[Molecular Medicine and Medicinal Chemistry] Alzheimer's Disease: Insights into Low Molecular Weight...

November 26, 2012 10:0 9in x 6in Alzheimer’s Disease: Insights Into Low Molecular … b1377-chA3

3Biophysical Characterization

of Aβ Assembly

Eric Y. Hayden∗ and David B. Teplow∗

3.1 Preface

The amyloid β-protein (Aβ) is a seminal neuropathogenetic agent inAlzheimer’s disease (AD). The pathologic properties of Aβ derive from thepeptide’s ability to self-associate into a variety of oligomeric and polymericforms. Understanding how these structures form and cause neurotoxiceffects is critical to the development of therapeutic approaches with thepotential to prevent, ameliorate, or cure AD. In addition, Aβ is an archetypefor the family of amyloid-forming proteins and peptides that are involvedin other neurodegenerative and systemic amyloidotic disorders. Knowledgegleaned in studies of Aβ will probably be applicable in these other systems.We review here results of experimental and computational studies seekingto understand the biophysics of Aβ. This biophysics encompasses Aβ

conformational dynamics, self-assembly, and biological activity across theentire Aβ assembly spectrum, from the nascent Aβ monomer to the end-stage amyloid fibril.

∗Department of Neurology, David Geffen School of Medicine at UCLA, Brain ResearchInstitute and Molecular Biology Institute, University of California. Los Angeles, CA 90095,USA.

83

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.


84 E. Y. Hayden and D. B. Teplow

3.2 Introduction

Alois Alzheimer described the case of the dementia patient Auguste D.over 100 years ago (Alzheimer, 1906), a description that would leadEmil Kraeplin, in 1910, to name the associated disease “Alzheimer’s dis-ease” (Kraepelin, 1910). AD, like most neurodegenerative disorders, is a dis-ease of aging. Advances in healthcare have led to increased lifespans, alongwith a concurrent increase in AD cases (Brookmeyer et al., 2007). In theUS alone, ≈ 5.3 million suffer from AD. The current worldwide incidenceof dementia is estimated to be 35.6 million, the majority of which are ADcases (Prince and Jackson, 2009). This number is estimated to double every≈ 20 years, to reach 65.7 million in 2030 (Prince and Jackson, 2009). Thereis no cure for AD and the available treatments provide, at best, modest, tem-porary improvements in clinical status. If effective treatments for AD are tobe developed, a deeper understanding of disease mechanism is obligatory.

Two pathognomonic histopathologic features of AD are parenchymalsenile (neuritic) plaques and neurofibrillary tangles, which are composedof Aβ protein and the microtubule-associated protein tau, respectively (fora review, see Selkoe, 2001). Amyloid fibrils of Aβ also can accumulate inthe walls of blood vessels in the brain, a condition termed cerebral amyloidangiopathy (CAA) (Revesz et al., 2002; Murakami et al., 2003), where theycause hemorrhagic forms of AD (Van Broeckhoven et al., 1990).

Current working hypotheses of AD etiology posit a seminal role forAβ. Aβ is a product of a larger precursor protein, the amyloid β-proteinprecursor (AβPP), which is a ubiquitous type I transmembrane proteinthat undergoes a series of endoproteolytic cleavage events to form multipleproducts (Chow et al., 2010), one of which is Aβ (Kang et al., 1987; Tanziet al., 1987). AβPP is cleaved at the plasma membrane or in intracellularorganelles by β-secretase (Vassar et al., 1999), releasing a large N-terminaldomain and creating the Aβ N-terminus. The C-terminal fragment of 99amino acids remains membrane bound, where it is cleaved within its intra-membrane region by γ-secretase to release Aβ and the C-terminal domain.The predominant cleavage products of γ-secretase, Aβ40 and Aβ42, are40 or 42 amino acids long, respectively.

Three genes have been linked to AD, the AβPP gene on chromosome 21(Levy et al., 1990; Goate et al., 1991), presenilin 1 (Kamino et al., 1992),

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.


Biophysical Characterization of Aβ Assembly 85

and presenilin 2 (Scheuner et al., 1996). Down’s syndrome, which iscaused by a triplication of chromosome 21, invariably produces AD-likepathology in patients surviving into their fourth decade (Wisniewski et al.,1985; Lai and Williams, 1989). This is thought to be a gene dosage effectdue to increased AβPP expression. Presenilin is a component of a four-protein complex that comprises γ-secretase (Haass and Steiner, 2002).Mutations in the AβPP or presenilin genes cause autosomal dominantforms of the disease, known as familial AD (FAD), that account for 10–15%of cases (Goate et al., 1991). Mutations cause FAD by increasing AβPPexpression or by increasing the concentration ratio of Aβ42: Aβ40, factsthat emphasize the critical role of Aβ in AD pathogenesis (Selkoe andPodlisny, 2002).

The centrality of Aβ aggregation in AD pathogenesis was recognizedby the National Institutes of Health in the US, which in 2004 funded thelargest collaborative research effort in its history, the Alzheimer’s DiseaseNeuroimaging Initiative (ADNI), on AD biomarkers, imaging, and diseaseprognostication. Using data from patient information entered into theADNI databases, amyloid plaque pathology (as measured by Pittsburghcompound B (PIB) positron emission tomography imaging) was comparedwith hippocampus volume and episodic memory in normal controls (age-,gender-, and education-matched) as compared with PIB-positive mildcognitive impairment (MCI) subjects. Significant correlations were foundbetween episodic memory and hippocampus volume, but the PIB index wasnot found to be significantly associated. These findings support a modelin which Aβ deposition, hippocampal atrophy, and impaired episodicmemory occur sequentially, with Aβ deposition as the primary event in thiscascade. This sequence of events suggests a seminal neuropathogenetic rolefor Aβ in AD (Mormino et al., 2009). The ADNI collaboration reportedthat decreased Aβ42 levels, combined with increased total tau levels, incerebrospinal fluid (CSF) are both early changes that predict progressionfrom amnestic MCI to AD (Khachaturian, 2010). Another recent studyrevealed that the brains of normal adults with low Aβ42 CSF levels (38% ofthe group) shrank twice as much as those with normal Aβ42 levels. Thesepatients were five times more likely to have the APOE4 risk gene and hadhigher levels of tau (Schott et al., 2010).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Figure 3.1. Aβ assembly. Assembly proceeds from Aβ monomers, which exist as anequilibrium mixture of many different conformers, through an oligomer stage (duringwhich precursors that are “on-pathway” and “off-pathway” for fibril formation exist), toprotofibrils (the penultimate precursors of fibrils), and then to fibrils and macroscopic fibrilaggregates. A multitude of amyloid-forming species typically exist at the same time and theprogression or “pathway” towards higher-order assemblies is not necessarily linear.

The ADNI studies, and many others, have led researchers to target Aβ

aggregation and deposition in clinical trials of therapeutics for AD (Yaminet al., 2008). Unfortunately, more than 200 such trials have yielded noeffective therapeutic agents (Mangialasche et al., 2010). The most likelyexplanation for these failures is drug targeting and design. No consensusexists as to the identity of the proximate neurotoxin in AD. Is it the amyloidfibril? Is it a prefibrillar form of Aβ? Is it an Aβ oligomer? Is it a combinationof different types of Aβ assemblies? Answering these questions requiresformal determination of structure–neurotoxicity relationships. We discusshere recent studies of Aβ structure and dynamics, which have revealedunexpected complexity in Aβ assembly (Fig. 3.1). We begin with fibrils,the historical start for the field, continuing with progressively less complexassemblies, and ending with the Aβ monomer.

3.3 The Amyloid Fibril

A natural starting point for studies of AD pathobiology was the amyloidfibril. The partial determination of the primary structure of vascularamyloid formed by Aβ (Glenner and Wong, 1984) led to the cloning ofthe AβPP gene (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al.,

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



1987; Tanzi et al., 1987). This established the complete sequence of Aβ,opening the entire field of study of AβPP and Aβ. Five years later, Hardy andHiggins proposed the “amyloid cascade hypothesis” (Hardy and Higgins,1992). This hypothesis, which posited that amyloid fibrils caused AD,became the primary working hypothesis for the field. As such, tremendousefforts have been and continue to be expended to understand the fibrilformation process and to target therapeutic approaches to it (Yamin et al.,2008). Amyloid fibrils are defined by specific morphological, tinctorial,and structural properties. These properties are a consequence of specificsecondary, tertiary, and quaternary structure characteristics of amyloidfibrils, which we now discuss.

3.3.1 Fibril morphology

Amyloid fibrils typically appear in negative stain transmission electronmicroscopy (TEM) studies as straight, unbranched filaments that are≈ 10 nm in width (Fig. 3.2a). Fibril lengths vary, but often can exceed 1 µm.Fibrils may be composed of multiple filaments, often with helical twists.TEM, combined with solid-state nuclear magnetic resonance spectroscopy(SS-NMR) measurements, have revealed that Aβ40 fibrils exist withmultiple morphologies. For unagitated samples, a periodic twisted fibrilwas observed (50–200 nm period and 12 ± 1 nm maximum width).The predominant morphology for agitated samples was a filament withno resolvable twist (5.5 ± 0.5 nm width) and a tendency to associatelaterally into difilar or oligofilar assemblies. Two-dimensional solid-state13C nuclear magnetic resonance (NMR) spectra of unagitated and agitatedfibrils show pronounced differences in cross-peak patterns by isotropic13C chemical shifts, demonstrating clear differences in core structures ofthe fibrils formed under different conditions. Distinct structures produceddistinct neurotoxic activities, as the unagitated fibrils were significantlymore toxic than the agitated fibrils when tested in cultures of primaryembryonic rat hippocampal neurons (Petkova et al., 2005).

3.3.2 X-ray fiber diffraction

It has been difficult to study amyloid fibrils formed by full-length proteinsusing classical X-ray crystallization approaches because the fibrils do

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Figure 3.2. (a) Amyloid fibrils are composed of long filaments that are visible in negativelystained transmission electron micrographs. (b) The schematic diagram of the cross-β-sheetsin a fibril, with the backbone hydrogen bonds represented by dashed lines, showing therepetitive spacings that give rise to (c) the typical fiber diffraction pattern with a meridionalreflection at ≈ 4.7 Å (black dashed box) and an equatorial reflection at ≈ 6–11 Å (whitedashed box). Used with permission from Greenwald and Riek (2010).

not form crystals. However, fiber diffraction studies have proved useful,revealing that a defining characteristic of amyloid fibrils is the presenceof the cross-β structural motif (Pauling and Corey, 1951; Eanes andGlenner, 1968) (Fig. 3.2c). This motif produces two primary reflections,a strong, sharp, meridional reflection at ≈ 4.7 Å and a slightly more diffuseequatorial reflection at ≈ 10–11 Å. The former reflects the interstrand dis-tances of peptide chains oriented perpendicular to the fibril axis andstabilized by H bonding. The latter corresponds to the spacing between β-sheets (Bonar et al., 1969). Thus, amyloid fibrils are composed of extendedβ-sheets arranged parallel to the fibril axis with their component β-strandsarranged perpendicular to the axis (“cross-β”)(Fig. 3.2b).

3.3.3 X-ray crystallography

Although full-length amyloid proteins have not been crystallized, theEisenberg group has shown that many amyloid peptide fragments can formmicrocrystals that produce high-resolution X-ray diffraction data (Nelsonet al., 2005; Sawaya et al., 2007). Using small amyloidogenic peptide repeats

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



from the yeast prion Sup35, GNNQQNY and NNQQNY, reflections wereobserved for β-strands ≈ 5 Å apart that are perpendicular to the fibril axis,and extended β-sheets ≈ 10 Å apart that are parallel to the fibril axis, bothconsistent with the cross-β-organization of fibrils (Nelson et al., 2005). Thisfinding was significant in that it provided a detailed structure consisting ofa pair of β-sheets whose side-chains interdigitated in an anhydrous crystalcore, which was termed a “steric zipper” (Nelson and Eisenberg, 2006)(Fig. 3.3).

These studies were followed by a tour de force in which Eisenberg’s groupreported studies of 13 amyloid-forming peptide segments derived fromAβ(12-40), tau, prion protein, insulin, islet amyloid polypeptide, lysozyme,myoglobin, α-synuclein, and β2-microglobulin (Sawaya et al., 2007). High-resolution structures obtained from the microcrystals all resembled theone observed in the GNNQQNY and NNQQNY microcrystals, i.e. a stericzipper.

