Chem Soc Rev - Structure, Dynamics, and Function of ...

Q1Implications of peptide assemblies in amyloid diseases

Pu Chun Ke, a Marc-Antonie Sani, b Feng Ding, c Aleksandr Kakinen, a

Ibrahim Javed, a Frances Separovic, b Thomas P. Davis ad andRaffaele Mezzenga *e

Neurodegenerative disorders and type 2 diabetes are global epidemics compromising the quality of life of millions

worldwide, with profound social and economic implications. Despite the significant differences in pathology –

much of which are poorly understood – these diseases are commonly characterized by the presence of cross-bamyloid fibrils as well as the loss of neuronal or pancreatic b-cells. In this review, we document research progress

on the molecular and mesoscopic self-assembly of amyloid-beta, alpha synuclein, human islet amyloid

polypeptide and prions, the peptides and proteins associated with Alzheimer’s, Parkinson’s, type 2 diabetes and

prion diseases. In addition, we discuss the toxicities of these amyloid proteins based on their self-assembly as well

as their interactions with membranes, metal ions, small molecules and engineered nanoparticles. Through this

presentation we show the remarkable similarities and differences in the structural transitions of the amyloid

proteins through primary and secondary nucleation, the common evolution from disordered monomers to alpha-

helices and then to b-sheets when the proteins encounter the cell membrane, and, the consensus (with a few

exceptions) that off-pathway oligomers, rather than amyloid fibrils, are the toxic species regardless of the

pathogenic protein sequence or physicochemical properties. In addition, we highlight the crucial role of molecular

self-assembly in eliciting the biological and pathological consequences of the amyloid proteins within the context

of their cellular environments and their spreading between cells and organs. Exploiting such structure–function–

toxicity relationship may prove pivotal for the detection and mitigation of amyloid diseases.

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Cite this: DOI: 10.1039/c7cs00372b

a ARC Center of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville,VIC 3052, Australia

b School of Chemistry, Bio21 Institute, The University of Melbourne, 30 Flemington Rd, Parkville, VIC 3010, Australiac Department of Physics and Astronomy, Clemson University, Clemson, SC 29634, USAd Department of Chemistry, University of Warwick, Gibbet Hill, Coventry, CV4 7AL, UKe ETH Zurich, Department of Health Science & Technology, Schmelzbergstrasse 9, LFO, E23, 8092 Zurich, Switzerland. E-mail: [email protected]

Pu Chun Ke

Pu Chun Ke is a Senior Fellow atCBNS, Monash University,Australia. He was a CSIRODistinguished Visiting Scientist(Melbourne, 2014) and tenuredAssociate Professor (2009–2013)and Assistant Professor (2003–2009) at Clemson University, USA.Dr Ke was recipient of the FacultyAchievement in the Sciences Award(Clemson, 2012), CAREER Award(US National Science Foundation,2008) and LJIS Postdoctoral Fellow-ship in Biophysics (Burroughs Well-

come Fund through University of California, San Diego, 2001–2003). Hehas authored 108 papers on protein aggregation, protein corona, singlemolecules, and environmental health and safety of nanomaterials, high-lighted by MRS Bulletin, Science Daily, Live Science, and Research.gov.

Marc-Antonie Sani

Marc-Antoine Sani is a ResearchFellow at the School of Chemistry– Bio21 Institute, The Universityof Melbourne. He graduated witha PhD degree from Umeå Univer-sity and the University of Bor-deaux in 2008. His field ofexpertise is Biophysics with spe-cial interest in biological solid-state NMR spectroscopy, withaim to develop in situtechniques to study living cellsand elucidate the role ofparticular lipids in the

molecular mechanism of diseases. He has authored 57 researcharticles with recent work in JACS and Angewandte Chemie relatedto protein–lipid complex interactions.

Received 23rd May 2017

DOI: 10.1039/c7cs00372b

rsc.li/chem-soc-rev

This journal is !c The Royal Society of Chemistry 2017 Chem. Soc. Rev., 2017, 00, 1"41 | 1

Chem Soc Rev

REVIEW ARTICLE

http://orcid.org/0000-0003-2134-0859

http://orcid.org/0000-0003-3284-2176

http://orcid.org/0000-0003-1850-6336

http://orcid.org/0000-0003-1547-2855

http://orcid.org/0000-0003-1101-5614

http://orcid.org/0000-0002-6484-2763

http://orcid.org/0000-0003-2581-4986

http://orcid.org/0000-0002-5739-2610

1. IntroductionMolecular self-assembly is a ubiquitous phenomenon across allliving systems: from the polymerization of tubulins and actinsinto microtubules and actin filaments, to the organization oflipids, transmembrane/peripheral proteins and ion channelsinto cell membranes, to the assembly of DNA and histones intochromatin fibers and solenoids, and to the aggregation ofpeptides and proteins intra- or extracellularly evolving fromfunctional monomers to toxic oligomers, amyloid fibrils andplaques. Fundamental to these processes are interactionsbetween the molecular constituents of the assemblies, as wellas interactions between the molecular constituents and their

associated chaperones, ligands, ions, molecular complexes andorganizations, driven by kinetic and thermodynamic processesto elicit desirable biological functions or malfunctions anddiseases.

In this review, we attempt to draw parallels from the atomicand mesoscopic structures of five major classes of amyloidproteins in self-assembly, namely, amyloid-beta (Ab), tau,alpha-synuclein (aS), prions, and human islet amyloid polypep-tide (IAPP), as well as the biological and pathological endpointsthese assemblies elicit in host systems (Fig. 1). The amyloidaggregation of these peptides has been implicated in Alzhei-mer’s, Parkinson’s, prion diseases and type 2 diabetes mellitus,or generically referred to as neurodegenerative disorders and

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Feng Ding

Feng Ding is an AssociateProfessor of Physics at ClemsonUniversity, USA. He obtained hisPhD from Boston University(2004) and worked as apostdoctoral fellow (2004–2006),research associate (2006–2008),and Research Assistant Professor(2008–2012) at the University ofNorth Carolina at Chapel Hillbefore being hired as an AssistantProfessor at Clemson University(2012–2017). His researchfocuses on understanding the

structure, dynamics, and function interrelationship of biomoleculesand molecular complexes. He was recipient of a PostdoctoralAward for Research Excellence (UNC, 2005), CAREER Award (USNational Science Foundation, 2016) and Outstanding YoungResearcher Award (Clemson, 2017).

Aleksandr Kakinen

Aleksandr Kakinen is aPostdoctoral Research Fellow atCBNS, Monash University,Australia, working with Pu ChunKe and Tom Davis. While workingat the National Institute ofChemical Physics and Biophysics,Tallinn, Estonia, he obtained hisPhD degree (2014) at TallinnUniversity of Technology,Estonia. His research interestsinclude protein–protein inter-action, protein corona, nano- andecotoxicology.

Ibrahim Javed

Ibrahim Javed is a PhD student atCBNS, Monash University,Australia, working with Pu ChunKe and Tom Davis. He obtainedhis PharmD from the Universityof Sargodha and MPhil(Pharmaceutical Chemistry) fromBahauddin Zakariya University,Pakistan. His research is focusedon designing nanostructuredmaterials and their interfacingwith proteins and other classesof bio-macromolecules forpharmaceutical andtoxicological applications.

Frances Separovic

Frances Separovic is aBiophysical Chemist, deputydirector of the Bio21 Instituteand former Head of Chemistry,University of Melbourne. Whileworking at CSIRO, Sydney, sheobtained a PhD at UNSW and apostdoctoral fellowship atNational Institutes of Health(USA). Frances joined theUniversity of Melbourne in 1996and specializes in thedetermination of peptidestructures within membranes

using solid-state NMR. She is a Fellow of the Biophysical Society,ISMAR Fellow and the first female chemist elected as Fellow of theAustralian Academy of Science (2012). In 2017 Frances received anIUPAC Distinguished Women of Chemistry/ChemicalEngineering award.

2 | Chem. Soc. Rev., 2017, 00, 1"41 This journal is !c The Royal Society of Chemistry 2017

Review Article Chem Soc Rev

T2D that debilitate hundreds of millions of people worldwide.In vitro, such peptides/proteins fibrillate on the timescales oftens of minutes for IAPP to days for Ab and aS, characterized bya sigmoidal kinetic curve consisting of a lag phase, an elonga-tion phase, and a saturation phase.1 The lag phase is where

nucleation is initiated through protein misfolding and whereintrinsic seeds and/or oligomers are formed, the elongationphase corresponds to the addition of monomers to growingprotofilaments, while the saturation phase is where protofila-ments associate through self-assembly to render amyloid

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Fig. 1 Scope of the present review, highlighting protein self-assembly, its biological and pathological implications, theranostics and prevention. Ab:amyloid-beta; IAPP: islet amyloid polypeptide; aS: alpha-synuclein; PrP: prion protein; CD: circular dichroism spectroscopy; FTIR: Fourier transforminfrared spectroscopy; ThT: thioflavin T assay; NMR: nuclear magnetic resonance; HD: hydrogen–deuterium exchange; SDSL: site directed spin labelling;EPR: electron paramagnetic resonance; TEM: transmission electron microscopy; AFM: atomic force microscopy. ER: endoplasmic reticulum; ROS:reactive oxygen species.

Raffaele Mezzenga

Raffaele Mezzenga finished hisPhD at EPFL Lausanne (2001)and a postdoc at UCSB SantaBarbara, before joining in 2003the Nestle Research Center inLausanne. In 2005 he was hiredas Associate Professor in Physicsat the University of Fribourg, andhe then joined ETH Zurich on2009 as Full Professor. Hisresearch focuses on thefundamental understanding ofself-assembly processes in softcondensed matter. His work has

been internationally recognized by several distinctions such as theBiomacromolecules/Macromolecules Young Investigator Award(2013, ACS), the Dillon Medal (2011, APS), and the YoungScientist Research Award (2011, AOCS).

Thomas P. Davis

TomQ3 Davis is the inauguralMonash–Warwick Professor.Tom is the Director of the Austra-lian Research Council (ARC) Cen-tre of Excellence in ConvergentBio-Nano Science andTechnology. He is an AustralianLaureate Fellow. Prior to hisappointment at Monash andWarwick he spent 21 years as asenior academic at the Universityof New South Wales in Sydney.Tom’s research focuses on theapplication of polymer science

and nanotechnology to therapeutic applications. Tom is a Fellowof the Australian Academy of Science. He has authored 450+ peer-reviewed papers and his work has been cited in excess of29 000 times.


Chem Soc Rev Review Article

fibrils. In addition to primary nucleation, secondary nucleationthrough the combination of both monomeric and aggregatedspecies is also feasible.2,3 It has been suggested that theamyloid state is perhaps available to any polypeptide chain4–9

and represents the energetically most favorable state evencompared to native proteins.1 In vivo, however, the develop-ment of amyloids and plaques in the brain or pancreatic isletsoften takes decades, or B10 000 times longer. Such drasticdifferences in fibrillization may originate from the crowdedhierarchical cellular environments, where amyloid proteins aresynthesized and then translocate and spread through inter- andintra-molecular assembly, chaperoned by proteins (e.g. insulinfor IAPP) or modulated by pH and ionic strength. Accordingly,while the main purpose of this review is to highlight thestructure–function–toxicity triangle of a selected few amyloidproteins, another goal of this presentation is to draw thereader’s attention from focusing exclusively on amyloid pro-teins to the environments of the culprits at large, whichundoubtedly also contribute to the pathologies of the amyloiddiseases. Such perspective may prove beneficial to the develop-ment of mitigation strategies and theranostics against amyloi-dogenesis that has become increasingly perilous to modernsociety.

In terms of content, this review consists of 6 sections:Section 1 offers an introduction to protein self-assembly andamyloid diseases; Sections 2–5 review the structure, functionand toxicity characteristics of Ab, tau, IAPP, aS and prions,loosely following their increasing number of amino acids(residues); Section 6 provides a summary. Ab and tau, despitetheir great contrast in chain length, both contribute to the ADpathology and hence are presented together. Although Ab isslightly longer than IAPP in chain length, Ab is the moststudied of all amyloid proteins10 and is therefore discussed inan early section of this review.

2. Ab, tau and Alzheimer’s diseaseThe hallmark of Alzheimer’s disease (AD) is the accumulation oftoxic aggregates that impair synaptic function and induce cog-nitive decline. The first reported occurrence of cognitive disorderlinked to AD was in 1907 by Alzheimer, who observed two typesof abnormality in a brain autopsy that he attributed to be thecause of an unusual type of dementia.11 The discovery of neuriticplaques (or miliary foci) and neurofibrillary tangles (NFTs) wasimmediately linked to the dystrophic neuronal process, and laterAb fibrils12,13 and hyperphosphorylated tau tangles14 were iso-lated and characterized (and proposed to cause dementia).Characterizations of the monomeric forms of the moleculesfound in these neurotoxic deposits have led to greater under-standing of the pathways leading to AD, with a particular focuson the structure–function relationship between aggregates andneurotoxicity. However, almost all drugs tested thus far inclinical trials have failed or shown limited impact on AD.

Tau is a neuronal protein associated with microtubules andmay regulate neuron morphology. There are six main tau

isoforms in the brain and central nervous system (CNS). Thelongest human isoform has 441 residues with a high propor-tion of phosphorylable residues (serine and threonine) and alow proportion of hydrophobic amino acids. Tau protein insolution is considered an intrinsically disorder protein (IDP)and behaves as a random coil,15 although modifications byphosphorylation may lead to an increase in a-helix or b-sheetregions. Aggregation of totally or partially disordered proteinsis associated with many neurodegenerative diseases, includingAD.16 However, the molecular mechanism of aggregation andthe structure of the aggregated form remain controversial. Tauis mainly an axonal protein but in AD and other tauopathies itis also present at dendritic spines and may play a toxic role. Thetau hypothesis of AD considers that excessive phosphorylationof tau protein can result in the self-assembly of tangles ofpaired helical filaments (PHFs) and straight filaments whichare involved in the pathogenesis of AD and other tauopathies.These NFTs are insoluble structures that impair axonal trans-port and lead to cell death. The molecular structures of PHFsand tau protein are not well defined.

NMR data17 have revealed that 343 of the 441 amino acids intau are disordered with six segments of the sequence displayingpropensity to form b-strands, three segments showing poly-Prohelices and two segments with a transient a-helix structure. Inparticular, aggregation of tau is believed to be strongly asso-ciated with two short residue sequences:18–21 the first in thethird repeat fragment (R3, i.e. VQIVYKPVDLSKVTSKCGSLG-NIHHK) of the microtubule binding domain of tau, VQIVYK,or the mutant VQIINK, in the second repeat fragment. Theaggregation of the R3 fragment has been extensively studied inthe presence of polyanions, such as heparin, pointing at theformation of fibrillar structures with similar features as thoseassembled from pristine tau protein.22 In the absence ofheparin, however, the same R3 fragment has been shown toself-assemble into giant amyloid ribbons of remarkable aspectratios.23

The tau protein is a highly dynamic structure. An NMR studyof a peptide derived from tau showed that phosphorylationstabilized the a-helix structure,24 suggesting a possible highercontent of a-helices in hyperphosphorylated tau in PHFs. Tauprotein can form dimers, oligomers and larger aggregates andfibrils. However, in this review we focus on the Ab peptiderather than tau aggregates, as greater structural details areavailable for the former.

2.1 Role of APP and production of Ab

Ab peptides are produced by an intrinsic cleavage of theamyloid precursor protein (APP) that is an integral membraneprotein encoded on chromosome 21 by the APP gene.25 It isaccepted that patients with trisomy 21 (Down syndrome) over-express APP and develop AD-like senile plaques in theirbrain.26,27 Yet, the physiological function of APP remainsuncertain, mostly because APP is part of a gene family withoverlapping function (e.g. producing the amyloid precursor-likeproteins APLP1 and APLP2) and is subject to various post-expression modifications.28 Still, only APP generates the Ab

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fragment. APP modulates critical features in brain developmentsince APP knock-out mice are viable but exhibit reduced bodyweight and brain mass29 with increased brain levels ofcopper,30 cholesterol and sphingolipid.31 Interestingly, reintro-ducing the APP ectodomain, which is produced by cleavage ofthe membrane-anchored APP, improved cognitive function andsynaptic density32,33 and acted as an apoptosis modulatorthrough caspases activations.34 The intracellular C-terminaldomain also has a functional role in sorting APP and, inparticular, the highly conserved YENPTY cytoplasmic sequenceis prone to interaction with other proteins, such as X11 andFe65, which are postulated to regulate APP internalization.35

The location and sequence of the proteolytic cleavage of APPare critical to AD. APP is primarily translocated to the cellsurface (short residence time) where a-secretase and then g-secretase produce APPsa, p3 and AICD fragments, which arenot amyloidogenic. However, when APP is relocated throughendocytosis (rapid turnover due to the YENPTY sequence) intoendosomes containing the b-secretase (also called BACE1) andthe g-secretase, then APPsb, the toxic Ab peptides and AICDfragments are produced (Fig. 2). BACE1 is an aspartyl proteasethat has optimum efficiency at pH 4.5.36 Interestingly, acid pHcan promote greater aggregation rate of Ab peptides37 due tothe protonation state of the three histidines (His6, His13 andHis14), and also attenuate lysosomal degradation of Abpeptides.38 Furthermore, if APP is relocated to the trans Golginetwork instead of the ER, BACE1 can produce N-truncated Abpeptides which are prone to rapid pyroglutamylation.39 These

species have been characterized as highly toxic40 and found inintracellular, extracellular and vascular Ab deposits in AD braintissue,41 while unmodified peptides are primarily located inendosomal compartments and are eventually exocytosed intothe extracellular space.

It is noteworthy that intracellular pools of Ab peptides arepointed as the most toxic species causing the death ofneurons.42,43 The physiological function of these Ab peptides,usually 39–43 residues in length, is unconfirmed. However,during excitatory neuronal activity, an increase in excretion ofAb peptides is observed44 with the effect of downregulatingexcitatory synaptic transmission.45 Thus, Ab peptides are animportant modulator of memory, since inhibition of peptideproduction impairs learning.46 Ab(1–40) is the most abundantAb isoform found in its soluble form in plasma, cerebrospinalfluid and brain interstitial fluid47 but is also a major compo-nent in amyloid plaques. Interestingly, the level of the fast-aggregating isoform Ab(1–42) is a biomarker for detectingamyloid pathologic changes in the brain and cerebralvessels48 and, moreover, the relative Ab(1–42)/Ab(1–40) ratio ismarkedly increased in AD.49 Overall, both the concentrationand location of Ab peptides are critical for brain function,thereby complicating therapeutic strategies against AD.

2.2 Atomic structures of Ab40 & Ab42, post-modifications andamyloid fibrils

Ab peptides vary in length due to the multiple cleavage sitesrecognized by the secretases, but the most abundant species are

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55Fig. 2 (a) Non-amyloidogenic pathway triggered by the location of APP at the plasma membrane interface; and (b) amyloidogenic pathway inducedthrough APP endocytosis into endosomal vesicles containing the protease BACE1. Ab peptides are then prone to aggregation and can be either secretedextracellularly or remain in the intracellular space to target other organelles, such as mitochondria, or be degraded by proteases such as cathepsin B.



Ab(1–40) and Ab(1–42), whose sequences are shown in Fig. 3.Furthermore, Ab peptides can be degraded by proteases such asinsulin degrading enzymes,50 neprilysin51 and cathepsin B,52

which render the fragments non-amyloidogenic. Ab peptidescan be subject to post-translational modifications includingpyroglutamate formation (Glu3, 11 and 22),53 phosphorylation(Ser8 and 26),54 dityrosine formation (Tyr10)55 and oxidation(Met35)56 (see Fig. 3).

The Ab peptide primary sequence exhibits two stretches ofhydrophobic residues (17–21 and 32 until C-terminus), whichare predicted to adopt a b-sheet conformation.57 Two turnregions are also predicted between residues His6 and Ser8,and between Asp23 and Asn27. Finally, the hydrophilic patchesbetween Asp1 and Lys16 and between Glu22 and Lys28 haveeither b-sheet or a-helical propensity.58,59

A missing piece in the AD puzzle is the secondary structuresof Ab peptides immediately after cleavage by the g-secretase.Firstly, b-CTF is likely to remain structured after cleavage byBACE1, at least up to the transmembrane a-helical segmentthat contains part of Ab sequence (Ala28 until C-terminus).Secondly, cleavage by the g-secretase occurs at intra-membraneand Ab peptides have a demonstrated affinity for lipid mem-branes. Thus immediate trafficking is unclear: do Ab peptidesremain in, on or away from the membrane interface? Sincelipid membranes modulate the aggregation kinetics,60 this stepcould play a critical role in subsequent trafficking and ADpathology.

Several Ab peptide structures have been compiled in the pasttwo decades,61 with a consensus that an unstructured to b-sheettransition first occurs followed by a seeded aggregation processto form oligomeric structures that eventually proceeds to matureamyloid fibrils of 70–120 Å in diameter and an indeterminatelength according to electron microscopy.62 Determination of the

initial structure of Ab peptides in native conditions is challen-ging since the rapid self-aggregation rate accompanied by poorsolubility prevents the application of high-resolution techniquessuch as solution NMR. Nevertheless, several structures of themonomeric peptides have been determined in either organicsolvents (dimethylsulfoxide, hexafluoroisopropanol, trifluor-oethanol), aqueous solution or detergent micelles (sodiumdodecyl sulfate or SDS). In general, Ab peptides adopt helicalconformations with unstructured termini and various turnregions in organic solvents,63–65 aqueous buffer66 and inmembrane mimetic detergent micelles.67,68 Interestingly, moststructural studies show that physiological pH,69 low saltconcentration70 and higher temperature71 could heavily modu-late the peptide conformational transition to b-sheet structure,thereby promoting rapid self-aggregation. The a-helical confor-mation is proposed as a transient on-pathway intermediateduring the complex amyloid fibril formation.72 Indeed, themultistep kinetics of amyloid assembly comprise a lag phase,during which little or no fibril material is formed, followed by anexponential growth of b-sheet-rich aggregates that propagateinto amyloid fibrils.1 Increasing evidence suggests that thenative partly helical intermediates form early nucleation seedsduring the lag phase.73 The intramolecular interactions stabiliz-ing the b-sheet structure are shown in Fig. 3. In both Abisoforms, the turn conformation is stabilized by hydrophobicinteractions and by a salt bridge between Asp23 and Lys28. Manyside chain contacts are observed, in particular between Phe19and Ile32, Leu34 and Val36, and between pairs Gln15–Val36 andHis13–Val40.57,74

Phosphorylation of Ab peptides, however, does not modifytheir primary unstructured conformation but does lead to a 5-fold reduction in the lag phase due to a faster transition to b-sheet structures, more efficient nucleation and a greater num-ber of oligomeric seeds.75 N-truncated and/or pyroglutamate-modified Ab peptides form b-sheet structures76 with fasteraggregation kinetics than the corresponding full-length pep-tides, which suggests they could be potential seeding speciesfor aggregate formation. More remarkably, the pyroglutamate-modified Ab peptides also inhibit the full-length peptide fibril-logenesis and lead to a greater content of small oligomericspecies77 that have been demonstrated as most toxic.

2.3 Ab aggregation kinetics and amyloid fibril formation

Knowledge of Ab aggregation kinetics and mechanisms hasbeen acquired mainly through in vitro studies using syntheticpeptides. The kinetics of fibril formation depends on severalintrinsic and extrinsic factors. The primary sequence of thepeptide modulates the propensity to aggregate into maturefibrils. Post-modifications promote faster aggregation kinetics,as does the Ab(1–42) sequence compared to the shorter Ab(1–40) peptide. Extrinsic factors, such as interaction with lipidmembranes, can have either a slowing or accelerating effect,rendering determination of a generic model nontrivial.

The lag phase is a period of slow self-aggregation andstructural change, likely from helical to b-sheet structures,and characterized by a combination of multiple nucleation

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Fig. 3 (a) Ab peptide sequence (CINEMA color code), potential post-modification sites and physicochemical properties; and (b) intramolecularinteractions stabilizing the typical hairpin b-sheet structure. Red andorange dashes: molecular contacts. Blue dashes: side-chain packing.Green: hydrophobic residues. Black: a salt bridge. Reproduced withpermission from ref. 57, copyright 2010 Nature Publishing Group.



and elongation phases2,78,79 leading to a large number ofoligomeric species. Primary nucleation is a fast process (milli-seconds) producing the first seeds that are elongated furtherinto fibrils by the addition of monomers. The formation of newaggregates is thought to be dominated by a second nucleationphase where existing fibrils are fragmented to expose new seedseither co-aggregating or recruiting monomers. Interestingly,changes in the primary nucleation rate do not affect theelongation phase while secondary nucleation and fragmenta-tion modify the lag and elongation phases.80 The difference inaggregation rate between the amyloid peptide species, however,may be related to their primary nucleation rate. In fact, Ab(1–40) monomers, in comparison to Ab(1–42), exhibit a slowernucleation rate inducing (or caused by) a shift towards nuclea-tion on the fibril surface rather than accumulation of smalloligomeric species.79 These fibril-catalyzed secondary nuclea-tion and elongation processes could be a critical difference inrelation to the trafficking and toxicity of the Ab peptidevariants.81 Notably, measuring the kinetics of aggregation ischallenged by the difficulty in sample preparation,81 especiallywith regard to starting an experiment without any preformedseeds or a controlled amount of seeds.

The elongation phase is due to the addition of oligomers/monomers onto protofibrils (Fig. 4) or association of protofi-brils, in competition with fragmentation of the protofibrils. It isoften typified by the half time of the aggregation reaction wheremonomers and protofibrils are near equimolar. However,intrinsic and extrinsic factors modulate the stability of theoligomeric species and can template seeds, thereby shifting

the kinetic rate towards primary nucleation with a fasteraggregation rate. The stationary phase represents a steady statewhere the monomer concentration has reached an equilibriumvalue and the fibrils are the prevalent species. Notably, AFMstudies show that fibrils of different amyloid-forming peptideswith diverse macroscopic structures/polymorphism (i.e.,ribbon-like versus nanotube-like packing) have a similarYoung’s modulus, and thus all Ab peptides are anticipated toexhibit similar mechanical strength.82

Intrinsic and extrinsic factors also play a critical role in themodulation of the lag and elongation phases by changing theconcentration of free monomers in solution and/or acting asseeding interfaces. The molecular factors influencing the aggre-gation kinetic of Ab peptides are various and difficult to assignto a particular microscopic event (primary versus secondarynucleation, fragmentation, etc.), although some properties aremore straightforward to correlate; for instance, the effect of pHas electrostatic interactions mediate either attraction or repul-sion of the monomers.78

2.4 Mesoscopic structures of Ab amyloid fibrils

An original molecular model of Ab(1–40) fibrils83 based onsolid-state NMR data shows the first B10 residues as structu-rally disordered while residues 12–24 and 30–40 adopt b-strandconformations and form parallel b-sheets through intermole-cular hydrogen bonding. A bend at residues 25–29 brings thetwo b-sheets in contact through sidechain–sidechain interac-tions. The cross-b motif common to all amyloid fibrils is adouble-layered structure, with in-register parallel b-sheets.83

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55Fig. 4 Ab aggregation pathways from monomer to fibril formation and their toxic outcomes.



