeabilityandimpairthefunctionofspecific membrane-boundproteins and signaling pathways(Houet al., 2005;Mattson,1999).Theroleofcholesterolandgangliosidesas modulatorsnotonlyofAbetapeptidegenerationandaggregation but also of oligomer interaction with the membrane and internalization has been extensively studied as well (Par. 3). Recently, it has been reported that pre-fibrillar aggregates supplementedtotheculture mediadisplayreduced interaction withthecells,internalizationandcytotoxicityuponenrichingin cholesterol the cell membrane whereas opposite effects were foundin cholesterol-depletedcells(Cramerietal.,2006;Cecchi etal.,2007,2008). Althoughrequiring moreextensiveresearch, thesedatasupporttheideathat,ingeneral,ahighermembrane rigidity following increased cholesterol content can hinder aggregateinteractionwiththecellmembranesandinternalization thus improving membrane resistance against disassembly. On turn,oligomerinteractioncanbyitselfprovidetoxicitybyaltering membranefluidity(Houetal.,2005)andintegrity,withpossible phospholipidpull-outfromthebilayer.

Amyloid interaction with thecell membrane raises another questiondebatedsincelongtime:arethereonthecellmembrane specific receptors for amyloids responsible for the amyloid– membrane interaction? The surface of the cell membrane is crowded of protein molecules. It has been estimated that the average plasma membrane surface is around2000

m

m2 _witha

densityofmembraneproteinsaveragingabout20,000molecules/

m

m2_{,accountingfora total40}

106_{proteinmoleculespercell}

surface(SimonsandEhahalt,2002).Itisthereforeconceivablethat amyloid oligomers may interact more or less specifically with someofthemembraneproteinstheycontact.

Inthepast,severalcellsurfaceproteinshavebeenconsideredas possiblecandidate receptors of A

b

aggregates. These receptors couldbespecificforthesharedcross-betafoldratherthanforany peculiarstructuralfeatureoftheA

b

peptidesalthough,insome cases,theycouldalsobemonomer-specific,asintheA

b

-APP,A

b

-TNFR1orA

b

-PrPinteractionsproposedtobeattheoriginofA

b

cytotoxicity(Shadeket al., 2006;Heet al., 2004;Laure´n etal., 2009). Since 1996, the receptor for advanced glycation end products (RAGE) has been proposed as a major candidate as amyloidreceptor(Yanetal.,1996).RAGEisincreasedinsystemic amyloidoses, is ableto interactwithamyloidassemblies made

fromserumamyloidA,amylinand prion-derivedpeptides(Yan etal.,2000)andappearsinvolvedinAlzheimer’sand Creutzfeldt-Jacobdiseases(Yanetal.,1998;Sasakietal.,2002).Bycompeting forligandbindingwithcell-surfaceRAGE,itsplasmasolubleform, sRAGE,mighttrapcirculatingligandspreventingtheirinteraction with cell surface receptors. Actually, sRAGE appears protective againstcytotoxicityoftransthyretinaggregates(Monteiroetal., 2006)anditshighplasmalevelsareassociatedwithareducedrisk of several diseases including AD. Increasing plasma sRAGE is therefore considered a promising therapeutic target potentially preventingvasculardamageandneurodegeneration(Geroldietal., 2006).

In additionto RAGE, several cell surface proteins, including voltage-gated(Houetal.,2007)orligand-gatedcalciumchannels suchastheglutamateNMDAandAMPAreceptors(Hsiehetal., 2006;DeFeliceetal.,2007;Pellistrietal.,2008)havealsobeen considered aspossiblereceptorsorspecificinteractionsites for amyloids.Inaddition,tissue-typeplasminogenactivator(tPA)has beenproposedasamultiligandspecificforthecross-

b

structure (Kranenburgetal.,2002).Finally,increasingevidencesuggeststhat additionalneuronalbindingsites,includinganioniclipidclusters (seeabove),couldbeinvolvedintheinteractionwiththeplasma membrane ofamyloidaggregates madefromdifferentpeptides andproteins(Lacoretal.,2004).Suchanideaissupportedbythe findingthatanyriseofthecontentofnegatively chargedlipids resultsinincreasedchannel formationbyamyloidsinsynthetic lipidbilayers(Jayakumaretal.,2004).

Thepresenceofspecificeffectsmediatedbythepreferential,or evenspecific,interactionwithmembraneproteinscould,atleastin part,explainthevariablevulnerabilitytoamyloidsofdifferentcell types(Cecchietal.,2005).However,inspiteoftheseandotherdata on specificinteractionsites foramyloids, thetendencyofearly amyloid aggregates tointeractwithsyntheticlipid membranes supports the idea that the interactioncan be non-specific but, possibly,modulatedbythemembranelipidcontent(Par.3),such aninteractioncanalso,byitself,impaircellviabilitybyaltering membranestructureandpermeability.

The‘‘channelhypothesis’’ofamyloidtoxicity,proposedsince 1993,statesthattoxicamyloidaggregatesformnon-specific pore-likechannelsinthemembranesoftheexposedcells(Arispeetal., 1993,reviewedinKourieandHenry,2002).Thisproposalisnow supportedbystudiescarriedoutbothonsyntheticphospholipid bilayers and on cell membranes showing that the function of specificmembrane proteinsis impairedby theinteractionwith misfoldedspeciesortheiroligomers(Houetal.,2005;Mattson, 1999,reviewedinKourieandHenry,2002).Forexample,the size-dependentpermeabilizationofartificialvesiclesbyprotofibrillar

a

-synuclein suggests that vesicle permeabilization may occur mainly asa resultof a specificmembrane perturbationviathe formation of pores at least 2.5nm in diametercoexisting with fibrils,raisingthepossibilitythat,attheconditionsfoundinthe cytoplasm, these‘‘pores’’ may bestable enough tobe thetrue pathogenicspeciesinParkinson’sdisease(Lashueletal.,2002).The ability of most amyloidogenic peptides and proteins to form ‘‘pores’’intheiraggregationpathsupportstheideathatmembrane permeabilizationcanresulteveninvivofromthepresenceofsuch species and be the key trigger of the sufferance and death of exposedcells(reviewedinCaugheyandLansbury,2003).Overall, the channel hypothesis and the data showing that protein aggregationat,andoligomerinteractionwith,thecellmembrane candisassemblemembranestructuralorganizationdestroyingits selective permeability (see below), further confirm the key importance,foramyloidcytotoxicity,oftheearlycellmembrane perturbationbyamyloids.

The presenceof toxic aggregates inside oroutside the cells, togetherwiththeirinteractionwithcellmembranescanimpaira