A number of different steric zipper organizations may exist, dependingon whether: (1) their sheets are parallel or antiparallel; (2) sheets packwith the same (face-to-face) or different (face-to-back) surfaces adjacent toone another; or (3) the sheets are oriented parallel (up–up) or antiparallel(up–down) with respect to one another. Combinations of these three struc-tural arrangements yield eight possible classes of steric zippers. Examples offive classes were observed in the 13 microcrystal structures. The definition ofthis common motif extends our understanding of the atomic organizationof the classic cross-β-structure and reveals, at the atomic level, that theredoes not exist a “generic” amyloid structure, as some have argued (Dobson,2003; Kayed et al., 2007).

3.3.4 Nuclear magnetic resonance spectroscopy

NMR studies have been particularly informative with respect toAβ structural organization. An early study by Lansbury et al.(1995) examined the structure of the Aβ(34-42) nonapeptide usingSS-NMR. Their data were consistent with a fibril structure in which the Aβ

peptide backbone existed in an antiparallel β-sheet. Subsequently, evidencefor cross-β-structures with parallel β-strands was provided in studies offibrils formed by Aβ(10-35) (Benzinger et al., 1998; Burkoth et al., 2000).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Figure 3.3. Aβ steric zipper. (a) A pair-of-sheets structure, showing the backbone ofeach β-strand, with side-chains protruding. (b) A steric zipper viewed edge on (down thea-axis), showing the interdigitation of the side-chains from each sheet. (c) The GNNQQNYcrystal viewed down the sheets. Six rows of β-sheets run horizontally. Peptide moleculesare shown in black and water molecules are red plus signs. The atoms in the lower left unitcell are shown as spheres representing van der Waals radii. (d) Close-up view of a pair ofGNNQQNY molecules in the steric zipper conformation, showing the remarkable shapecomplementarity of the Asn and Gln side-chains protruding into the dry interface. Usedwith permission from Nelson et al. (2005).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Tycko and co-workers also applied SS-NMR to the Aβ fibril organizationproblem, observing nearest-neighbor intermolecular distances of 4.8 ±0.5 Å for carbon sites from residues 12-39. These data were consistentwith parallel, in-register β-strands. Additionally, one-dimensional 13Cmagic angle spinning NMR spectra of labeled Aβ40 samples indicatedstructural order at the same core sites. Both experiments revealed structuraldisorder in the N-terminal segment of Aβ40, which included the first nineresidues (Balbach et al., 2002). A key finding was the existence of a turnbetween residues 25 and 29. This turn region also displayed Coulombicinteractions between the Nε cation of Lys28 and the carboxylate anion ofAsp23 (Petkova et al., 2002; Tycko and Ishii, 2003; Tycko, 2006a).

Lührs et al. (2005) proposed a similar model based on hydrogen-bonding constraints from quenched hydrogen/deuterium (H/D) exchangeNMR and side-chain packing constraints from pairwise mutagenesisstudies. This model comprised a β-strand–turn–β-strand motif containingtwo intermolecular, parallel, in-register β-sheets formed by residues 18-26and 31-42. At least two molecules of Aβ42 were required to produce therepeating structure of a protofilament. The N-terminal peptide segment 1-17 was disordered. An important difference between the Tycko and Lührsmodels was the position of the Lys28–Asp23 salt bridge, which in the formermodel was intramolecular but in the latter model was intermolecular.

3.3.5 Hydrogen/deuterium exchange

One of the biological consequences of the cross-β-structure of amyloidfibrils is their exceptional physical stability. This is true of the fibrils ofAβ and those of other amyloid proteins. The archetypal cross-β-proteins,the spider silks, have a tensile strength comparable to that of high-gradesteel (1,500 MPa) (Griffiths and Salanitri, 1980). In the case of the prionagent, which readily forms amyloid fibrils, common methods for steriliza-tion and disinfection cannot inactivate this protein-only pathogen (Gileset al., 2007). However, exceptional stability also provides a basis for thesuccessful implementation of structure determination strategies, such asH/D exchange, that reveal stable and unstable peptide segments withinfolded proteins and protein assemblies. Peptide segment stability is directlyrelated to the ability of the segment to participate in the formation of stable

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



structural elements, including turns, helices, sheets, and intermolecularinterfaces, thus these techniques can provide useful information aboutprotein topology.

The H/D exchange data for Aβ40 fibrils have shown that ≈50% ofbackbone amide protons are highly protected from exchange (Kheterpalet al., 2003). This result is consistent with SS-NMR-derived models in whichtwo β-strand segments span residues 12-24 and 30-40, and the remainingresidues exist in disordered or loop segments. In proteolysis experimentsin vitro, trypsin and chymotrypsin digestion of Aβ40 fibrils showedthat the F4–R5, R5–H6, and Y10–E11 peptide bonds are susceptible toproteolysis (Kheterpal et al., 2001). These results are consistent with theH/D exchange and SS-NMR data described above, with regard to regionsof predicted susceptibility based on the structural model of fibrils.

3.3.6 Secondary structure of fibrils

The existence of extended, oriented β-sheets in the amyloid fibrilcan be detected tinctorially. A number of dyes, including thioflavin T(ThT), thioflavin S, and Congo red, change their chromogenic properties(absorption and emission spectrum changes) when bound to amyloidfibers (Vassar and Culling, 1959; Saeed and Fine, 1967). In addition,a characteristic birefringence (Maltese cross) is observed when cross-polarizers are employed in the fluorescence microscope (Howie and Brewer,2009). These characteristics have also been incorporated into strategies forthe development of assays for amyloid fibril formation (Kirschner et al.,2008). Until recently, our understanding of the physical interaction and ori-entation of amyloidophilic dyes with fibrils has been poor. However, recentexperimental and computational studies have suggested different bindingmodes, including a model wherein the dye molecule binds preferentiallyin parallel with the long axis of the β-sheet (Krebs et al., 2005), which isconsistent with the results of earlier spectroscopic studies (Jin et al., 2003).More recently, Biancalana et al. (2009) found that a five-residue ladder ofamino acids is necessary for achieving amyloid fiber-like affinity for ThT,and a stretch of four amino acids constitutes a weak binding site. Interest-ingly, this residue requirement is consistent with the tip-to-tail length ofThT of 15 Å, and the Cα–Cα distance of a four-residue ladder (14 Å).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



The β-sheet structure of Aβ fibrils has also been revealed by Fourier-transform infrared (FTIR) absorption spectroscopy (Hilbich et al., 1991)and circular dichroism spectroscopy (CD). However, neither techniquereveals exclusively β-structure. For example, CD studies of Aβ40 and Aβ42following fibril assembly in vitro show that levels were ≈ 10% for α-helix,≈ 66% for β-strand, ≈ 5% for β-turn, and ≈ 20% for statistical coil (Majiet al., 2009) (Fig. 3.6).

3.3.7 Toxicity of fibrils

The amyloid cascade hypothesis posited the primacy of fibrils in ADpathogenesis. For this reason, early studies examined fibril toxicity usingcultured neurons or neuronal cell lines. In primary cultures of ratneurons, Aβ fibrils were toxic to both immature and mature hippocampalneurons (Ueda et al., 1994). Aβ40 fibrils were 10-fold more potent thanunassembled Aβ40. In this system, the toxicity of fibrils was also comparedwith that of amorphous aggregates of Aβ, thought to be analogous to diffuseplaques. Diffuse plaques are histologically defined areas of the brain thatstain with antibodies to Aβ, but not tau, and are found predominantlyin the cerebral cortex. The fibrils were toxic but the amorphous materialwas not. Fibrils caused significant loss of synapses in viable neurons,whereas amorphous Aβ had no effect on synapse number (Lorenzo andYankner, 1994). Further evidence of fibril-linked pathogenesis comes fromthe finding that AD lesions have been found immunohistochemically tocontain proteins involved in inflammatory responses (Eikelenboom andStam, 1982; McGeer and Rogers, 1992). Neuritic plaque formation islikely to be a multistep process occurring over many years; thus chronicinflammation resulting from this slow, progressive process could play asignificant role in AD pathogenesis. Although the importance of oligomers(see below) is currently a predominant theme in AD research, the role offibrils, be they toxic or benign, remains undetermined.

3.4 Protofibrils

The existence of fibrils, and their linkage to the neuropathological lesions(senile plaques) present in the AD brain, provided impetus to studiesseeking to determine how the Aβ monomer polymerized into fibrils. Early

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



mechanistic ideas included “one-dimensional crystallization” (Jarrett andLansbury, 1993). This term actually was a misnomer with respect to the Aβ

system because Aβ forms non-crystalline assemblies. However, the termaccurately reflected the fact that, like crystallization, fibril formation isa nucleation-dependent polymerization process (Andreu and Timasheff,1986; Jarrett and Lansbury, 1992; Tobacman and Korn, 1983). Monitoringassembly size by quasi-elastic light-scattering spectroscopy, or secondarystructure by CD, FTIR, or ThT fluorescence, reveals a lag phase, a transitionphase, and a plateau phase (Lomakin et al., 1996, 1997; Kirkitadze et al.,2001; Fezoui and Teplow, 2002; Naiki et al., 1989). During the lag phase,fibril nuclei form. During the transition stage, peptide monomers add tothese nuclei increasing assembly size, and the β-sheet secondary structureincreases. These increases reflect fibril formation and elongation that, inclosed systems, slows and then finally ends when the concentration of freemonomers reaches the critical concentration for fibril formation Cr = 1/K ,where K is the equilibrium constant for fibrilogenesis.

3.4.1 Protofibril structure and growth

Walsh et al. 1997 applied the technique of size-exclusion chromatography(SEC) to the problem of Aβ fibril formation, reasoning that periodicsampling of fibril assembly reactions might reveal states intermediatebetween monomers and fibrils. Using a Superdex 75 column, they observeda continuum of assemblies spanning the separation range of the column(3–70 kDa for globular proteins and 0.5–30 kDa for dextrans) (Fig. 3.4).The most prominent feature of the chromatogram, other than a peakcorresponding to monomers, was a peak eluting just after the void volume,with an apparent mass >100 kDa. The size of this peak increased duringthe initial stages of fibril elongation (Fig. 3.4).

Examination of the components of this peak by TEM of eithernegatively stained or rotary shadowed preparations revealed relatively short(150 nm), flexible, narrow (5 nm) assemblies that often had a beadedmorphology (Walsh et al., 1997, 1999) (Fig. 3.5). These assemblies werenamed “protofibrils”. Harper et al. (1997) using atomic force microscopy(AFM) techniques, contemporaneously reported the identification of

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Figure 3.4. Characterization of Aβ assembly by size-exclusion chromatography. Examina-tion of fibril assembly reactions during the initial growth phase revealed two predominantpeaks, one corresponding to low molecular weight species (monomers and low-orderoligomers, termed “low molecular weight” Aβ) and one comprising protofibrils that elutednear the void volume (V0 of the column). For Aβ40 the low molecular weight peak elutes at≈ 12.4 ml, whereas the gel-excluded peak elutes at ≈ 7.2 ml. Elution positions of molecularweight standards are indicated by arrows, molecular masses in kDa. From Walsh et al. (1997).

Figure 3.5. Protofibril morphology. Transmission electron microscopy of negativelystained protofibrils, isolated by size exclusion chromatography. Scale bar 100 nm.From Walsh et al. (1997).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



protofibrils. They found that Aβ42 formed protofibrils much more rapidlythan did Aβ40, consistent with its connection to early-onset AD.

The kinetics of protofibril formation and disappearance were consistentwith protofibrils being intermediates in the evolution of amyloid fibrils.Protofibrils were observed during the assembly of Aβ40, Aβ42, and theDutch mutant [Gln22]Aβ40. Protofibril formation has since been deter-mined to be an obligate part of Aβ fibril assembly (Kirkitadze et al., 2001)and also to occur during the assembly of other amyloid proteins, includingα-synuclein (Conway et al., 2000, 2001), ABri (the amyloid of familialBritish dementia) (El-Agnaf et al., 2001), ADan (the amyloid protein offamiliar Danish dementia) (Vidal et al., 2000), transthyretin (Lashuel et al.,1998), superoxide dismutase 1 (Rodriguez et al., 2002), and prions (Fischeret al., 1996).