However, several studies have shown that Ab peptides formpolymorphic fibrils depending on growth conditions and var-ious oligomeric aggregates. Thus it is unlikely that amyloidfibrils formed in vitro resemble those in the brain. Tycko andco-workers84 seeded fibril growth from brain extract and usedsolid-state NMR and electron microscopy to gain structuraldetails of the Ab fibrils. Using tissue from two AD patients theyfound a single Ab40 fibril structure for each patient emphasiz-ing the critical role of the seeding process. The molecularstructure for Ab40 fibrils from one patient (Fig. 5) revealeddifferences from in vitro fibrils. The authors then proposed thatfibrils may spread from a single nucleation site and thatstructural variations may correlate with variations in AD.

In comparison with Ab40, Ab42 is more neurotoxic and theirdifferences in behaviour may be due to intrinsic differences instructure. An atomic resolution structure of a single form ofAb42 amyloid fibrils has been derived from high field magicangle spinning NMR spectra.85 The structure shows a dimer ofAb42 molecules, each containing four b-strands in an S-shapedamyloid fold (Fig. 6). The dimer is arranged to form twohydrophobic cores, capped by a salt bridge at the end with ahydrophilic outer surface. The monomer interface within thedimer shows contacts between M35 of one molecule and L17and Q15 of the second. Intermolecular constraints show thatthe amyloid fibrils are parallel in-register. Interestingly, Ishiiand co-workers obtained a similar S-shape arrangement (Fig. 6)using ultra-fast spinning solid-state NMR techniques.86

Although knowing atomic details of the fibril may be usefulfor drug design, nevertheless, the oligomer species are gener-ally accepted as the toxic species.87

2.5 Extrinsic factors modulating Ab structure, aggregationkinetic and toxicity

2.5.1 Ab–metal interactions. The role of transition metalsin AD is highly debated and a recent literature search usingmeta-analysis and systemic review methodologies identified awidespread misconception that iron and, to a lesser degree,

zinc and copper levels are increased in AD brain.88 Metals wereprimarily thought to be accumulated in AD brain tissue due topositive staining but quantitative analysis failed to confirm asignificant increase,89 and more recent studies have confirmedthe artefacts in quantitation due to tissue fixation prior toanalysis.90 Qualitative ex vivo and in vitro studies havedemonstrated that Ab peptides recruit iron, zinc and copperwith high affinity91 and, more dramatically, induce a redoxcomplex with oxidative stress properties92 that may be relatedto the toxicity of Ab peptides and has been widely accepted as apotential toxic mechanism in AD.93 Two binding sites wereidentified: the Met35 mediating the Fenton reaction throughthe electron donor sulfide group;94 and the N-terminal regionforming a chelating domain95 of Asp1, His6, His13 and His14,which undergoes a major structural rearrangement during theredox cycle of ROS production.96 Interestingly, in vitroexperiments have also shown that metal binding noticeablyextends the lag time by stabilizing oligomeric and amorphousaggregates,97 which may explain poor in vivo detection of thepeptide amyloids. Ab–copper complexes have also been shownto promote lipid peroxidation, in particular within the poly-unsaturated chains of membrane lipids, which is anotherpotential toxic mechanism due to neuronal membranedisruption.98

2.5.2 Ab–membrane interactions. The role of lipids in ADwas first suggested by Alzheimer when he discovered adiposeinclusions and alterations of lipid composition in braintissue.11 Several classes of lipids have been investigated fortheir specific interactions with Ab peptides, such as cholesterol,gangliosides or anionic phospholipids.99 The lipid membraneinterface itself is proposed to be a heterogeneous nucleationsite, which modulates Ab peptide folding kinetics and pathwaysby reducing the seeding mechanism to a two-dimensionalsystem.100,101 To date, there is a consensus that lipid bilayerplays a role in Ab aggregation and may be involved inneurotoxicity. Different model membranes influence thestructure and size of Ab fibrils based on the charge andhydrophobicity of the membrane.60,102 Membrane-attachedoligomers of Ab40 displayed a b-turn, flanked by two b-sheetregions or an anti-parallel beta-hairpin conformation by Raman

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Fig. 6 Two recent solid-state NMR Ab42 fibril structures identifyingdifferent assemblies by (left) Griffin and co-workers (PDB: 5kk3)85 and(right) Ishii and co-workers (2MXU).86 High similarity is apparent with the b-sheet domain (purple ribbons) and the unstructured strand (gray ribbons)forming an S-shape. The hydrophobic surfaces are based on Kyte–Doo-little scale (red: hydrophobic, white: neutral, blue: hydrophilic).

Fig. 5 Ab40 structural polymorphism depending on experimental condi-tions. Rendered from PDB 1BA4, 1AML, 2MVX and 2MJ4.



spectroscopy and solid-state NMR.103 In contrast to the mature, less-toxic Ab fibrils, the membrane-attached oligomer appeared to form ab-barrel or ‘porin’-like structure (also refer to Fig. 15b in Section 4 foraS), which may account for a mechanism for Ab toxicity.

Cholesterol is proposed to be related to AD pathology althoughcholesterol stabilizes phospholipid bilayers against Ab.104 Lipid‘rafts’ or domains in the membrane enriched in cholesterol andsphingolipids could modulate Ab production, aggregation andtoxicity.105 Sanders and co-workers106 showed that the C99 segmentof APP bound to cholesterol and proposed that APP might act as acholesterol sensor critical for the trafficking of APP to cholesterol-rich membrane domains. Cholesterol increases the thickness ofphospholipid bilayers and may influence the proteolytic processingof APP and proportion of Ab40 to Ab42 produced. Lipid membranesare also susceptible to oxidative stress, as mentioned above as amechanism for neurodegeneration in AD.98

2.6 Ab toxicity and Alzheimer’s disease

The physiological markers of AD are progressive cognitive decline,synaptic loss, presence of extracellular b-amyloid plaques andintracellular neurofibrillary tangles ultimately leading to neuronalcell death and a massive brain cell mass loss. To date, there is nodrug that can prevent AD neurodegeneration probably becausemany pathways are activated during the uncontrolled production ofAb peptides, although several candidates are in ongoing clinicaltrials. Indeed, it has been demonstrated that Ab peptides accumu-late at synapses, thereby disrupting the whole neuronal network.107

More specifically, complex interactions between Ab peptides andboth synaptic ion channels and mitochondria alter their physiolo-gical activities. Ab peptides and, more particularly, the oligomers ofAb have affinity for the glutamate108 and acetylcholine109 receptors,mediating the influx/efflux rate of critical mediators such ascalcium ions. Ab-mediated deregulation of these receptors – parti-cularly NMDAR and AMPAR – has been linked to the impairmentof plasticity and degeneration of synapses during AD.110

The observation that Ab oligomers are able to co-localizewithin mitochondria has exposed another potential neurotoxicpathway.111 Ab oligomers are able to alter the function of proteinsinvolved in the mitochondrial fusion/fission process, which causestheir fragmentation leading to the loss of neuron viability.112

Moreover, accumulation of Ab peptides in synaptic mitochondriahas been shown to decrease mitochondrial respiration and keyrespiratory enzyme activity, elevate oxidative stress, compromisecalcium-handling capacity, and trigger apoptotic signals.113,114

Finally, intracellular accumulation of Ab peptides drasticallyreduces the lysosomal efficiency in removing damaged organellesand unfolded proteins, such as tau.115 Better understanding of thecell biology of the downstream effects of Ab oligomers may uncoverpotential therapeutic targets for the prevention of AD.

2.7 Mitigation strategies and theranostics

With increased knowledge of the mechanism of fibril formationfrom the cleavage of APP to the kinetic modulation by extrinsicfactors, several strategies to mitigate AD have emerged. Stabiliz-ing the monomeric form of Ab peptides is a direct strategy tolimit the formation of oligomeric species. Peptides that

specifically interact with the pro-aggregating domains have beendeveloped, as recently shown with a cyclopeptide, to inhibit Abamyloidogenesis.116 Antibody-based immunotherapy is anotherstrategy to mitigate AD. For instance, a promising candidate,aducanumab, has been shown highly selective against aggre-gated Ab, inducing significant reduction of insoluble and inso-luble Ab population and slowing clinical decline, although theoutcome of ongoing phase 3 clinical trials is needed to confirmthese promising observations.117 The affinity of Ab peptides fortransition metals was seen as another area for potential devel-opment of AD therapeutics, but so far chelators, such as D-penicillamine, have not produced any clinical improvement.118

After drugs (e.g. bapineuzumab and solanezumab) whichsought to lower existing Ab loads had failed, increasing attentionwas paid to BACE drugs that interfere with the process thatcreates Ab. However, Merck recently closed its trial for the BACEinhibitor, verubecestat, in mild-to-moderate AD after concludingthat the drug had little chance of success.119 A particular focushas been to decrease the production of apparently toxic Abpeptides by inhibiting BACE1 activity.120 For instance, thecholesterol-rich endosomal environment, which promotes selec-tive processing of APP by BACE1, has been pursued as a targetusing a membrane-anchored BACE1 transition-state inhibitorlinked to a sterol moiety to generate highly effective BACE1inhibitors.121 Treatment with BACE inhibitor IV, which does notchange the APP concentration level, was shown to preventmitochondrial abnormalities caused by Ab.122 Reducing theactivation of caspases, such as caspase 3, can improve neuronalgrowth and decrease abnormal tau species, which may be aninteresting therapeutic pathway for the treatment of AD.123

Since the approval of memantine in 2003, no new AD drugcandidate has passed the FDA approval, with an alarming failurerate of 99.6%, the highest in all serious disease researchprograms.124 A growing strategy in integrating therapeutics andpersonalized diagnostics has recently emerged as a promisingroute. Based on nanomedicine, small molecules – necessary toovercome prerequisite to cross the brain blood barrier – have beendeveloped to label and simultaneously inhibit oligomerization ofAb peptides.125 The term theranostic has thus been coined tocharacterize these new inhibitor-biomarkers, many based on scaf-folds of fluorescent probes such as ThT, to detect fibril formationin vivo and alter their accumulation.126 These new strategies havebeen made possible by improved understanding of the assemblymechanism of Ab at the molecular level, which will continue toguide rational drug design against AD.

3. IAPP and type 2 diabetes3.1 Function of IAPP

Human islet amyloid polypeptide (IAPP, a.k.a. amylin) is a 37-residue peptide hormone co-secreted with insulin from pan-creatic b-cell islets. The IAPP physiology has been recentlyreviewed by Westermark et al.127 Briefly, the peptide is synthe-sized from a 67-residue precursor peptide, proIAPP, by proteo-lysis and posttranslational modifications, such as the C-

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terminal amidation and a disulfide bond formation betweenresidues 2 and 7 (Fig. 7a).128,129 Both IAPP and insulin areregulated by similar factors with a common regulatory promo-ter motif.130 Before secreting to the circulation, IAPP is storedtogether with insulin inside the b-cell granules at high con-centrations. IAPP functions as a synergistic partner of insulin tocontrol the blood glucose level by slowing down gastric empty-ing, inhibiting digestive secretion, and promoting satiety.131,132

IAPP is also known to play a role in bone metabolism alongwith calcitonin and calcitonin gene-related peptides.133

A hallmark of type 2 diabetes (T2D) is the formation of IAPP-enriched amyloid plaques found in the pancreas of patients.Insulin resistance in T2D leads to increased production ofinsulin and also IAPP by b-cells because of their shared synthesisand secretion pathways. Since IAPP is one of the most amyloido-genic peptides known, over-production of IAPP in b-cells pro-motes the accumulation of toxic aggregates. Other studies alsosuggested that insufficient process of proIAPP and accumulationof intermediately processed peptides might promote the for-mation of amyloid fibrils, but the detailed molecular mechan-isms remain unclear. The disease progression is marked by b-cell death and loss of b-cell functions, resulting in insulindeficiency and diabetic dependence on external insulin sources.

3.2 Atomic structures of IAPP and IAPP amyloid fibrils

Structural characterization of IAPP monomers is extremely challen-ging due to the high aggregation propensity of the peptide. Byreducing IAPP aggregation with detergent micelles, solution NMRstudies have been used to study the structure of IAPPmonomers.137–139 It has been shown that SDS micelles stabilize

IAPP in a highly helical form (Fig. 7b–d). At low pH, the peptideassumes an extended alpha-helix. At neutral pH, the peptide hasbeen found to form a kinked helix around residue H18. Suchstructural difference is likely due to the electrostatic interaction ofthe protonated His18 at low pH with the anionic SDS. Combing lowpH, low temperature, and low peptide concentrations to hinderIAPP aggregation in solution, an NMR study has recently revealedthat the N-terminus of IAPP remains alpha-helical while the C-terminus is unstructured, which is consistent with moleculardynamics (MD) simulations of isolated IAPP monomers.140

The fibril aggregates of IAPPs share the same characteristiccross-beta structures of known fibrils.141 Although the atomicstructure of full-length IAPP amyloid fibrils is not available,several model structures have been proposed based on variousexperimental methods. Using constraints derived from solid-state NMR, Tycko et al. proposed a U-shaped fibrils modelwhere residues 8–17 and 26–37 form two beta-sheets(Fig. 7e).134 Based on X-ray microcrystallography structures oftwo short peptides, Eisenberg et al. reconstructed a similarfibril model with main differences in the side-chain packing(Fig. 7f).135 Recently, EPR studies of disulfide-reduced IAPP ledto a different fibril model (Fig. 7g), where the peptide stilladopted a U-shape with two strands separated by a longerdistance.136 The two strands in a single peptide had to bestaggered with respect to each other to have the appropriatedinter b-sheet packing and distances.

3.3 Mesoscopic structure of IAPP amyloids

The morphology of IAPP amyloid fibril has been studied byboth TEM and AFM.142,143 IAPP fibrils at the mesoscopic scale

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Fig. 7 Structural studies of IAPP. (a) The primary structure of IAPP peptide. Solution NMR structures of IAPP monomers stabilized by SDS micelle at (b)pH 4.2 (PDB: 2KB8) and (c) pH 7.3 (PDB: 2L86). (d) Solution NMR structure of IAPP whose aggregation is reduced at pH 5.3, 4 1C, and 100 mM inconcentration (PDB: 5MGQ). Residues 1–19 are colored purple and His18 is in sticks. The overall U-shaped IAPP fibril models are derived fromexperimental constraints by (e) solid-state NMR134 and (f) X-ray crystallography of short peptides.135 Reproduced with permission from ref. 134, copyright2007 American Chemical Society. Reproduced with permission from ref. 135, copyright 2008 John Wiley & Sons. In panels (e and f), two peptides in thefibril cross-section are shown in sticks viewed along the fibril axis. (g) EPR constraints were applied to reconstruct the fibril model of disulfide reducedIAPP. The sub-panels A and B correspond to views along and perpendicular to the fibril axis, and sub-panels C and D are the accordingly reconstructedfibril models with two different views perpendicular to the fibril axis.136 Reproduced with permission from ref. 136, copyright 2012 American Society forBiochemistry and Molecular Biology.



displayed significant structural polymorphism, includingribbon-like, sheet-like and helical fibril morphologies (Fig. 8).The ribbons and sheets were formed by lateral association of5 nm wide protofibrils (Fig. 8a). Most of the fibrils were foundin left-handed coil morphology with cross-over periodicities ofeither B25 nm or B50 nm (Fig. 8b). Based on these observa-tions, Goldsbury et al. proposed that the building block of IAPPfibrils is the 5 nm protofibril which can either self-assemblelaterally into ribbon-like or sheet-like arrays or coiled fibrils.143

The atomic models of IAPP fibrils are consistent with theseTEM and AFM observations.

3.4 IAPP toxicity and type 2 diabetes

Mounting evidence suggests that IAPP aggregation and therelated toxicity are associated with T2D. IAPP variants fromdiabetes-prone primates and cats formed amyloid aggregatesreadily in vitro, while those from diabetes-free rodents and pigsfeatured significantly lower aggregation propensities.144 Anaturally-occurring polymorphic S20G mutation rendered IAPPmore aggregation prone;145 and an Asian subpopulation carry-ing this mutation is subjected to early onset of T2D.146 IAPPaggregated rapidly upon transplanting human islets into nudemice, and the aggregation process occurred before the recur-rence of hyperglycermia and was correlated with b-celldeath.147,148 Transgenic mice expressing human IAPP variantstarted to develop diabetes.149 Moreover, as with other amyloidproteins,150,151 IAPP amyloid aggregates are toxic to pancreaticislet cells.152 Therefore, amyloid aggregation of IAPP is relatedto b-cell death in T2D.153

3.4.1 Oligomers vs. amyloids. Amyloid aggregation is anucleation process, featuring a characteristic all-or-none sig-moidal kinetics. The final mature amyloid fibrils have beenfound relatively inert and have no significant cell toxicity. Incontrast, freshly dissolved IAPP has been found to be highlytoxic to islet cells and also cause membrane instabilityin vitro,154 where the small and soluble aggregation intermedi-ates of IAPP are expected to accumulate before the rapid fibrilgrowth. IAPP oligomers have also been found to disrupt cellcoupling, induce apoptosis, and impair insulin secretion inisolated human islets.155 Additional evidence includes trans-genic mice studies,149,156 where amyloid deposits were notalways observed under optical microscopy in animals startingto show diabetic symptoms, and there was a lack of autocorre-lation between beta cell loss and amyloid deposits in thesemodels.157 In addition, inhibition of the formation of insolubleIAPP aggregates but not oligomers by either small molecules158

or proteins159 did not reduce the cytotoxicity. Hence, theseresults among many others led to the toxic oligomer hypothesisin T2D.160,161

As the aggregation intermediate species, IAPP oligomers arenot well-defined and are extremely challenging to characterizedue to their transient and heterogeneous nature. Many in vitrostudies support the accumulation of helical intermediatespopulated along the aggregation pathway.162–164 It has beensuggested that the N-terminal helixes of soluble IAPPs (Fig. 7d)are amphiphilic and hydrophobic interactions drive the helixassociation, which in turn increases the local concentration ofthe C-terminus containing the amyloidogenic sequence 20–29.165 Both discrete molecular simulations (DMD) of IAPP

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Fig. 8 Morphology of IAPP amyloid fibrils. (a) Lateral association of ribbon-like IAPP protofibrils revealed by TEM of freeze-dried tungsten-shadowedsamples. Subpanels a–d depict ribbons assembled by lateral association of 1 to 4 protofibrils. Ribbons with multiple protofibrils often crossed over in aleft-handed sense at moderately regular intervals. Subpanel e corresponds to lateral assembly of protofibrils into single-layered, sheet-like arrays. Scalebar: 100 nm. (b) IAPP fibrils with coiled morphologies.142,143 Reproduced with permission from ref. 142, copyright 1999 Elsevier. Reproduced withpermission from ref. 143, copyright 1997 Elsevier. Subpanels a and b denote coiled fibrils visualized by TEM and AFM, respectively. Arrows point to a left-handed fibril with a 25 nm cross-over periodicity. Longer periodicities of approximately 50 nm can also be seen in both subpanels. Subpanel c shows theAFM height distribution, and d compares the 25 nm periodicity fibril in TEM and AFM. Scale bars: 100 nm in subpanels a and b and 50 nm in d.



dimers140 and X-ray crystallography study of IAPP fused to amaltose-binding protein164 supported this scenario. On theother hand, ion mobility mass spectroscopy (IM-MS) combinedwith MD simulations pointed to a different model of earlyintermediate states with beta-hairpin dimers.166 The differenceis possibly due to the enhanced sampling method – replicaexchange167 – used in the MD study, which reduced the freeenergy barrier of helix unfolding in the N-terminus. Furtherresearch is necessary to fully understand the structure anddynamics of IAPP oligomers in order to identify the toxicspecies and the molecular mechanism of IAPP toxicity.

3.4.2 The endogenous inhibition of IAPP aggregation. IAPPis highly aggregation prone and readily forms amyloid fibrilsin vitro at mM concentrations within hours.168 However, beforeits secretion to the bloodstream IAPP is stored inside b-cellgranules at mM concentrations for hours without apparentformation of toxic aggregates in healthy individuals.169 There-fore, the physiological environment inside b-cell granulesnatively inhibits the formation of toxic IAPP aggregates whiledisruption to the native inhibition environment may lead toamyloid aggregation of IAPP, causing b-cell death.

Islet b-cell granules have a distinct cellular environment.170

First, the pH value inside the granules is 5.5, which is below thephysiological pH of 7.4. Second, b-cell granules have one of thehighest concentrations of Zn2+ ions in the entire human body. Thehigh concentration of zinc in b-cell granules, maintained by a b-cell-specific zinc transporter—ZnT8,171 is believed to be importantfor the efficient storage of insulin in b-cell granules: zinc coordi-nates the formation of insulin hexamers, which form insulincrystals as the dense core of b-cell granules.172 Third, beside IAPPpeptides b-cell granules also have other molecules in large quan-tities, including insulin and proinsulin C-peptide. Insulin is co-secreted with IAPP by b-cells at a ratio of B100 : 1 in healthyindividuals, and such a high insulin-to-IAPP ratio is reduced toB20 : 1 in T2D patients.173 The C-peptide is a part of the proinsulinsequence connecting A- and B-chains of insulin. Protease-processing of proinsulin results into mature insulin and C-peptide with an equal molar concentration inside b-cell granules.

Low pH. Inhibition of IAPP aggregation at low pH has beenobserved in vitro. At low pH, an increase in the lag time and adecrease in the growth rate of IAPP fibrillization wasobserved.174,175 The electrostatic repulsion between IAPPs withprotonated histidine18 (His18) is responsible for inhibiting theself-association of IAPP at low pH,176 supported by DMDsimulations of IAPP dimerization with and without protonationof His18.177 However, since the pH value inside b-cell granulesis close to the isoelectric point of His18174,178 and a significantportion of IAPP is still unprotonated, interactions of IAPP withother granule components are necessary for natively inhibitingthe peptide amyloid aggregation at high concentrations.

Insulin. In vitro experiments have revealed that insulin is apotent IAPP aggregation inhibitor, which can significantly slowdown aggregation at sub-stoichiometry concentrations.180 Sev-eral studies, including peptide mapping,181 IMS-MS combined

with MD simulations,182 and DMD studies140 suggested that theB-chain of insulin can bind IAPP. Computational studies withatomistic DMD simulations showed that both insulin monomersand dimers (but not the zinc-bound hexamer as the IAPP-binding interface is buried) could bind IAPP monomer andinhibit IAPP self-association by competing with the amyloido-genic regions important for aggregation, subsequently prevent-ing amyloid aggregation (Fig. 9a and b). The preferred binding ofinsulin with the amyloidogenic region in the beta-strand con-formation (Fig. 9a) suggests that insulin can also cap the fibrilgrowth, consistently with the observed sub-stoichiometric inhi-bition of IAPP aggregation by insulin. Comparing to high zincconcentrations where insulin is insoluble in the crystal form,183

zinc-deficiency due to loss-of-function mutations of ZnT8 shiftsthe insulin oligomer/crystallization equilibrium toward solublemonomers and dimers, which can efficiently inhibit IAPP aggre-gation and reduce T2D risk in the subpopulation carrying thesemutations.184 However, since IAPP is found almost exclusively inthe soluble halo fraction of b-cell granules while insulin ismostly insoluble in the core, the balance of other granulecomponents such as Zn2+ and/or C-peptide co-localized withIAPP appears crucial for maintaining the native state of IAPP.

Zinc. In an early study by Steiner and co-workers where ZnCl2

was added to B250 mM IAPP solution, aggregation promotionwas observed.185 This promotion effect leveled off till B1 mMzinc ion was added, but no data at higher salt concentrationswas reported. In later experimental studies, IAPP aggregationinhibition was observed at low zinc concentrations (5 and 10mM, but relatively high zinc/IAPP stoichiometry), followed by apartial recovery of aggregation at very high stoichiometry (B50–100).186,187 A ‘‘two-site binding’’ model, where a high affinitybinding with His18 stabilized non-aggregating oligomers butan unknown weaker secondary binding promoted amyloidfibril formation, was proposed.187 However, this model cannotaccount for aggregation-promotion at low ion/proteinstoichiometry185 (e.g., in the case of 10 mM of IAPP there wasa single data point with increased aggregation at B25 mM ofzinc186). Combining DMD simulations with experimentalcharacterizations,179 Govindan et al. developed an alternativemodel that was consistent with the experimentally-observedconcentration-dependent effect of zinc on IAPP aggregation. Atlow zinc/IAPP stoichiometry, the IAPP oligomers cross-linkedby zinc were aggregation-prone due to high local peptideconcentrations (Fig. 9c). As ion/protein stoichiometryincreased, each IAPP tended to bind only one zinc ion atHis18. The electrostatic repulsion between the bound zinc ions(+2e) inhibited IAPP aggregation, similarly to the low pHcondition where IAPP aggregation was inhibited by protonatedHis18 (+1e).176 With zinc concentration kept increasing, thescreening effect due to high salt concentrations reduced elec-trostatic repulsion, and allowed for the aggregation to recover(Fig. 9d).186,187

C-peptide. Without zinc binding, C-peptide is disordered inwater and weakly helical in trifluoroethanol (TFE) solution.188

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The peptide contains five acidic amino acids. Alanine scancoupled with MS experiments suggest that all these acidicamino acids bind zinc ions and the binding is 1 : 1 instoichiometry.189 It has been found that zinc-binding mayinduce structural changes.190 It was hypothesized that multiplenegatively charged acidic amino acids in C-peptide allow thebinding with multiple IAPP peptides, locally increasing IAPPconcentration and subsequently promoting IAPP aggregation.Upon binding zinc, C-peptide adopts specific secondary andtertiary structures with reduced net charges, which might bindand stabilize IAPP peptides in the aggregation-incompetentstate. In addition, other granule molecules includingproIAPP191 and proInsulin may also contribute to native inhibi-tion of IAPP aggregation and cytotoxicity in beta-cells and aresubject to future investigations.

3.4.3 IAPP–membrane interactions. It has been proposedthat IAPP exerts cytotoxicity by membrane disruption.154,192,193

The positively charged IAPP can bind anionic cell membranesand lipid micelles, and the peptide conformational andaggregation propensities change upon binding alsodepending on the membrane curvature.192 Binding of IAPPwith small micelles was found to stabilize the peptide inhelical conformation (Fig. 7), while absorption of IAPP on flatmembrane accelerated the peptide aggregation.194 Using alipophilic Laurdan dye for examining MIN6 cell membranesupon exposure to freshly dissolved IAPP as well as matureamyloid fibrils, Pilkington et al. found that all species,especially fresh IAPP, enhanced membrane fluidity andcaused losses in cell viability.195 The cell generation of ROS,

however, was the most pronounced with mature amyloidfibrils. This study suggests a correlation of cytotoxicity withchanges in membrane fluidity rather than ROS production.

The exact mechanism by which IAPP oligomers disrupt thecell membrane is under active investigation. Pore formation byamyloid peptides has been suggested important for membranedisruption.196–198 The amyloid pore model is strongly sup-ported by single channel recordings of IAPP on planarmembranes.196,199,200 A detergent-like mechanism has alsobeen advocated, where the mosaic-like opening and closing oftransient defects within the membrane (also see Fig. 15d inSection 4 for aS) was supported by AFM studies showing large-scale defects in the lipid bilayer upon prolonged exposure toIAPP.201 However, the strong correlation between fibril for-mation and membrane disruption by this mechanism202 isinconsistent with the toxic oligomer hypothesis. Recently,biophysical measurements in conjunction with cytotoxicitystudies showed that nonamyloidogenic rat IAPP was as effectiveas IAPP at disrupting standard anionic model membranesunder conditions where rat IAPP did not induce cellular toxi-city, suggesting that there is no direct relationship betweendisruption of model membranes and induction of cellulartoxicity.203 Therefore, the connection between IAPP cytotoxicityand membrane disruption remains inconclusive.

