Current Alzheimer Research, 2008, 5, 319-341 319
1567-2050/08 $55.00+.00 ©2008 Bentham Science Publishers Ltd.
Structure–Function Relationships of Pre-Fibrillar Protein Assemblies in
Alzheimer's Disease and Related Disorders
F. Rahimi, A. Shanmugam and G. Bitan§,†,*
Department of Neurology, David Geffen School of Medicine, §Brain Research Institute, and †Molecular Biology Insti-
tute, University of California at Los Angeles, Neuroscience Research Building 1, Room 451, 635 Charles E. Young
Drive South, Los Angeles, CA 90095-7334, USA
Abstract: Several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases, are
characterized pathognomonically by the presence of intra- and/or extracellular lesions containing proteinaceous aggre-
gates, and by extensive neuronal loss in selective brain regions. Related non-neuropathic systemic diseases, e.g., light-
chain and senile systemic amyloidoses, and other organ-specific diseases, such as dialysis-related amyloidosis and type-2
diabetes mellitus, also are characterized by deposition of aberrantly folded, insoluble proteins. It is debated whether the
hallmark pathologic lesions are causative. Substantial evidence suggests that these aggregates are the end state of aberrant
protein folding whereas the actual culprits likely are transient, pre-fibrillar assemblies preceding the aggregates. In the
context of neurodegenerative amyloidoses, the proteinaceous aggregates may eventuate as potentially neuroprotective
sinks for the neurotoxic, oligomeric protein assemblies. The pre-fibrillar, oligomeric assemblies are believed to initiate the
pathogenic mechanisms that lead to synaptic dysfunction, neuronal loss, and disease-specific regional brain atrophy.
The amyloid ?-protein (A?), which is believed to cause Alzheimer's disease (AD), is considered an archetypal amyloi-
dogenic protein. Intense studies have led to nominal, functional, and structural descriptions of oligomeric A? assemblies.
However, the dynamic and metastable nature of A? oligomers renders their study difficult. Different results generated us-
ing different methodologies under different experimental settings further complicate this complex area of research and
identification of the exact pathogenic assemblies in vivo seems daunting.
Here we review structural, functional, and biological experiments used to produce and study pre-fibrillar A? assemblies,
and highlight similar studies of proteins involved in related diseases. We discuss challenges that contemporary researchers
are facing and future research prospects in this demanding yet highly important field.
Keywords: Amyloid, neurodegeneration, Alzheimer's disease, amyloid ?-protein, protein misfolding, pre-fibrillar assemblies,
The amyloid-cascade hypothesis , suggesting that
amyloid ?-protein (A?) fibril formation and plaque deposi-
tion lead to neuronal dysfunction, dementia, and death in
Alzheimer's disease (AD), had guided scientific research into
discovery of etiologic and pathogenic mechanisms of AD.
However, this hypothesis has been contentiously debated
because: 1) fibrillar amyloid burden does not correlate well
with neurological dysfunction , 2) cognitive impairment
in transgenic murine models of AD is observed before and/or
independently of amyloid plaque formation , 3) plaque-
independent pathology can be explained by the neurotoxicity
of soluble A? assembly intermediates, 4) oligomer-induced
memory dysfunction occurs before neuronal death, and 5)
brain, plasma, and cerebrospinal fluid (CSF) concentrations
of soluble A? oligomers correlate with neurodegeneration
better than those of fibrils . These observations have led
to a burgeoning yet encompassing alternative paradigm hy-
pothesizing that soluble pre-fibrillar protein assemblies,
*Address correspondence to this author at the Department of Neurology,
David Geffen School of Medicine, Brain Research Institute, and Molecular
Biology Institute, University of California at Los Angeles, Neuroscience
Research Building 1, Room 451, 635 Charles E. Young Drive South, Los
Angeles, CA 90095-7334, USA; E-mail: firstname.lastname@example.org
rather than mature fibrillar deposits, act as proximate neuro-
toxins that cause synaptic dysfunction, neuronal loss, demen-
tia, and death [4-11]. This new hypothesis has been sup-
ported by discovery of toxic pre-fibrillar protein assemblies
involved in other protein-misfolding diseases, such as, Park-
inson's disease, Huntington's disease, transmissible spongi-
form encephalopathies, amyotrophic lateral sclerosis, poly-
glutamine diseases, type-2 diabetes mellitus (T2D), and sys-
temic amyloidoses [7, 12-15].