Advances in AFM enabled real-time observation of fibril growth insitu (Goldsbury et al., 1999). These techniques were applied to Aβ assemblyand revealed that Aβ first forms small globular aggregates ≈ 4.5 nmin height that grow slowly and then rapidly disappear in concert withprototypical amyloid fibrils appearing (Blackley et al., 2000; Mastrangeloet al., 2006). These studies suggested that the formation of protofibrilsoccurs by the joining of two single oligomer units of Aβ. Elongation thenproceeds by the addition of more oligomers. Finally, development of theprotofibrils into mature fibrils occurs either by the further addition ofsingle oligomer units of Aβ, addition of Aβ monomers, or addition of otherprotofibrils (Blackley et al., 2000). The maturation process may also involvethe winding of protofibrils and further conformational changes (Harperet al., 1997).

Protofibrils isolated by SEC and examined immediately by CD revealeda spectrum characteristic for β-sheet structure (Walsh et al., 1999).Deconvolution of the spectrum revealed 47% β-structure (β-sheet orβ-turn), 40% random coil, and 13% α-helix. The level of β-sheet isconsistent with, but slightly lower than, that found in fibrils � 50%. Notsurprisingly, protofibrils bound Congo red and ThT in a concentration-dependent manner. A decade ago, when the aforementioned studies wereperformed, the conformational transition thought to operate in Aβ fibrilformation was statistical coil (SC) → β-sheet. The observation that α-helix

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



existed in protofibrils led Kirkitadze et al. (2001) to study the conforma-tional dynamics of 18 physiologically relevant Aβ alloforms. Using CD, theyshowed that a transitory α-helix-containing stage was an obligatory featureof the fibril formation process for all peptides studied. α-Helix formationoccurred just before the stage of assembly during which rapid increases inβ-sheet structure are observed (Fig. 3.6). These data are consistent with theidea that protofibrils are the immediate precursors of fibrils.

3.4.2 Biological activity of protofibrils

To determine whether protofibrils were biologically active, structure–activity studies must be performed rapidly, over a time scale of minutesto hours, before protofibrils mature into fibrils. The (3-(4,5-Dimethyl-thiazol-2-y6)-2,5-diphenylterazoliu bromide (MTT) assay allows the deter-mination, with an assay period of a few hours, of physiologic effectsinduced by treatment of cells with exogenous agents (Shearman et al.,1994). Protofibrils isolated by SEC, and then added to cultured primary

Figure 3.6. Conformational dynamics of Aβ assembly. During Aβ oligomerizationand higher-order assembly, the Aβ monomer undergoes a SC → α-helix → β-sheetconformational transition. Used with permission from Kirkitadze et al. (2001).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



rat cortical neurons, caused a significant (P <0.01) reduction in cellularMTT metabolism, demonstrating that these assemblies were potent neuro-toxins (Walsh et al., 1999). Subsequent electrophysiologic studies confirmedthese results (Hartley et al., 1999).

Strong clinical evidence for Aβ protofibrils as neurotoxic agents inAD is provided by the Arctic mutation, E22G, found in a Swedish FADfamily (Nilsberth et al., 2001). The Arctic mutation has been shown tocause enhanced formation of protofibrils in vitro, both for Aβ40 (Nilsberthet al., 2001) and Aβ42 (Johansson et al., 2006). The mutation also acceleratesintraneuronal Aβ accumulation, which increases with age and predates Aβ

plaque formation in vivo (Lord et al., 2006).

3.5 Oligomers

The discovery and characterization of protofibrils raised the question ofwhether additional, preprotofibrillar intermediates existed. SEC studiesduring fibril formation revealed a continuum of species rather than oneor more stable intermediates (Fig. 3.4). This was not surprising becausenucleation-dependent polymerization processes produce nuclei that arevery short-lived and to which monomers attach very rapidly, resulting infibril elongation and the production of polydisperse populations. However,this polydispersity could be related simply to fibril length, or it could beindicative of species distinct from fibrils, namely oligomers.

3.5.1 Oligomers and sodium dodecyl sulfate-polyacrylamidegel electrophoresis

One of the most difficult aspects of efforts to establish structure–activitycorrelations of Aβ assemblies is the metastability of the assemblies (Teplow,2006). This metastability is a problem from at least two perspectives:(1) large assemblies in equilibrium with smaller assemblies and monomerswill dissociate when isolated from them, according to Le Châtelier’sprinciple (Le Châtelier, 1984); and (2) small assemblies can associate duringexperiments, thus producing effects that may be caused by large assembliesnot originally present. These facts complicate studies seeking to define andcharacterize Aβ oligomers.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



As an example, sodium dodecyl sulfate (SDS) may cause oligomerdissociation, or conversely, peptide oligomerization and aggregation (Bitanet al., 2003a). Aβ is amphipathic and has been shown to form SDS-stable oligomers (Walsh et al., 1997). Nearly identical monomer–trimer–tetramer distributions are observed for Aβ42, regardless of whether itsstarting state is “monomeric”, oligomeric, or fibrillar, when analyzed bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Bitan et al., 2003a,2005; Hepler et al., 2006). Therefore, the use of SDS-PAGE for determiningoligomer distributions is not recommended, unless the assemblies arecovalently associated. Experimental misinterpretation resulting from SDS-gel use is illustrated by structure–activity relationship (SAR) work on Aβ

dimers. In 2007, Shankar et al. suggested that Aβ dimers and trimerswere mediators of hippocampal synapse loss in AD (Shankar et al.,2007). This suggestion was based on determinations of oligomer orderusing SDS-PAGE. Subsequent studies that carefully examined the detailsof the experimental protocol used in the original study revealed thatneurotoxic activity was actually caused protofibrils that formed after dimerisolation (O’Nuallain et al., 2010).

3.5.2 Quantitative determination of the oligomer size frequencydistribution

The identification of postnucleation intermediates required the develop-ment of methods to stabilize the non-covalently associated, metastable Aβ

assemblies. This goal was achieved using the method of photo-inducedcross-linking of unmodified proteins (PICUP). PICUP is a gentle (neutralpH and visible light), rapid (<1 s), efficient (up to ≈ 90%), photochemicalcross-linking method that produces C–C bonds between adjacent, native(no pre-facto chemical modification) peptides (Bitan and Teplow, 2004).Quantitative determination of the oligomer size frequency distribution canthen be done by combining SDS-PAGE, silver staining, and densitometricanalysis.

PICUP was used to determine the initial oligomerization statesof Aβ40 and Aβ42 (Fig. 3.7). Aβ42 formed pentamer/hexamer units,termed paranuclei, that self-associated to form higher-order, protofibril-like oligomers (Bitan et al., 2003a). Aβ40 did not form the pentamer

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Figure 3.7. Non-cross-linked (lanes 1 and 3) or cross-linked (lanes 2 and 4) Aβ40 andAβ42. Intensity profiles of lanes 2 (cross-linked Aβ40) and 4 (cross-linked Aβ42) areshown (left and right of the gel, respectively). Molecular mass standards are shown onthe left in kDa, and arrows on the right indicate oligomer order. From Bitan et al. (2003a).

and hexamer units, instead it was found to exist as a mixture comprisingpredominately monomer, dimer, trimer, and tetramer (Bitan et al., 2003a).The unique ability of Aβ42 to form paranuclei offered an explanation forits strong etiologic linkage to AD (Bitan et al., 2003a).

To understand the effects of primary structure on oligomerization,the effects of amino acid substitutions in various regions of Aβ wereexamined (Bitan et al., 2003b). Aβ42 was most sensitive to changes at theC-terminus. The side-chain of residue 41 was critical for effective formationof paranuclei as well as for self-association of paranuclei into largeroligomers. The side-chain of residue 42 and the C-terminal carboxyl groupfacilitated paranucleus self-association. Substitutions for Phe19 or Ala21appeared to facilitate paranucleus formation in Aβ42 but not in Aβ40.Interestingly, Aβ40 oligomerization was most sensitive to substitutions ofGlu22 or Asp23 and to truncation of the N-terminus. In contrast, Aβ42oligomerization was largely unaffected by substitutions at positions 22 or23 or by N-terminal truncations (Bitan et al., 2003b).

3.5.3 Studying Aβ assembly in the gas phase

Ion mobility spectrometry-mass spectroscopy (IMS-MS) is rapidly becom-ing the method of choice for studies of protein oligomerization. IMS-MS isable to determine oligomer size at high resolution, allowing determinationof the structures of different ions, and even of ions of identical m/z butdifferent structures (Wyttenbach and Bowers, 2003). IMS-MS can be

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



conceptualized as an in vacuo equivalent of SEC or gel electrophoresis,wherein molecules of different size, under the influence of a constantfluid flow or electric field, respectively, move through matrices of definedporosity at different rates (Teplow et al., 2006). “Size”, for IMS-MS, isdetermined as collision cross-section. This parameter is related directlyto the amount of time that it takes from the injection of sample into themass spectrometer source to the time at which the ion is detected at theend of the instrument, the arrival time. Mass spectroscopy data, along witharrival time distributions, allow determination of oligomer order.

Using IMS-MS, Bernstein et al. (2009) have shown that freshlyprepared, aggregate-free preparations of Aβ42 produce monomers, dimers,tetramers, hexamers, and dodecamers. This suggested that the first stepsin protofibril formation could be hexamerization, followed by dihexamerformation. An alloform of Aβ42, [Pro19]Aβ42, which has been shownto assemble very poorly compared with wild-type (WT) peptide (Teplowet al., 1997; Wood et al., 1995), produced abundant monomers, dimers,trimers, and tetramers, but no large oligomers. Recent studies confirmedand extended these results by demonstrating that Aβ40 assembly producesmonomers, dimers, and tetramers, but no larger oligomers (Bernsteinet al., 2009). The experimental cross-sections for the hexamer wereconsistent with a quasi-planar ring structure. Dodecamers could bemodeled most accurately as stacked planar hexamer rings. It is reasonable topostulate that previously described hexamer/dodecamer assemblies, such asparanuclei (Bitan et al., 2003a), Aβ∗56 (Lesné et al., 2006), and Aβ-deriveddiffusible ligands (ADDLs) (Lambert et al., 1998), possess these structures.

3.5.4 Oligomer structure–activity relationships

To rigorously establish the oligomer SAR for Aβ oligomers, Ono et al. tookadvantage of the PICUP approach to produce pure populations of stableoligomers of defined order and then determined the relative neurotoxicactivities of each population (Ono et al., 2009; Rosensweig et al., 2012). Toobtain pure oligomer populations, a method was developed to fractionateand extract PICUP-stabilized oligomers from SDS gels (Ono et al., 2009;Rosensweig et al., 2012). Purities of Aβ40 monomer, dimer, trimer, andtetramer populations ranged from 94 to 99%.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



The CD spectroscopy of the pure oligomer populations revealed anorder-dependent increase in the wavelength of the spectrum minimum.A single minimum at ≈ 210 nm was observed for the Aβ monomerpopulation. The wavelength of this minimum increased to ≈212, ≈214,and ≈ 216 nm for the dimer, trimer, and tetramer populations, respec-tively. Deconvolution of the spectra revealed a direct correlation betweenoligomer order and β-sheet content.

The Aβ assembly morphology was determined using electronmicroscopy (EM) and AFM. EM showed that purified, cross-linkedmonomers displayed irregular thread-like and globular shapes that hadaverage diameters of 1.15–1.30 nm. As oligomer order increased, the relativeamounts of thread-like structures decreased and the amounts of quasi-spherical structures increased. The diameter increased progressively witholigomer order, from 1.78 nm for dimers to 11.00 nm for tetramers. Therank order of heights of monomer, dimer, trimer, and tetramer in AFMstudies (0.24, 0.53, 0.94, and 1.51 nm) was identical to that of the diametersin EM. The magnitude of the size increase observed in both EM andAFM between the monomer and dimer states was disproportionatelylarge compared with those observed for the higher-order transitions.This disproportionate increase was also observed for oligomer secondarystructure. The data suggest that dimerization is a process fundamentallydifferent from trimerization and higher-order assembly (Ono et al., 2009).

A fundamental question in any nucleated assembly process is whetherparticular assemblies can act as nuclei, i.e. can they eliminate the lag phasein the fibril elongation process. We found that purified oligomers as smallas dimers can seed fibril formation. However, they are not as effective astrimers or tetramers, which have an effectiveness closer to that of fibrilsthan to that of monomers (Ono et al., 2009).