3.5 Mitigation strategies and theranostics

As with other amyloid diseases,204–206 inhibition of IAPP aggre-gation is an attractive therapeutic strategy to prevent b-celldeath207 and halt the progression of diabetic conditions in T2D.

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Fig. 9 Effects of b-cell granule components on IAPP aggregation. (a) A representative IAPP–insulin complex from DMD simulations,140 where theamyloidogenic residues of IAPP (residues 22–29) are shown in orange. Reproduced with permission from ref. 140, copyright 2015 Nature PublishingGroup. The residues in the B-chain of insulin important for binding IAPP are highlighted in stick representation. (b) The residues of an insulin monomerare colored according to IAPP binding frequencies (red-blue: high to low frequencies) in the structure of an insulin hexamer. The view with an 1801rotation is also presented. The residues with strong IAPP-binding are located at the insulin monomer–monomer interface.140 Reproduced withpermission from ref. 140, copyright 2015 Nature Publishing Group. (c) A representative IAPP tetramer with His18 (highlighted as sticks in pink) coordinatedby a Zn2+ (blue sphere) from DMD simulations.179 Reproduced with permission from ref. 179, copyright 2015 American Chemical Society. Theamyloidogenic sequences from each IAPP monomer are highlighted in rainbow colors. (d) A mechanistic scheme demonstrating the dependence of IAPPaggregation on relative zinc concentration.



Various approaches have been explored to reduce aggregation-induced IAPP cytotoxicity, through the use of peptides, peptide-mimetics,208–211 small molecules,212–223 and nanoparticles(NPs).224–226 Non-amyloidogenic sequence variants of IAPPincluding rat IAPP227 have been found to inhibit the fibrilformation of human IAPP,208,209 and the inhibition efficaciescan be improved by synthesizing peptide mimetics with con-formational restraints.210,211 Targeting the early helical inter-mediate states of IAPP aggregation,162–164 small moleculepeptidomimetics212,213 have been designed to mimic helixesthat complementarily bind to the N-terminal helix of IAPP.Another attractive set of amyloid aggregation inhibitors aresmall-molecule polyphenols221 such as epigallocatechin gallate(EGCG),228 curcumin,219,220 and resveratrol,229 which inhibitaggregation and reduce the related cytotoxicity of IAPP230 as

well as other proteins and peptides such as Ab.231 Thesepolyphenols have the advantage of being naturally occurring,and are non-toxic at moderate concentrations. Despite well-known therapeutic benefits of small molecules,232 however,pharmacological applications of these polyphenols are limiteddue to some common issues, such as their poor watersolubility.233

Several studies have examined the anti-amyloid mechan-isms of small molecules and NPs. For example, IMS-MS experi-ments showed that EGCG exerted an inhibitory effect on IAPPaggregation through direct binding of EGCG to the peptide215

and alternating the aggregation pathways.228 Using simulationsof the amyloidogenic segment of IAPP, resveratrol was found tobind and prevent the lateral growth of the fibril-like b-sheets.235

In another work, resveratrol was found to bind weakly to IAPP

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Fig. 10 Inhibition of IAPP aggregation. (a) Left: High-throughput dynamic light scattering measurement of IAPP size distributions with and withoutresveratrol (2 : 1 ligand/IAPP ratio). Right: Distribution of IAPP aggregates of different molecular weights with and without resveratrol in silico. Stable IAPP/resveratrol oligomer has the resveratrol molecules forming a nano-sized core and IAPP peptides a corona, which prevents aggregation.234 Reproducedwith permission from ref. 234, copyright 2016 Nature Publishing Group. (b) Left: A typical IAPP dimer in DMD simulations. Right: Binding to a PAMAM-OHdendrimer (spheres) inhibits self-association of the amyloidogenic sequences (yellow region) between two IAPP peptides.224 Reproduced withpermission from ref. 224, copyright 2016 John Wiley & Sons. The peptides are shown in cartoon representation with rainbow color from blue (N-terminus) to red (C-terminus).



and reduce inter-peptide contacts.236 A recent computationalstudy showed that resveratrol altered the structure of an IAPPpentamer,237 which was modelled by the amyloid fibril structurederived from solid-state NMR.134 By modelling the effects ofpolyphenols like resveratrol and curcumin on the initial self-association and aggregation of IAPP in DMD simulation,234

Nedumpully-Govindan et al. showed that these polyphenolsinhibited IAPP aggregation by promoting ‘‘off-pathway’’ oligo-mers with the hydrophobic polyphenols forming the core(Fig. 10a). The peptides were stabilized in the aggregation-incompetent helix-rich state by burying their hydrophobic resi-dues inside the core and exposing the hydrophilic residues.Graphene oxide nanosheets displayed strong inhibition effectson IAPP aggregation and associated cytotoxicity because strongbinding affinity rendered the peptides to bind with thenanosheets rather than between themselves.225 OH-terminatedpolyamyloidoamine (PAMAM-OH) dendrimers inhibited IAPPaggregation and cytotoxicity, where the polymeric NPs encapsu-lated and stabilized monomeric IAPP in their hydrophobicinterior (Fig. 10b).224 In general, these inhibitors all reducedthe population of the oligomeric species, thereby reducing IAPPtoxicity.

4. Alpha-synuclein and Parkinson’sdisease4.1 Function of alpha-synuclein

Alpha synuclein (aS) is a 140-residue small protein highlyconcentrated in presynaptic terminals,238 making up as muchas 1% of all proteins in the cytosol of brain cells. Small traces ofaS are also found in the heart, gut,239 muscles and othertissues, reminiscent of the confounding bodily distributionsof Ab and IAPP beyond their purported origins. In the intra-neuronal space, aS assumes an equilibrium between anunfolded monomeric conformation and a membrane-bound

state that is rich in alpha helices.240 The precise physiologicalrole of aS is unclear, but is relevant to the modulation ofneurotransmitter dopamine release, ER/Golgi trafficking, andsynaptic vesicles.241 The membrane-bound aS influences lipidpacking and induces vesicle clustering through physical andphysicochemical interactions, while aS in the multimeric formhas been shown to promote SNARE complex assembly duringsynaptic exocytosis.240

Aggregated aS mediates dopaminergic neurotoxicityin vivo.244 However, the precise mechanisms by which aS lendstoxicity to host cells remain unclear. Pathologically, aS is amajor component of Lewy bodies and neurites, the intracellularprotein aggregates first identified by Spillantini et al. in 1997(Fig. 11)242 and hallmarks of Parkinson’s disease (PD), Parkin-son’s disease dementia (PDD) and dementia with Lewy bodies(DLB). Compared with the ambiguous pathology of aS, theneuritic pathology of b and g synuclein homologs does notappear widespread, and both neuroprotective and neurotoxicpotentials of b synuclein have been reported.245

4.2 Atomic structures of alpha-synuclein and alpha-synucleinamyloid fibrils

The sequence of aS is encoded by the SNCA gene and can bedivided into three distinct domains: (a) the amphipathic N-terminal domain (1–60), which contains consensus KTKEGVrepeats and has alpha-helical propensity, (b) the centraldomain (61–95) or the non-amyloid-beta component (NAC) thatis highly hydrophobic and amyloidogenic, and (c) the acidic C-terminal domain (96–140) which contains negatively chargedand proline residues to afford protein flexibility but no appar-ent structural propensity.246 High resolution ion-mobility massspectroscopy has revealed that HPLC-purified aS is autoproteo-lytic, giving rise to a number of small molecular weight frag-ments upon incubation. In particular, the fragment of residues72–140 contains majority of the NAC region and aggregates

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Fig. 11 (a) (Large image) Pigmented nerve cells containing aS-positive Lewy body (thin arrows) and Lewy neurites (thick arrow).242 Reproduced withpermission from ref. 242, copyright 1997 Nature Publishing Group. Small image: a pigmented nerve cell with two aS-positive Lewy bodies. Scale bar: 8mm. (b) Hypothesized aS toxicity and spread of pathology in Parkinson’s disease (PD) and Parkinson’s disease dementia (PDD). UPS: ubiquitin proteasomesystem.243 Reproduced with permission from ref. 243, copyright 2013 Nature Publishing Group.



faster than full-length aS.247 These autoproteolytic productsmay serve as intermediates or cofactors in the aggregation of aSin vivo.

The atomic structures of fragmental and full-length aS in thefibrillar form have been elucidated over the past decade(Fig. 12). Using quenched HD exchange Vilar et al. identifiedfive b-strands within the fibril core comprising residues 35–96and with solid-state NMR spectroscopy the presence of b-sheetsecondary structure within the fibril core of residues 30–110.241

This study has further detailed the mesoscopic features of aSfibrils, as we will visit in the next sub-section.

Based on micro-electron diffraction Rodriguez et al. revealedsmall crystal structures of the toxic NAC core (68–78, orNACore) and the preNAC segment (47–56) of aS, at spatialresolution of 1.4 Å (Fig. 13a).248 The NACore strands stackedin-register into b-sheets. The sheets were paired, forming steric-zipper protofilaments as observed for other types of amyloido-genic proteins. Notably, each pair of the sheets contained twowater molecules, and each was associated with a threonine sidechain within the interface. X-ray fiber diffraction patternsfurther revealed a similarity of the NACore to full-length aSfibrils.248

In a more recent study, Tuttle et al. established the atomicstructure of full-length aS fibrils based on 68 spectra, using 2Dand 3D ssNMR.249 The fibrils were collected from cell cultureand shown to adopt a b-serpentine arrangement (Fig. 13b–e).The fold exhibited hydrogen bonds in register along the fibrilaxis, orthogonal to the hydrogen bond geometry in a standardGreek-key motif unseen for other fibrils (Fig. 13d).249 Theinnermost b-sheet contained amyloidogenic residues 71–82,while the sidechains in the core were tightly packed(Fig. 13e). Compact residues facilitated a close backbone-

backbone interaction: A69–G93 bridged the distal loops of theGreek key, and G47–A78 rendered a stable intermolecular saltbridge between E46 and K80. Hydrophobic sidechain packingamong I88, A91 and F94 established the innermost portion ofthe Greek key. Residues 55–62 were disordered, consistent withthat reported by Comellas et al.250 Collectively, the stericzippers, glutamine ladder and intermolecular salt bridge con-tributed to the structural complexity and stability of the fibril.However, it remains uncertain whether such atomic structurereflects that of aS fibrils extracted directly from PD patients.

4.3 Mesoscopic structure of alpha-synuclein amyloids

The morphology of aS fibrils has been examined withAFM251–253 and cryoelectron microscopy.241 A hierarchicalassembly model (HAM) was proposed by Inonescu-Zanettiet al.254 to describe the architecture of immunoglobulin light-chain protein SMA fibrils assembled from smaller subspeciesand has shown general applicability to the nanoscale assem-blies of Ab, aS and IAPP as well as SH3 domain, lysozyme, SMA,b2-microglobulin and beta-lactoglobulin.142,251,252,255–260 Alter-natively, a new packing model was proposed by Sweers et al.,261

in attempt to reconcile the morphological and mechanical dataobserved for two distinct fibril species of E46K, a mutant of aS.Nonetheless, according to the HAM, protofilaments are estab-lished by the nucleated polymerization kinetic model, in whichthe protofilaments elongate by the addition of monomeric,partially folded intermediates to their growing ends. The pro-tofilaments then interact with each other to form protofibrils,each consisting of twisted 2–3 protofilaments, and two proto-fibrils entwine to form mature fibrils and, eventually, plaques.The driving force for such stepwise assembly is both electro-static and hydrophobic.254

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Fig. 12 Landmark studies concerning the structures of aS fragments with respect to its full 140 residues consisting of N terminus, NAC and Cterminus.248 Reproduced with permission from ref. 248, copyright 2015 Nature Publishing Group. The research teams are chronicled on the left while theemployed techniques are abbreviated on the right. EPR: electron paramagnetic resonance; ssNMR: solid-state nuclear magnetic resonance; HD:hydrogen–deuterium exchange; SDSL: site directed spin labelling.



The HAM model predicts the occurrence of periodicity forprotofilaments and fibrils, which assume twisted morpholo-gies. Such periodicity is driven by a balance between mechan-ical forces dominated by the protofilament elasticity andelectrostatic forces due to the distribution of hydrophobicregions and charge along the protofilament backbone,253 aswell as by the inherent chirality of constituting amino acids andb-sheets/helices of the fibrils. The average heights of aS proto-filaments and fibrils were 3.8 nm and 6.5 nm, respectively,while the periodicity of aS fibrils ranged from 100–150 nm asdetermined by AFM (Fig. 14a).253 These parameters are consis-tent with immunoelectron microscopy of filaments extracted

from the brains of patients with DLB and multiple systematrophy,262 and agree with high-resolution cryoelectron micro-scopy where twisted protofilament of B2 # 3.5 nm in bound-aries and 120 $ 10 nm in periodicity were observed leading tothe proposal of a folded aS fibril model (Fig. 14b).241 The cross-section of individual aS monomers in the fold was trapezoidinstead of circular, resulting in a two-fold increase in momentof inertia (Sweers 2012).263 Though not substantiated, suchnon-circular packing of monomers could also hold true forother b-sheet folded proteins.261,263 In addition, curly aS fibrilsprepared by filtration-like steps during aggregation possessed apersistence length of 170 nm, while straight aS fibrils from

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Fig. 13 (a) Top and side views of the structures of NACore (orange; residues 68–78, sequence also see Fig. 12) and PreNAC segments (blue; residues47–56, sequence also see Fig. 12). The A53T mutation in PreNAC is shown in black.248 Reproduced with permission from ref. 248, copyright 2015 NaturePublishing Group. (b–e) Three-dimensional structure of a full aS fibril. (b) A central monomer from residues 44 to 96 looking down the fibril axis showingthe Greek-key motif of the fibril core. (c) Stacked monomers showing the sidechain alignment between each monomer down the fibril axis. (d) Residues25 to 105 of 8 monomers displaying the b-sheet alignment of each monomer in the fibril and the Greek-key topology of the core. (e) Overlaid ten lowestenergy structures, showing sidechain positions within the core. Residues 51–57 are indicated in red with side chains removed.249 Reproduced withpermission from ref. 249, copyright 2016 Nature Publishing Group.



unperturbed aggregation displayed persistence lengths of up to140 mm.264

4.4 Alpha-synuclein toxicity and Parkinson’s

4.4.1 Oligomers vs. amyloids. Natively unfolded aS under-goes a transition to partially folded intermediates prior to fibrilformation.265 Such partially folded conformations are favored bymutations266 or changes in pH, ionic strength and temperature andare thought to be critical intermediates in the transition to amyloidfibrils.253 Clearly, such dynamic transition has an important bear-ing on aS toxicity, as evidenced by a body of literature focused onthe complex roles of aS oligomers and amyloids.

The aggregation of aS follows a nucleation polymerizationpathway involving prefibrillar species of remarkable conforma-tional plasticity,267 both transient and stable. Specifically, it ispostulated that aS aggregation takes place in the cytoplasma orin association with the cellular membrane. In the cytosol,soluble monomers interact to form unstable dimers, whichdevelop into oligomers and, subsequently, fibrils.268 The cur-rent understanding concerning aS toxicity follows the narrativeof the ‘‘toxic oligomer hypothesis’’,251 in that the oligomericspecies are more toxic than the fibrillar form,251,269–272 assimilarly proposed for Ab273–279 and IAPP.280 However, due tothe different structural characteristics and aggregation rates,different cellular environments, as well as prion-like cell to cellspreading and crosstalk of proteins of different origins andpathologies, this generalization remains putative.281,282

In an early in vitro study, Conway et al. compared the rates ofdisappearance of monomeric aS and appearance of fibrillar aSfor wide-type and two mutant proteins A53T and A30P.251 Thedifferences between the trends suggested the occurrence ofnonfibrillar aS oligomers. Using sedimentation and gel filtra-tion chromatography, the researchers identified spheres (rangeof heights: 2–6 nm), chains of spheres (protofibrils), and rings/annulars (heights: B4 nm) from fibrils (B8 nm in diameter) byAFM. For a comprehensive account of aS oligomers and theirin vitro preparation protocols, readers may refer to a recentreview by van Diggelen et al.283

Using attenuated total reflection-Fourier-transform infrared(ATR-FTIR) spectroscopy Celej et al. revealed that isolated aSoligomers adopted an antiparallel b-sheet structure, whereas fibrilsassumed a parallel arrangement.284 Notably, antiparallel b-sheetstructures have also been reported for the oligomeric structures ofAb, b2-microglobulin and human prion peptide PrP82–146.284 Suchcontrasting features in secondary structure between the oligomersand fibrils entail differences in conformational change, affinity andmode of interaction when binding with the cell membrane, furthercompounded by the differences in aspect ratio and surface hydro-phobicity between the two species. The toxicities of aS oligomersand amyloid protein oligomers in general have been postulated asan inherent property.285 Unlike amyloid fibrils, the oligomers sharesimilar structural properties273,286 and possess higher portions ofrandom coils and helical structures. Consequently, the exposedhydrophobic surfaces of the oligomers could mediate interactionswith intracellular proteins to trigger aberrant cellular pathways.

Celej et al. found that purified aS oligomers spheroidal andpolydisperse (10–60 nm), while aS fibrils were unbranched of 6–10 nm in diameter and micrometers long when examined underelectron microscopy.287 These isolated oligomers were on-pathway intermediates sharing the same structural motif withother prefibrillar oligomers and possessing no canonical cross-bfibril structure.284 Curiously, the aS oligomers were recognizedby A11 antibody, which also targeted the oligomeric but notmonomeric or fibrillar forms of Ab, prions, and IAPP.273,288

While it remains debatable whether aS oligomers are inter-mediates in the process of amyloid formation, or precursors tofibrils, or byproducts of fibril elongation, or associated with apathway of aggregation different from the standard amyloidfibrillization,269 there is little ambiguity that aS oligomers aretoxic, as validated by in vitro and animal models.272,273,289,290

4.4.2 Alpha-synuclein–membrane interactions. Towardsunderstanding the origin of amyloid protein toxicity, muchresearch over the past two decades has been focused on theinteractions of the proteins as well as their aggregationproducts with cell membranes, model lipid vesicles, or lipidrafts. This focus is especially justified for aS considering its

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Fig. 14 (a) AFM image showing a periodicity of 100–150 nm along an aS protofibril. The peak (red arrow) to trough (blue arrow) differs by B1 nm inheight. (inset) A section of a protofilament with an average height of 3.8 nm.253 Reproduced with permission from ref. 253, copyright 2003 Elsevier. (b)Proposed fold of an aS fibril. A monomeric aS within a protofilament (center). Incorporation of protofilaments into a straight or twisted fibril is illustrated inthe left and right panel, respectively.241 Reproduced with permission from ref. 241, copyright 2008 National Academy of Sciences.



strong functional association with synaptic vesicles and its cell-to-cell spreading.291 From a biophysical standpoint suchinteractions may be understood as a manifestation of thestructural attributes of aS (sequence, charge andhydrophobicity), as well as the changing properties of aS fromsoluble and disordered monomers to soluble and less randomoligomers, and to waxy and highly ordered fibrils and plaques.

Research concerning protein–membrane interaction should takeinto consideration of two convoluting aspects: protein aggregationmodulated by a model lipid bilayer or cell membrane, andmembrane integrity perturbed by protein aggregation. Numerousstudies have confirmed that lipid membranes can speed up theprocess of protein fibrillization due to the amphiphilicity of bothinteractants.292,293 Specifically, the N-terminal region of aS, contain-ing 7 amphiphilic imperfect repeats each of 11-residues, can initiateelectrostatic interaction with anionic lipid head groups. The NACregion of the protein can establish hydrophobic interaction withlipid fatty acyl tails to promote membrane partitioning.294 Uponmembrane exposure, the protein concentration at the membranesurface is abruptly increased due to the 3D to 2D transition.Consequently, protein conformational entropy is reduced to favoraggregation.294,295 Specifically, the rate of aS primary nucleation wasenhanced by three orders of magnitude when exposed to smallunilamellar vesicles (SUVs, 20–100 nm in diameter) of 1,2-dimyristoyl-sn-glycerol-3-phospho-L-serine (DMPS).296

Upon adsorption onto lipid membranes, monomeric amyloidproteins adopt an a helical state, followed by a conversion to b-sheet rich oligomers and amyloid fibrils modulated by thecurvature and charge of the membranes, presence of metal ions,peptide to lipid ratio, and ganglioside clusters, cholesterols andlipid rafts.299 aS assumes a fully extended a helical state cominginto contact with larger vesicles, likely representative of theprotein conformation in vivo.300,301 In contrast, smaller vesicles

with greater curvatures and smaller surface areas are associatedwith proteins in bent a helices or antiparallel helix-turn-helixconformation to maximize protein–membrane binding.293

A high peptide/lipid ratio favors protein crowding on themembrane surface to induce nucleation.302 Binding of aS (iso-electric point of 4.74)303 with membranes is elevated withincreased acidic phospholipid content.304–306 aS oligomers alsoshow propensity for the liquid disordered phase of anionicvesicles.307 The exact mechanism of aS association with lipidrafts is unclear, but is linked to the high lipid packing densityof anionic head groups in the rafts. Such specific bindingbetween aS and lipid rafts may be essential to both the normalcellular function of aS and its role in PD pathology.299

Elevated levels of metal ions have been found in the substantianigra of PD patients.308 Addition of metal cations of Cu2+, Fe3+ orCo3+ induced secondary structure in aS and accelerated proteinaggregation in vitro,265 through metal ion-mediated amyloid protein–membrane interaction. Although Ca2+ (of B300 mM) in the ER servesto facilitate protein folding, addition of Ca2+ and other heavy metalions to monomeric aS rapidly produced annular oligomers,309 whiledivalent metal ions also enabled the clustering of aS on the surfacesof anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(10-rac-glycerol)/phosphatidylcholine bilayers.306 It is possible that metal cationsenabled the interaction of the likely charged C-terminus of aS andmembranes through charge neutralization. Such strong metal-hosting capacity of amyloid proteins has been utilized in entirelydifferent contexts from amyloidogenesis, such as purification ofwastewater and in vitro iron fortification using functional b-lactolglobulin amyloids.310–312

The adsorption of aS has been shown to compromisemembrane permeability.282 One mechanism proposed for suchperturbation is pore formation by the protein oligomers(Fig. 15a–c).271,284,293,297 In combination with biochemical

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Fig. 15 Proposed mechanisms of membrane damage induced by aS aggregation. (a) Projection averages of annular oligomers formed by aS mutantsA53T and A30P.271 Reproduced with permission from ref. 271, copyright 2002 Nature Publishing Group. (b) aS oligomer spans the membrane in a porin-like fashion to induce toxicity.284 Reproduced with permission from ref. 284, copyright 2012 Portland Press. (c) Oligomers but not monomers or fibrilsinduced frequent channel formation in planar lipid bilayers formed from diphytanoylphosphatidylcholine dissolved in n-decane in 1 M KCl, at a bias of+100 mV.297 Reproduced with permission from ref. 297, copyright 2009 American Chemical Society. (d) (top panel) Monomeric aS adsorbed to a lipidbilayer. (middle panel) Aggregation of aS monomers causes membrane thinning and lipid extraction. (lower panel) Further incubation results in assemblyof mature aS fibrils and disassembly of the lipid membrane.298 Reproduced with permission from ref. 298, copyright 2011 American Chemical Society.



and ultrastructural analysis, Tsigelny et al. revealed throughMD simulations and docking that aS monomers, upon adsorp-tion onto lipid membranes through their N-termini, assembledinto homodimers of both propagating (head to head) and non-propagating (head to tail) conformations. The propagatingform docked on the membrane surface to recruit additionalaS molecules, rendering pentamers and hexamers to form ring-like structures partitioning in the membrane.313 Consistently,addition of stable aS oligomers has been shown to induce ion-channel activity (Fig. 15c),297 while Ca2+ and dopamine exhib-ited much higher leakage rates than polymers of cytochrome cand fluorescein isothiocyanate–dextran from anionic vesicles inthe presence of oligomeric A30P and A53T, two major aSmutations.314 Under conditions in which vesicular membraneswere less stable due to the lack of counter-ion Ca2+, aS permea-tion was less size selective and monomeric aS permeated via adetergent-like mechanism.293

Another mechanism proposed for aS-membrane interactionis illustrated in Fig. 15d.298 Here the presence of a supportedlipid bilayer facilitates the conversion of aS from randomlystructured monomers to alpha helices (top panel), whichfurther aggregate into oligomers and fibrils while strippinglipids off the bilayer (middle and lower panels). Membranethinning and depolarization, changing fluidity, lipid flip–flop,calcium leakage, and disruption of ionic homeostasis are plau-sible consequences of aS membrane adsorption, aS self-assembly, and aS assembly with membrane lipids, throughhydrophobic and electrostatic interactions as well as lipid micel-lar encasing of the protein species (i.e., the carpet model315).This mechanism is supported by experimental studies employ-ing giant vesicles as well as reporters of ThT, calcein, Ca2+ andfluorescence recovery after photobleaching,316–319 to name a few.

In close connection with aS toxicity and aS–membraneinteraction, a body of literature has revealed links between aSoligomers and mitochondrial dysfunction, cytoskeleton defor-mation, enhanced ROS production, neuroinflammation, ERstress, as well as impaired protein degradation systems.320–328

An analysis of wide-type aS and two mutational variants A30Pand E46K interacting with synaptic-like SUVs suggested amechanism by which a single aS binds to two different synapticvesicles via the NAC to promote their assembly and vesicleclustering.329 In addition, promotion of SNARE-complex for-mation has been found to be associated with aS assembly intohigh-order multimers upon their binding with plasma mem-branes, suggesting that aS may act as a SNARE chaperone at thepresynaptic terminal against neurodegeneration.240

4.4.3 Parkinson’s disease, mitigation strategies and theranos-tics. Synucleinopathies refer to a family of neurodegenerativediseases including PD, PDD and DLB, where inclusions of Lewybodies and neurites are located within the neuronal cells (Fig. 11a).Multiple system atrophy (MSA) is a special type of synucleinopathy,since aS-positive inclusions are found in oligodendroglia instead ofin neurons. In these diseases, aS pathology in the substantia nigra isclosely correlated with motor symptoms and death of SN dopami-nergic neurons stimulates the striatum.282 The PD pathologyinvolves progressive neuronal accumulation of aggregated aS, and

formation of Lewy bodies affects various functional structuresthroughout the human nervous system to compromisemovement.330

Exogenous aS fibrils seeded Lewy body- and Lewy neurite-likeinclusions in cell culture models, and direct neuron to neuron aStransmission throughout the brain propagated PD-likepathology.291,331,332 Failure of the protein quality control systems,especially lysosomes, promoted accumulation of transmitted aS andinclusion formation. Cells exposed to neuron-derived aS displayedsigns of apoptosis, such as nuclear fragmentation and caspase 3activation, both in vitro and in vivo.291 Inoculation of aS fibrils intowide-type non-transgenic mice seeded aggregation of endogenousmouse aS and reproduced key features of the neurodegenerativecascade.249 A molecular level understanding of the pathologicalspreading of aS in PD is lacking, but growing evidence suggests itsorigin lies in protein self-assembly through templated seeding,where the imported aS aggregates catalyze the conversion of localsoluble protein molecules into their aggregated forms. A recent studyhas revealed regulation of motor deficits and neuroinflammation byintestinal microbiota in a PD model,239 suggesting a role for micro-bial signals in PD.333 Multiplication of the protein aggregates byrecruiting additional aS en route has been proposed as an additionalmechanism to templated seeding, to ensure sustainable concen-tration of the aggregates spreading from cell to cell.332 However,multiplication of aS at neutral pH has not been observed, pointing tothe involvement of other cellular processes in enabling the prion-likeaS spreading.