Diversity and sometimes inaccuracy in nominal defini-
tions, and in structural/functional descriptions of soluble pre-
fibrillar A? assemblies, along with different methodologies
to generate and study these assemblies, are confounding fac-
tors in this already-vast and complex research area. Various
forms of soluble pre-fibrillar A? assemblies (reviewed in
[10, 16-18]) including monomeric A? conformers , se-
creted cell- and brain-derived low-order oligomers [20-25],
A?-derived diffusible ligands (ADDLs) [26, 27], protofibrils
(PF) [28-31], A?*56 , paranuclei [33-35], amylosphe-
roids , annular assemblies , amyloid pores [18, 37,
38], and ?amy balls  have been described. However,
despite a global concerted scientific effort, the relationships
amongst these A?-derived assemblies and their relevance to
AD pathogenesis are unclear and the fundamental quest for a
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320 Current Alzheimer Research, 2008, Vol. 5, No. 3 Rahimi et al.
unanimous pathogenic "equivalent" active in AD-afflicted
brain is ongoing .
We begin our discussion in the first step of studying pre-
fibrillar A? assemblies—protein preparation.
SOURCES AND METHODS OF A? PREPARATION
The low physiological concentration and the difficulty of
procuring highly pure and homogeneous tissue-derived A?
have precluded its routine use in experimental studies in vi-
tro. Therefore, synthetic A? preparations have emerged as
alternatives. Synthetic A? is produced either by standard
solid-phase peptide synthesis (SPPS) [40, 41] or by recom-
binant DNA technology [42-45].
The A? sequence is recognized as a "difficult" target for
SPPS owing to its high hydrophobicity and innate propensity
to aggregate [46, 47]. To overcome these issues, various de-
protecting agents , novel solvent systems for coupling
, and solid-support modifications  have been em-
ployed to augment synthetic yield and improve purity of
crude A? peptides. Recently, application of an O-N acyl mi-
gration reaction, also called "O-acyl isopeptide" chemistry
was proposed as an efficient alternative SPPS route for ob-
taining A? with increased solubility and purity [49, 50].
Similarly, tailored recombinant expression systems have
been used to produce A? in high yields. A strategy to in-
crease solubility and facilitate purification is production of
A? peptides as fusions with sequences affording high solu-
bility, followed by cleavage of the fusion protein and purifi-
cation by high-performance liquid chromatography (HPLC)
Because self-association of A? is believed to be central in
the pathogenesis of AD, extensive research has been dedi-
cated to developing methodologies to characterize A? as-
semblies structurally and biologically . Multiple studies
have shown that many of the neurotoxic effects of A? as-
semblies can be recapitulated by synthetic A? in vitro and in
vivo . However, differences in peptide quality, presence
of trace contaminants in A? preparations from different
sources, and compositional variation of A? preparations,
even from the same source, have been a serious problem
leading to irreproducible or discrepant study outcomes [54-
56]. For example, under identical conditions, an A? oli-
gomer-specific monoclonal antibody was shown to react
only with oligomers derived from recombinant A? but not
those derived from chemically synthesized A? . In our
hands, photo-induced cross-linking of unmodified proteins
(PICUP) using A? obtained from different sources, but pre-
pared identically, yielded distinct results (Fig. (1)).
Neither SPPS nor recombinant methods can produce
100% pure A?. Failure sequences, oxidation of Met35, and
racemization may occur during various SPPS steps . In
recombinant preparations where a fusion protein is enzy-
matically cleaved to release A?, it is important to verify that
the cleavage product is not contaminated with the uncleaved
fusion protein, the cleaving enzyme, or adventitious prote-
olytic fragments . Practically, it is important that the
researcher verifies chemical purity of the preparation and
ensures removal of residual components which could com-
plicate solvation and stock preparation, potentially alter the
biophysical and biological features of the peptide, and render
concentration measurements error-prone .
Fig. (1). Comparison of photo-cross-linking using A? peptides
from different sources. Synthetic A? from Global Peptide (G) and
the UCLA Biopolymers Laboratory (U), and recombinant A? from
rPeptide (R) were prepared in 10 mM sodium phosphate, pH 7.4 at
2 mg/ml nominal concentration and filtered through a 10-kDa mo-
lecular-weight cut-off filter . Each filtered peptide was cross-
linked using PICUP . The resulting cross-linked oligomers were
subjected to SDS-PAGE and silver-staining. The data suggest that
A?40 from Global peptide contained contaminants that prevented
cross-linking and that A?42 from rPeptide aggregated during the
filtration step and was hardly detectable.
METHODS USED TO STUDY PRE-FIBRILLAR A?