To establish rigorous SAR, Ono et al. (2009) first ensured that oligomersto be used in cell culture studies existed in the medium in the samestate throughout the assay period (<2 d). To do so, each pure oligomerpopulation was incubated in cell culture medium for up to 2 d. ThT studiesof the populations revealed that low initial ThT fluorescence intensities wereobserved that did not change significantly over the 2 d period. SDS-PAGEstudies showed that no oligomer dissociation occurred. A small percentage(<10%) of oligomers of order twice that of the original population

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



(e.g. tetramers in the case of the dimer population) was observed bySDS-PAGE. Whether these “2×” oligomers existed natively in the cellculture medium or were formed during the electrophoresis procedure isnot known. Then both MTT and lactate dehydrogenase assays were carriedout to quantify oligomer toxicity. All oligomers were toxic, and their toxicitywas directly related to their order. Larger oligomers were significantly moretoxic than smaller oligomers (Ono et al., 2009).

3.5.5 The “oligomer cascade hypothesis”

Studies in transgenic mouse models of AD and in humans have providedevidence that oligomers are the primary neurotoxins in AD (McLean et al.,1999; Näslund et al., 2000; Wang et al., 2002; Yang et al., 2005; Fagan et al.,2009). Evaluation of neuronal function in transgenic mice expressing Aβ

has revealed neurological deficits prior to amyloid deposition, suggestingthat soluble Aβ assemblies were neurotoxic (Oddo et al., 2003). Subsequentstudies in humans have shown that Aβ is detectable in the brain andCSF and that levels of soluble Aβ measured in the CSF directly predictthe occurrence of AD (Schott et al., 2010). In biomarker studies, it hasbeen shown that levels of CSF tau and ptau181, but not Aβ42, correlatedinversely with the whole-brain volume in very mild and mild dementia ofthe Alzheimer type. However, levels of CSF Aβ42, but not tau or ptau181,were positively correlated with the whole-brain volume in non-dementedcontrol subjects (Fagan et al., 2009).

A variety of neurotoxic oligomeric assemblies have been reportedthat have molecular weights consistent with dodecamers, includingADDLs (Lambert et al., 1998), Aβ∗56 (Lesné et al., 2006), and paranu-clei (Bitan et al., 2003a). ADDLs are 5–6 nm tall globular structures thathave been observed in vitro as dodecamers and detected at high levels inthe brains of patients with AD (Lambert et al., 1998; Gong et al., 2003).ADDLs have been found to be more toxic to PC12 cells than Aβ42 fibrils.In the Tg2576 transgenic mouse model of AD, Aβ∗56, a soluble 56-kDaAβ assembly, is present when neurological dysfunction exists (Lesné et al.,2006). This assembly could be purified from brain extracts and shownto disrupt memory when administered to young rats (Lesné et al., 2006).Paranuclei appear to be the smallest quasi-stable Aβ assemblies formed

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



in vitro (Bitan et al., 2003a). Paranuclei form quickly within aggregate-freeAβ42 solutions, have been shown to multimerize into dodecamers andoctadecamers, and, as discussed in Section 3.5.3, both the paranucleus(hexamer) and “diparanucleus” (dodecamer) forms of Aβ42 are readilyobserved in IMS-MS experiments. (It should be noted, in the case ofdodecameric assemblies, that a consideration of thermodynamics, andOccam’s razor (Re and Bellini, 2002), suggests that ADDLs, Aβ∗56, andparanuclei may be identical.)

Taken together with our own SAR work (Ono et al., 2009), we havepostulated that Aβ oligomers are the proximate neurotoxins in AD and thatthe “amyloid cascade hypothesis” should be supplanted by the “oligomercascade hypothesis” (Ono et al., 2009).

Support for this hypothesis comes from studies that have revealed amultitude of oligomeric Aβ assemblies that are neurotoxic (for a recentreview, see Roychaudhuri et al., 2009). One interesting finding has been theobservation of oligomeric pores and channels formed from Aβ (for a recentreview, see Kagan, 2011). Arispe et al. reported in 1993 that Aβ40 couldform Ca2+-selective ion channels incorporated in lipid bilayers (Arispeet al., 1993). Subsequent work has shown that channels can be formedby Aβ42 (Bhatia et al., 2000), as well as by other amyloid to formproteins. Pore formation, and the consequent disruption Ca2+ homeostasis,may be a common mechanism of neurotoxicity among amyloid proteinoligomers (Quist et al., 2005). Single particle averaging of TEM data fromWT and the Arctic mutant showed distinct sizes of pore-like species,and scanning TEM data revealed that spherical protofibrils, includingannular and large globular structures, showed a size from 80 to 400 kDa,corresponding to 18–93 monomers (Lashuel et al., 2003). Pores formed byα-synuclein and Aβ have similar outer (≈ 7–10 nm) and inner (1.5–2.0 nm)diameters (Lashuel et al., 2002).

Other forms of oligomers have been described. Globulomers areAβ42 oligomers produced by incubating Aβ42 in the presence of deter-gent (Barghorn et al., 2005). This suggests that they are generated byinteraction of Aβ42 with micelles of SDS or fatty acids. Aβ42 globulomerswere neurotoxic to rat brain slices, and globulomer-specific antibodiesstained dendritic processes of neurons but not glia in hippocampal cellcultures (Barghorn et al., 2005). Larger oligomers, termed amylospheroids

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



(ASPDs) are spherical structures of diameter 10–15 nm that have beenfound in vitro and in patients with AD (Hoshi et al., 2003). ASPDs <10 nmin diameter were not toxic, whereas those >10 nm were highly toxic. Aβ42formed ASPDs more rapidly and these ASPDs were more toxic (≈ 100-fold) to neurons at lower concentrations than were ASPDs formed byAβ40 (Hoshi et al., 2003).

3.5.6 Statistical biophysics and “the most toxic oligomer”

One of the most significant questions in the field is, “what is the most toxicoligomer”? Much depends on the answer to this question, including thedetermination of an appropriate therapeutic target for medicinal chemists,a disease marker for AD, and an immunogen for immunotherapeutics.Which of the many structures is the proximate neurotoxin in AD?Attempts to answer such a question have been reported in the prionfield (Silveira et al., 2005). However, for AD (and probably prions as well),we argue that the question is “ill-posed”, i.e. there does not exist a singlesolution (Hadamard, 1901, 1902). Furthermore, even if such an entity didexist, it would be unlikely to be the most attractive therapeutic target. Why?The answer lies in the necessity to consider the Aβ system, and AD, in thecontext of statistical distributions and not in terms of simple unimodalcause and effect relationships.

It is typical to interpret Aβ SAR experiments using the total concentra-tion of Aβ in a preparation as one metric and the magnitude of an effectas the other metric. Interpretations derived from this type of analysis aremeaningful, in some respects, but not in the respect that they reflect realityin systems of equivalent concentration but different oligomer composition.This is because the same concentration of total Aβ in a preparation ofmonomers produces twice the number concentration as does a solution ofAβ dimers, and so forth as oligomer order increases. When we consider thissimple fact and examine results of studies of the toxicity of Aβ oligomers, wefind that dimers are ≈ 3-fold more toxic than monomers, and tetramers are≈13-fold more toxic. The “specific activity” of Aβ assemblies thus dependsnon-linearly on oligomer order.

We cannot, at this point, create a mathematical formulation definingtoxicity vs. oligomer order. Are tetramers the most toxic assembly? Aredodecamers the most toxic? We do not know. Determination of a complete

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



toxicity/order correlation is not yet feasible because of the progressivedecrease in occurrence frequency of higher-order oligomers in our in vitrosystems. However, and of great significance, is the fact that this decreasein occurrence frequency would also be expected in vivo. Simple chemicalkinetics predicts that the occurrence frequency fi = kpi for an oligomerof order i, in a unitary system, where p represents the probability of aneffective interaction between two monomers (they stick together) and kis a proportionality constant. In the complex milieu of the brain, thisrelationship also must hold, but with many factors modifying it, includingdifferences in local (intracellular, extracellular, tissue type, CSF, plasma)Aβ concentration, molecular chaperones, catabolic processes, molecularcrowding, and the propensity for higher order assembly (resulting indecreasing oligomer concentration). Given these latter considerations,no single neurotoxin exists. Instead, in a given micromilieu in the body,toxicity T = ∑n

i=1 fi ti , where ti is the specific toxicity of an assembly oforder i.

When one considers that the occurrence frequency of an oligomerhas a negative exponential dependence on oligomer order, and that smallassemblies can nucleate rapid Aβ polymerization, the steady-state levels ofany oligomer must be relatively low. This logic suggests that a strategytargeting low-order oligomers, e.g. dimers to hexamers, is reasonablebecause these targets probably contribute most substantially to ADneurotoxicity. The larger of these assemblies is significantly more toxic thanthe smaller, but the concentration of these highly toxic larger oligomers willbe lower, so long as they are not covalently associated. It must be noted,however, that fibrils (very high i), even though some consider them to bebenign (Haass and Selkoe, 2007), are the most abundant Aβ assembliespresent in the AD brain. Thus, even with a low level of toxicity, andbecause they remain in the brain parenchyma for years to decades, theycould produce significant damage.

3.6 Conformational Dynamics of the Aβ Monomer

We end this chapter at the beginning, namely with a consideration ofthe Aβ monomer, from which all higher-order assemblies evolve. The Aβ

monomer exhibits the greatest conformational diversity among all the Aβ

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



assemblies. NMR studies have shown that the monomer in solution ishighly disordered, existing largely as a series of loops and turns (Zhanget al., 2000; Petkova et al., 2002). Careful analysis of NMR data onAβ(10-35) revealed a central hydrophobic cluster (CHC) from Leu17–Ala21. This hydrophobic cluster is at the center of an extended core, butsolvent shielding for the hydrophobic surfaces of the residues within thehydrophobic cluster is relatively incomplete. Toward the amino terminus,residues 14-17 form a loop, whereas residues 10-14 are extended into astrand folding back toward the hydrophobic cluster. The partial orderingseems to result from general partitioning of hydrophobic residues awayfrom the solvent, along with the extension of polar side-chains toward thewater phase (Zhang et al., 2000). Differences do apparently exist betweenAβ40 and Aβ42, the latter peptide displaying decreased flexibility at itsC-terminus (Lazo et al., 2005). More recent studies also confirmed therigidity of the Aβ42 C-terminus as compared with Aβ40 (Yan and Wang,2006). The inclusion of detergents, such as SDS, can produce helicalconformers (Shao et al., 1999), but these conformers are unlikely to occurin vivo in the neuronal cytoplasm or the extracellular milieu, unless thepeptide is integrally associated with membranes or lipoprotein particles.

The CD experiments also reveal coil structure in the Aβ monomer(Kirkitadze et al., 2001; Maji et al., 2009). CD data are population averagedata. They comprise the summation of the spectra for each individualpeptide and cannot provide single-molecule information. However, unlikeNMR studies of Aβ aggregation, secondary structure changes associatedwith Aβ conformational organization and assembly can be monitoredfairly easily by CD. As discussed in Section 3.4.1, Aβ undergoes anSC → α-helix → β-sheet conformational transition during assembly intofibrils (Kirkitadze et al., 2001). The α-helix stage is transitory and occurscoincident with the formation of high-order oligomers that precede fibrils.

3.6.1 The Aβ(21-30) turn region

Lazo et al. took a non-spectrometric approach towards elucidatingmonomer structure by using the technique of limited proteolysis (Lazoet al., 2005). This approach has proven useful in domain analysis of proteinswith native folds (Teplow et al., 1988) and in studies of time-dependent

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



changes in peptide chain organization (Fontana et al., 2004). These exper-iments involved brief treatment of Aβ populations with endoproteinasesunder non-denaturing conditions at low enzyme : substrate ratios. Massspectrometric determination of the resulting endoproteolytic productsrevealed regions that were resistant or sensitive to protease action. Theinterpretation of these data was that resistant regions were partially or fullyfolded, whereas the sensitive regions were not. The Ala21–Ala30 region inboth Aβ40 and Aβ42 peptide was resistant to seven different proteases. Inaddition, and surprisingly considering its small size, the correspondingdecapeptide was also protease resistant. Solution-state NMR studies ofthe Aβ(21-30) decapeptide revealed a turn structure involving residues24-28 (Lazo et al., 2005). This structure was stabilized by Coulombicinteractions between the Lys28 Nε cation and the carboxylate groups ofeither Glu22 or Asp23, in addition to hydrophobic interactions between theisopropyl and n-butyl side-chains of Val24 and Lys28, respectively. Basedon these data, the authors hypothesized that this turn structure served as amonomer folding nucleus.