The ambiguities concerning the natural state, toxicity andaggregation pathway of aS have hindered the development ofmitigation and theranostics against PD. The currentapproaches, still very much in the incubation stage, aim atexploiting the structural, functional and toxicological proper-ties of aS, or the self-assembly of the protein and its structuraland pathological characteristics for therapeutic efficacy.268

Stabilization of the native aS structure from misfolding is alogical strategy. This intervention may also help resolve thecontroversy concerning a tetrameric initial state of aS.334 Onepromising approach to slow down aS synucleinopathies is tolimit the role of extracellular aS in disease progression, frominterfering with aS secretion to neuronal uptake. Removal of aSfrom the extracellular space to minimize inflammations may beachieved with immunotherapy, as immunization with humanaS suppressed protein aggregation and decreased neurodegen-eration in transgenic mice overexpressing the protein.335–337

The use of small molecules and mutation is another feasibleapproach for stabilizing oligomeric species and amelioratingtoxicity. The antioxidation and anti-inflammatory properties ofthe small molecules – often polyphenols or their structuralderivatives with the capacity of interfering with protein aggre-gation through competing H-bonding, hydrophobic interactionand p-stacking with the protein – may counteract the toxicityelicited by the oligomeric species.338–340 The presence of smallmolecules and other aggregation antagonists may also reduceaccessibility to the oligomers by environmental chaperones,ligands and molecular organizations, thereby driving the aggre-gation off pathway to halt aS pathology.

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5. Prions and prion diseasesTransmissible spongiform encephalopathies (TSEs), also known asprion diseases, are a family of rare fatal neurodegenerative dis-orders associated with prion protein (PrP), and arise in severalmammalian species by sporadic, inherited, or infectious means.Kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Straussler–Scheinker (GSS), fatal familial insomnia (FFI) and fatal sporadicinsomnia (FSI) are PrP-related human disorders,341 whereas scra-pie, bovine spongiform encephalopathy (BSE) and chronic wastingdisease (CWD) are known sheep,342 cattle343 and cevids344 priondiseases. The main characteristic symptoms of TSEs are brainvacuolation, astrogliosis and neuronal apoptosis,345,346 which areassociated with accumulation of extracellular PrP amyloid depositsin the CNS.347–350 Despite shared sequence between cellular non-pathological PrP (PrPC) and misfolded PrP (PrPSc),351 pathologicalPrPSc aggregates are proteinase K resistant352 and have a b-enrichedsecondary structure.353–355

The most distinct feature of TSEs, unique among diseasesrelated to protein misfolding, is the infectivity of the patho-genic agent. Procedures that hydrolyze or modify proteinsreduce scrapie infectivity, whereas procedures that alter nucleicacids have no effect.356–359 The ‘‘protein-only hypothesis’’ hasnow been widely accepted,360–363 contending that a proteinstructure can be replicated without the use of nucleic acidsand the infectious pathogen is the misfolded PrPSc.356,357,360–365

In addition, prion diseases progress in host without any sign ofimmune responses to a ‘‘foreign infectious agent’’.341 When theprotein requirement for infectivity was established, prions weredefined as proteinaceous infectious particles that resistedinactivation by procedures that modified nucleic acids.341

Since prion pathology and infectivity366 are closely related to aprotein existing in two different conformations, much research inthe last decade has been dedicated to understanding the structuresof native PrPC 367 and pathological PrpSc.368 PrPSc is believed to act asa structural template, inducing conversion of other PrPC moleculesinto the pathological form.341 Understanding PrP conformational

structures is therefore essential for describing protein misfoldingand the specific role of PrP in prion pathology.

5.1 Function of PrP

PrP is encoded by gene (PRNP) found in chromosome 20 (inhuman)369 and expressed in many tissues, including the brain,circulating lymphocytes, heart, kidney, skin, digestive tract, endothe-lia, mammary gland and muscle. The physiological role of nativePrPC remains unclear.370 It has been shown that the protein isinvolved in several cellular processes including neuroprotectionagainst excitotoxicity and serum starvation,371 proliferation andcell–cell adhesion,370,372 formation of synapses373 and ligandbinding.374,375 PrP can protect cells against heavy metal overloadingand subsequent oxidative stress by binding divalent ions of copper,zinc, manganese and nickel.375 Due to the ability of PrP to modulatecell proliferation and apoptosis it is believed to play a role on cancerdevelopment.376 Indeed, increased PrPC level has been found ingastric cancer,377 colorectal cancer378 and skin cancer.379

A common approach to study the function of PrP is using PrPknockout transgenic mice (Prnp"/"). The major finding in Prnp"/"

mice was the lack of developmental differences and resistance toprion diseases.380 However, Prnp"/" mice have shown cognitiveabnormalities370 such as depressive-like behavior, anxiety-relateddisorders and alterations in circadian activity.381 In addition,decreased spatial learning of Prnp"/" mice has been noticed.382

Using three Prnp knockout mice lines Firestein and colleaguesfound that PrPC was important in the normal processing of sensoryinformation by the olfactory system.383

5.2 Atomic structures of prions and prion amyloids

The atomic structures of full-length and truncated PrPC were mostlysolved by NMR.384–388 Notably, X-ray crystallography studies wererestricted to the C-terminal domain of PrP, suggesting an intrinsictendency of the protein to avoid crystallization.389,390

Proto-protein of human PrP (huPrP) is 253 residues long(Fig. 16a). After translation to mature form, the first 22 residues

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55Fig. 16 (a) Overview of the PrP sequence and architecture.341,389 The residue numbering refers to human PrP. (b) 3D representation of the secondarystructure of mouse PrPC.384 Reproduced with permission from ref. 384, copyright 1996 Nature Publishing Group. The unordered N-terminus is omittedand the sulphur bridge between Cys179 and Cys214 is indicated in yellow.



are removed and the last 23 residues are cleaved off prior to theaddition of a glycosyl phosphatidylinositol (GPI) anchor toSer230. PrP is attached to the outer surface of the cellularmembrane by a GPI anchor within the raft domains. Thesequence of PrP is highly conserved amongst mammals:391,392

human PrP shares 99.2%, 94.9% and 92.8% of identicalsequences with the proteins from chimpanzee, sheep andcow, respectively.

PrPC has two regions with distinct structural and dynamicalproperties.367 In mammals, depending on the organism, the N-terminus contains a variable number of amino-terminal octa-peptide repeats with sequence PHGGGWGQ. Each octarepeat isable to generate a divalent metals-binding domain via nitrogenatoms in the histidine imidazole side-chains.393 The N-terminus is up to residue 120 and this region has been shownto constitute a pH-dependent folding: at pH 4.5 it is flexiblydisordered,394 however at pH 6.2 residues 61–84 of the octar-epeats adopt a loop and a b-turn like conformation.386 Incontrast, the C-terminus of PrPC is structured, containing threea-helices (H1, H2 and H3) and a short, two-stranded, antipar-allel b-sheet (S1, S2)367 (Fig. 16b). A disulphide bridge isbetween Cys179 and Cys214, which anchors H2 and H3 helices.This disulphide bridge is one of the major determinants of thetertiary structure of PrP. FTIR study of PrP secondary structurerevealed 42% of a-helices, 3% of b-sheets, 32% of turns and23% of coils, respectively.395

The physicochemical properties of PrPSc and PrPC greatlydiffer. Spectroscopic measurements indicated that PrPSc containsabout 34–43% of b-sheet structure,395,396 significantly higher thanthat of PrPC.395 X-ray fiber diffraction of infectious prions revealedthe presence of cross-b diffraction patterns. Meridional diffractionat 4.8 Å specified the presence of b-strands, characteristic of astacked-sheet amyloid structure. Thus, b-enriched structure of PrPSc

results from misfolding and self-assembly of protein PrPC intoproteinase K-resistant amyloid-like aggregates. However, the high-resolution structures of infectious prions are not yet solved, asconventional structural methods have been hindered by the largeand insoluble aggregates of PrP.

Several structural models of PrPSc self-assembly have beenproposed based on information derived from biophysical tech-niques. Parallel left-handed b-helical structure is the modelproposed by Cohen and colleagues and based on electronmicroscopy analysis of 2D crystals368 (Fig. 17a). The authorsconstructed a trimeric model of PrP 27–30 from a study of 119all-b folds globular proteins. PrP 27–30 is a protease-resistant27–30 kDa core of PrPSc (Fig. 16), and it retains prioninfectivity.353,397 According to the b-helical model the N-terminal residues of PrP 27–30 form left-handed b-helices thatare horizontally stacked, whereas the C-terminus maintains a-helical secondary structure as in native PrPC. Larger aggregatesare formed by vertically stacking of PrP trimers along the b-helical axis (Fig. 17a).

Based on MD simulations of PrP 27–30 conformationalfluctuations under amyloidogenic conditions, DeMarco andDaggett proposed the b-spiral model398 (Fig. 17b). Similarly tothe b-helical model the C-terminal a-helical characteristics of

PrPC remain unchanged and natively unfolded N-terminusadopt a b-structure. The core structure is comprised of threeshort b-strands spanning 116–119, 129–132 and 160–164residues.

Surewicz and colleagues proposed the in-register b-sheetmodel of PrPSc using site-directed spin labelling and EPRspectroscopy399 (Fig. 17c). In contrast to the other models, theyobserved that the refolding of PrPC involved major refolding ofthe C-terminal a-helical region. According to this model PrPSc

structure possesses no a-helices, consisting mainly of singlemolecules stacked on top of one another with parallel, in-register b-strands. Using MD simulations, Caughey and collea-gues suggested that linear PrPSc fibrils possessed a parallel in-register b-sheet structure400 (Fig. 18e).

In addition, a number of structures have been proposed formammalian401 and fungal402 prion protein segments. It isdifficult to determine which of the proposed models is theclosest to the PrPSc structure, as they were established based onlow-resolution experimental data. The diversity of the modelscould originate from the specimens used. For example, Willeand colleagues compared natural brain-delivered PrPSc andsynthetic bacteria-expressed recombinant PrP (with the samesequence) amyloid structure and found substantial differencesin structure, heterogeneity and infectivity.403 In addition, theexistence of PrP tertiary structural diversity and prion strainshave been experimentally proven,404–408 including the for-mation of new strains during the passage of prions throughanimals with different PrP sequences.409,410 For instance, mul-tiple scrapie prion strains were isolated with different incuba-tion times and neuropathology.411 However, these prion strainswere encoded by the same PrP primary structure and werepropagated in mice with the same PrP gene. Despite this,limited proteolysis generated different PrPSc fragments, sug-gesting that these prion strains possessed differentconformations.352

5.3 Mesoscopic structures of prions

The morphology of prions has been examined with TEM412–414

and AFM.414 Usually PrPSc isolated from brain appears as largeamorphous highly insoluble aggregates (Fig. 18a). Individualprion fibrils, termed prion rods (Fig. 18b), are not always visibleprobably because of heavy surface glycosylation415 (Fig. 18c).Each PrP monomer has up to two large sugar moieties linked tothe N-terminus to obscure the fibril core. Deficiencies inglucans and GPI archorless PrP have been found suitable foranalyzing the structural features of prion protofilaments(Fig. 18d), while neither glycosylation416,417 nor the GPIanchor348,418 is required for the infectivity of PrPSc.

Majority of PrP fibrils, either wild-type, anchorless, or ofdifferent strains, possess a twisted morphology. Prion fibrilscan be either left-handed or right-handed,414 consisting of twoor more protofilaments.412 However, some fibrils also containstraight, parallel protofilaments.414 In addition, fibrils occa-sionally resemble celery stalks or half-pipes414 (Fig. 18c). In arecent study the gap previously thought to be the spacingbetween two protofilaments of celery stalk fibrils was assigned

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Fig. 18 Electron microscopy of prion fibrils. (a) Aggregates of wild-type 22L scrapie prion aggregates.414 Reproduced with permission from ref. 414,copyright 2009 Elsevier. (b) Prion rods of PrP 27–30.341 Reproduced with permission from ref. 341, copyright 1998 National Academy of Sciences. (c) Wildtype RML scrapie prion structure obscured by non-fibrillar material, while (d) anchorless RML fibril morphology was much cleaner.414 Reproduced withpermission from ref. 414, copyright 2009 Elsevier. (e) Celery stalk-like brain-derived GPI-anchorless 22L fibril414 and proposed parallel in-register b-sheetmodel of PrP (90–231) octametric segment.400 Reproduced with permission from ref. 414, copyright 2009 Elsevier. Reproduced with permission fromref. 400, copyright 2014 American Society for Biochemistry and Molecular Biology. Scale bars: 100 nm.

Fig. 17 Structural models for the PrPSc aggregates: (a) in the b-helical model the N-terminal region (90–177 residues, light green) of PrP 27–30 refoldsinto a b-helix motif and the C-terminal region (residues 178–230, dark green) maintains a-helical secondary structure as in native PrPC.368 Reproducedwith permission from ref. 368, copyright 2004 National Academy of Sciences. (b) The b-spiral model consists of a spiralling core of extended sheetsconsisting of short b-strands, comprising residues 116–119, 129–132 and 160–164. The three a-helices in C-terminus maintain this conformationalmotif.398 Reproduced with permission from ref. 398, copyright 2004 National Academy of Sciences. (c) The parallel in-register extended b-sheet modelof PrPSc, where PrPC refolds into a structure consisting mainly of b-sheets.399 Reproduced with permission from ref. 399, copyright 2007 NationalAcademy of Sciences.



to represent the trough between the major hairpins. Accord-ingly, an in-register b-sheet PrP amyloid model wasproposed.400 The periodicity of PrP fibrils, on the other hand,ranged between 40 nm to 133 nm412,414,419 while the width ofeach PrP protofilament varied from 3.1 $ 0.7 nm414 to 6.9nm.368

5.4 Transmission of prions

Epidemiological transmission of PrP diseases is via the expo-sure of PrPC to PrPSc. However, point mutations in the PRNPgene at K200E, D178N, L102P and V117A codons were observedin families initially diagnosed with vCJD and GSS.420–422 Lineartransmission in human has an early history in the fore peopleof Papua New Guinea who suffered from Kuru: a human variantof PrP disease with clinical symptoms of ataxia, shivering anddeath within year of manifestation. Although the endemic isceased by terminating the ritual cannibalism in 1950s, Kuru iswaning gradually due to long sub-clinical incubation period i.e.450 years.423 In modern days, the inter-human transmission isvia blood transfusions as person infected with vCJD carry PrPSc

load in all blood components with transmission efficiency ofWBCs 4 platelets 4 RBCs 4 plasma and shed PrPSc in saliva,urine and other bodily fluids.424,425 Less common ways oftransmission in humans are surgical instruments and humanderived growth hormones.426

The clinical symptoms of PrP diseases originate from patho-logical changes in CNS such as vacuolization, astrogilosis andneuronal apoptosis. However, once prion replication in CNSreaches its peak, the PrPSc is disseminated centrifugally to theperipheral secretory organs and lymphoid tissues. PrPSc

excretes are detected in blood, urine, saliva, milk and bone–meat meal (MBM) of infected animal even at sub-clinical stage.The titre from urine and saliva of CWD infected cervid was ableto reproduce infectivity in naıve cervid and transgenic micemodels.427–429 Salivary expression can contaminate drinkingwater and pose a risk of transmission to human and otheranimals.430 Scrapie infected sheep shed PrPSc in all compo-nents of colostrum and milk i.e. cells, cream and casein/wheyproteins, which carried infectivity to lambs and dairyproducts.431,432 The titre of infectivity per mL of milk wasequivalent to 6 mg of brain homogenate from terminallyscrapie-infected sheep.433 Bone and meat materials, eitherdecaying in the soil or processed into MBM for cattle feeds,had PrPSc attached to its particles. PrPSc attached to MBM orsoil particles had higher transmission efficiency.434 Infectivitywas retained and transmissible to animals even after proces-sing of MBM for biodiesel productions.435 Once attached to thesoil particles, PrPSc not detachable via surfactants and soilcould retain infectivity up to 19 years.436–438 However,hyperthermophilic bacteria were able to digest the PrPSc parti-cles from soil by secreting keratins and b-sheets proteases.439

Rasmussen et al. first showed that hamster PrPSc were ableto bind with wheat grass roots, from soil and brain homoge-nate, but neither absorbed in roots nor detected in areal partsof the plants.440 Pritzkow et al. used protein misfolded cyclicamplification (PMCA) as a more sensitive detection method

and transmitted hamster 263K PrPSc to wheat grass roots viainfected brain homogenate, excreta, contaminated soils anddirect spray of PrP on areal parts.441 The 263K PrPSc were able toadsorb from the sources to the roots and travelled in the arealparts, which were further able to reproduce the infectivity innaıve hamster. Apart from the extraneous PrPSc in plants, aprotein named luminidependens from Arabidopsis thaliana wastransformed and propagated like PrP when injected in yeastcells.442

5.5 Conversion and replication of prions

The molecular interaction between PrPSc and PrPC is based onself-assembly driven by hydrogen bonding and p-stacking of thetyrosine residues. Two initial models described the mechanismof PrP replication.443 The ‘‘template directed’’ model describedPrPSc as the more stable but thermodynamically inaccessibleform of PrPC. In contrast, the ‘‘seeded nucleation’’ modeldescribed the contact of small oligomers of PrPSc with PrPC:the seeds of PrPSc recruit PrPC into conformationally changedform, and the growing fibril is broken down into various smallseeds acting as nuclei for further recruitment. The PrPSc

monomers are less stable but become stabilized when joinedin the seeded oligomer form.444,445 The seeded nucleationmodel was supported by later experiments where smallamounts of preformed PrPSc oligomers converted large quan-tities of PrPC as in PMCA, where seeds shredding was inducedby sonication and the conversion process was amplified.446

Makarava et al. refined the conversion phenomenon by study-ing the conformation switch (R and S) within single, mouse-hamster cross-seeded PrP amyloids and introduced the con-cepts of catalytic versus templated conversion and amyloidflexibility.447 Hamster PrP (S conformation), when incubatedwith mouse PrP monomers, catalyzed the conversion by accel-erating the fibrillation rate and shortening the lag phase, butthe newly formed daughter fibrils retained R conformationonly. In contrast, when hamster PrP seeds were introduced tohamster PrP monomers, it accelerated fibrillation and tem-plated the same S conformation in daughter fibrils. Molecularevents occurring in template-directed PrP conversion startedfrom p–p interaction of PrPC and PrPSc in 6 different bindingand conversion domains (BCD) of PrP. In the absence of PrPSc,when human and hamster PrPC BCD were probed with mono-clonal antibody (mAb), it resulted in structural denaturation ofPrPC, regional loss of tertiary structure, dissociation of b-sheets,and exposure of bityrosine regions (YYR) at a1 and a2 helices.The exposure of YYR regions was confirmed with binding ofanti-YYR mAb in these lose regions.448 In contrast to mAb, PrPSc

binding induced melting and exposure of YYR regions from b2–a2, a2–a3 and a1 regions.448–450 The exposed YYR could be thesite of further PrPC attachment and connected the oligomerand monomer.450 The loose structure induced by mAb was notable to acquire any conformation or secondary structure frommAb. However, when PrPSc oligomers induced this structuralloosening, it acquired b-sheets from oligomer’s hydrogenbonded backbone and stabilized the whole fibril column.447

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5.6 Co-factors in prion assembly

Co-factors, initially recognized as ‘‘Protein X’’ by Prusiner,stabilize the PrPC–PrPSc assembly and may further facilitatethe spontaneous conversion of PrPC into protease resistantform.451 Biomacromolecules of polysaccharides, sphingolipids,phospholipids, cholesterol, detergents like SDS, lipopolysac-charides from bacterial membranes, and polyanions like RNAhave been found to interact with PrPC and are co-localized withPrPSc from infected animals.452–454 The interaction with co-factors melted the secondary structure of PrPC and converted itinto protease resistant but non-infectious b-sheet structure,differently from the b-sheets of PrPSc, even though PrPSc andco-factors exposed YYR from the same regions of PrPC.449,450

5.7 Transmission barriers: sequence and conformation

A transmission barrier appears when there is no clinicalneuropathology of spongiform encephalopathies upon inocula-tion of PrPSc from infected species A into the naıve species B.The molecular etiology for the transmission barrier is attribu-ted to (i) difference in the sequences of host and donor PrP, (ii)conformational misfit during assembly, and (iii) post conver-sion maturation in host. The exact residual regions responsiblefor the transmission barrier are (i) 165–175 (b2–a2) withswitches at 170 (S/N), 174 (N/T), 169 (Y/G), (ii) 138–143 (b1–a1)

with switches at 139 (M/I) and (iii) 129 (b1) for M/V switch(Fig. 19).455–458 Sheep’s scrapie and cattle’s BSE are inter-transmissible with clinical symptoms as both are 170S homo-zygous. However, transmission between mice (170S) and ham-ster (170N) does not produce clinical disease.459,460 Sigurdsonet al. demonstrated the 170 S/N and 174 N/T switches at themolecular level.458 Inoculation of deer scrapie PrPSc (170N,174N) into wild type tg20 mice (170S, 174N) didn’t expressclinical symptoms at first passage but inoculation in rigid looptg1020 mice (S170N, N174T) produced terminal symptoms in74 days. Furthermore, hamster PrPSc (170N, 174T) acceleratedclinical disease in tg1020 mice (S170N, N174T) but not in tg20mice (170S, 174N). In contrast, cattle and sheep PrPSc (170S,174N) produced disease in tg20 mice (170S, 174N) but not intg1020 mice (S170N, N174T).458 Similarly, 139I in humans andmice PrPSc induced parallel b-sheet stacking and R conforma-tion in fibril while 139M in hamster induced anti-parallel b-sheet stacking and S conformation.447,456 The region 138–143has been demonstrated as a steric zipper in stabilizing PrPSc

fibril by hydrogen bonding between inter-monomeric b-sheets.Incompatibility at the steric zipper also erects a cross-speciesbarrier.461 The tyrosine residue at 169 is responsible for initialp–p interaction between PrPSc oligomers and host PrPC mono-mers and is conserved in all mammalian PrP. Eliminatingtyrosine or replacing it with glycine completely blocked PrPSc

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Fig. 19 Amino acid sequence and 3D structural comparison of b-sheet stacking from steric zones of PrPSc in different mammalian species.Superimposition of mouse (grey) and hamster (blue) PrPSc with 165–172 backbone fold (a). Amino acid sequence from 170–175 backbone region(b).463 Reproduced with permission from ref. 463, copyright 1999 American Chemical Society. Cyan highlights human while orange highlights elk specificresidues. Stick representation of steric zipper interfacing b-sheet back bone region for human (c) and both alignments of elk (d and e). X-raycrystallographic atomic structures from barrier determining steric zippers from human, mouse and hamster, side view for single b-sheet stacking (f, g andh) and top view of steric zipper (i, j and k).456 Reproduced with permission from ref. 456, copyright 2011 American Chemical Society. Sequencedifferences at molecular switches, defining the conformational and transmission barrier between different species (l).456,463 Reproduced with permissionfrom ref. 456, copyright 2011 American Chemical Society. Reproduced with permission from ref. 463, copyright 1999 American Chemical Society. Greyand red indicate transmission and barrier while cyan at 139 presents molecular switch for parallel or anti-parallel sheet stacking in human, mouse andhamster.



interaction with PrPC and halted the conversion.457 Anothermolecular switch between human TSE and mad cow BSE is at129 residue (M/V). vCJD infected human possessed 129Mhomologues with bovine 129M. However, 129V PrP peptidewas still converted to the PrPSc form in vitro, but at a lowefficiency. Hence the possibility of bovine 129M infecting 129Vheterozygous human cannot be excluded.455,462

5.8 PrP evolution and conformational adaptation: sub-clinicalstages

Cross-species transmission of PrPSc infection produced noclinical symptoms in first passage due to dissimilar residualsequence and thus conformation as discussed above. However,the clinically silent phase led to the understanding of (i) longsub-clinical stage, with no disease symptoms but undergoingPrP replication in brain, spleen and lymphoid tissues and (ii)concepts of PrPSc maturation, stability, selection andevolution.464,465 First passage of cattle BSE to human PrPSc

transgenic mice produced only a 0.6% attack rate after 739 daysbut a 75% attack rate after 639 days on second passage.466 Thedecrease in incubation time and increase in attack rate werealso observed by passaging the inoculum in vitro with PCMArounds, which led to the emergence of mutated and phenoty-pically distinct PrP strains.467–469 Kimberlin et al. inoculated139A PrPSc from mouse to hamster and then back to mouseresulting in 139H/M strain.470 Colby et al. infected mice withrPrP of different conformational stability and resulted in phe-notypically different strains with different physical morpholo-gies, shorter incubation time, higher attack rate and varyingclinical pathologies.471 It was hypothesized that the originalinoculum consisted of different rPrP strains and upon infectinginto the host, the strain having close conformational fit withhost PrP replicated at a faster rate, induced the clinical diseaseand subsequently appeared in animal tissues.471 Similarly,Bruce et al. raised 22C natural PRNPa strain in PRNPb miceand ended up with 22H strain. However, upon passaging therecombinant and pure 22C strain, only 22C was resulted in thehost indicating the presence of both 22C and 22H in naturalinoculum but 22H with shorted incubation time and close fitwith host PrP was able to replicate at a faster rate.472 Makaravaet al. annealed hamster rPrPSc with normal hamster brainhomogenate and inoculated in naıve hamster. On first passage,50% attack was observed but all hamster succumbed to diseaseon second passage and clinical symptoms were different fromoriginal rPrPSc which was used to prepare the inoculum.464 Theexplanation is that (i) original inoculum lacked the GPI anchorand failed to penetrate the cell to initiate neurotoxicity, (ii)conformational adaptation and stability were observed uponserial PCMA and (iii) rPrPSc-NBH failed to acquire co-factors,which it acquired on subsequent passages in the host.464 Serialpassages of donor PrPSc with host PrPC by PCMA in vivo orin vitro also changed the biochemical parameters like electro-phoretic mobility, protease digestion and degree ofglycosylation.473 The efficiency and properties of evolutionaryadaptation was also found to be tissue dependant, i.e.,

occurring at a faster rate in spleen than brain and cell andbrain adapting different strains.465,474