Important biological functions of oligomeric A? assem-
blies have spurred extensive efforts to characterize them
structurally. The non-crystalline nature of the oligomers and
their slow tumbling time in aqueous solutions preclude high-
resolution structural determination by X-ray crystallography
and solution-state NMR, respectively. Moreover, the metas-
table nature of A? oligomers and their existence in rapidly
changing mixtures have made their structural characteriza-
tion particularly difficult. To address these issues, multiple
low-resolution methodologies have been used to assess vari-
ous structural features of pre-fibrillar A? assemblies. Here,
we outline some of the key methods used to provide struc-
tural and biophysical information on pre-fibrillar A? assem-
Solution-state NMR is a powerful high-resolution tech-
nique for determining peptide and protein structure in solu-
tion. Typically, the structure is calculated based on distances
and angles obtained through measurements of nuclear Over-
hauser effect and spin-spin scalar coupling interactions as
constraints for computer-generated models. As mentioned
above, peak broadening due to slow tumbling times currently
precludes solving the structure of A? oligomers using solu-
tion-state NMR. However, multiple NMR studies have as-
sessed structural properties of A? monomers. For instance,
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Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 321
studies by Lee et al. introduced the concept of "plaque com-
petence," which defines the propensity of near-physiological
concentrations of soluble A? to deposit onto authentic amy-
loid plaques in vitro . The plaque competence assay
identified a central 26-residue fragment (Tyr10–Met35) which
was deemed necessary to mimic plaque-deposition character-
istics of the full-length A? . Preliminary NMR confor-
mational analysis showed that this 26-residue fragment had a
different conformation from a plaque-incompetent fragment
(Asp1–Lys28) . Further NMR studies also confirmed that
the central 26 residues of A? were sufficient to mimic amy-
loidogenic properties of A? . It was reported that the
central hydrophobic cluster of full-length A?, and A?(10-
35), both adopted well-defined, albeit irregular, conforma-
tions in solution, whereas the C- and N-terminal flanking
regions of the full-length A? were partially disordered .
NMR studies also have highlighted differences between
A?40 and A?42. Solution-state NMR studies of non-
oxidized  or Met-oxidized [61, 62] A?40 and A?42 show
that the C-terminus of A?42 is more rigid compared to that
of A?40, likely due to the extended hydrophobic C-terminus
of A?42. Similarly, a study combining molecular dynamics
and NMR experiments, showed that the C-terminus of A?42
is more structured than that of A?40 . NMR studies also
revealed that common C-terminal peptide segments within
A?40 and A?42 have distinct structures, which may be rele-
vant to the strong disease-association of elevated A?42 pro-
X-ray crystallography examines atomic structures of
crystals by X-ray diffraction techniques (reviewed in ).
The signal is intensified by the coherent alignment and lat-
tice repeat of the crystal. The wavelength of the light used in
X-ray crystallography is usually around 1.5 Å, about the
length of a C–C bond. Use of X-rays with this wavelength
theoretically allows resolution of individual atoms. Recently,
Sawaya et al. reported that 33 peptide segments derived from
14 different amyloidogenic proteins formed amyloid-like
fibrils, microcrystals, or both  and used X-ray crystallog-
raphy to examine the atomic organization of molecules
within microcrystals of these peptides. Microcrystals of 2 A?
segments were resolved, Gly37–Ala42 and Met35–Val40. The
authors suggested that the structural organization of these
peptides within the crystals is similar to those of A? fibrils
and concluded that the fundamental unit of amyloid-like fi-
brils is a steric zipper arrangement formed by two tightly
interdigitated ?-sheets .
Hydrogen–deuterium exchange is a powerful probe of
protein structure and dynamics. The method involves the
study of exchange rates of labile protons in proteins with
deuterons from the solvent, typically D2O. Labile protons are
those bonded to nitrogen, sulfur, or oxygen. These protons
can exchange with solvent hydrogen or deuterium cations.
Labile protons that are solvent-exposed and are not involved
in hydrogen bonding exchange rapidly, whereas buried or
hydrogen-bonded protons exchange at substantially slower
rates. This makes hydrogen–deuterium exchange sensitive to
structural rearrangements occurring during protein aggrega-
tion. Thus, amide protons buried in the core of oligomeric
and higher-order assemblies or hydrogen-bonded in helices
and sheets do not exchange readily with solvent deuterons.
The exchange rate is detected using NMR and/or mass spec-
trometry. For study of rapidly changing assemblies, mass
spectrometric detection of exchange may be advantageous
because NMR requires longer times (hours) to record the
spectra, making the study of short-lived oligomers difficult.