A test of the hypothesis was to determine if mutations linked to FADor CAA, and that cause amino acid substitutions in the 21-30 region ofAβ, altered fold structure or stability. To explore this question, Grantet al. (2007) studied Aβ peptides containing amino acid substitutions atAla21, Glu22, or Asp23. These included the Flemish (A21G) (Hendrikset al., 1992), Arctic (E22G) (Kamino et al., 1992; Nilsberth et al., 2001),Dutch (E22Q) (Levy et al., 1990; Van Broeckhoven et al., 1990), Italian(E22K) (Tagliavini et al., 1999), and Iowa (D23N) (Grabowski et al.,2001) amino acid substitutions. In addition, an [Orn23]Aβ(21-30) peptidewas synthesized to model the Italian (E22K) substitution within anAsp side-chain. Protease sensitivity was distributed among three groups:Asp23Orn, Glu22Gly, Asp23Gly > Asp23Asn, Glu22Lys > Glu22Gln, WT,Ala21Gly (Grant et al., 2007). NMR studies revealed that, to various degrees,all of the amino acid substitutions increased Lys28 side-chain mobility, aproxy for turn destabilization.

Additional experimental support for the structural organization anddynamics of the 21-30 turn region has come recently from studies ofAβ(18-41) crystallized as a fusion protein within the CDR3 loop regionof a shark immunoglobulin single variable domain (Streltsov et al., 2011).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



The unit cell was a dimer of tightly associated dimers (a tetramer). Eachof the monomers within the tetramer displayed a 24VGSNK28 turn thatwas stabilized by hydrophobic, Coulombic, and H bond interactions. TheLys28 side-chain was oriented on one side of the plane of the loop orthe other, depending on its location within the tetramer. This Lys28 side-chain “flipping” had been observed dynamically in simulations of the21–30 turn (Borreguero et al., 2005). It is interesting to consider whatthe Aβ monomer structure would be in a construct extending to Ala42.Each of the last two residues of Aβ42 is critical in controlling peptidefolding and oligomerization (Bitan et al., 2003a), thus it is possible thatsignificant differences might exist in an Aβ(18-42)-containing crystal. Ofcourse, the effects of “caging” an Aβ peptide in a fusion protein crystal alsohave the potential of producing structures not present within the solubleoligomer.

3.6.2 Differences in Aβ40 and Aβ42 monomer dynamics

Replica-exchange molecular dynamics simulations were performed todetermine free energy surfaces for Aβ40 and Aβ42 (Yang and Teplow,2008). The surfaces of both peptides were characterized by two large basins,comprising conformers with either substantial α-helix or β-sheet content.Conformational transitions within and between these basins were rapid.The two additional hydrophobic residues at the Aβ42 C-terminus increasedcontacts within the C-terminus and led to more contact between the C-terminus and the central hydrophobic cluster (Leu17–Ala21). This madethe β-structure of Aβ42 more stable than that of Aβ40, thus shifting theconformational equilibrium in Aβ42 towards β-structure.

Other studies, both experimental and computational, have suggestedthat the conformational dynamics of Aβ40 and Aβ42 are similar, withthe exception of the dynamics at their C-termini (Sgourakis et al., 2007;Hou et al., 2004; Yan and Wang, 2006). It has been reported that the C-terminus of Aβ42 is more rigid than that of Aβ40 (Yan and Wang, 2006),consistent with the increase in intramolecular contact frequency that weobserved (Yang and Teplow, 2008) (Fig. 3.8). An important observationfrom limited proteolysis studies of full-length Aβ40 and Aβ42 was thatthe Val39–Val40 peptide bond was protease sensitive in Aβ40, but not

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Figure 3.8. The energy surfaces for (a) Aβ40 and (b) Aβ42, and representative structuresfor various regions. Used with permission from Yang and Teplow (2008).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



in Aβ42 (Lazo et al., 2005). The Val40–Ile41 peptide bond in Aβ42 wasalso protease resistant, but could be cleaved in the presence the chaotropeGu·HCl. Substantial support therefore exists for the hypothesis that a C-terminal fold requiring stabilization by Ile41–Ala42 is responsible for thedistinct oligomerization differences between Aβ40 and Aβ42, and as aconsequence, their differing contributions to AD pathogenesis.

From the preceding discussion, and from first principles, it is clear thatthe C-terminus of Aβ is critical in determining peptide monomer foldingand self-association behavior, which determines the subsequent biologicalactivity.

3.6.3 Scanning Tyr mutagenesis

However, the C-terminus does not exist in isolation. It is a part of the full-length Aβ peptide and it can interact with other peptide regions. This isclear from NMR studies of Aβ fibrils, but it is also clear from the fact thatthe monomer folding nucleus folds the monomer into a hairpin that hasthe potential to place the C-terminus in proximity to other portions of thepeptide.

Maji et al. (2005) used intrinsic Tyr fluorescence (Lakowicz, 1999)to monitor assembly-dependent changes in Aβ structure (Maji et al.,2005). Ring fluorescence can be quenched by exposure to hydrated peptidecarbonyl groups and through hydrogen bond formation with peptidecarbonyls or with carboxylate side-chains of aspartic or glutamic acid (Rosset al., 1992; Cowgil, 1976). By using Tyr as a probe of Aβ structure, no non-native amino acids need to be incorporated into the sequence. Probingthe local environment at position 10 thus can be done using the nativeprotein, and with periodic substitutions of Tyr from the N-terminus to theC-terminus, distinct regions of each Aβ isoform can be probed.

Although the sequences of Aβ40 and Aβ42 are identical throughVal40, the fluorescence intensities of the peptides in which single Tyrsubstitutions were made at positions 1, 10, 20, and 30 were significantlydifferent between the isoforms. With the exception of the WT peptides andthe C-terminally labeled peptides [Tyr40]Aβ40 and [Tyr42]Aβ42, whichproduced similar initial intensities, all of the Aβ42 peptides fluoresced withintensities 2–2.5-fold higher than those of the Aβ40 series. During assembly,

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



all forms except [Tyr20]Aβ40 displayed modest monotonic decreases influorescence intensity. [Tyr20]Aβ40 displayed a sigmoidal increase influorescence intensity that was not seen with [Tyr20]Aβ42. Instead, allAβ42 peptides exhibited a temporal decrease in fluorescence intensity, butone that was significantly faster than that of the Aβ40 peptides. These datashow that substantial conformational differences exist between the Aβ40and Aβ42 peptide families in regions throughout the peptide, not only atthe C-terminus (Maji et al., 2005).

The CD studies of the assembly of these peptides showed that allunderwent SC → α/β →β secondary structure transitions (Maji et al.,2009). However, substitution-dependent alterations in assembly kineticsand conformer complexity were observed. Tyr1-substituted homologuesof Aβ40 and Aβ42 assembled the slowest and yielded unusual patternsof oligomer bands in gel electrophoresis experiments, suggesting oligomercompaction had occurred. Consistent with this suggestion was the observa-tion of relatively narrow [Tyr1]Aβ40 fibrils by EM. Substitution of Aβ40 atthe C-terminus decreased the population conformational complexity andsubstantially extended the highest order of oligomers observed. This lattereffect was observed in both Aβ40 and Aβ42 as the Tyr substitution positionnumber increased.

The ability of a single amino acid substitution at the N-terminus toalter the oligomer frequency distribution and the assembly kinetics suggeststhat the N-terminus is not a peptide sub-region uninvolved in peptideassembly, as might be inferred from studies of fibril structure (Tycko,2006b). Rather, the contribution of this region in the WT peptide to theoverall assembly energetics is significant but differs in magnitude from thecontributions of the CHC and C-terminus. In the PICUP experiments,increased frequencies were observed for higher-order oligomers formed bythe peptides in which the Tyr was substituted at Ala30, Val40, or Ala42.These data could be explained by the effect of increased hydrophobicityof the substituted Tyr residue relative to the Ala and Val residues replaced,which results in increased stability of the C-terminal turn or of the structureformed through interaction of the central hydrophobic cluster and theC-terminus. This explanation also provides a mechanistic rationale forthe increased oligomerization potential of the N-terminally substituted

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



peptides, in which Tyr replaced Asp. This substitution produces a morehydrophobic N-terminus, the increased solvophobic nature of which wouldfavor intramolecular interactions with the apolar central hydrophobiccluster (Urbanc et al., 2004; Maji et al., 2009).

References

Alzheimer, A. (1906). Über einen eigenartigen schweren Erkrankungsprozeß derHirnrinde, Neurol. Centralblatt., 23, 1129–1136.

Andreu, J.M. and Timasheff, S.N. (1986). The measurement of cooperative proteinself-assembly by turbidity and other techniques, Methods Enzymol., 130,47–59.

Arispe, N., Rojas, E. and Pollard, H.B. (1993). Alzheimer disease amyloid β proteinforms calcium channels in bilayer membranes: blockade by tromethamineand aluminum, Proc. Natl. Acad. Sci. U.S.A., 90, 567–571.

Balbach, J.J., Petkova, A.T., Oyler, N.A., Antzutkin, O.N., Gordon, D.J., Mered-ith, S.C. and Tycko, R. (2002). Supramolecular structure in full-lengthAlzheimer’s β-amyloid fibrils: evidence for a parallel β-sheet organiza-tion from solid-state nuclear magnetic resonance, Biophys. J., 83 (2),1205–1216.

Barghorn, S., Nimmrich, V., Striebinger, A., Krantz, C., Keller, P., Janson, B., Bahr,M., Schmidt, M., Bitner, R.S., Harlan, J., Barlow, E., Ebert, U. and Hillen, H.(2005). Globular amyloid β-peptide1−42 oligomer — a homogenous andstable neuropathological protein in Alzheimer’s disease, J. Neurochem., 95(3), 834–847.

Benzinger, T.L., Gregory, D.M., Burkoth, T.S., Miller-Auer, H., Lynn, D.G.,Botto, R.E. and Meredith, S.C. (1998). Propagating structure of Alzheimer’sβ-amyloid(10-35) is parallel β-sheet with residues in exact register, Proc.Natl. Acad. Sci. U.S.A., 95 (23), 13407–13412.

Bernstein, S.L., Dupuis, N.F., Lazo, N.D.,Wyttenbach, T., Condron, M.M., Bitan, G.,Teplow, D.B., Shea, J.-E., Ruotolo, B.T., Robinson, C.V. and Bowers, M.T.(2009). Amyloid-β protein oligomerization and the importance of tetramersand dodecamers in the aetiology of Alzheimer’s disease, Nat. Chem., 1 (4),326–331.

Bhatia, R., Lin, H. and Lal, R. (2000). Fresh and globular amyloid β protein (1-42) induces rapid cellular degeneration: evidence for AβP channel-mediatedcellular toxicity, FASEB J., 14, 1233–1243.

Biancalana, M., Makabe, K., Koide, A. and Koide, S. (2009). Molecular mechanismof thioflavin-T binding to the surface of β-rich peptide self-assemblies, J.Mol. Biol., 385 (4), 1052–1063.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Bitan, G. and Teplow, D.B. (2004). Rapid photochemical cross-linking a new toolfor studies of metastable, amyloidogenic protein assemblies, Acc. Chem. Res.,37 (6), 357–364.

Bitan, G., Kirkitadze, M.D., Lomakin, A., Vollers, S.S., Benedek, G.B. and Teplow,D.B. (2003a). Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerizethrough distinct pathways, Proc. Natl. Acad. Sci. U.S.A., 100 (1), 330–335.

Bitan, G., Vollers, S.S. and Teplow, D.B. (2003b). Elucidation of primary structureelements controlling early amyloid β-protein oligomerization, J. Biol. Chem.,278 (37), 34882–34889.

Bitan, G., Fradinger, E.A., Spring, S.M. and Teplow, D.B. (2005). Neurotoxicprotein oligomers what you see is not always what you get, Amyloid, 12 (2),88–95.

Blackley, H.K., Sanders, G.H., Davies, M.C., Roberts, C.J., Tendler, S.J. andWilkinson, M.J. (2000). In-situ atomic force microscopy study of β-amyloidfibrillization, J. Mol. Biol., 298 (5), 833–840.

Bonar, L., Cohen, A.S. and Skinner, M.M. (1969). Characterization of the amyloidfibril as a cross-β protein, Proc. Soc. Exp. Biol. Med., 131 (42), 1373–1375.