5.9 Toxicity of prions and mitigation

Two and a half decades on, the mechanism of prion toxicity hasbeen narrowed down to the distortion of neuronal cell mem-branes due to the assembly of PrPSc oligomers with GPIanchored PrPC.475,476 Deletion of GPI anchored PrPC from themembrane or expression of anchorless PrPC in transgenic miceresulted a minimum infectivity or reversal of clinical symptomsin infected mice, which implicated anchored PrPC forneurotoxicity.348,477 However, the extracellular accumulationof PrPSc continued as plaques as in terminally-ill wild-typemice.78,79 Apart from PrPSc, when anchorless PrPC was exposedto lipid membrane or expressed in transgenic mice theyadhered to the membranes, underwent conformationalchanges into the protease resistant form, oligomerized locally,and caused membrane disruption and ion channelformation.478–480 Although PrPC has a neuroprotective roleagainst cellular stress, it also intervenes toxic signals to neuro-nal cell and initiates an apoptotic cascade when probed byPrPSc, b-sheet conformers, yeast prions, Ab or other amyloidoligomers and even anti-PrPC antibodies.481–483 The mecha-nism is postulated as either through blocking the physiologicalbinding domains of PrPC or disruption of neuronal membranesby PrPC–PrPSc oligomer adducts.484,485 The adduct formed onthe membrane can be internalized and disrupt endosomaltrafficking or distort the local fluidity, structure and functionof lipid bilayers like channel formation in GSS.476,486 In addi-tion to adduct formation, PrPSc oligomers can independentlyinteract with membranes via their own GPI anchors, which theytend to develop during sequel passages.487 The oligomer formof PrPSc has been shown to be the toxic species, other than themonomers or amyloids.480 PrPSc oligomers possess the neces-sary hydrogen bonding backbone running up and own in thecolumn to induce conformational change in PrPC and recruitthe latter at the growing end.447 PrPSc oligomers corrupt PrPC

function and deliver a neurotoxic signal.476

Initial therapeutic strategies considered silencing of thePrPC gene. Silencing was well tolerated in animals apart fromminor disturbance in sleep cycle and electrophysiology ofhippocampus.500 However, as the PrPC is associated withneuroprotection excitotoxicity outcomes are being predictedfor silencing PrPC in human.501 RNA is physiologically foundco-localized with PrPSc which triggered research for RNA as ananti PrP drug.452 RNA based aptamers can bind PrPC to preventPrPSc-induced conversion or with PrPSc oligomers to block theiractivity.502 The subsequent interaction of PrPC with the sameaptamer increases the binding efficiency due to the adaptationflexibility of PrPC.503 Different ways of delivering aptamer RNAacross the blood brain barrier (BBB) include conjugation withtransferrin, cell penetrating peptides, NPs, liposomes anddendrimer.503 Polyethylene glycol-conjugated polycyanoacrylateNPs were able to penetrate the brain and spleen of scrapieinfected animals, however their ability to delivery therapeuticcargo and mode of interaction with PrPSc fibrils are

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questioned.504 Branched polyamines degraded PrPSc amyloidsto undetectable levels and reversed PrPSc toxicity in neuroblas-toma cell culture (Fig. 20h).499 Pre-treatment of PrPSc amyloidswith polyamines rendered them susceptible to proteolyticdigestion. Tran et al. used polyallylamine (+) and polystyrene-sulfonate (") as two oppositely charged polyamines for layer-by-layer coating of gold NPs (AuNPs) (Fig. 20c). The AuNPstranslocated across the BBB, disrupted the PrPSc amyloids,and mitigated the toxicity in scrapie-infected cells. Nanomolarconcentrations of AuNPs, with poly(allylamine) as the outer-most layer, prolonged incubation time and delayed the diseaseonset in infected mice.497 Polyamine-based dendrimers coatedwith maltose or maltotriose stimulated PrP fibrillization atlower concentrations by breaking long fibrils into small seeds,but at higher concentrations blocked the fibrillization bystabilizing individual seeds (Fig. 20h).491

NP–PrP interactions have been explored for diagnostic andsensing applications. Monothiolation of RNA aptamers makes

them a good ligand to cap AuNPs or AgNPs. These NPs thenspecifically interact with PrPSc and sequester the latter on theirsurfaces. The binding of PrPSc or cell bound PrPC is sensed in aconcentration dependant manner via changes in the surfaceplasmon resonance or Raman signals of the AuNPs/nanorods(NRs) and aptamer ligated AgNPs (Fig. 20j).489,495 PrP bindingwith aptamer-conjugated NPs induces controlled aggregationwhich can be sensed via resonance light scattering of metal NPsaggregation (Fig. 20b).488 Henry et al. employed fluorescenceturn-on and turn-off sensing for PrP detection.498 The fluores-cence of fluorescein-GABA-QYQRES-COOH peptide bound toantibody-conjugated AuNPs (turned-off) was turned-on byreplacing the peptide with competitive binding of PrPSc withthe antibody (Fig. 20a). The fluorescence property of biotin–avidin or monoclonal antibody bound quantum dots (QDs) hasalso been explored for the detection of PrP in vitro and in vivo(Fig. 20g).490 Xiao et al. used the dual-aptamer technique byligating aptamer 1 on Fe magnetic NPs (MNPs) and aptamer 2

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Fig. 20 Prion diagnostics and therapeutics at the nano and medicinal chemistry fronts.488–499 Compilated from ref. 488–499. PrPSc can be sensed byturning on/off the fluorescence of fluorescein-AuNPs (a), free QDs (g) or QD-FeNP sandwiches (f), or by resonance light scattering (RLS) of lipoic acid-AuNP aggregates (b). Reproduced with permission from ref. 488, copyright 2014 American Chemical Society. Quantitative sensing can be performed byRaman spectroscopy of Au nanorods (j). Reproduced with permission from ref. 495, copyright 2011 National Academy of Sciences. PrPSc can be capturedby AuNPs with polyamines and sulphonates surface layers of (c) or by FeNPs with mercaptopropionic acid, aspartic acid (d) or RNA aptamer surface layers(e). Cell-bound PrPSc can be captured by aptamer–AgNP conjugates (i) while complete denaturation of PrPSc can be observed with 5G polyaminedendrimers (h). Reproduced with permission from ref. 499, copyright 2001 American Society for Microbiology.



on QDs. MNPs and QDs sandwiched PrPSc or PrPC in betweenand the technique was used to detect and isolate PrP byfluorescent QDs and magnetic NPs even from 0.1% infectedbrain homogenate (Fig. 20f).496 MNPs directly capped withaspartic acid or Au-mercaptopropionic acid were able to seques-ter PrP via carbodiimide coupling (Fig. 20d).505 Miller et al.engaged the aptamer-ligated MNPs to capture and clear PrPSc

from solution. The sequestered PrPSc on MNPs were able to actas seeds in PCMA and enabled the detection and amplificationof small quantities of PrPSc (Fig. 20e).506

On the medicinal chemistry front, amphotericin B,507,508

quinacrine, dimeric analogues of statins, pyrazolones and pyridylhydrozones are available drugs for prolonging the lifespan of PrPinfected animals.493,509 Tacrolimus and astemizole reduce the PrPexpression on cell membranes and inhibit PrPSc replication.510

Drug discovery for small organic molecules led to 2-aminothiazoles which cap PrPSc seeds and inhibit their replicationactivity.511 Lipoic acid, an endogenous anti-oxidant compoundsand when conjugated with acridine and quinolone, inhibited PrPSc

fibril formation.494 A structure–activity relationship study of thepyrazole derivative of carbazole led to the understanding that atricyclic aromatic ring with hydroxyl and amino groups inhibitedPrPSc fibrillization in PrPSc-infected neuronal cells.492

5.10 Prions versus other neurodegenerative disorders

Cross-seeding and mutual stimulation of amyloid fibrils haverevealed possible links between AD, PD and T2D.512,513 Mou-genot et al. injected the PrPSc from cattle BSE, human BSE andscrapie into mice over-expressing aS. The incubation time wasreduced significantly and mice died of cerebral spongiformpathologies of PrPSc without accumulating insoluble fibrils ofaS.514 AD and PD have different neuronal pathologies than PrPand their ability to transmit and infect like prions isinconclusive.515,516 More in vitro and in vivo cross seedingstudies are necessary to elucidate the mechanisms and relation-ships between these diseases of different origins.

6. SummaryA survey of the literature has revealed striking similarities in thecross-b motifs of amyloid fibrils held together by H-bonding,regardless of the sequence and origin of the proteins. However,a recent study reported a cross-a amyloid structure associatedwith PSMa3, a 22-residue functional amyloid peptide secretedby Staphylococcus aureus for inflammatory response stimula-tion, human cell lysis and biofilm formation, representing asurprising departure from the common amyloid structure.517

The suprastructure of amyloid fibrils – including that of all (S)Ab1–40 and hen egg lysozyme – has been shown as predomi-nantly left handed,518 originated from the inherent left-handedchirality of the (S) amino acids. However, right-handed amy-loids have been reported for truncated serum amyloid A (SAA)peptides (o12 residues), resulting from the occurrence of bhelices in SAA protofilaments prior to their assembly intofibrils.519 A recent study on serum albumin amyloids, has

revealed that handedness can be inverted from left to righthanded, upon lateral addition of protofilaments of amyloidfibrils of a lower hierarchical level.520

There is compelling evidence that amyloid proteins canspread from cell to cell and cross talk in vivo to either speedup or slow down aggregation of the host protein.291,519,521–523

Furthermore, aggregates of non-amyloidogenic proteins, suchas bovine PI3-SH3 domain and E. coli HypF domain, can serveas seeds to promote cytotoxicity in brain cells. This phenom-enon suggests a generic origin of protein misfolding diseasesresulting from the emergence of trace amounts of aggregates,either introduced intracellularly through misfolding or muta-tions or externally through cross seeding.285

The development of neurodegenerative disorders appears tobe correlated with aging, where misfolding of proteins downthe free energy landscape towards the amyloid state is likelyprevented by metal ions (such as Ca2+ in the ER), molecularchaperones, ubiquitination enzymes and proteasomes, whichkinetically trap the aggregating proteins off pathway.285,524

Compared with Ab or IAPP, the tremendous plasticity of aSand PrP may originate from their much longer chain lengthsand, therefore, greater populations of misfolded intermediates.

Although controversies remain, the observations that oligo-mers are more toxic than their fibrillar counterparts appearpervasive to amyloid proteins. In addition, oligomer-specificantibody developed for Ab also bound the oligomers of IAPP,lysozyme, prion106–126, human insulin, polyglutamine and aS,suggesting a common tertiary structure273 as well as a commonmechanism of pathogenesis beyond the individuality of theproteins and compositions of their molecular chaperones andcellular environments. As amyloid proteins fibrillate along thekinetic pathway, both their solubility and reactivity appear todecline, consequently impacting protein self-assembly andtheir engagement with environmental ligands, proteins, cellmembranes and organelles to elicit toxicity. While such condi-tions can be manipulated in vitro, such as through the regula-tion of temperature and pH or the introduction of metal ions,small molecules or engineered NPs, how to create in vivoconditions that prohibit trace amounts of aggregates fromactivating primary and/or secondary nucleation remains atremendous challenge. Despite the complexity of protein struc-ture, function and toxicity, as revealed by intensive researchspanning the past two decades and highlighted in this review,protein aggregation through self-assembly and interaction withcellular environments constitutes hallmarks of neuronal andpancreatic b-cell degeneration. Consequently, understandingand exploiting molecular assembly under physiological condi-tions could make inroads on the development of therapeuticsand diagnostics against amyloid diseases.

List of abbreviationsAb Amyloid-betaAD Alzheimer’s diseaseAFM Atomic force microscopy

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APP Amyloid precursor proteinaS Alpha-synucleinATR-FTIR Attenuated total reflection-Fourier-transform

infraredBBB Blood brain barrierBCD Binding and conversion domainsBSE Bovine spongiform encephalopathyCD Circular dichroism spectroscopyCJD Creutzfeldt–Jakob diseaseCNS Central nervous systemCWD Chronic wasting diseaseDLB Dementia with Lewy bodiesDMD Discrete molecular simulationsEGCG Epigallocatechin gallateEPR Electron paramagnetic resonanceER Endoplasmic reticulumFFI Fatal familial insomniaFSI Fatal sporadic insomniaFTIR Fourier transform infrared spectroscopyGPI anchor Glycosyl phosphatidylinositolGSS Gerstmann–Straussler–ScheinkerHAM Hierarchical assembly modelHD Hydrogen–deuterium exchangehuPrP Human PrPIAPP Islet amyloid polypeptideIDP Intrinsically disorder proteinIM-MS Ion mobility mass spectroscopymAb Monoclonal antibodyMBM Bone–meat mealMD Molecular dynamicsMNPs Fe magnetic NPsNAC Non-amyloid-beta componentNFTs Neurofibrillary tanglesNMR Nuclear magnetic resonanceNSF N-Ethylmaleimide-sensitive factorNPs NanoparticlesPD Parkinson’s diseasePDD Parkinson’s disease dementiaPHFs Paired helical filamentsPMCA Protein misfolded cyclic amplificationPrP Prion proteinPrPC Non-pathological PrPPrPSc Misfolded PrPROS Reactive oxygen speciesSAA Serum amyloid ASDS Sodium dodecyl sulfateSDSL Site directed spin labellingSNARE Soluble NSF attachment protein receptorssNMR Solid-state nuclear magnetic resonanceSUVs Small unilamellar vesiclesT2D Type 2 diabetesTEM Transmission electron microscopyThT Thioflavin T assayTSEs Transmissible spongiform encephalopathiesUPS Ubiquitin proteasome systemYYR Bityrosine regions

AcknowledgementsThe authors thank the support of ARC Project CE140100036(Davis), DP160100959 (Separovic), NSF CAREER CBET-1553945(Ding), NIH MIRA 1R35GM119691 (Ding) and Monash Instituteof Pharmaceutical Sciences (Ke). Davis is thankful for anAustralian Laureate Fellowship from the ARC. We apologizeto those whose works are not cited in this review due to thetremendous amount of the relevant literature.

References1 T. P. J. Knowles, M. Vendruscolo and C. M. Dobson, Nat.

Rev. Mol. Cell Biol., 2014, 15, 384–396.2 S. I. A. Cohen, S. Linse, L. M. Luheshi, E. Hellstrand,

D. A. White, L. Rajah, D. E. Otzen, M. Vendruscolo,C. M. Dobson and T. P. J. Knowles, Proc. Natl. Acad. Sci.U. S. A., 2013, 110, 9758–9763.

3 T. P. J. Knowles, C. A. Waudby, G. L. Devlin, S. I. A. Cohen,A. Aguzzi, M. Vendruscolo, E. M. Terentjev, M. E. Wellandand C. M. Dobson, Science, 2009, 326, 1533–1537.

4 J. I. Guijarro, M. Sunde, J. A. Jones, I. D. Campbell andC. M. Dobson, Proc. Natl. Acad. Sci. U. S. A., 1998, 95,4224–4228.

5 F. Chiti, P. Webster, N. Taddei, A. Clark, M. Stefani,G. Ramponi and C. M. Dobson, Proc. Natl. Acad. Sci. U. S.A., 1999, 96, 3590–3594.

6 B. Urbanc, L. Cruz, S. Yun, S. V. Buldyrev, G. Bitan,D. B. Teplow and H. E. Stanley, Proc. Natl. Acad. Sci. U. S.A., 2004, 101, 17345–17350.

7 S. Auer, F. Meersman, C. M. Dobson and M. Vendruscolo,PLoS Comput. Biol., 2008, 4, e1000222.

8 B. N. Ratha, A. Ghosh, J. R. Brender, N. Gayen, H. Ilyas,C. Neeraja, K. P. Das, A. K. Mandal and A. Bhunia, J. Biol.Chem., 2016, 291, 23545–23556.

9 I. W. Hamley, Angew. Chem., Int. Ed., 2007, 46, 8128–8147.10 I. W. Hamley, Chem. Rev., 2012, 112, 5147–5192.11 A. Alzheimer, R. A. Stelzmann, H. N. Schnitzlein and

F. R. Murtagh, Clin. Anat., 1995, 8, 429–431.12 G. G. Glenner and C. W. Wong, Biochem. Biophys. Res.

Commun., 1984, 120, 885–890.13 G. Blessed, B. E. Tomlinson and M. Roth, Br. J. Psychiatry,

1968, 114, 797–811.14 I. Grundke-Iqbal, K. Iqbal, Y. C. Tung, M. Quinlan,

H. M. Wisniewski and L. I. Binder, Proc. Natl. Acad. Sci.U. S. A., 1986, 83, 4913–4917.

15 R. Skrabana, J. Sevcik and M. Novak, Cell. Mol. Neurobiol.,2006, 26, 1085–1097.

16 J. Avila, N. Pallas, M. Bolos, C. L. Sayas and F. Hernandez,Expert Opin. Ther. Targets, 2016, 20, 653–661.

17 M. D. Mukrasch, S. Bibow, J. Korukottu, S. Jeganathan,J. Biernat, C. Griesinger, E. Mandelkow andM. Zweckstetter, PLoS Biol., 2009, 7, e34.

18 P. Ganguly, T. D. Do, L. Larini, N. E. LaPointe, A. J. Sercel,M. F. Shade, S. C. Feinstein, M. T. Bowers and J. E. Shea,J. Phys. Chem. B, 2015, 119, 4582–4593.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



19 W. Li and V. M. Lee, Biochemistry, 2006, 45, 15692–15701.20 E. Mandelkow, M. von Bergen, J. Biernat and

E. M. Mandelkow, Brain Pathol., 2007, 17, 83–90.21 M. von Bergen, P. Friedhoff, J. Biernat, J. Heberle,

E. M. Mandelkow and E. Mandelkow, Proc. Natl. Acad.Sci. U. S. A., 2000, 97, 5129–5134.

22 K. Minoura, T. M. Yao, K. Tomoo, M. Sumida, M. Sasaki,T. Taniguchi and T. Ishida, Eur. J. Biochem., 2004, 271,545–552.

23 J. Adamcik, A. Sanchez-Ferrer, N. Ait-Bouziad,N. P. Reynolds, H. A. Lashuel and R. Mezzenga, Angew.Chem., Int. Ed., 2016, 55, 618–622.

24 N. Sibille, I. Huvent, C. Fauquant, D. Verdegem, L. Amniai,A. Leroy, J. M. Wieruszeski, G. Lippens and I. Landrieu,Proteins, 2012, 80, 454–462.

25 R. Sandbrink, C. L. Masters and K. Beyreuther, Neurobiol.Dis., 1994, 1, 13–24.

26 R. E. Tanzi, A. I. McClatchey, E. D. Lamperti, L. Villa-Komaroff, J. F. Gusella and R. L. Neve, Nature, 1988, 331,528–530.

27 P. C. Burger and F. S. Vogel, Am. J. Pathol., 1973, 73, 457–476.28 D. J. Selkoe, Annu. Rev. Cell Biol., 1994, 10, 373–403.29 H. Zheng, M. Jiang, M. E. Trumbauer, D. J. Sirinathsinghji,

R. Hopkins, D. W. Smith, R. P. Heavens, G. R. Dawson,S. Boyce, M. W. Conner, K. A. Stevens, H. H. Slunt,S. S. Sisoda, H. Y. Chen and L. H. Van der Ploeg, Cell,1995, 81, 525–531.

30 A. R. White, R. Reyes, J. F. Mercer, J. Camakaris, H. Zheng,A. I. Bush, G. Multhaup, K. Beyreuther, C. L. Masters andR. Cappai, Brain Res., 1999, 842, 439–444.

31 M. O. Grimm, H. S. Grimm, A. J. Patzold, E. G. Zinser,R. Halonen, M. Duering, J. A. Tschape, B. De Strooper,U. Muller, J. Shen and T. Hartmann, Nat. Cell Biol., 2005, 7,1118–1123.

32 H. Meziane, J. C. Dodart, C. Mathis, S. Little, J. Clemens,S. M. Paul and A. Ungerer, Proc. Natl. Acad. Sci. U. S. A.,1998, 95, 12683–12688.

33 J. M. Roch, E. Masliah, A. C. Roch-Levecq, M. P. Sundsmo,D. A. Otero, I. Veinbergs and T. Saitoh, Proc. Natl. Acad. Sci.U. S. A., 1994, 91, 7450–7454.

34 A. Nikolaev, T. McLaughlin, D. D. O’Leary and M. Tessier-Lavigne, Nature, 2009, 457, 981–989.

35 S. Ring, S. W. Weyer, S. B. Kilian, E. Waldron,C. U. Pietrzik, M. A. Filippov, J. Herms, C. Buchholz,C. B. Eckman, M. Korte, D. P. Wolfer and U. C. Muller,J. Neurosci., 2007, 27, 7817–7826.

36 R. Vassar, B. D. Bennett, S. Babu-Khan, S. Kahn,E. A. Mendiaz, P. Denis, D. B. Teplow, S. Ross,P. Amarante, R. Loeloff, Y. Luo, S. Fisher, J. Fuller,S. Edenson, J. Lile, M. A. Jarosinski, A. L. Biere,E. Curran, T. Burgess, J. C. Louis, F. Collins, J. Treanor,G. Rogers and M. Citron, Science, 1999, 286, 735–741.

37 O. O. Olubiyi and B. Strodel, J. Phys. Chem. B, 2012, 116,3280–3291.

38 S. Caglayan, S. Takagi-Niidome, F. Liao, A. S. Carlo,V. Schmidt, T. Burgert, Y. Kitago, E. M. Fuchtbauer,

A. Fuchtbauer, D. M. Holtzman, J. Takagi andT. E. Willnow, Sci. Transl. Med., 2014, 6, 223ra220.

39 J. T. Huse, K. Liu, D. S. Pijak, D. Carlin, V. M. Lee andR. W. Doms, J. Biol. Chem., 2002, 277, 16278–16284.

40 K. Liu, I. Solano, D. Mann, C. Lemere, M. Mercken,J. Q. Trojanowski and V. M. Lee, Acta Neuropathol., 2006,112, 163–174.

41 R. Hosoda, T. C. Saido, L. Otvos, Jr., T. Arai, D. M. Mann,V. M. Lee, J. Q. Trojanowski and T. Iwatsubo,J. Neuropathol. Exp. Neurol., 1998, 57, 1089–1095.

42 P. Gomez-Ramos and M. Asuncion Moran, J. Alzheimer’sDis., 2007, 11, 53–59.

43 O. Wirths, G. Multhaup, C. Czech, V. Blanchard,S. Moussaoui, G. Tremp, L. Pradier, K. Beyreuther andT. A. Bayer, Neurosci. Lett., 2001, 306, 116–120.

44 J. R. Cirrito, K. A. Yamada, M. B. Finn, R. S. Sloviter,K. R. Bales, P. C. May, D. D. Schoepp, S. M. Paul,S. Mennerick and D. M. Holtzman, Neuron, 2005, 48,913–922.

45 F. Kamenetz, T. Tomita, H. Hsieh, G. Seabrook,D. Borchelt, T. Iwatsubo, S. Sisodia and R. Malinow, Neu-ron, 2003, 37, 925–937.

46 J. E. Morley, S. A. Farr, W. A. Banks, S. N. Johnson,K. A. Yamada and L. Xu, J. Alzheimer’s Dis., 2010, 19,441–449.

47 J. Ghiso and B. Frangione, Adv. Drug Delivery Rev., 2002, 54,1539–1551.

48 D. Strozyk, K. Blennow, L. R. White and L. J. Launer,Neurology, 2003, 60, 652–656.

49 D. Scheuner, C. Eckman, M. Jensen, X. Song, M. Citron,N. Suzuki, T. D. Bird, J. Hardy, M. Hutton, W. Kukull,E. Larson, E. Levy-Lahad, M. Viitanen, E. Peskind,P. Poorkaj, G. Schellenberg, R. Tanzi, W. Wasco,L. Lannfelt, D. Selkoe and S. Younkin, Nat. Med., 1996, 2,864–870.

50 I. V. Kurochkin and S. Goto, FEBS Lett., 1994, 345, 33–37.51 N. Iwata, S. Tsubuki, Y. Takaki, K. Watanabe, M. Sekiguchi,

E. Hosoki, M. Kawashima-Morishima, H. J. Lee, E. Hama,Y. Sekine-Aizawa and T. C. Saido, Nat. Med., 2000, 6,143–150.

52 S. Mueller-Steiner, Y. Zhou, H. Arai, E. D. Roberson, B. Sun,J. Chen, X. Wang, G. Yu, L. Esposito, L. Mucke and L. Gan,Neuron, 2006, 51, 703–714.

53 T. L. Tekirian, T. C. Saido, W. R. Markesbery, M. J. Russell,D. R. Wekstein, E. Patel and J. W. Geddes, J. Neuropathol.Exp. Neurol., 1998, 57, 76–94.

54 N. Rezaei-Ghaleh, M. Amininasab, S. Kumar, J. Walter andM. Zweckstetter, Nat. Commun., 2016, 7, 11359.

55 Y. K. Al-Hilaly, T. L. Williams, M. Stewart-Parker, L. Ford,E. Skaria, M. Cole, W. G. Bucher, K. L. Morris, A. A. Sada,J. R. Thorpe and L. C. Serpell, Acta Neuropathol. Commun.,2013, 1, 83.

56 J. Naslund, A. Schierhorn, U. Hellman, L. Lannfelt,A. D. Roses, L. O. Tjernberg, J. Silberring, S. E. Gandy,B. Winblad and P. Greengard, et al., Proc. Natl. Acad. Sci. U.S. A., 1994, 91, 8378–8382.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



57 M. Ahmed, J. Davis, D. Aucoin, T. Sato, S. Ahuja, S. Aimoto,J. I. Elliott, W. E. Van Nostrand and S. O. Smith, Nat. Struct.Mol. Biol., 2010, 17, 561–567.

58 D. A. Kirschner, H. Inouye, L. K. Duffy, A. Sinclair, M. Lindand D. J. Selkoe, Proc. Natl. Acad. Sci. U. S. A., 1987, 84,6953–6957.

59 C. Soto, M. C. Branes, J. Alvarez and N. C. Inestrosa,J. Neurochem., 1994, 63, 1191–1198.

60 M. A. Sani, J. D. Gehman and F. Separovic, FEBS Lett., 2011,585, 749–754.

61 L. C. Serpell, Biochim. Biophys. Acta, 2000, 1502, 16–30.62 T. Shirahama and A. S. Cohen, J. Cell Biol., 1967, 33,

679–708.63 S. Tomaselli, V. Esposito, P. Vangone, N. A. van Nuland,

A. M. Bonvin, R. Guerrini, T. Tancredi, P. A. Temussi andD. Picone, ChemBioChem, 2006, 7, 257–267.

64 C. J. Barrow and M. G. Zagorski, Science, 1991, 253,179–182.

65 O. Crescenzi, S. Tomaselli, R. Guerrini, S. Salvadori,A. M. D’Ursi, P. A. Temussi and D. Picone, Eur.J. Biochem., 2002, 269, 5642–5648.

66 S. Vivekanandan, J. R. Brender, S. Y. Lee andA. Ramamoorthy, Biochem. Biophys. Res. Commun., 2011,411, 312–316.

67 M. Coles, W. Bicknell, A. A. Watson, D. P. Fairlie andD. J. Craik, Biochemistry, 1998, 37, 11064–11077.

68 H. Shao, S. Jao, K. Ma and M. G. Zagorski, J. Mol. Biol.,1999, 285, 755–773.

69 S. J. Wood, B. Maleeff, T. Hart and R. Wetzel, J. Mol. Biol.,1996, 256, 870–877.

70 S. Narayanan and B. Reif, Biochemistry, 2005, 44,1444–1452.

71 J. Danielsson, J. Jarvet, P. Damberg and A. Graslund, FEBSJ., 2005, 272, 3938–3949.

72 Y. Fezoui and D. B. Teplow, J. Biol. Chem., 2002, 277,36948–36954.

73 A. Abedini and D. P. Raleigh, Phys. Biol., 2009, 6, 015005.74 R. Tycko, Q. Rev. Biophys., 2006, 39, 1–55.75 S. Kumar, N. Rezaei-Ghaleh, D. Terwel, D. R. Thal,

M. Richard, M. Hoch, J. M. Mc Donald, U. Wullner,K. Glebov, M. T. Heneka, D. M. Walsh, M. Zweckstetterand J. Walter, EMBO J., 2011, 30, 2255–2265.

76 W. He and C. J. Barrow, Biochemistry, 1999, 38,10871–10877.

77 C. D’Arrigo, M. Tabaton and A. Perico, Biopolymers, 2009,91, 861–873.

78 P. Hortschansky, V. Schroeckh, T. Christopeit,G. Zandomeneghi and M. Fandrich, Protein Sci., 2005, 14,1753–1759.

79 G. Meisl, X. Yang, E. Hellstrand, B. Frohm, J. B. Kirkegaard,S. I. Cohen, C. M. Dobson, S. Linse and T. P. Knowles, Proc.Natl. Acad. Sci. U. S. A., 2014, 111, 9384–9389.