Borreguero, J.M., Urbanc, B., Lazo, N.D., Buldyrev, S.V., Teplow, D.B. andStanley, H.E. (2005). Folding events in the 21–30 region of amyloid β-protein(Aβ) studied in silico, Proc. Natl. Acad. Sci. U.S.A., 102 (17), 6015–6020.

Brookmeyer, R., Johnson, E., Ziegler-Graham, K. and Arrighi, H.M. (2007).Forecasting the global burden of Alzheimer’s disease, Alzheimers Dement.,3 (3), 186–191.

Burkoth, T.S., Benzinger, T.L.S., Urban, V., Morgan, D.M., Gregory, D.M., Thiya-garajan, P., Botto, R.E., Meredith, S.C. and Lynn, D.G. (2000). Structure ofthe β-amyloid(10−35) fibril, J. Am. Chem. Soc., 122, 7883–7889.

Chow, V.W., Mattson, M.P., Wong, P.C. and Gleichmann, M. (2010). An overviewof APP processing enzymes and products, Neuromolec. Med., 12 (1), 1–12.

Conway, K.A., Harper, J.D. and Lansbury, P.T. (2000). Fibrils formed in vitro fromalpha-synuclein and two mutant forms linked to Parkinson’s disease aretypical amyloid, Biochemistry, 39 (10), 2552–2563.

Conway, K.A., Rochet, J.C., Bieganski, R.M. and Lansbury, P.T. (2001). Kineticstabilization of the α-synuclein protofibril by a dopamine-α-synucleinadduct, Science, 294 (5545), 1346–1349.

Cowgil, R.W. (1976). Biochemical Fluorescence, Marcel Dekker, New York.Dobson, C.M. (2003). Protein folding and misfolding, Nature, 426 (6968),

884–890.Eanes, E.D. and Glenner, G.G. (1968). X-ray diffraction studies on amyloid

filaments, J. Histochem. Cytochem., 16 (11), 673–677.Eikelenboom, P. and Stam, F.C. (1982). Immunoglobulins and complement factors

in senile plaques. An immunoperoxidase study, Acta Neuropathol., 57 (2–3),239–242.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



El-Agnaf, O.M., Nagala, S., Patel, B.P. and Austen, B.M. (2001). Non-fibrillaroligomeric species of the amyloid ABri protein, implicated in familial Britishdementia, are more potent at inducing apoptotic cell death than protofibrilsor mature fibrils, J. Mol. Biol., 310, 157–168.

Fagan, A.M., Head, D., Shah, A.R., Marcus, D., Mintun, M., Morris, J.C. andHoltzman, D.M. (2009). Decreased cerebrospinal fluid Aβ(42) correlateswith brain atrophy in cognitively normal elderly, Ann. Neurol., 65 (2),176–183.

Fezoui, Y. and Teplow, D.B. (2002). Kinetic studies of amyloid β-protein fibrilassembly. Differential effects of alpha-helix stabilization, J. Biol. Chem., 277(40), 36948–36954.

Fischer, M., Rülicke, T., Raeber, A., Sailer, A., Moser, M., Oesch, B., Brandner, S.,Aguzzi, A. and Weissmann, C. (1996). Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie,EMBO J., 15 (6), 1255–1264.

Fontana, A., de Laureto, P.P., Spolaore, B., Frare, E., Picotti, P. and Zambonin, M.(2004). Probing protein structure by limited proteolysis, Acta Biochim. Pol.,51 (2), 299–321.

Giles, K., Supattapone, S., Peretz, D., Glidden, D.V., Baron, H. and Prusiner, S.B.(2007). Disinfection of Prisons, in Zhu, P.C. (ed) New Biocides Development,Oxford University Press, New York, pp. 52–74.

Glenner, G.G. and Wong, C.W. (1984). Alzheimer’s disease: initial report of thepurification and characterization of a novel cerebrovascular amyloid protein,Biochem. Biophys. Res. Commun., 120, 885–890.

Goate, A., Chartier-Harlin, M.C., Mullan, M., Brown, J., Crawford, F., Fidani, L.,Giuffra, L., Haynes, A., Irving, N. and James, L. (1991). Segregation ofa missense mutation in the amyloid precursor protein gene with familialAlzheimer’s disease, Nature, 349 (6311), 704–706.

Goldgaber, D., Lerman, M.I., McBridge, O.W., Saffiotti, V. and Gajdusek, D.C.(1987). Characterization and chromosomal localization of a cDNA encodingbrain amyloid of Alzheimer’s disease, Science, 235 877–880.

Goldsbury, C., Kistler, J., Aebi, U., Arvinte, T. and Cooper, G.J. (1999). Watchingamyloid fibrils grow by time-lapse atomic force microscopy, J. Mol. Biol., 285(1), 33–39.

Gong, Y., Chang, L., Viola, K.L., Lacor, P.N., Lambert, M.P., Finch, C.E., Krafft, G.A.and Klein, W.L. (2003). Alzheimer’s disease-affected brain: presence ofoligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversiblememory loss, Proc. Natl. Acad. Sci. U.S.A., 100 (18), 10417–10422.

Grabowski, T.J., Cho, H.S., Vonsattel, J.P.G., Rebeck, G.W. and Greenberg, S.M.(2001). Novel amyloid precursor protein mutation in an Iowa family withdementia and severe cerebral amyloid angiopathy, Ann. Neurol., 49 (6),697–705.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Grant, M.A., Lazo, N.D., Lomakin, A., Condron, M.M., Arai, H., Yamin, G.,Rigby, A.C. and Teplow, D.B. (2007). Familial Alzheimer’s disease mutationsalter the stability of the amyloid β-protein monomer folding nucleus, Proc.Natl. Acad. Sci. U.S.A., 104, 16522–16527.

Greenwald, J. and Riek, R. (2010). Biology of amyloid: structure, function, andregulation, Structure, 18 (10), 1244–1260.

Griffiths, J.R. and Salanitri, V.R. (1980). The strength of spider silk, J. Mat. Sci., 15,491–496.

Haass, C. and Selkoe, D.J. (2007). Soluble protein oligomers in neurodegeneration:lessons from the Alzheimer’s amyloid β-peptide, Nat. Rev. Mol. Cell. Biol., 8(2), 101–112.

Haass, C. and Steiner, H. (2002). Alzheimer disease γ-secretase: a complex story ofGxGD-type presenilin proteases, Trends Cell. Biol., 12(12), 556–562.

Hadamard, J. (1901). Sur l’iteration et les solutions asymptotiques des equationsdifferentielles, Bull. Soc. Math., 29, 224–228.

Hadamard, J. (1902). Sur les problèmes aux dérivés partielles et leur significationphysique, Princeton Univ. Bull., 13, 49–52.

Hardy, J.A. and Higgins, G.A. (1992). Alzheimer’s disease: the amyloid cascadehypothesis, Science, 256, 184–185.

Harper, J.D., Lieber, C.M. and Lansbury, P.T. (1997). Atomic force microscopicimaging of seeded fibril formation and fibril branching by the Alzheimer’sdisease amyloid-β protein, Chem. Biol., 4 (12), 951–959.

Hartley, D.M., Walsh, D.M., Ye, C.P.P., Diehl, T., Vasquez, S., Vassilev, P.M.,Teplow, D.B. and Selkoe, D.J. (1999). Protofibrillar intermediates of amy-loid β-protein induce acute electrophysiological changes and progressiveneurotoxicity in cortical neurons, J. Neurosci., 19 (20), 8876–8884.

Hendriks, L., van Duijn, C.M., Cras, P., Cruts, M., Van Hul, W., van Harskamp, F.,Warren, A., McInnis, M.G., Antonarakis, S.E., Martin, J.J., Hofman, A. andVan Broeckhoven, C. (1992). Presenile dementia and cerebral haemorrhagelinked to a mutation at codon 692 of the β-amyloid precursor protein gene,Nat. Genet., 1 (3), 218–221.

Hepler, R.W., Grimm, K.M., Nahas, D.D., Breese, R., Dodson, E.C., Acton, P.,Keller, P.M., Yeager, M., Wang, H., Shughrue, P., Kinney, G. and Joyce, J.G.(2006). Solution state characterization of amyloid β-derived diffusibleligands, Biochemistry, 45 (51), 15157–15167.

Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C.L. and Beyreuther, K. (1991).Aggregation and secondary structure of synthetic amyloid β A4 peptides ofAlzheimer’s disease, J. Mol. Biol., 218 (1), 149–163.

Hoshi, M., Sato, M., Matsumoto, S., Noguchi, A., Yasutake, K., Yoshida,N. and Sato, K. (2003). Spherical aggregates of β-amyloid (amylospheroid)show high neurotoxicity and activate tau protein kinase I/glycogen synthasekinase-3β, Proc. Natl. Acad. Sci. U.S.A., 100 (11), 6370–6375.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Hou, L., Shao, H., Zhang, Y., Li, H., Menon, N.K., Neuhaus, E.B., Brewer, J.M.,Byeon, I.-J.L., Ray, D.G.,Vitek, M.P., Iwashita, T., Makula, R.A., Przybyla, A.B.and Zagorski, M.G. (2004). Solution NMR studies of the Aβ(1-40) and Aβ(1-42) peptides establish that the Met35 oxidation state affects the mechanismof amyloid formation, J. Am. Chem. Soc., 126 (7), 1992–2005.

Howie, A.J. and Brewer, D.B. (2009). Optical properties of amyloid stained byCongo red: history and mechanisms, Micron., 40 (3), 285–301.

Jarrett, J.T. and Lansbury, P.T. (1992). Amyloid fibril formation requires a chemi-cally discriminating nucleation event: studies of an amyloidogenic sequencefrom the bacterial protein OsmB, Biochemistry, 31 (49), 12345–12352.

Jarrett, J.T. and Lansbury, P.T. (1993). Seeding “one-dimensional crystallization” ofamyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie? Cell.,73, 1055–1058.

Jin, L.-W., Claborn, K.A., Kurimoto, M., Geday, M.A., Maezawa, I., Sohraby, F.,Estrada, M., Kaminksy, W. and Kahr, B. (2003). Imaging linear birefringenceand dichroism in cerebral amyloid pathologies, Proc. Natl. Acad. Sci. U.S.A.,100 (26), 15294–15298.

Johansson, A.-S., Berglind-Dehlin, F., Karlsson, G., Edwards, K., Gellerfors, P.and Lannfelt, L. (2006). Physiochemical characterization of the Alzheimer’sdisease-related peptides Aβ1-42Arctic and Aβ1-42wt, FEBS J., 273 (12),2618–2630.

Kagan, B.L. (2011). Membrane pores in the pathogenesis of neurodegenerativedisease, in Teplow, D.B. (ed) Molecular Biology of Neurodegenerative Diseases,Academic Press, San Diego, CA, pp. 295–326.

Kamino, K., Orr, H.T., Payami, H., Wijsman, E.M., Alonso, M.E., Pulst, S.M.,Anderson, L., O’dahl, S., Nemens, E. and White, J.A. (1992). Linkage andmutational analysis of familial Alzheimer disease kindreds for the APP generegion, Am. J. Hum. Genet. 51 (5), 998–1014.

Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik,K.H., Multhaup, G., Beyreuther, K. and Muller-Hill, B. (1987). The precursorof Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor,Nature, 325, 733–736.

Kayed, R., Head, E., Sarsoza, F., Saing, T., Cotman, C.W., Necula, M., Margol, L.,Wu,J., Breydo, L., Thompson, J.L., Rasool, S., Gurlo, T., Butler, P. and Glabe, C.G.(2007). Fibril specific, conformation dependent antibodies recognize ageneric epitope common to amyloid fibrils and fibrillar oligomers that isabsent in prefibrillar oligomers, Mol. Neurodegener., 2, 18.

Khachaturian, Z.S. (2010). Perspective on the Alzheimer’s disease neuroimaginginitiative: progress report and future plans, Alzheimers Dement., 6 (3),199–201.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Kheterpal, I., Williams, A., Murphy, C., Bledsoe, B. and Wetzel, R. (2001).Structural features of the Aβ amyloid fibril elucidated by limited proteolysis,Biochemistry, 40, 11757–11767.

Kheterpal, I., Lashuel, H.A., Hartley, D.M., Walz, T., Lansbury, P.T. and Wetzel, R.(2003). Aβ protofibrils possess a stable core structure resistant to hydrogenexchange, Biochemistry, 42, 14092–14098.