80 P. Arosio, T. P. Knowles and S. Linse, Phys. Chem. Chem.Phys., 2015, 17, 7606–7618.

81 G. Meisl, X. Yang, B. Frohm, T. P. Knowles and S. Linse,Sci. Rep., 2016, 6, 18728.

82 J. Adamcik, C. Lara, I. Usov, J. S. Jeong, F. S. Ruggeri,G. Dietler, H. A. Lashuel, I. W. Hamley and R. Mezzenga,Nanoscale, 2012, 4, 4426–4429.

83 A. T. Petkova, Y. Ishii, J. J. Balbach, O. N. Antzutkin,R. D. Leapman, F. Delaglio and R. Tycko, Proc. Natl. Acad.Sci. U. S. A., 2002, 99, 16742–16747.

84 J. X. Lu, W. Qiang, W. M. Yau, C. D. Schwieters,S. C. Meredith and R. Tycko, Cell, 2013, 154, 1257–1268.

85 M. T. Colvin, R. Silvers, Q. Z. Ni, T. V. Can, I. Sergeyev,M. Rosay, K. J. Donovan, B. Michael, J. Wall, S. Linse andR. G. Griffin, J. Am. Chem. Soc., 2016, 138, 9663–9674.

86 Y. Xiao, B. Ma, D. McElheny, S. Parthasarathy, F. Long,M. Hoshi, R. Nussinov and Y. Ishii, Nat. Struct. Mol. Biol.,2015, 22, 499–505.

87 D. M. Walsh, I. Klyubin, J. V. Fadeeva, M. J. Rowan andD. J. Selkoe, Biochem. Soc. Trans., 2002, 30, 552–557.

88 M. Schrag, C. Mueller, U. Oyoyo, M. A. Smith andW. M. Kirsch, Prog. Neurobiol., 2011, 94, 296–306.

89 B. Hallgren and P. Sourander, J. Neurochem., 1960, 5,307–310.

90 M. Schrag, A. Dickson, A. Jiffry, D. Kirsch, H. V. Vinters andW. Kirsch, Biometals, 2010, 23, 1123–1127.

91 A. I. Bush, W. H. Pettingell, G. Multhaup, M. D. Paradis,J. P. Vonsattel, J. F. Gusella, K. Beyreuther, C. L. Mastersand R. E. Tanzi, Science, 1994, 265, 1464–1467.

92 W. R. Markesbery, Arch. Neurol., 1999, 56, 1449–1452.93 A. I. Bush, Curr. Opin. Chem. Biol., 2000, 4, 184–191.94 D. A. Butterfield and R. Sultana, J. Amino Acids, 2011,

2011, 198430.95 L. E. Cassagnes, V. Herve, F. Nepveu, C. Hureau, P. Faller

and F. Collin, Angew. Chem., Int. Ed., 2013, 52, 11110–11113.96 S. Parthasarathy, B. Yoo, D. McElheny, W. Tay and Y. Ishii,

J. Biol. Chem., 2014, 289, 9998–10010.97 S. Keskitalo, M. Farkas, M. Hanenberg, A. Szodorai,

L. Kulic, A. Semmler, M. Weller, R. M. Nitsch andM. Linnebank, Front. Aging Neurosci., 2014, 6, 237.

98 D. G. Smith, R. Cappai and K. J. Barnham, Biochim.Biophys. Acta, 2007, 1768, 1976–1990.

99 G. P. Eckert, W. G. Wood and W. E. Muller, Curr. ProteinPept. Sci., 2010, 11, 319–325.

100 C. Aisenbrey, T. Borowik, R. Bystrom, M. Bokvist,F. Lindstrom, H. Misiak, M. A. Sani and G. Grobner, Eur.Biophys. J., 2008, 37, 247–255.

101 R. Bystrom, C. Aisenbrey, T. Borowik, M. Bokvist,F. Lindstrom, M. A. Sani, A. Olofsson and G. Grobner, CellBiochem. Biophys., 2008, 52, 175–189.

102 D. K. Weber, J. D. Gehman, F. Separovic and M. A. Sani,Aust. J. Chem., 2012, 65, 472–479.

103 D. Bhowmik, K. R. Mote, C. M. MacLaughlin, N. Biswas,B. Chandra, J. K. Basu, G. C. Walker, P. K. Madhu andS. Maiti, ACS Nano, 2015, 9, 9070–9077.

104 T. L. Lau, J. D. Gehman, J. D. Wade, C. L. Masters,K. J. Barnham and F. Separovic, Biochim. Biophys. Acta,2007, 1768, 3135–3144.

105 D. A. Hicks, N. N. Nalivaeva and A. J. Turner, Front.Physiol., 2012, 3, 189.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



106 A. J. Beel, C. K. Mobley, H. J. Kim, F. Tian,A. Hadziselimovic, B. Jap, J. H. Prestegard andC. R. Sanders, Biochemistry, 2008, 47, 9428–9446.

107 P. N. Lacor, M. C. Buniel, L. Chang, S. J. Fernandez,Y. Gong, K. L. Viola, M. P. Lambert, P. T. Velasco,E. H. Bigio, C. E. Finch, G. A. Krafft and W. L. Klein,J. Neurosci., 2004, 24, 10191–10200.

108 O. Garaschuk, R. Schneggenburger, C. Schirra, F. Tempiaand A. Konnerth, J. Physiol., 1996, 491(Pt 3), 757–772.

109 H. Y. Wang, D. H. Lee, M. R. D’Andrea, P. A. Peterson,R. P. Shank and A. B. Reitz, J. Biol. Chem., 2000, 275, 5626–5632.

110 E. M. Snyder, Y. Nong, C. G. Almeida, S. Paul, T. Moran, E. Y.Choi, A. C. Nairn, M. W. Salter, P. J. Lombroso, G. K. Gourasand P. Greengard, Nat. Neurosci., 2005, 8, 1051–1058.

111 C. A. Hansson Petersen, N. Alikhani, H. Behbahani,B. Wiehager, P. F. Pavlov, I. Alafuzoff, V. Leinonen,A. Ito, B. Winblad, E. Glaser and M. Ankarcrona, Proc.Natl. Acad. Sci. U. S. A., 2008, 105, 13145–13150.

112 M. Manczak, M. J. Calkins and P. H. Reddy, Hum. Mol.Genet., 2011, 20, 2495–2509.

113 H. Du, L. Guo, S. Yan, A. A. Sosunov, G. M. McKhann andS. S. Yan, Proc. Natl. Acad. Sci. U. S. A., 2010, 107,18670–18675.

114 M. D’Amelio, V. Cavallucci, S. Middei, C. Marchetti,S. Pacioni, A. Ferri, A. Diamantini, D. De Zio, P. Carrara,L. Battistini, S. Moreno, A. Bacci, M. Ammassari-Teule,H. Marie and F. Cecconi, Nat. Neurosci., 2011, 14, 69–76.

115 Z. Berger, B. Ravikumar, F. M. Menzies, L. G. Oroz,B. R. Underwood, M. N. Pangalos, I. Schmitt, U. Wullner,B. O. Evert, C. J. O’Kane and D. C. Rubinsztein, Hum. Mol.Genet., 2006, 15, 433–442.

116 G. Wen, W. Qin, D. Chen, Y. Wang, X. Yue, Z. Liu, Y. Cao,J. Du, B. Zhou and X. Bu, Chem. Commun., 2017, 53,3886–3889.

117 J. Sevigny, P. Chiao, T. Bussiere, P. H. Weinreb,L. Williams, M. Maier, R. Dunstan, S. Salloway, T. Chen,Y. Ling, J. O’Gorman, F. Qian, M. Arastu, M. Li, S. Chollate,M. S. Brennan, O. Quintero-Monzon, R. H. Scannevin,H. M. Arnold, T. Engber, K. Rhodes, J. Ferrero, Y. Hang,A. Mikulskis, J. Grimm, C. Hock, R. M. Nitsch andA. Sandrock, Nature, 2016, 537, 50–56.

118 R. Squitti, D. Lupoi, P. Pasqualetti, G. Dal Forno,F. Vernieri, P. Chiovenda, L. Rossi, M. Cortesi,E. Cassetta and P. M. Rossini, Neurology, 2002, 59,1153–1161.

119 J. Carroll, Science, 2017, DOI: 10.1126/science.aal0759Q4 .120 R. Vassar, P. H. Kuhn, C. Haass, M. E. Kennedy,

L. Rajendran, P. C. Wong and S. F. Lichtenthaler,J. Neurochem., 2014, 130, 4–28.

121 L. Rajendran, A. Schneider, G. Schlechtingen, S. Weidlich,J. Ries, T. Braxmeier, P. Schwille, J. B. Schulz, C. Schroeder,M. Simons, G. Jennings, H. J. Knolker and K. Simons,Science, 2008, 320, 520–523.

122 X. Wang, B. Su, S. L. Siedlak, P. I. Moreira, H. Fujioka,Y. Wang, G. Casadesus and X. Zhu, Proc. Natl. Acad. Sci. U.S. A., 2008, 105, 19318–19323.

123 W. Noble, C. Garwood, J. Stephenson, A. M. Kinsey,D. P. Hanger and B. H. Anderton, FASEB J., 2009, 23, 739–750.

124 J. L. Cummings, T. Morstorf and K. Zhong, Alzheimer’s Res.Ther., 2014, 6, 37.

125 H. Fu, M. Cui, L. Zhao, P. Tu, K. Zhou, J. Dai and B. Liu,J. Med. Chem., 2015, 58, 6972–6983.

126 M. L. Bolognesi, A. Gandini, F. Prati and E. Uliassi, J. Med.Chem., 2016, 59, 7759–7770.

127 P. Westermark, A. Andersson and G. T. Westermark, Phy-siol. Rev., 2011, 91, 795–826.

128 L. Marzban, G. Trigo-Gonzalez, X. Zhu, C. J. Rhodes,P. A. Halban, D. F. Steiner and C. B. Verchere, Diabetes,2004, 53, 141–148.

129 J. Wang, J. Xu, J. Finnerty, M. Furuta, D. F. Steiner andC. B. Verchere, Diabetes, 2001, 50, 534–539.

130 M. S. German, L. G. Moss, J. Wang and W. J. Rutter, Mol.Cell. Biol., 1992, 12, 1777–1788.

131 T. A. Lutz, Physiol. Behav., 2006, 89, 465–471.132 A. Young, Adv. Pharmacol., 2005, 52, 99–121.133 D. Naot and J. Cornish, Bone, 2008, 43, 813–818.134 S. Luca, W.-M. Yau, R. Leapman and R. Tycko, Biochem-

istry, 2007, 46, 13505–13522.135 J. J. W. Wiltzius, S. A. Sievers, M. R. Sawaya, D. Cascio,

D. Popov, C. Riekel and D. Eisenberg, Protein Sci., 2008, 17,1467–1474.

136 S. Bedrood, Y. Li, J. M. Isas, B. G. Hegde, U. Baxa,I. S. Haworth and R. Langen, J. Biol. Chem., 2012, 287,5235–5241.

137 S. M. Patil, S. Xu, S. R. Sheftic and A. T. Alexandrescu,J. Biol. Chem., 2009, 284, 11982–11991.

138 R. P. R. Nanga, J. R. Brender, S. Vivekanandan andA. Ramamoorthy, Biochim. Biophys. Acta, 2011, 1808,2337–2342.

139 D. C. R. Camargo, K. Tripsianes, K. Buday, A. Franko,C. Gobl, C. Hartlmuller, R. Sarkar, M. Aichler,G. Mettenleiter, M. Schulz, A. Boddrich, C. Erck,H. Martens, A. K. Walch, T. Madl, E. E. Wanker,M. Conrad, M. H. d. Angelis and B. Reif, Sci. Rep., 2017,7, 44041.

140 P. Nedumpully-Govindan and F. Ding, Sci. Rep., 2015,5, 8240.

141 O. Sumner Makin and L. C. Serpell, J. Mol. Biol., 2004, 335,1279–1288.

142 C. Goldsbury, J. Kistler, U. Aebi, T. Arvinte andG. J. S. Cooper, J. Mol. Biol., 1999, 285, 33–39.

143 C. S. Goldsbury, G. J. S. Cooper, K. N. Goldie, S. A. Muller,E. L. Saafi, W. T. M. Gruijters, M. P. Misur, A. Engel,U. Aebi and J. Kistler, J. Struct. Biol., 1997, 119, 17–27.

144 S. Chakraborty, B. Chatterjee and S. Basu, Biophys. Chem.,2012, 168–169, 1–9.

145 S. Sakagashira, H. J. Hiddinga, K. Tateishi, T. Sanke,T. Hanabusa, K. Nanjo and N. L. Eberhardt, Am.J. Pathol., 2000, 157, 2101–2109.

146 S. Seino and M. Study Group of Comprehensive Analysis ofGenetic Factors in Diabetes, Diabetologia, 2001, 44,906–909.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



147 P. Westermark, D. L. Eizirik, D. G. Pipeleers,C. Hellerstrom and A. Andersson, Diabetologia, 1995, 38,543–549.

148 J. Udayasankar, K. Kodama, R. L. Hull, S. Zraika, K. Aston-Mourney, S. L. Subramanian, J. Tong, M. V. Faulenbach,J. Vidal and S. E. Kahn, Diabetologia, 2009, 52, 145–153.

149 J. Janson, W. C. Soeller, P. C. Roche, R. T. Nelson,A. J. Torchia, D. K. Kreutter and P. C. Butler, Proc. Natl.Acad. Sci. U. S. A., 1996, 93, 7283–7288.

150 B. A. Yankner, L. R. Dawes, S. Fisher, L. Villa-Komaroff,M. L. Oster-Granite and R. L. Neve, Science, 1989, 245,417–420.

151 B. A. Yankner, L. K. Duffy and D. A. Kirschner, Science,1990, 250, 279–282.

152 A. Lorenzo, B. Razzaboni, G. C. Weir and B. A. Yankner,Nature, 1994, 368, 756–760.

153 A. Lorenzo and B. A. Yankner, Ann. N. Y. Acad. Sci., 1996,777, 89–95.

154 J. Janson, R. H. Ashley, D. Harrison, S. McIntyre andP. C. Butler, Diabetes, 1999, 48, 491–498.

155 R. A. Ritzel, J. J. Meier, C.-Y. Lin, J. D. Veldhuis andP. C. Butler, Diabetes, 2007, 56, 65–71.

156 W. P. S. Wong, D. W. Scott, C.-L. Chuang, S. Zhang, H. Liu,A. Ferreira, E. L. Saafi, Y. S. Choong and G. J. S. Cooper,Diabetes, 2008, 57, 2737–2744.

157 A. E. Butler, J. Janson, W. C. Soeller and P. C. Butler,Diabetes, 2003, 52, 2304–2314.

158 J. J. Meier, R. Kayed, C.-Y. Lin, T. Gurlo, L. Haataja,S. Jayasinghe, R. Langen, C. G. Glabe and P. C. Butler,Am. J. Physiol.: Endocrinol. Metab., 2006, 291, E1317–1324.

159 E. H. Pilkington, Y. Xing, B. Wang, A. Kakinen, M. Wang,T. D. Davis, F. Ding and P. C. Ke, Sci. Rep., 2017, DOI:10.1038/s41598-017-02597-0.

160 L. Haataja, T. Gurlo, C. J. Huang and P. C. Butler, Endocr.Rev., 2008, 29, 303–316.

161 S. Zraika, R. L. Hull, C. B. Verchere, A. Clark, K. J. Potter,P. E. Fraser, D. P. Raleigh and S. E. Kahn, Diabetologia,2010, 53, 1046–1056.

162 J. A. Williamson and A. D. Miranker, Protein Sci., 2007, 16,110–117.

163 J. A. Williamson, J. P. Loria and A. D. Miranker, J. Mol.Biol., 2009, 393, 383–396.

164 J. J. W. Wiltzius, S. A. Sievers, M. R. Sawaya andD. Eisenberg, Protein Sci., 2009, 18, 1521–1530.

165 P. Cao, A. Abedini and D. P. Raleigh, Curr. Opin. Struct.Biol., 2013, 23, 82–89.

166 N. F. Dupuis, C. Wu, J.-E. Shea and M. T. Bowers, J. Am.Chem. Soc., 2011, 133, 7240–7243.

167 Y. Okamoto, J. Mol. Graphics Modell., 2004, 22, 425–439.168 S. B. Padrick and A. D. Miranker, Biochemistry, 2002, 41,

4694–4703.169 J. C. Hutton, Diabetologia, 32, 271–281Q5 .170 A. S. DeToma, S. Salamekh, A. Ramamoorthy and

M. H. Lim, Chem. Soc. Rev., 2012, 41, 608–621.171 F. Chimienti, A. Favier and M. Seve, Biometals, 2005, 18,

313–317.

172 K. Lemaire, M. A. Ravier, A. Schraenen, J. W. M. Creemers,R. Van de Plas, M. Granvik, L. Van Lommel, E. Waelkens,F. Chimienti, G. A. Rutter, P. Gilon, P. A. in’t Veld andF. C. Schuit, Proc. Natl. Acad. Sci. U. S. A., 2009, 106,14872–14877.

173 R. L. Hull, G. T. Westermark, P. Westermark andS. E. Kahn, J. Clin. Endocrinol. Metab., 2004, 89, 3629–3643.

174 S. Jha, J. M. Snell, S. R. Sheftic, S. M. Patil, S. B. Daniels,F. W. Kolling and A. T. Alexandrescu, Biochemistry, 2014,53, 300–310.

175 L. Khemtemourian, E. Domenech, J. P. F. Doux,M. C. Koorengevel and J. A. Killian, J. Am. Chem. Soc.,2011, 133, 15598–15604.

176 A. Abedini and D. P. Raleigh, Biochemistry, 2005, 44,16284–16291.

177 P. Nedumpully-Govindan, D. B. Jemec and F. Ding,J. Chem. Inf. Model., 2015, 56(6), 1042–1052.

178 J. C. Hutton, Biochem. J., 1982, 204, 171–178.179 P. Nedumpully-Govindan, Y. Yang, R. Andorfer, W. Cao

and F. Ding, Biochemistry, 2015, 54, 7335–7344.180 J. L. Larson and A. D. Miranker, J. Mol. Biol., 2004, 335, 221–231.181 S. Gilead, H. Wolfenson and E. Gazit, Angew. Chem., Int.

Ed., 2006, 45, 6476–6480.182 A. C. Susa, C. Wu, S. L. Bernstein, N. F. Dupuis, H. Wang,

D. P. Raleigh, J.-E. Shea and M. T. Bowers, J. Am. Chem.Soc., 2014, 136, 12912–12919.

183 G. Dodson and D. Steiner, Curr. Opin. Struct. Biol., 1998, 8,189–194.

184 J. Flannick, G. Thorleifsson, N. L. Beer, S. B. R. Jacobs,N. Grarup, N. P. Burtt, A. Mahajan, C. Fuchsberger,G. Atzmon, R. Benediktsson, J. Blangero, D. W. Bowden,I. Brandslund, J. Brosnan, F. Burslem, J. Chambers,Y. S. Cho, C. Christensen, D. A. Douglas, R. Duggirala,Z. Dymek, Y. Farjoun, T. Fennell, P. Fontanillas, T. Forsen,S. Gabriel, B. Glaser, D. F. Gudbjartsson, C. Hanis, T. Hansen,A. B. Hreidarsson, K. Hveem, E. Ingelsson, B. Isomaa,S. Johansson, T. Jørgensen, M. E. Jørgensen, S. Kathiresan,A. Kong, J. Kooner, J. Kravic, M. Laakso, J.-Y. Lee, L. Lind,C. M. Lindgren, A. Linneberg, G. Masson, T. Meitinger,K. L. Mohlke, A. Molven, A. P. Morris, S. Potluri,R. Rauramaa, R. Ribel-Madsen, A.-M. Richard, T. Rolph,V. Salomaa, A. V. Segre, H. Skarstrand, V. Steinthorsdottir,H. M. Stringham, P. Sulem, E. S. Tai, Y. Y. Teo, T. Teslovich,U. Thorsteinsdottir, J. K. Trimmer, T. Tuomi, J. Tuomilehto,F. Vaziri-Sani, B. F. Voight, J. G. Wilson, M. Boehnke,M. I. McCarthy, P. R. Njølstad, O. Pedersen, T. D. C. Go, T.d.-G. Consortium, L. Groop, D. R. Cox, K. Stefansson andD. Altshuler, Nat. Genet., 2014, 46, 357–363.

185 P. Westermark, Z.-C. Li, G. T. Westermark, A. Leckstromand D. F. Steiner, FEBS Lett., 1996, 379, 203–206.

186 J. R. Brender, K. Hartman, R. P. R. Nanga, N. Popovych,R. de la Salud Bea, S. Vivekanandan, E. N. G. Marsh andA. Ramamoorthy, J. Am. Chem. Soc., 2010, 132, 8973–8983.

187 S. Salamekh, J. R. Brender, S.-J. Hyung, R. P. R. Nanga,S. Vivekanandan, B. T. Ruotolo and A. Ramamoorthy,J. Mol. Biol., 2011, 410, 294–306.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



188 C. E. Munte, L. Vilela, H. R. Kalbitzer and R. C. Garratt,FEBS J., 2005, 272, 4284–4293.

189 Z. Keltner, J. A. Meyer, E. M. Johnson, A. M. Palumbo,D. M. Spence and G. E. Reid, Analyst, 2010, 135, 278–288.

190 A. W. Giebink, P. A. Vogel, W. Medawala and D. M. Spence,Diabetes, 2013, 29, 44–52.

191 F. Meng, A. Abedini, B. Song and D. P. Raleigh, Biochem-istry, 2007, 46, 12091–12099.

192 J. R. Brender, S. Salamekh and A. Ramamoorthy, Acc.Chem. Res., 2012, 45, 454–462.

193 L. Khemtemourian, J. A. Killian, J. W. M. Hoppener andM. F. M. Engel, Exp. Diabetes Res., 2008, 2008, 421287.

194 J. A. Hebda and A. D. Miranker, Annu. Rev. Biophys., 2009,38, 125–152.

195 E. H. Pilkington, E. N. Gurzov, A. Kakinen, S. A. Litwak,W. J. Stanley, T. P. Davis and P. C. Ke, Sci. Rep., 2016, 6, 21274.

196 T. A. Mirzabekov, M. C. Lin and B. L. Kagan, J. Biol. Chem.,1996, 271, 1988–1992.

197 N. B. Last, E. Rhoades and A. D. Miranker, Proc. Natl. Acad.Sci. U. S. A., 2011, 108, 9460–9465.

198 M. Anguiano, R. J. Nowak and P. T. Lansbury, Biochemistry,2002, 41, 11338–11343.

199 Y. Hirakura, W. W. Yiu, A. Yamamoto and B. L. Kagan,Amyloid, 2000, 7, 194–199.

200 A. Quist, I. Doudevski, H. Lin, R. Azimova, D. Ng,B. Frangione, B. Kagan, J. Ghiso and R. Lal, Proc. Natl.Acad. Sci. U. S. A., 2005, 102, 10427–10432.

201 J. D. Green, L. Kreplak, C. Goldsbury, X. Li Blatter, M. Stolz,G. S. Cooper, A. Seelig, J. Kistler and U. Aebi, J. Mol. Biol.,2004, 342, 877–887.

202 M. F. M. Engel, L. Khemtemourian, C. C. Kleijer,H. J. D. Meeldijk, J. Jacobs, A. J. Verkleij, B. de Kruijff,J. A. Killian and J. W. M. Hoppener, Proc. Natl. Acad. Sci. U.S. A., 2008, 105, 6033–6038.

203 P. Cao, A. Abedini, H. Wang, L.-H. Tu, X. Zhang,A. M. Schmidt and D. P. Raleigh, Proc. Natl. Acad. Sci. U.S. A., 2013, 110, 19279–19284.

204 F. E. Cohen and J. W. Kelly, Nature, 2003, 426, 905–909.205 T. Klabunde, H. M. Petrassi, V. B. Oza, P. Raman,

J. W. Kelly and J. C. Sacchettini, Nat. Struct. Biol., 2000,7, 312–321.

206 H. Wang, M. L. Duennwald, B. E. Roberts, L. M. Rozeboom,Y. L. Zhang, A. D. Steele, R. Krishnan, L. J. Su, D. Griffin,S. Mukhopadhyay, E. J. Hennessy, P. Weigele,B. J. Blanchard, J. King, A. A. Deniz, S. L. Buchwald,V. M. Ingram, S. Lindquist and J. Shorter, Proc. Natl. Acad.Sci. U. S. A., 2008, 105, 7159–7164.

207 F. Meng, A. Abedini, A. Plesner, C. T. Middleton,K. J. Potter, M. T. Zanni, C. B. Verchere andD. P. Raleigh, J. Mol. Biol., 2010, 400, 555–566.

208 A. Abedini, F. Meng and D. P. Raleigh, J. Am. Chem. Soc.,2007, 129, 11300–11301.

209 D. Sellin, L.-M. Yan, A. Kapurniotu and R. Winter, Biophys.Chem., 2010, 150, 73–79.

210 L.-M. Yan, A. Velkova, M. Tatarek-Nossol, E. Andreetto andA. Kapurniotu, Angew. Chem., Int. Ed., 2007, 46, 1246–1252.

211 L.-M. Yan, M. Tatarek-Nossol, A. Velkova, A. Kazantzis andA. Kapurniotu, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,2046–2051.

212 I. Saraogi, J. A. Hebda, J. Becerril, L. A. Estroff,A. D. Miranker and A. D. Hamilton, Angew. Chem., Int.Ed., 2010, 49, 736–739.

213 J. A. Hebda, I. Saraogi, M. Magzoub, A. D. Hamilton andA. D. Miranker, Chem. Biol., 2009, 16, 943–950.

214 F. Meng, A. Abedini, A. Plesner, C. B. Verchere andD. P. Raleigh, Biochemistry, 2010, 49, 8127–8133.

215 L. M. Young, J. C. Saunders, R. A. Mahood, C. H. Revill,R. J. Foster, L. H. Tu, D. P. Raleigh, S. E. Radford andA. E. Ashcroft, Nat. Chem., 2015, 7, 73–81.

216 H. Noor, P. Cao and D. P. Raleigh, Protein Sci., 2012, 21,373–382.

217 C. Zelus, A. Fox, A. Calciano, B. S. Faridian, L. A. Nogaj andD. A. Moffet, Open Biochem. J., 2012, 6, 66–70.

218 L.-H. Tu, H. Noor, P. Cao and D. P. Raleigh, ACS Chem.Biol., 2014, 9, 1632–1637.

219 M. Daval, S. Bedrood, T. Gurlo, C.-J. Huang, S. Costes,P. C. Butler and R. Langen, Amyloid, 2010, 17, 118–128.

220 S. Sparks, G. Liu, K. J. Robbins and N. D. Lazo, Biochem.Biophys. Res. Commun., 2012, 422, 551–555.

221 B. Cheng, H. Gong, X. Li, Y. Sun, X. Zhang, H. Chen, X. Liu,L. Zheng and K. Huang, Biochem. Biophys. Res. Commun.,2012, 419, 495–499.

222 S. Rigacci, V. Guidotti, M. Bucciantini, M. Parri,C. Nediani, E. Cerbai, M. Stefani and A. Berti, J. Nutr.Biochem., 2010, 21, 726–735.

223 R. Mishra, D. Sellin, D. Radovan, A. Gohlke and R. Winter,ChemBioChem, 2009, 10, 445–449.

224 E. N. Gurzov, B. Wang, E. H. Pilkington, P. Chen,A. Kakinen, W. J. Stanley, S. A. Litwak, E. G. Hanssen,T. P. Davis, F. Ding and P. C. Ke, Small, 2016, 12, 1615–1626.

225 P. Nedumpully-Govindan, E. N. Gurzov, P. Chen,E. H. Pilkington, W. J. Stanley, S. A. Litwak, T. P. Davis,P. C. Ke and F. Ding, Phys. Chem. Chem. Phys., 2016, 18,94–100.

226 C. Cabaleiro-Lago, F. Quinlan-Pluck, I. Lynch,K. A. Dawson and S. Linse, ACS Chem. Neurosci., 2010, 1,279–287.

227 C. T. Middleton, P. Marek, P. Cao, C.-c. Chiu, S. Singh,A. M. Woys, J. J. de Pablo, D. P. Raleigh and M. T. Zanni,Nat. Chem., 2012, 4, 355–360.

228 D. E. Ehrnhoefer, J. Bieschke, A. Boeddrich, M. Herbst,L. Masino, R. Lurz, S. Engemann, A. Pastore andE. E. Wanker, Nat. Struct. Mol. Biol., 2008, 15, 558–566.

229 A. R. A. Ladiwala, J. C. Lin, S. S. Bale, A. M. Marcelino-Cruz,M. Bhattacharya, J. S. Dordick and P. M. Tessier, J. Biol.Chem., 2010, 285, 24228–24237.

230 R. Mishra, B. Bulic, D. Sellin, S. Jha, H. Waldmann andR. Winter, Angew. Chem., Int. Ed., 2008, 47, 4679–4682.

231 F. Yang, G. P. Lim, A. N. Begum, O. J. Ubeda,M. R. Simmons, S. S. Ambegaokar, P. P. Chen, R. Kayed,C. G. Glabe, S. A. Frautschy and G. M. Cole, J. Biol. Chem.,2005, 280, 5892–5901.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



232 C. Adessi and C. Soto, Curr. Med. Chem., 2002, 9, 963–978.233 A. C. Santos, F. Veiga and A. J. Ribeiro, Expert Opin. Drug

Delivery, 2011, 8, 973–990.234 P. Nedumpully-Govindan, A. Kakinen, E. H. Pilkington,

T. P. Davis, P. Chun Ke and F. Ding, Sci. Rep., 2016,6, 19463.

235 P. Jiang, W. Li, J. E. Shea and Y. Mu, Biophys. J., 2011, 100,1550–1558.

236 F. Lolicato, A. Raudino, D. Milardi and C. La Rosa, Eur.J. Med. Chem., 2015, 92, 876–881.

237 Q. Q. Wang, L. L. Ning, Y. Z. Niu, H. X. Liu and X. J. Yao,J. Phys. Chem. B, 2015, 119, 15–24.

238 L. Maroteaux, J. Campanelli and R. Scheller, J. Neurosci.,1988, 8, 2804–2815.

239 T. R. Sampson, J. W. Debelius, T. Thron, S. Janssen,G. G. Shastri, Z. E. Ilhan, C. Challis, C. E. Schretter,S. Rocha, V. Gradinaru, M.-F. Chesselet, A. Keshavarzian,K. M. Shannon, R. Krajmalnik-Brown, P. Wittung-Stafshede, R. Knight and S. K. Mazmanian, Cell, 2016,167, 1469–1480.

240 J. Burre, M. Sharma and T. C. Sudhof, Proc. Natl. Acad. Sci.U. S. A., 2014, 111, E4274–E4283.

241 M. Vilar, H.-T. Chou, T. Luhrs, S. K. Maji, D. Riek-Loher,R. Verel, G. Manning, H. Stahlberg and R. Riek, Proc. Natl.Acad. Sci. U. S. A., 2008, 105, 8637–8642.

242 M. G. Spillantini, M. L. Schmidt, V. M. Y. Lee,J. Q. Trojanowski, R. Jakes and M. Goedert, Nature, 1997,388, 839–840.

243 D. J. Irwin, V. M. Y. Lee and J. Q. Trojanowski, Nat. Rev.Neurosci., 2013, 14, 626–636.

244 M. Periquet, T. Fulga, L. Myllykangas,M. G. Schlossmacher and M. B. Feany, J. Neurosci., 2007,27, 3338–3346.

245 L. Stefanis, Cold Spring Harbor Perspect. Med., 2012,2, a009399.

246 A. Villar-Pique, T. Lopes da Fonseca and T. F. Outeiro,J. Neurochem., 2016, 139, 240–255.

247 C. Vlad, K. Lindner, C. Karreman, S. Schildknecht,M. Leist, N. Tomczyk, J. Rontree, J. Langridge, K. Danzer,T. Ciossek, A. Petre, M. L. Gross, B. Hengerer andM. Przybylski, ChemBioChem, 2011, 12, 2740–2744.

248 J. A. Rodriguez, M. I. Ivanova, M. R. Sawaya, D. Cascio,F. E. Reyes, D. Shi, S. Sangwan, E. L. Guenther,L. M. Johnson, M. Zhang, L. Jiang, M. A. Arbing,B. L. Nannenga, J. Hattne, J. Whitelegge, A. S. Brewster,M. Messerschmidt, S. Boutet, N. K. Sauter, T. Gonen andD. S. Eisenberg, Nature, 2015, 525, 486–490.

249 M. D. Tuttle, G. Comellas, A. J. Nieuwkoop, D. J. Covell,D. A. Berthold, K. D. Kloepper, J. M. Courtney, J. K. Kim,A. M. Barclay, A. Kendall, W. Wan, G. Stubbs,C. D. Schwieters, V. M. Y. Lee, J. M. George andC. M. Rienstra, Nat. Struct. Mol. Biol., 2016, 23, 409–415.

250 G. Comellas, L. R. Lemkau, A. J. Nieuwkoop,K. D. Kloepper, D. T. Ladror, R. Ebisu, W. S. Woods,A. S. Lipton, J. M. George and C. M. Rienstra, J. Mol. Biol.,2011, 411, 881–895.

251 K. A. Conway, S.-J. Lee, J.-C. Rochet, T. T. Ding,R. E. Williamson and P. T. Lansbury, Proc. Natl. Acad.Sci. U. S. A., 2000, 97, 571–576.

252 J.-C. Rochet, K. A. Conway and P. T. Lansbury, Biochemistry,2000, 39, 10619–10626.

253 R. Khurana, C. Ionescu-Zanetti, M. Pope, J. Li, L. Nielson,M. Ramırez-Alvarado, L. Regan, A. L. Fink and S. A. Carter,Biophys. J., 2003, 85, 1135–1144.

254 C. Ionescu-Zanetti, R. Khurana, J. R. Gillespie, J. S. Petrick,L. C. Trabachino, L. J. Minert, S. A. Carter and A. L. Fink,Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 13175–13179.

255 J. D. Harper, C. M. Lieber and P. T. Lansbury, Chem. Biol.,1997, 4, 951–959.

256 W. B. Stine, S. W. Snyder, U. S. Ladror, W. S. Wade,M. F. Miller, T. J. Perun, T. F. Holzman and G. A. Krafft,J. Protein Chem., 1996, 15, 193–203.

257 H. K. L. Blackley, G. H. W. Sanders, M. C. Davies,C. J. Roberts, S. J. B. Tendler and M. J. Wilkinson, J. Mol.Biol., 2000, 298, 833–840.

258 A. K. Chamberlain, C. E. MacPhee, J. Zurdo,L. A. Morozova-Roche, H. A. Hill, C. M. Dobson andJ. J. Davis, Biophys. J., 2000, 79, 3282–3293.

259 N. M. Kad, N. H. Thomson, D. P. Smith, D. A. Smith andS. E. Radford, J. Mol. Biol., 2001, 313, 559–571.

260 J. Adamcik, J.-M. Jung, J. Flakowski, P. De Los Rios, G. Dietlerand R. Mezzenga, Nat. Nanotechnol., 2010, 5, 423–428.

261 K. K. M. Sweers, I. M. J. Segers-Nolten, M. L. Bennink andV. Subramaniam, Soft Matter, 2012, 8, 7215–7222.

262 R. A. Crowther, S. E. Daniel and M. Goedert, Neurosci. Lett.,2000, 292, 128–130.

263 K. K. M. Sweers, M. L. Bennink and V. Subramaniam,J. Phys.: Condens. Matter, 2012, 24, 243101.

264 G. Bhak, S. Lee, J. W. Park, S. Cho and S. R. Paik, Bioma-terials, 2010, 31, 5986–5995.

265 V. N. Uversky, J. Li and A. L. Fink, J. Biol. Chem., 2001, 276,10737–10744.

266 L. Xu, B. Ma, R. Nussinov and D. Thompson, ACS Chem.Neurosci., 2017, 8, 837–849.

267 A. Deleersnijder, M. Gerard, Z. Debyser and V. Baekelandt,Trends Mol. Med., 2013, 19, 368–377.

268 H. A. Lashuel, C. R. Overk, A. Oueslati and E. Masliah, Nat.Rev. Neurosci., 2013, 14, 38–48.

269 N. Lorenzen, S. B. Nielsen, A. K. Buell, J. D. Kaspersen,P. Arosio, B. S. Vad, W. Paslawski, G. Christiansen,Z. Valnickova-Hansen, M. Andreasen, J. J. Enghild,J. S. Pedersen, C. M. Dobson, T. P. J. Knowles andD. E. Otzen, J. Am. Chem. Soc., 2014, 136, 3859–3868.

270 L. Giehm, D. I. Svergun, D. E. Otzen and B. Vestergaard,Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 3246–3251.

271 H. A. Lashuel, D. Hartley, B. M. Petre, T. Walz andP. T. Lansbury, Nature, 2002, 418, 291.

272 B. Winner, R. Jappelli, S. K. Maji, P. A. Desplats, L. Boyer,S. Aigner, C. Hetzer, T. Loher, M. Vilar, S. Campioni,C. Tzitzilonis, A. Soragni, S. Jessberger, H. Mira,A. Consiglio, E. Pham, E. Masliah, F. H. Gage andR. Riek, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 4194–4199.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



273 R. Kayed, E. Head, J. L. Thompson, T. M. McIntire,S. C. Milton, C. W. Cotman and C. G. Glabe, Science,2003, 300, 486–489.

274 D. M. Walsh and D. J. Selkoe, J. Neurochem., 2007, 101,1172–1184.

275 L. M. Luheshi, G. G. Tartaglia, A.-C. Brorsson, A. P. Pawar,I. E. Watson, F. Chiti, M. Vendruscolo, D. A. Lomas,C. M. Dobson and D. C. Crowther, PLoS Biol., 2007, 5, e290.

276 C. M. Dobson, Nature, 2003, 426, 884–890.277 S. Lesne, M. T. Koh, L. Kotilinek, R. Kayed, C. G. Glabe,

A. Yang, M. Gallagher and K. H. Ashe, Nature, 2006, 440,352–357.

278 C. Nilsberth, A. Westlind-Danielsson, C. B. Eckman,M. M. Condron, K. Axelman, C. Forsell, C. Stenh,J. Luthman, D. B. Teplow, S. G. Younkin, J. Naslund andL. Lannfelt, Nat. Neurosci., 2001, 4, 887–893.

279 S. L. Bernstein, N. F. Dupuis, N. D. Lazo, T. Wyttenbach,M. M. Condron, G. Bitan, D. B. Teplow, J.-E. Shea,B. T. Ruotolo, C. V. Robinson and M. T. Bowers, Nat.Chem., 2009, 1, 326–331.

280 L. Haataja, T. Gurlo, C. J. Huang and P. C. Butler, Endocr.Rev., 2008, 29, 303–316.

281 P. Krotee, J. A. Rodriguez, M. R. Sawaya, D. Cascio, F. E. Reyes,D. Shi, J. Hattne, B. L. Nannenga, M. E. Oskarsson, S. Philipp,S. Griner, L. Jiang, C. G. Glabe, G. T. Westermark, T. Gonen andD. S. Eisenberg, eLife, 2017, 6, e19273.

282 H. Roberts and D. Brown, Biomolecules, 2015, 5, 282–305.283 F. van Diggelen, A. W. J. W. Tepper, M. M. Apetri and

D. E. Otzen, Isr. J. Chem., 2017, DOI: 10.1002/ijch.201600116.284 Marıa S. Celej, R. Sarroukh, E. Goormaghtigh,

Gerardo D. Fidelio, J.-M. Ruysschaert and V. Raussens,Biochem. J., 2012, 443, 719–726.

285 M. Bucciantini, E. Giannoni, F. Chiti, F. Baroni,L. Formigli, J. Zurdo, N. Taddei, G. Ramponi,C. M. Dobson and M. Stefani, Nature, 2002, 416, 507–511.

286 B. Bolognesi, J. R. Kumita, T. P. Barros, E. K. Esbjorner,L. M. Luheshi, D. C. Crowther, M. R. Wilson,C. M. Dobson, G. Favrin and J. J. Yerbury, ACS Chem. Biol.,2010, 5, 735–740.

287 L. C. Serpell, J. Berriman, R. Jakes, M. Goedert andR. A. Crowther, Proc. Natl. Acad. Sci. U. S. A., 2000, 97,4897–4902.

288 C. G. Glabe, J. Biol. Chem., 2008, 283, 29639–29643.289 K. M. Danzer, D. Haasen, A. R. Karow, S. Moussaud,

M. Habeck, A. Giese, H. Kretzschmar, B. Hengerer andM. Kostka, J. Neurosci., 2007, 27, 9220–9232.

290 D. P. Karpinar, M. B. G. Balija, S. Kugler, F. Opazo,N. Rezaei-Ghaleh, N. Wender, H.-Y. Kim,G. Taschenberger, B. H. Falkenburger, H. Heise,A. Kumar, D. Riedel, L. Fichtner, A. Voigt, G. H. Braus,K. Giller, S. Becker, A. Herzig, M. Baldus, H. Jackle,S. Eimer, J. B. Schulz, C. Griesinger and M. Zweckstetter,EMBO J., 2009, 28, 3256–3268.

291 P. Desplats, H.-J. Lee, E.-J. Bae, C. Patrick, E. Rockenstein,L. Crews, B. Spencer, E. Masliah and S.-J. Lee, Proc. Natl.Acad. Sci. U. S. A., 2009, 106, 13010–13015.

292 A. Relini, O. Cavalleri, R. Rolandi and A. Gliozzi, Chem.Phys. Lipids, 2009, 158, 1–9.

293 S. M. Butterfield and H. A. Lashuel, Angew. Chem., Int. Ed.,2010, 49, 5628–5654.

294 C. Aisenbrey, T. Borowik, R. Bystrom, M. Bokvist,F. Lindstrom, H. Misiak, M.-A. Sani and G. Grobner, Eur.Biophys. J., 2008, 37, 247–255.

295 J. A. Hebda and A. D. Miranker, Annu. Rev. Biophys., 2009,39, 125–152.

296 C. Galvagnion, A. K. Buell, G. Meisl, T. C. T. Michaels,M. Vendruscolo, T. P. J. Knowles and C. M. Dobson, Nat.Chem. Biol., 2015, 11, 229–234.

297 H.-Y. Kim, M.-K. Cho, A. Kumar, E. Maier, C. Siebenhaar,S. Becker, C. O. Fernandez, H. A. Lashuel, R. Benz, A. Langeand M. Zweckstetter, J. Am. Chem. Soc., 2009, 131,17482–17489.

298 N. P. Reynolds, A. Soragni, M. Rabe, D. Verdes, E. Liverani,S. Handschin, R. Riek and S. Seeger, J. Am. Chem. Soc.,2011, 133, 19366–19375.

299 D. L. Fortin, M. D. Troyer, K. Nakamura, S.-i. Kubo,M. D. Anthony and R. H. Edwards, J. Neurosci., 2004, 24,6715–6723.

300 E. R. Georgieva, T. F. Ramlall, P. P. Borbat, J. H. Freed andD. Eliezer, J. Am. Chem. Soc., 2008, 130, 12856–12857.

301 A. J. Trexler and E. Rhoades, Biochemistry, 2009, 48,2304–2306.

302 P. T. Wong, J. A. Schauerte, K. C. Wisser, H. Ding, E. L. Lee,D. G. Steel and A. Gafni, J. Mol. Biol., 2009, 386, 81–96.

303 A. M. Morris and R. G. Finke, Biophys. Chem., 2009, 140, 9–15.304 L. Kjaer, L. Giehm, T. Heimburg and D. Otzen, Biophys. J.,

2009, 96, 2857–2870.305 D. P. Smith, D. J. Tew, A. F. Hill, S. P. Bottomley,

C. L. Masters, K. J. Barnham and R. Cappai, Biochemistry,2008, 47, 1425–1434.

306 A. P. Pandey, F. Haque, J.-C. Rochet and J. S. Hovis,Biophys. J., 2009, 96, 540–551.

307 B. D. van Rooijen, M. M. A. E. Claessens andV. Subramaniam, FEBS Lett., 2008, 582, 3788–3792.

308 E. C. Hirsch, J. P. Brandel, P. Galle, F. Javoy-Agid andY. Agid, J. Neurochem., 1991, 56, 446–451.

309 D. L. Pountney, N. H. Voelcker and W. P. Gai, Neurotoxic.Res., 2005, 7, 59–67.

310 S. Bolisetty and R. Mezzenga, Nat. Nanotechnol., 2016, 11,365–371.

311 Y. Shen, L. Posavec, S. Bolisetty, F. M. Hilty, G. Nystrom,J. Kohlbrecher, M. Hilbe, A. Rossi, J. Baumgartner,M. B. Zimmermann and R. Mezzenga, Nat. Nanotechnol.,2017, DOI: 10.1038/nnano.2017.58.

312 G. Wei, Z. Su, N. P. Reynolds, P. Arosio, I. W. Hamley,E. Gazit and R. Mezzenga, Chem. Soc. Rev., 2017, DOI:10.1039/C6CS00542J.

313 I. F. Tsigelny, P. Bar-On, Y. Sharikov, L. Crews,M. Hashimoto, M. A. Miller, S. H. Keller, O. Platoshyn,J. X. J. Yuan and E. Masliah, FEBS J., 2007, 274, 1862–1877.

314 M. J. Volles and P. T. Lansbury, Biochemistry, 2002, 41,4595–4602.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



315 B. Bechinger and K. Lohner, Biochim. Biophys. Acta, Bio-membr., 2006, 1758, 1529–1539.

316 J. Kiskis, I. Horvath, P. Wittung-Stafshede and S. Rocha, Q.Rev. Biophys., 2017, 50.

317 H. Chaudhary, A. N. D. Stefanovic, V. Subramaniam andM. M. A. E. Claessens, FEBS Lett., 2014, 588, 4457–4463.

318 E. Monsellier, L. Bousset and R. Melki, Sci. Rep., 2016,6, 19180.

319 A. Iyer, N. Schilderink, M. M. A. E. Claessens andV. Subramaniam, Biophys. J., 2016, 111, 2440–2449.

320 L. Devi, V. Raghavendran, B. M. Prabhu, N. G. Avadhaniand H. K. Anandatheerthavarada, J. Biol. Chem., 2008, 283,9089–9100.

321 E. S. Luth, I. G. Stavrovskaya, T. Bartels, B. S. Kristal andD. J. Selkoe, J. Biol. Chem., 2014, 289, 21490–21507.

322 L. Chen, J. Jin, J. Davis, Y. Zhou, Y. Wang, J. Liu,P. J. Lockhart and J. Zhang, Biochem. Biophys. Res. Com-mun., 2007, 356, 548–553.

323 N. Cremades, S. I. Cohen, E. Deas, A. Y. Abramov, A. Y. Chen,A. Orte, M. Sandal, R. W. Clarke, P. Dunne, F. A. Aprile,C. W. Bertoncini, N. W. Wood, T. P. Knowles, C. M. Dobsonand D. Klenerman, Cell, 2012, 149, 1048–1059.

324 E. Junn and M. M. Mouradian, Neurosci. Lett., 2002, 320,146–150.

325 D.-H. Choi, A. C. Cristovao, S. Guhathakurta, J. Lee,T. H. Joh, M. F. Beal and Y.-S. Kim, Antioxid. RedoxSignaling, 2012, 16, 1033–1045.

326 H. A. Lashuel, B. M. Petre, J. Wall, M. Simon, R. J. Nowak,T. Walz and P. T. Lansbury Jr, J. Mol. Biol., 2002, 322,1089–1102.

327 J. J. M. Hoozemans, E. S. van Haastert, D. A. T. Nijholt,A. J. M. Rozemuller and W. Scheper, Neurodegener. Dis.,2012, 10, 212–215.

328 H. Snyder, K. Mensah, C. Theisler, J. Lee, A. Matouschekand B. Wolozin, J. Biol. Chem., 2003, 278, 11753–11759.

329 G. Fusco, T. Pape, A. D. Stephens, P. Mahou, A. R. Costa,C. F. Kaminski, G. S. Kaminski Schierle, M. Vendruscolo,G. Veglia, C. M. Dobson and A. De Simone, Nat. Commun.,2016, 7, 12563.

330 L. S. Forno, J. Neuropathol. Exp. Neurol., 1996, 55, 259–272.331 A. Recasens, B. Dehay, J. Bove, I. Carballo-Carbajal,

S. Dovero, A. Perez-Villalba, P.-O. Fernagut, J. Blesa,A. Parent, C. Perier, I. Farinas, J. A. Obeso, E. Bezard andM. Vila, Ann. Neurol., 2014, 75, 351–362.

332 M. Iljina, G. A. Garcia, M. H. Horrocks, L. Tosatto,M. L. Choi, K. A. Ganzinger, A. Y. Abramov, S. Gandhi,N. W. Wood, N. Cremades, C. M. Dobson, T. P. J. Knowlesand D. Klenerman, Proc. Natl. Acad. Sci. U. S. A., 2016, 113,E1206–E1215.

333 H. Malkki, Nat. Rev. Neurol., 2017, 13, 66–67.334 T. Bartels, J. G. Choi and D. J. Selkoe, Nature, 2011, 477,

107–110.335 O. Marques and T. F. Outeiro, Cell Death Dis., 2012, 3, e350.336 E. Masliah, E. Rockenstein, A. Adame, M. Alford, L. Crews,

M. Hashimoto, P. Seubert, M. Lee, J. Goldstein, T. Chilcote,D. Games and D. Schenk, Neuron, 46, 857–868.

337 E. Valera and E. Masliah, Pharmacol. Ther., 2013, 138,311–322.

338 S. Di Giovanni, S. Eleuteri, K. E. Paleologou, G. Yin,M. Zweckstetter, P.-A. Carrupt and H. A. Lashuel, J. Biol.Chem., 2010, 285, 14941–14954.

339 M. Masuda, N. Suzuki, S. Taniguchi, T. Oikawa, T. Nonaka,T. Iwatsubo, S.-i. Hisanaga, M. Goedert and M. Hasegawa,Biochemistry, 2006, 45, 6085–6094.

340 Y. Porat, A. Abramowitz and E. Gazit, Chem. Biol. Drug Des.,2006, 67, 27–37.

341 S. B. Prusiner, Proc. Natl. Acad. Sci. U. S. A., 1998, 95,13363–13383.

342 Z. Huang, S. B. Prusiner and F. E. Cohen, Folding Des.,1996, 1, 13–19.

343 P. N. Campbell, IUBMB Life, 2005, 57, 273–276.344 C. J. Sigurdson and A. Aguzzi, Biochim. Biophys. Acta, 2007,

1772, 610–618.345 D. Westaway, C. Cooper, S. Turner, M. Da Costa,

G. A. Carlson and S. B. Prusiner, Proc. Natl. Acad. Sci. U.S. A., 1994, 91, 6418–6422.

346 J. Mastrianni, F. Nixon, R. Layzer, S. J. DeArmond andS. B. Prusiner, Neurology, 1997, 48, A296.

347 P. Westermark, M. D. Benson, J. N. Buxbaum, A. S. Cohen,B. Frangione, S.-I. Ikeda, C. L. Masters, G. Merlini,M. J. Saraiva and J. D. Sipe, Amyloid, 2005, 12, 1–4.

348 B. Chesebro, M. Trifilo, R. Race, K. Meade-White, C. Teng,R. LaCasse, L. Raymond, C. Favara, G. Baron, S. Priola,B. Caughey, E. Masliah and M. Oldstone, Science, 2005,308, 1435.

349 A. Aguzzi, Science, 2005, 308, 1420.350 A. Aguzzi and M. Heikenwalder, Nat. Rev. Microbiol., 2006,

4, 765–775.351 I. V. Baskakov, G. Legname, M. A. Baldwin, S. B. Prusiner

and F. E. Cohen, J. Biol. Chem., 2002, 277, 21140–21148.352 R. A. Bessen and R. F. Marsh, J. Virol., 1994, 68, 7859–7868.353 M. P. McKinley, R. K. Meyer, L. Kenaga, F. Rahbar,

R. Cotter, A. Serban and S. B. Prusiner, J. Virol., 1991, 65,1340–1351.

354 M. Sunde and C. C. Blake, Q. Rev. Biophys., 1998, 31, 1–39.355 K.-W. Leffers, H. Wille, J. Stohr, E. Junger, B. Prusiner

Stanley and D. Riesner, Biol. Chem., 2005, 386, 569.356 J. G. Safar, K. Kellings, A. Serban, D. Groth, J. E. Cleaver,

S. B. Prusiner and D. Riesner, J. Virol., 2005, 79,10796–10806.

357 B. Caughey and G. S. Baron, Nature, 2006, 443, 803–810.358 L. Manuelidis, Virulence, 2013, 4, 373–383.359 F. O. Bastian, Arch. Pathol. Lab. Med., 1979, 103, 665–669.360 J. S. Griffith, Nature, 1967, 215, 1043–1044.361 S. B. Prusiner, Annu. Rev. Microbiol., 1989, 43, 345–374.362 C. Soto and J. Castilla, Nat. Med., 2004, 10, 63–67.363 K. Abid and C. Soto, Cell. Mol. Life Sci., 2006, 63,

2342–2351.364 M. Stefani and C. M. Dobson, J. Mol. Med., 2003, 81,

678–699.365 V. Novitskaya, O. V. Bocharova, I. Bronstein and

I. V. Baskakov, J. Biol. Chem., 2006, 281, 13828–13836.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



366 R. Chiesa, P. Piccardo, E. Quaglio, B. Drisaldi, S. L. Si-Hoe,M. Takao, B. Ghetti and D. A. Harris, J. Virol., 2003, 77,7611–7622.

367 K. Wuthrich and R. Riek, Adv. Protein Chem., 2001, 57,55–82.

368 C. Govaerts, H. Wille, S. B. Prusiner and F. E. Cohen, Proc.Natl. Acad. Sci. U. S. A., 2004, 101, 8342–8347.

369 R. S. Sparkes, M. Simon, V. H. Cohn, R. E. Fournier, J. Lem,I. Klisak, C. Heinzmann, C. Blatt, M. Lucero andT. Mohandas, Proc. Natl. Acad. Sci. U. S. A., 1986, 83,7358–7362.

370 R. Linden, V. R. Martins, M. A. M. Prado, M. Cammarota,I. Izquierdo and R. R. Brentani, Physiol. Rev., 2008, 88,673–728.