Kirkitadze, M.D., Condron, M.M. and Teplow, D.B. (2001). Identification and char-acterization of key kinetic intermediates in amyloid β-protein fibrillogenesis,J. Mol. Biol., 312 (5), 1103–1119.

Kirschner, D.A., Gross, A.A.R., Hidalgo, M.M., Inouye, H., Gleason, K.A., Abdel-sayed, G.A., Castillo, G.M., Snow, A.D., Pozo-Ramajo, A., Petty, S.A. andDecatur, S.M. (2008). Fiber diffraction as a screen for amyloid inhibitors,Curr. Alzheimer Res., 5 (3), 288–307.

Kraepelin, E. (1910). Ein Lehrbuch f’́ur Studierende und ’́Arzte. II. Band, KlinischePsychiatrie, Psychiatrie., 2 (8th edn). Verlay, Barth, Leipzig.

Krebs, M.R.H., Bromley, E.H.C. and Donald, A.M. (2005). The binding ofthioflavin-T to amyloid fibrils: localisation and implications, J. Struct. Biol.,149 (1), 30–37.

Lai, F. and Williams, R.S. (1989). A prospective study of Alzheimer disease in Downsyndrome, Arch. Neurol., 46 (8), 849–853.

Lakowicz, J.R. (1999). Principles of Fluorescence Spectroscopy, 2nd ed. KluwerAcademic/Plenum Publishers, New York.

Lambert, M.P., Barlow, A.K., Chromy, B.A., Edwards, C., Freed, R., Liosatos, M.,Morgan, T.E., Rozovsky, I., Trommer, B., Viola, K.L., Wals, P., Zhang, C.,Finch, C.E., Krafft, G.A. and Klein, W.L. (1998). Diffusible, nonfibrillar lig-ands derived from Aβ(1-42) are potent central nervous system neurotoxins,Proc. Natl. Acad. Sci. U.S.A., 95 (11), 6448–6453.

Lansbury, P.T., Costa, P.R., Griffiths, J.M., Simon, E.J., Auger, M., Halverson, K.J.,Kocisko, D.A., Hendsch, Z.S., Ashburn, T.T. and Spencer, R.G. (1995).Structural model for the β-amyloid fibril based on interstrand alignment ofan antiparallel-sheet comprising a C-terminal peptide, Nature Struct. Biol.,2 (11), 990–998.

Lashuel, H.A., Lai, Z. and Kelly, J.W. (1998). Characterization of the transthyretinacid denaturation pathways by analytical ultracentrifugation: implicationsfor wild-type, V30M, and L55P amyloid fibril formation, Biochemistry, 37(51), 17851–17864.

Lashuel, H.A., Hartley, D., Petre, B.M., Walz, T. and Lansbury, P.T. (2002).Neurodegenerative disease: amyloid pores from pathogenic mutations,Nature, 418 (6895), 291.

Lashuel, H.A., Hartley, D.M., Petre, B.M., Wall, J.S., Simon, M.N., Walz, T. andLansbury, P.T. (2003). Mixtures of wild-type and a pathogenic (E22G) form

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



of Aβ40 in vitro accumulate protofibrils, including amyloid pores, J. Mol.Biol., 332 (4), 795–808.

Lazo, N.D., Grant, M.A., Condron, M.C., Rigby, A.C. and Teplow, D.B. (2005). Onthe nucleation of amyloid β-protein monomer folding, Protein Sci., 14 (6),1581–1596.

Le Châtelier, H. (1884). Sur un énoncé général des lois des équilibres chimiques,Comptes rendus de l’Academie des Sciences, 99, 786–789.

Lesné, S., Koh, M.T., Kotilinek, L., Kayed, R., Glabe, C.G., Yang, A., Gallagher, M.and Ashe, K.H. (2006). A specific amyloid-β protein assembly in the brainimpairs memory, Nature, 440 (7082), 352–357.

Levy, E., Carman, M.D., Fernandez-Madrid, I.J., Power, M.D., Lieberburg, I., vanDuinen, S.G., Bots, G.T.A.M., Luyendijk, W. and Frangione, B. (1990).Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebralhemorrhage, Dutch-type, Science, 248, 1124–1126.

Lomakin, A., Chung, D.S., Benedek, G.B., Kirschner, D.A. and Teplow, D.B. (1996).On the nucleation and growth of amyloid β-protein fibrils: detection ofnuclei and quantitation of rate constants, Proc. Natl. Acad. Sci. U.S.A., 93 (3),1125–1129.

Lomakin, A., Teplow, D.B., Kirschner, D.A. and Benedek, G.B. (1997). Kinetictheory of fibrillogenesis of amyloid β-protein, Proc. Natl. Acad. Sci. U.S.A.,94, 7942–7947.

Lord, A., Kalimo, H., Eckman, C., Zhang, X.-Q., Lannfelt, L. and Nilsson, L.N.G.(2006). The Arctic Alzheimer mutation facilitates early intraneuronal Aβ

aggregation and senile plaque formation in transgenic mice, Neurobiol. Aging,27 (1), 67–77.

Lorenzo, A. and Yankner, B.A. (1994). β-Amyloid neurotoxicity requires fibrilformation and is inhibited by Congo red, Proc. Natl. Acad. Sci. U.S.A., 91,12243–12247.

Lührs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Döbeli, H.,Schubert, D. and Riek, R. (2005). 3D structure of Alzheimer’s amyloid-β(1-42) fibrils, Proc. Natl. Acad. Sci. U.S.A., 102, 17342–17347.

Maji, S.K., Amsden, J.J., Rothschild, K.J., Condron, M.M. and Teplow, D.B. (2005).Conformational dynamics of amyloid β-protein assembly probed usingintrinsic fluorescence, Biochemistry, 44 (40), 13365–13376.

Maji, S.K., Loo, R.R.O., Inayathullah, M., Spring, S.M.,Vollers, S.S., Condron, M.M.,Bitan, G., Loo, J.A. and Teplow, D.B. (2009). Amino acid position-specificcontributions to amyloid β-protein oligomerization, J. Biol. Chem., 284 (35),23580–23591.

Mangialasche, F., Solomon, A., Winblad, B., Mecocci, P. and Kivipelto, M. (2010).Alzheimer’s disease: clinical trials and drug development, Lancet Neurol., 9(7), 702–716.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Mastrangelo, I.A., Ahmed, M., Sato, T., Liu, W., Wang, C., Hough, P. and Smith, S.O.(2006). High-resolution atomic force microscopy of soluble Aβ42 oligomers,J. Mol. Biol., 358 (1), 106–119.

McGeer, P.L. and Rogers, J. (1992). Anti-inflammatory agents as a therapeuticapproach to Alzheimer’s disease, Neurology, 42 (2), 447–449.

McLean, C.A., Cherny, R.A., Fraser, F.W., Fuller, S.J., Smith, M.J., Beyreuther, K.,Bush, A.I. and Masters, C.L. (1999). Soluble pool of Aβ amyloid as adeterminant of severity of neurodegeneration in Alzheimer’s disease, Ann.Neuro., 46 (6), 860–866.

Mormino, E.C., Kluth, J.T., Madison, C.M., Rabinovici, G.D., Baker, S.L., Miller,B.L., Koeppe, R.A., Mathis, C.A., Weiner, M.W., Jagust, W.J. and theAlzheimer’s Disease Neuroimaging Initiative (2009). Episodic memory lossis related to hippocampal-mediated β-amyloid deposition in elderly subjects,Brain, 132 (5), 1310–1323.

Murakami, K., Irie, K., Morimoto, A., Ohigashi, H., Shindo, M., Nagao, M.,Shimizu, T. and Shirasawa, T. (2003). Neurotoxicity and physicochemicalproperties of A β mutant peptides from cerebral amyloid angiopathy:implication for the pathogenesis of cerebral amyloid angiopathy andAlzheimer’s diseases, J. Biol. Chem., 278 (46), 46179–46187.

Naiki, H., Higuchi, K., Hosokawa, M. and Takeda, T. (1989). Fluorometricdetermination of amyloid fibrils in vitro using the fluorescent dye, thioflavinT1, Anal. Biochem., 177 (2), 244–249.

Näslund, J., Haroutunian, V., Mohs, R., Davis, K.L., Davies, P., Greengard, P. andBuxbaum, J.D. (2000). Correlation between elevated levels of amyloid β-peptide in the brain and cognitive decline, JAMA., 283 (12), 1571–1577.

Nelson, R. and Eisenberg, D. (2006). Recent atomic models of amyloid fibrilstructure, Curr. Opin. Struc. Biol., 16, 260–265.

Nelson, R., Sawaya, M.R., Balbirnie, M., Madsen, A.O., Riekel, C., Grothe, R. andEisenberg, D. (2005). Structure of the cross-β spine of amyloid-like fibrils,Nature, 435 (7043), 773–778.

Nilsberth,C.,Westlind-Danielsson,A.,Eckman,C.B.,Condron,M.M.,Axelman,K.,Forsell, C., Stenh, C., Luthman, J., Teplow, D.B.,Younkin, S.G., Naslund, J. andLannfelt, L. (2001). The “Arctic” APP mutation (E693G) causes Alzheimer’sdisease by enhanced Aβ protofibril formation, Nat. Neurosci., 4 (9),887–893.

Oddo, S., Caccamo, A., Shepherd, J.D., Murphy, M.P., Golde, T.E., Kayed, R.,Metherate, R., Mattson, M.P., Akbari, Y. and LaFerla, F.M. (2003). Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracel-lular Aβ and synaptic dysfunction, Neuron., 39 (3), 409–421.

Ono, K., Condron, M.M. and Teplow, D.B. (2009). Structure-neurotoxicityrelationships of amyloid β-protein oligomers, Proc. Natl. Acad. Sci. U.S.A.,106 (35), 14745–14750.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



O’Nuallain, B., Freir, D.B., Nicoll, A.J., Risse, E., Ferguson, N., Herron, C.E.,Collinge, J. and Walsh, D.M. (2010). Amyloid β-protein dimers rapidly formstable synaptotoxic protofibrils, J. Neurosci., 30 (43), 14411–14419.

Pauling, L. and Corey, R.B. (1951). Configurations of polypeptide chains withfavored orientations around single bonds: two new pleated sheets, Proc. Natl.Acad. Sci. U.S.A., 37, 729–740.

Petkova, A.T., Ishii, Y., Balbach, J.J., Antzutkin, O.N., Leapman, R.D., Delaglio, F.and Tycko, R. (2002). A structural model for Alzheimer’s β-amyloid fibrilsbased on experimental constraints from solid state NMR, Proc. Natl. Acad.Sci. U.S.A., 99 (26), 16742–16747.

Petkova, A.T., Leapman, R.D., Guo, Z., Yau, W.-M., Mattson, M.P. and Tycko, R.(2005). Self-propagating, molecular-level polymorphism in Alzheimer’s β-amyloid fibrils, Science, 307, 262–265.

Prince, M. and Jackson, J. (2009). Alzheimer’s Disease International World AlzheimerReport 2009, Tech. rep., Institute of Psychiatry, Kings College London.

Quist, A., Doudevski, I., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B.,Ghiso, J. and Lal, R. (2005). Amyloid ion channels: a common structurallink for protein-misfolding disease, Proc. Natl. Acad. Sci. U.S.A., 102, 10427–10432.

Re,V.L. and Bellini, L.M. (2002). William of Occam and Occam’s razor, Ann. Intern.Med., 136 (8), 634–635.

Revesz, T., Holton, J.L., Lashley, T., Plant, G., Rostagno, A., Ghiso, J. andFrangione, B. (2002). Sporadic and familial cerebral amyloid angiopathies,Brain Pathol., 12 (3), 343–357.

Robakis, N.K., Ramakrishna, N.,Wolfe, G. and Wisniewski, H.M. (1987). Molecularcloning and characterization of a cDNA encoding the cerebrovascular and theneuritic plaque amyloid peptides, Proc. Natl. Acad. Sci. U.S.A., 84, 4190–4194.

Rodriguez, J.A., Valentine, J.S., Eggers, D.K., Roe, J.A., Tiwari, A., Brown, R.H. andHayward, L.J. (2002). Familial amyotrophic lateral sclerosis-associated muta-tions decrease the thermal stability of distinctly metallated species of humancopper/zinc superoxide dismutase, J. Biol. Chem., 277 (18), 15932–15937.