371 F. Llorens and J. A. del Rıo, Prion, 2012, 6, 245–251.372 A. Aguzzi, M. Heikenwalder and M. Polymenidou, Nat. Rev.

Mol. Cell Biol., 2007, 8Q6 .373 J. Kanaani, S. B. Prusiner, J. Diacovo, S. Baekkeskov and

G. Legname, J. Neurochem., 2005, 95, 1373–1386.374 D. R. Brown, K. Qin, J. W. Herms, A. Madlung, J. Manson,

R. Strome, P. E. Fraser, T. Kruck, A. von Bohlen, W. Schulz-Schaeffer, A. Giese, D. Westaway and H. Kretzschmar,Nature, 1997, 390, 684–687.

375 M. Prcina, E. Kontsekova and M. Novak, Acta Virol., 2015,59, 179–184.

376 M. Mehrpour and P. Codogno, Cancer Lett., 2010, 290,1–23.

377 J. Du, Y. Pan, Y. Shi, C. Guo, X. Jin, L. Sun, N. Liu, T. Qiaoand D. Fan, Int. J. Cancer, 2005, 113, 213–220.

378 Q.-Q. Li, Y.-P. Sun, C.-P. Ruan, X.-Y. Xu, J.-H. Ge, J. He, Z.-D. Xu, Q. Wang and W.-C. Gao, Cancer Sci., 2011, 102,400–406.

379 M. de Wit, C. R. Jimenez, B. Carvalho, J. A. M. Belien,P. M. Delis-van Diemen, S. Mongera, S. R. Piersma,M. Vikas, S. Navani, F. Ponten, G. A. Meijer andR. J. A. Fijneman, Gut, 2012, 61, 855–864.

380 H. Bueler, A. Aguzzi, A. Sailer, R. A. Greiner, P. Autenried,M. Aguet and C. Weissmann, Cell, 1993, 73, 1339–1347.

381 A. D. Steele, S. Lindquist and A. Aguzzi, Prion, 2007, 1,83–93.

382 J. R. Criado, M. Sanchez-Alavez, B. Conti, J. L. Giacchino,D. N. Wills, S. J. Henriksen, R. Race, J. C. Manson,B. Chesebro and M. B. A. Oldstone, Neurobiol. Dis., 2005,19, 255–265.

383 C. E. Le Pichon, M. T. Valley, M. Polymenidou,A. T. Chesler, B. T. Sagdullaev, A. Aguzzi and S. Firestein,Nat. Neurosci., 2009, 12, 60–69.

384 R. Riek, S. Hornemann, G. Wider, M. Billeter,R. Glockshuber and K. Wuthrich, Nature, 1996, 382,180–182.

385 R. Zahn, A. Liu, T. Luhrs, R. Riek, C. von Schroetter,F. Lopez Garcıa, M. Billeter, L. Calzolai, G. Wider andK. Wuthrich, Proc. Natl. Acad. Sci. U. S. A., 2000, 97,145–150.

386 R. Zahn, P. Guntert, C. von Schroetter and K. Wuthrich,J. Mol. Biol., 2003, 326, 225–234.

387 L. Calzolai and R. Zahn, J. Biol. Chem., 2003, 278,35592–35596.

388 L. Calzolai, D. A. Lysek, P. Guntert, C. von Schroetter,R. Riek, R. Zahn and K. Wuthrich, Proc. Natl. Acad. Sci.U. S. A., 2000, 97, 8340–8345.

389 A. Pastore and A. Zagari, Prion, 2007, 1, 185–197.390 K. J. Knaus, M. Morillas, W. Swietnicki, M. Malone,

W. K. Surewicz and V. C. Yee, Nat. Struct. Mol. Biol.,2001, 8, 770–774.

391 F. Wopfner, G. Weidenhofer, R. Schneider, A. von Brunn,S. Gilch, T. F. Schwarz, T. Werner and H. M. Schatzl, J. Mol.Biol., 1999, 289, 1163–1178.

392 E. Rivera-Milla, B. Oidtmann, C. H. Panagiotidis, M. Baier,T. Sklaviadis, R. Hoffmann, Y. Zhou, G. P. Solis,C. A. O. Stuermer and E. Malaga-Trillo, FASEB J., 2005,DOI: 10.1096/fj.05-4279fje.

393 G. L. Millhauser, Annu. Rev. Phys. Chem., 2007, 58, 299–320.394 D. G. Donne, J. H. Viles, D. Groth, I. Mehlhorn, T. L. James,

F. E. Cohen, S. B. Prusiner, P. E. Wright and H. J. Dyson,Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 13452–13457.

395 K. M. Pan, M. Baldwin, J. Nguyen, M. Gasset, A. Serban,D. Groth, I. Mehlhorn, Z. Huang, R. J. Fletterick andF. E. Cohen, Proc. Natl. Acad. Sci. U. S. A., 1993, 90,10962–10966.

396 J. Safar, P. P. Roller, D. C. Gajdusek and C. J. Gibbs, J. Biol.Chem., 1993, 268, 20276–20284.

397 M. P. McKinley and S. B. Prusiner, Int. Rev. Neurobiol.,1986, 28, 1–57.

398 M. L. DeMarco and V. Daggett, Proc. Natl. Acad. Sci. U. S. A.,2004, 101, 2293–2298.

399 N. J. Cobb, F. D. Sonnichsen, H. McHaourab andW. K. Surewicz, Proc. Natl. Acad. Sci. U. S. A., 2007, 104,18946–18951.

400 B. R. Groveman, M. A. Dolan, L. M. Taubner, A. Kraus,R. B. Wickner and B. Caughey, J. Biol. Chem., 2014, 289,24129–24142.

401 M. R. Sawaya, S. Sambashivan, R. Nelson, M. I. Ivanova,S. A. Sievers, M. I. Apostol, M. J. Thompson, M. Balbirnie,J. J. W. Wiltzius, H. T. McFarlane, A. O. Madsen, C. Riekeland D. Eisenberg, Nature, 2007, 447, 453–457.

402 C. Wasmer, A. Lange, H. Van Melckebeke, A. B. Siemer,R. Riek and B. H. Meier, Science, 2008, 319, 1523–1526.

403 H. Wille, W. Bian, M. McDonald, A. Kendall, D. W. Colby,L. Bloch, J. Ollesch, A. L. Borovinskiy, F. E. Cohen,S. B. Prusiner and G. Stubbs, Proc. Natl. Acad. Sci. U. S.A., 2009, 106, 16990–16995.

404 A. G. Dickinson, V. M. H. Meikle and H. Fraser, J. Comp.Pathol., 1968, 78, 293–299.

405 M. E. Bruce and A. G. Dickinson, J. Gen. Virol., 1987, 68,79–89.

406 R. A. Bessen and R. F. Marsh, J. Gen. Virol., 1992, 73,329–334.

407 L. Monari, S. G. Chen, P. Brown, P. Parchi, R. B. Petersen,J. Mikol, F. Gray, P. Cortelli, P. Montagna and B. Ghetti,Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 2839–2842.

408 J. Collinge and A. R. Clarke, Science, 2007, 318, 930–936.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



409 M. R. Scott, D. Groth, J. Tatzelt, M. Torchia, P. Tremblay,S. J. DeArmond and S. B. Prusiner, J. Virol., 1997, 71,9032–9044.

410 R. H. Kimberlin, C. A. Walker and H. Fraser, J. Gen. Virol.,1989, 70, 2017–2025.

411 H. Fraser and A. G. Dickinson, J. Comp. Pathol., 1973, 83,29–40.

412 P. A. Merz, R. A. Somerville, H. M. Wisniewski and K. Iqbal,Acta Neuropathol., 1981, 54, 63–74.

413 S. B. Prusiner, M. P. McKinley, K. A. Bowman, D. C. Bolton,P. E. Bendheim, D. F. Groth and G. G. Glenner, Cell, 1983,35, 349–358.

414 V. L. Sim and B. Caughey, Neurobiol. Aging, 2009, 30,2031–2042.

415 R. J. Kascsak, R. Rubenstein, P. A. Merz, R. I. Carp,N. K. Robakis, H. M. Wisniewski and H. Diringer,J. Virol., 1986, 59, 676–683.

416 E. Neuendorf, A. Weber, A. Saalmueller, H. Schatzl,K. Reifenberg, E. Pfaff and M. H. Groschup, J. Biol. Chem.,2004, 279, 53306–53316.

417 G. Zanusso, A. Polo and A. Farinazzo, et al., Arch. Neurol.,2007, 64, 595–599.

418 P. A. Lewis, F. Properzi, K. Prodromidou, A. R. Clarke,J. Collinge and G. S. Jackson, Biochem. J., 2006, 395,443–448.

419 M. J. Stack, A. M. Aldrich, A. D. Kitching and A. C. Scott,Res. Vet. Sci., 1995, 59, 247–254.

420 K. Hsiao and S. B. Prusiner, Neurology, 1990, 40, 1820.421 S. B. Prusiner, Arch. Neurol., 1993, 50, 1129–1153.422 D. Gajdusek, Eur. J. Epidemiol., 1991, 7, 567–577.423 J. Collinge, J. Whitfield, E. McKintosh, J. Beck, S. Mead,

D. J. Thomas and M. P. Alpers, Lancet, 2006, 367,2068–2074.

424 S. McCutcheon, A. R. A. Blanco, E. F. Houston, C. de Wolf,B. C. Tan, A. Smith, M. H. Groschup, N. Hunter,V. S. Hornsey and I. R. MacGregor, PLoS One, 2011,6, e23169.

425 O. Andreoletti, C. Litaise, H. Simmons, F. Corbiere,S. Lugan, P. Costes, F. Schelcher, D. Vilette, J. Grassi andC. Lacroux, PLoS Pathog., 2012, 8, e1002782.

426 D. J. Bonda, S. Manjila, P. Mehndiratta, F. Khan,B. R. Miller, K. Onwuzulike, G. Puoti, M. L. Cohen,L. B. Schonberger and I. Cali, Neurosurg. Focus, 2016,41, E10.

427 C. K. Mathiason, J. G. Powers, S. J. Dahmes, D. A. Osborn,K. V. Miller, R. J. Warren, G. L. Mason, S. A. Hays, J. Hayes-Klug and D. M. Seelig, Science, 2006, 314, 133–136.

428 C. K. Mathiason, S. A. Hays, J. Powers, J. Hayes-Klug,J. Langenberg, S. J. Dahmes, D. A. Osborn, K. V. Miller,R. J. Warren and G. L. Mason, PLoS One, 2009, 4, e5916.

429 N. J. Haley, D. M. Seelig, M. D. Zabel, G. C. Telling andE. A. Hoover, PLoS One, 2009, 4, e4848.

430 C. DeJoia, B. Moreaux, K. O’Connell and R. A. Bessen,J. Virol., 2006, 80, 4546–4556.

431 T. Konold, S. J. Moore, S. J. Bellworthy and H. A. Simmons,BMC Vet. Res., 2008, 4, 14.

432 C. Ligios, C. J. Sigurdson, C. Santucciu, G. Carcassola,G. Manco, M. Basagni, C. Maestrale, M. G. Cancedda,L. Madau and A. Aguzzi, Nat. Med., 2005, 11, 1137–1139.

433 C. Lacroux, S. Simon, S. L. Benestad, S. Maillet, J. Mathey,S. Lugan, F. Corbiere, H. Cassard, P. Costes andD. Bergonier, PLoS Pathog., 2008, 4, e1000238.

434 C. J. Johnson, D. McKenzie, J. A. Pedersen and J. M. Aiken,J. Toxicol. Environ. Health, Part A, 2011, 74, 161–166.

435 C. E. Bruederle, R. M. Hnasko, T. Kraemer, R. A. Garcia,M. J. Haas, W. N. Marmer and J. M. Carter, PLoS One, 2008,3, e2969.

436 M. W. Miller, Emerging Infect. Dis., 2004, 10, 1003–1006.437 G. Georgsson, S. Sigurdarson and P. Brown, J. Gen. Virol.,

2006, 87, 3737–3740.438 S. E. Saunders, J. C. Bartz and S. L. Bartelt-Hunt, Environ.

Sci. Technol., 2009, 43, 5242.439 K. Tsiroulnikov, H. Rezai, E. Bonch-Osmolovskaya,

P. Nedkov, A. Gousterova, V. Cueff, A. Godfroy,G. Barbier, F. Metro and J.-M. Chobert, J. Agric. Food Chem.,2004, 52, 6353–6360.

440 J. Rasmussen, B. H. Gilroyed, T. Reuter, S. Dudas,N. F. Neumann, A. Balachandran, N. N. Kav, C. Graham,S. Czub and T. A. McAllister, Prion, 2014, 8, 136–142.

441 S. Pritzkow, R. Morales, F. Moda, U. Khan, G. C. Telling,E. Hoover and C. Soto, Cell Rep., 2015, 11, 1168–1175.

442 Y. O. Chernoff, Proc. Natl. Acad. Sci. U. S. A., 2016, 113,6097–6099.

443 S. Brandner, M. A. Klein, R. Frigg, V. Pekarik, P. Parizek,A. Raeber, M. Glatzel, P. Schwarz, T. Rulicke andC. Weissmann, Exp. Physiol., 2000, 85, 705–712.

444 A. Aguzzi and M. Polymenidou, Cell, 2004, 116, 313–327.445 A. Aguzzi and A. M. Calella, Physiol. Rev., 2009, 89,

1105–1152.446 C. Soto, L. Estrada and J. Castilla, Trends Biochem. Sci.,

2006, 31, 150–155.447 N. Makarava, V. G. Ostapchenko, R. Savtchenko and

I. V. Baskakov, J. Biol. Chem., 2009, 284, 14386–14395.448 L. Li, W. Guest, A. Huang, S. S. Plotkin and N. R. Cashman,

Protein Eng., Des. Sel., 2009, 22, 523–529.449 G. Legname, Prion, 2012, 6, 37–39.450 E. Paramithiotis, M. Pinard, T. Lawton, S. LaBoissiere,

V. L. Leathers, W.-Q. Zou, L. A. Estey, J. Lamontagne,M. T. Lehto and L. H. Kondejewski, Nat. Med., 2003, 9, 893–899.

451 K. Kaneko, L. Zulianello, M. Scott, C. M. Cooper,A. C. Wallace, T. L. James, F. E. Cohen andS. B. Prusiner, Proc. Natl. Acad. Sci. U. S. A., 1997, 94,10069–10074.

452 J. C. Geoghegan, P. A. Valdes, N. R. Orem, N. R. Deleault,R. A. Williamson, B. T. Harris and S. Supattapone, J. Biol.Chem., 2007, 282, 36341–36353.

453 I. V. Baskakov and L. Breydo, BBA, Mol. Basis Dis., 2007,1772, 692–703.

454 B. Caughey, Br. Med. Bull., 2003, 66, 109–120.455 M. Bishop, P. Hart, L. Aitchison, H. Baybutt, C. Plinston,

V. Thomson, N. Tuzi, M. Head, J. Ironside and R. Will,Lancet Neurol., 2006, 5, 393–398.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



456 M. I. Apostol, J. J. Wiltzius, M. R. Sawaya, D. Cascio andD. Eisenberg, Biochemistry, 2011, 50, 2456.

457 T. D. Kurt, C. Bett, N. Fernandez-Borges, S. Joshi-Barr,S. Hornemann, T. Rulicke, J. Castilla, K. Wuthrich,A. Aguzzi and C. J. Sigurdson, J. Neurosci., 2014, 34,1022–1027.

458 C. J. Sigurdson, K. P. R. Nilsson, S. Hornemann, G. Manco,N. Fernandez-Borges, P. Schwarz, J. Castilla, K. Wuthrichand A. Aguzzi, J. Clin. Invest., 2010, 120, 2590–2599.

459 A. F. Hill and J. Collinge, Trends Microbiol., 2003, 11,578–584.

460 C. Thuring, J. Erkens, J. Jacobs, A. Bossers, L. Van Keulen,G. Garssen, F. Van Zijderveld, S. Ryder, M. Groschup andT. Sweeney, J. Clin. Microbiol., 2004, 42, 972–980.

461 E. M. Jones and W. K. Surewicz, Cell, 2005, 121, 63–72.462 M. Jones, Emerging Infect. Dis., 2009.463 H. Liu, S. Farr-Jones, N. B. Ulyanov, M. Llinas, S. Marqusee,

D. Groth, F. E. Cohen, S. B. Prusiner and T. L. James,Biochemistry, 1999, 38, 5362–5377.

464 N. Makarava, G. G. Kovacs, O. Bocharova, R. Savtchenko,I. Alexeeva, H. Budka, R. G. Rohwer and I. V. Baskakov,Acta Neurol., 2010, 119, 177–187.

465 V. Beringue, L. Herzog, E. Jaumain, F. Reine, P. Sibille,A. Le Dur, J.-L. Vilotte and H. Laude, Science, 2012, 335,472–475.

466 D. Padilla, V. Beringue, J. C. Espinosa, O. Andreoletti,E. Jaumain, F. Reine, L. Herzog, A. Gutierrez-Adan,B. Pintado and H. Laude, PLoS Pathog., 2011, 7, e1001319.

467 J. Castilla, D. Gonzalez-Romero, P. Saa, R. Morales, J. DeCastro and C. Soto, Cell, 2008, 134, 757–768.

468 E. A. Asante, J. M. Linehan, M. Desbruslais, S. Joiner,I. Gowland, A. L. Wood, J. Welch, A. F. Hill, S. E. Lloydand J. D. Wadsworth, EMBO J., 2002, 21, 6358–6366.

469 J. D. Wadsworth, E. A. Asante, M. Desbruslais,J. M. Linehan, S. Joiner, I. Gowland, J. Welch, L. Stone,S. E. Lloyd and A. F. Hill, Science, 2004, 306, 1793–1796.

470 R. H. Kimberlin, S. Cole and C. A. Walker, J. Gen. Virol.,1987, 68, 1875–1881.

471 D. W. Colby, K. Giles, G. Legname, H. Wille, I. V. Baskakov,S. J. DeArmond and S. B. Prusiner, Proc. Natl. Acad. Sci. U.S. A., 2009, 106, 20417–20422.

472 M. E. Bruce, Br. Med. Bull., 2003, 66, 99–108.473 M. A. Barria, G. C. Telling, P. Gambetti, J. A. Mastrianni

and C. Soto, J. Biol. Chem., 2011, 286, 7490–7495.474 J. Li, S. Browning, S. P. Mahal, A. M. Oelschlegel and

C. Weissmann, Science, 2010, 327, 869–872.475 B. Caughey, G. S. Baron, B. Chesebro and M. Jeffrey, Annu.

Rev. Biochem., 2009, 78, 177–204.476 A. Aguzzi and J. Falsig, Nat. Neurosci., 2012, 15, 936–939.477 G. Mallucci, A. Dickinson, J. Linehan, P.-C. Klohn,

S. Brandner and J. Collinge, Science, 2003, 302, 871–874.478 R. Bahadi, P. V. Farrelly, B. L. Kenna, J. I. Kourie,

F. Tagliavini, G. Forloni and M. Salmona, Am. J. Physiol.:Cell Physiol., 2003, 285, C862–C872.

479 N. Sanghera and T. J. Pinheiro, J. Mol. Biol., 2002, 315,1241–1256.

480 S. Simoneau, H. Rezaei, N. Sales, G. Kaiser-Schulz,M. Lefebvre-Roque, C. Vidal, J.-G. Fournier, J. Comte,F. Wopfner and J. Grosclaude, PLoS Pathog., 2007, 3, e125.

481 U. K. Resenberger, A. Harmeier, A. C. Woerner,J. L. Goodman, V. Muller, R. Krishnan, R. M. Vabulas,H. A. Kretzschmar, S. Lindquist and F. U. Hartl, EMBO J.,2011, 30, 2057–2070.

482 J. W. Um, H. B. Nygaard, J. K. Heiss, M. A. Kostylev,M. Stagi, A. Vortmeyer, T. Wisniewski, E. C. Gunther andS. M. Strittmatter, Nat. Neurosci., 2012, 15, 1227–1235.

483 R. Linden, V. R. Martins, M. A. Prado, M. Cammarota,I. Izquierdo and R. R. Brentani, Physiol. Rev., 2008, 88,673–728.

484 M. Kristiansen, P. Deriziotis, D. E. Dimcheff, G. S. Jackson,H. Ovaa, H. Naumann, A. R. Clarke, F. W. van Leeuwen,V. Menendez-Benito and N. P. Dantuma, Mol. Cell, 2007,26, 175–188.

485 L. Westergard, H. M. Christensen and D. A. Harris, BBA,Mol. Basis Dis., 2007, 1772, 629–644.

486 M. Jeffrey, G. McGovern, C. M. Goodsir and L. Gonzalez,Brain Pathol., 2009, 19, 1–11.

487 B. Caughey, Trends Biochem. Sci., 2001, 26, 235–242.488 H.-J. Zhang, Y.-H. Lu, Y.-J. Long, Q.-L. Wang, X.-X. Huang,

R. Zhu, X.-L. Wang, L.-P. Liang, P. Teng and H.-Z. Zheng,Anal. Methods, 2014, 6, 2982–2987.

489 L. Zhan, L. Peng and C.-Z. Huang, Chin. Sci. Bull., 2014, 59,964–970.

490 P. Sobrova, I. Blazkova, J. Chomoucka, J. Drbohlavova,M. Vaculovicova, P. Kopel, J. Hubalek, R. Kizek andV. Adam, Prion, 2013, 7, 349–358.

491 M. F. Ottaviani, M. Cangiotti, L. Fiorani, A. Fattori,T. Wasiak, D. Appelhans and B. Klajnert, Curr. Med. Chem.,2012, 19, 5907–5921.

492 T. Kimura, J. Hosokawa-Muto, K. Asami, T. Murai andK. Kuwata, Eur. J. Med. Chem., 2011, 46, 5675–5679.

493 A. Gallardo-Godoy, J. Gever, K. L. Fife, B. M. Silber,S. B. Prusiner and A. R. Renslo, J. Med. Chem., 2011,54, 1010.

494 S. Bongarzone, H. N. A. Tran, A. Cavalli, M. Roberti,M. Rosini, P. Carloni, G. Legname and M. L. Bolognesi,ChemMedChem, 2011, 6, 601–605.

495 R. A. Alvarez-Puebla, A. Agarwal, P. Manna, B. P. Khanal,P. Aldeanueva-Potel, E. Carbo-Argibay, N. Pazos-Perez,L. Vigderman, E. R. Zubarev and N. A. Kotov, Proc. Natl.Acad. Sci. U. S. A., 2011, 108, 8157–8161.

496 S. J. Xiao, P. P. Hu, X. D. Wu, Y. L. Zou, L. Q. Chen, L. Peng,J. Ling, S. J. Zhen, L. Zhan and Y. F. Li, Anal. Chem., 2010,82, 9736–9742.

497 H. N. A. Tran, F. Sousa, F. Moda, S. Mandal, M. Chanana,C. Vimercati, M. Morbin, S. Krol, F. Tagliavini andG. Legname, Nanoscale, 2010, 2, 2724–2732.

498 J. Henry, A. Anand, M. Chowdhury, G. Cote, R. Moreira andT. Good, Anal. Biochem., 2004, 334, 1–8.

499 S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay,F. C. Szoka, F. E. Cohen, S. B. Prusiner and M. R. Scott,J. Virol., 2001, 75, 3453–3461.

1

5

10

15

20

25

30

35

40

45

50

55

1

5

10

15

20

25

30

35

40

45

50

55



500 A. Rangel, N. Madronal, R. Gavın, F. Llorens, L. Sumoy,J. M. Torres, J. M. Delgado-Garcıa and J. A. Del Rıo, PLoSOne, 2009, 4, e7592.

501 H. Khosravani, Y. Zhang, S. Tsutsui, S. Hameed, C. Altier,J. Hamid, L. Chen, M. Villemaire, Z. Ali and F. R. Jirik,J. Cell Biol., 2008, 181, 551–565.

502 S. Weiss, D. Proske, M. Neumann, M. H. Groschup,H. A. Kretzschmar, M. Famulok and E.-L. Winnacker,J. Virol., 1997, 71, 8790–8797.

503 A. S. Boese, A. Majer, R. Saba and S. A. Booth, Expert Opin.Drug Discovery, 2013, 8, 1265–1284.

504 P. Calvo, B. Gouritin, I. Brigger, C. Lasmezas, J.-P. Deslys,A. Williams, J. P. Andreux, D. Dormont and P. Couvreur,J. Neurosci. Methods, 2001, 111, 151–155.

505 G. K. Kouassi, P. Wang, S. Sreevatan and J. Irudayaraj,Biotechnol. Prog., 2007, 23, 1239–1244.

506 M. B. Miller and S. Supattapone, J. Virol., 2011, 85,2813–2817.

507 A. Mange, O. Milhavet, H. E. M. McMahon, D. Casanovaand S. Lehmann, J. Neurochem., 2000, 74, 754–762.

508 A. Mange, N. Nishida, O. Milhavet, H. E. M. McMahon,D. Casanova and S. Lehmann, J. Virol., 2000, 74,3135–3140.

509 A. Mange, N. Nishida, O. Milhavet, H. E. McMahon,D. Casanova and S. Lehmann, J. Virol., 2000, 74,3135–3140.

510 Y. E. Karapetyan, G. F. Sferrazza, M. Zhou, G. Ottenberg,T. Spicer, P. Chase, M. Fallahi, P. Hodder, C. Weissmannand C. I. Lasmezas, Proc. Natl. Acad. Sci. U. S. A., 2013, 110,7044–7049.

511 S. Ghaemmaghami, B. C. May, A. R. Renslo andS. B. Prusiner, J. Virol., 2010, 84, 3408–3412.

512 M. E. Oskarsson, J. F. Paulsson, S. W. Schultz,M. Ingelsson, P. Westermark and G. T. Westermark, Am.J. Pathol., 2015, 185, 834–846.

513 K. Ono, R. Takahashi, T. Ikeda and M. Yamada,J. Neurochem., 2012, 122, 883–890.

514 A.-L. J. Mougenot, A. Bencsik, S. Nicot, J. Vulin, E. Morignat,J. Verchere, D. Betemps, L. Lakhdar, S. Legastelois andT. G. Baron, J. Neuropathol. Exp. Neurol., 2011, 70, 377–385.

515 C. W. Olanow and S. B. Prusiner, Proc. Natl. Acad. Sci. U. S.A., 2009, 106, 12571–12572.

516 M. Beekes, A. Thomzig, W. J. Schulz-Schaeffer andR. Burger, Acta Neurol., 2014, 128, 463–476.

517 E. Tayeb-Fligelman, O. Tabachnikov, A. Moshe,O. Goldshmidt-Tran, M. R. Sawaya, N. Coquelle, J.-P. Colletier and M. Landau, Science, 2017, 355, 831–833.

518 N. Rubin, E. Perugia, M. Goldschmidt, M. Fridkin andL. Addadi, J. Am. Chem. Soc., 2008, 130, 4602–4603.

519 N. Rubin, E. Perugia, S. G. Wolf, E. Klein, M. Fridkin andL. Addadi, J. Am. Chem. Soc., 2010, 132, 4242–4248.

520 I. Usov, J. Adamcik and R. Mezzenga, ACS Nano, 2013, 7,10465–10474.

521 S.-J. Lee, P. Desplats, C. Sigurdson, I. Tsigelny andE. Masliah, Nat. Rev. Neurol., 2010, 6, 702–706.

522 K. Jackson, G. A. Barisone, E. Diaz, L.-w. Jin, C. DeCarli andF. Despa, Ann. Neurol., 2013, 74, 517–526.

523 I. Horvath and P. Wittung-Stafshede, Proc. Natl. Acad. Sci.U. S. A., 2016, 113, 12473–12477.

524 A. Horwich, J. Clin. Invest., 2002, 110, 1221–1232.

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