Rosensweig, C., Ono, K., Murakami, K., Lowenstein, D.K., Bitan, G. and Teplow,D.B. (2012). Preparation of stable amyloid β-protein oligomers of definedassembly order, in Sigurdsson, E.M., Calero, M. and Gasset, M. (eds) AmyloidProteins, Methods and Protocols, 2nd edn., Humana Press, New York.

Ross, J.B.A., Laws, W.R., Rousslang, K.W. and Wyssbrod, H.R. (1992). Topics inFluorescence Spectroscopy, Plenum Press, New York.

Roychaudhuri, R., Yang, M., Hoshi, M.M. and Teplow, D.B. (2009). Amyloid β-protein assembly and Alzheimer disease, J. Biol. Chem., 284 (8), 4749–4753.

Saeed, S.M. and Fine, G. (1967). Thioflavin-T for amyloid detection, Am. J. Clin.Pathol., 47 (5), 588–593.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Sawaya, M.R., Sambashivan, S., Nelson, R., Ivanova, M.I., Sievers, S.A., Apostol,M.I., Thompson, M.J., Balbirnie, M., Wiltzius, J.J.W., McFarlane, H.T.,Madsen, A.O., Riekel, C. and Eisenberg, D. (2007). Atomic structures ofamyloid cross-β spines reveal varied steric zippers, Nature, 447 (7143),453–457.

Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T.D.,Hardy, J., Hutton, M., Kukull, W., Larson, E., LevyLahad, E., Viitanen, M.,Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L.,Selkoe, D. and Younkin, S. (1996). Secreted amyloid β-protein similar tothat in the senile plaques of Alzheimer’s disease is increased in vivo by thepresenilin 1 and 2 and APP mutations linked to familial Alzheimer’s disease,Nat. Med., 2, 864–870.

Schott, J.M., Bartlett, J.W., Fox, N.C., Barnes, J. and the Alzheimer’s DiseaseNeuroimaging Initiative Investigators (2010). Increased brain atrophy ratesin cognitively normal older adults with low cerebrospinal fluid Aβ1-42, Ann.Neurol, 68 (6), 825–834.

Selkoe, D.J. (2001). Alzheimer’s disease: genes, proteins, and therapy, Physiol. Rev.,81 (2), 741–766.

Selkoe, D.J. and Podlisny, M.B. (2002). Deciphering the genetic basis of Alzheimer’sdisease, Annu. Rev. Genomics Hum. Genet., 3, 67–99.

Sgourakis, N.G., Yan, Y., McCallum, S.A., Wang, C. and Garcia, A.E. (2007). TheAlzheimer’s peptides Aβ40 and 42 adopt distinct conformations in water: acombined MD/NMR study, J. Mol. Biol., 368 (5), 1448–1457.

Shankar, G.M., Bloodgood, B.L., Townsend, M., Walsh, D.M., Selkoe, D.J. andSabatini, B.L. (2007). Natural oligomers of the Alzheimer amyloid-βprotein induce reversible synapse loss by modulating an NMDA-typeglutamate receptor-dependent signaling pathway, J. Neurosci., 27, 2866–2875.

Shao, H., Jao, S., Ma, K. and Zagorski, M.G. (1999). Solution structures of micelle-bound amyloid β-(1-40) and β-(1-42) peptides of Alzheimer’s disease, J.Mol. Biol., 285, (2), 755–773.

Shearman, M.S., Ragan, C.I. and Iversen, L.L. (1994). Inhibition of PC12 cell redoxactivity is a specific, early indicator of the mechanism of β-amyloid-mediatedcell death, Proc. Natl Acad. Sci. U.S.A., 91, 1470–1474.

Silveira, J.R., Raymond, G.J., Hughson, A.G., Race, R.E., Sim, V.L., Hayes, S.F. andCaughey, B. (2005). The most infectious prion protein particles, Nature, 437(7056), 257–261.

Streltsov, V.A., Varghese, J.N., Masters, C.L. and Nuttall, S.D. (2011). Crystalstructure of the amyloid-β p3 fragment provides a model for oligomerformation in Alzheimer’s diseases, J. Neurosci, 31 (4), 1419–1426.

Tagliavini, F., Rossi, G., Padovani, A., Magoni, M., Andora, G., Sgarzi, M., Bizzi, A.,Savioardo, M., Carella, F., Morbin, M., Giaccone, G. and Bugiani, O. (1999).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



A new βPP mutation related to hereditary cerebral hemorrhage, Alzheimer’sRep., 2 (Suppl.), S28.

Tanzi, R.E., Gusella, J.F., Watkins, P.C., Bruns, G.A.B., St. George-Hyslop, P.H.,Van Keuren, M.L., Patterson, D., Pagan, S., Kurnit, D.M. and Neve, R.L.(1987). Amyloid β-protein gene: cDNA, mRNA distribution, and geneticlinkage near the Alzheimer locus, Science, 235, 880–884.

Teplow,D.B. (2006). Preparation of amyloid β-protein for structural and functionalstudies, Meth. Enzymol., 413, 20–33.

Teplow, D.B., Nakayama, C., Leung, P.C. and Harshey, R.M. (1988). Structure–function relationships in the transposition protein B of bacteriophage Mu,J. Biol. Chem., 263 (22), 10851–10857.

Teplow, D.B., Lomakin, A., Benedek, G.B., Kirschner, D.A. and Walsh, D.M. (1997).Effects of β-protein mutations on amyloid fibril nucleation and elongation,in Iqbal, K., Winblad, B., Nishimura, T., Takeda, M. and Wisniewski, H.M.(eds) Alzheimer’s Disease: Biology, Diagnosis and Therapeutics, Wiley & SonsLtd, Chichester, England, 311–319.

Teplow, D.B., Lazo, N.D., Bitan, G., Bernstein, S., Wyttenbach, T., Bowers, M.T.,Baumketner, A., Shea, J.-E., Urbanc, B., Cruz, L., Borreguero, J. andStanley, H.E. (2006). Elucidating amyloid β-protein folding and assembly: amultidisciplinary approach, Acc. Chem. Res., 39 (9), 635–645.

Tobacman, L.S. and Korn, E.D. (1983). The kinetics of actin nucleation andpolymerization, J. Biol. Chem., 258 (5), 3207–3214.

Tycko, R. (2006a). Characterization of amyloid structures at the molecular levelby solid state nuclear magnetic resonance spectroscopy, Meth. Enzymol., 413,103–122.

Tycko, R. (2006b). Molecular structure of amyloid fibrils: insights from solid-stateNMR, Q. Rev. Biophys., 39 (1), 1–55.

Tycko, R. and Ishii, Y. (2003). Constraints on supramolecular structure in amyloidfibrils from two-dimensional solid-state NMR spectroscopy with uniformisotopic labeling, J. Am. Chem. Soc., 125 (22), 6606–6607.

Ueda, K., Fukui, Y. and Kageyama, H. (1994). Amyloid β protein-induced neuronalcell death: neurotoxic properties of aggregated amyloid β protein, Brain Res.,639 (2), 240–244.

Urbanc, B., Cruz, L.,Yun, S., Buldyrev, S.V., Bitan, G., Teplow, D.B. and Stanley, H.E.(2004). In silico study of amyloid β-protein folding and oligomerization,Proc.Natl Acad. Sci. U.S.A., 101 (50), 17345–17350.

Van Broeckhoven, C., Haan, J., Bakker, E., Hardy, J.A., Van Hul, W., Wehnert, A.,Vegter-Van der Vlis, M. and Roos, R.A. (1990). Amyloid β protein precursorgene and hereditary cerebral hemorrhage with amyloidosis (Dutch), Science,248 (4959), 1120–1122.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Vassar, P.S. and Culling, C.F. (1959). Fluorescent stains, with special reference toamyloid and connective tissues, Arch. Pathol., 68, 487–498.

Vassar, R., Bennett, B.D., Babu-Khan, S., Kahn, S., Mendiaz, E.A., Denis, P., Teplow,D.B., Ross, S.,Amarante, P., Loeloff, R., Luo,Y., Fisher, S., Fuller, J., Edenson, S.,Lile, J., Jarosinski, M.A., Biere, A.L., Curran, E., Burgess, T., Louis, J.C.,Collins, F., Treanor, J., Rogers, G. and Citron, M. (1999). β-Secretase cleavageof Alzheimer’s amyloid precursor protein by the transmembrane asparticprotease BACE, Science, 286 (5440), 735–741.

Vidal, R., Revesz, T., Rostagno, A., Kim, E., Holton, J.L., Bek, T., Bojsen-Mller, M.,Braendgaard, H., Plant, G., Ghiso, J. and Frangione, B. (2000). A decamerduplication in the 3′ region of the BRI gene originates an amyloid peptidethat is associated with dementia in a Danish kindred, Proc. Natl. Acad. Sci.U.S.A., 97 (9), 4920–4925.

Walsh, D.M., Lomakin, A., Benedek, G.B., Condron, M.M. and Teplow, D.B. (1997).Amyloid β-protein fibrillogenesis. Detection of a protofibrillar intermediate,J. Biol. Chem., 272 (35), 22364–22372.

Walsh, D.M., Hartley, D.M., Kusumoto,Y., Fezoui,Y., Condron, M.M., Lomakin, A.,Benedek, G.B., Selkoe, D.J. and Teplow, D.B. (1999). Amyloid β-protein fib-rillogenesis. Structure and biological activity of protofibrillar intermediates,J. Biol. Chem., 274 (36), 25945–25952.

Wang, H.W., Pasternak, J.F., Kuo, H., Ristic, H., Lambert, M.P., Chromy, B.,Viola, K.L., Klein, W.L., Stine, W.B., Krafft, G.A. and Trommer, B.L. (2002).Soluble oligomers of β amyloid (1-42) inhibit long-term potentiation butnot long-term depression in rat dentate gyrus, Brain Res., 924 (2), 133–140.

Wisniewski, K.E., Wisniewski, H.M. and Wen, G.Y. (1985). Occurrence ofneuropathological changes and dementia of Alzheimer’s disease in Down’ssyndrome, Ann. Neurol., 17 (3), 278–282.

Wood, S.J., Wetzel, R., Martin, J.D. and Hurle, M.R. (1995). Prolines andamyloidogenicity in fragments of the Alzheimer’s peptide β/A4, Biochemistry,34 (3), 724–730.

Wyttenbach, T. and Bowers, M.T. (2003). Gas-Phase Conformations: The IonMobility/Ion Chromatography Method, Topics in Current Chemistry, Springer,Berlin.

Yamin, G., Ono, K., Inayathullah, M. and Teplow, D.B. (2008). Amyloid β-proteinassembly as a therapeutic target of Alzheimer’s disease, Curr. Pharm. Des., 14(30), 3231–3246.

Yan, Y. and Wang, C. (2006). Aβ42 is more rigid than Aβ40 at the C ter-minus: implications for Aβ aggregation and toxicity. J. Mol. Biol., 364,853–862.

Yang, F., Lim, G.P., Begum, A.N., Ubeda, O.J., Simmons, M.R., Ambegaokar, S.S.,Chen, P.P., Kayed, R., Glabe, C.G., Frautschy, S.A. and Cole, G.M. (2005).

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.



Curcumin inhibits formation of amyloid β oligomers and fibrils, bindsplaques, and reduces amyloid in vivo, J. Biol. Chem., 280, 5892–5901.

Yang, M. and Teplow, D.B. (2008). Amyloid β-protein monomer folding: free-energy surfaces reveal alloform-specific differences, J. Mol. Biol., 384,450–464.

Zhang, S., Iwata, K., Lachenmann, M.J., Peng, J.W., Li, S., Stimson, E.R., Lu, Y.,Felix, A.M., Maggio, J.E. and Lee, J.P. (2000). The Alzheimer’s peptide a β

adopts a collapsed coil structure in water, J. Struct. Biol., 130 (2,3), 130–141.

Alz

heim

er's

Dis

ease

: Ins

ight

s in

to L

ow M

olec

ular

Wei

ght a

nd C

ytot

oxic

Agg

rega

tes

from

In

Vitr

o an

d C

ompu

ter

Exp

erim

ents

Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

UN

IVE

RSI

TY

OF

QU

EE

NSL

AN

D o

n 04

/30/

13. F

or p

erso

nal u

se o

nly.

[Molecular Medicine and Medicinal Chemistry] Alzheimer's Disease: Insights into Low Molecular Weight...

Documents

Transcript of [Molecular Medicine and Medicinal Chemistry] Alzheimer's Disease: Insights into Low Molecular Weight...