In addition, NMR requires prior assignment of the protons
and is generally limited to proteins smaller than 25 kDa. An
additional advantage of mass spectrometric detection is re-
quirement of substantially smaller amounts of protein. How-
ever, assignment of specific exchanging protons using mass
spectrometry requires tandem mass spectrometry and can be
a daunting task, whereas for a previously assigned NMR
spectrum, identification of exchanging protons is straight-
forward. Hydrogen–deuterium exchange coupled with mass
spectrometry was used to map structural differences in A?
PF and fibrils .
DETERMINATION OF OLIGOMER-SIZE DISTRIBU-
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electropho-
SDS-PAGE is a routine and inexpensive method enabling
separation of proteins based on their electrophoretic mobility
which is affected by a combination of the primary, secon-
dary, tertiary, and quaternary structures of proteins. In this
method, protein mixtures are electrophoresed after treatment
with SDS. SDS binds proteins via its hydrophobic dodecyl
tail, leaving its sulfate group solvent-exposed, and thus creat-
ing a negatively-charged envelope that "coats" protein mole-
cules . In most cases, SDS binding denatures secondary
and non-disulfide-linked tertiary structures, negatively
charging proteins approximately uniformly and proportion-
ally to their mass. Under these conditions, electrophoretic
migration of proteins through the gel matrix is governed di-
rectly by the molecular mass of the protein. Without SDS,
different proteins of the same mass may electrophorese dis-
tinctly due to differences in overall charge (different isoelec-
tric points) and folding.
Importantly, the effect of SDS on all proteins is not
equivalent. In many cases, SDS can induce or stabilize sec-
ondary and quaternary structures. SDS may cause dissocia-
tion of some oligomers or conversely induce oligomerization
and aggregation. Therefore, resolution of apparently mono-
meric or low-molecular-weight (LMW) oligomeric compo-
nents in a protein mixture does not necessarily indicate exis-
tence of such components under native conditions, i.e., with-
out SDS. A? is an amphipathic protein known to form SDS-
stable oligomers . Indeed, SDS-induced assembly of A?
into insoluble aggregates has been capitalized on to purify
A? from brain homogenates . When treated with SDS,
A? assembles rapidly into high-molecular-mass aggregates
. During electrophoresis of A?40, these aggregates dis-
sociate completely and only a monomer is observed follow-
ing staining, whereas electrophoresis of A?42 yields appar-
ent trimeric and tetrameric components . Essentially
identical monomer-trimer-tetramer distributions are observed
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322 Current Alzheimer Research, 2008, Vol. 5, No. 3 Rahimi et al.
when different preparations of A?42, including "mono-
meric", oligomeric, and fibrillar A?42 are analyzed by SDS-
PAGE . Thus, despite its wide use, SDS-PAGE is not a
reliable method for characterization and size determination
of non-covalently associated A? oligomers.
Photo-Induced Cross-Linking of Unmodified Proteins
PICUP is a method originally developed to study stable
protein homo- and hetero-oligomers . PICUP was used
by us and others to study oligomer size distribution of A?
 and a variety of other proteins, including amyloidogenic
proteins . PICUP generates covalent bonds between
closely interacting polypeptide chains within ?1 s exposure
to visible light without pre facto chemical modifications of
the native sequence and without using spacers. The cross-
linking is induced by rapid photolysis of a tris-bipyridyl
Ru(II) complex in the presence of an electron acceptor. Illu-
mination causes generation of a Ru(III) ion, which subse-
quently abstracts an electron from, and produces a carbon
radical within, the polypeptide. The radical reacts rapidly
with adjacent susceptible groups and forms covalent bonds.
Therefore, PICUP stabilizes oligomer populations by cova-
lent cross-linking, and "freezes" molecular interactions that
exist before cross-linking. The mechanism, protocol, and
limitations of PICUP were discussed in detail elsewhere [71,
Size-Exclusion Chromatography (SEC)
SEC (gel permeation chromatography) fractionates sol-
utes based on their Stokes (hydrodynamic) radii. When sol-
utes of different sizes pass through an SEC column packed
with porous material, larger molecules spend less time inter-
acting with the solid phase and elute faster, whereas smaller
molecules diffuse into the pores and therefore spend more
time interacting with the solid phase and elute later. SEC
affords an SDS-independent separation mechanism and cov-
ers a molecular mass range of ~103–106Da. However, SEC
provides lower resolution than SDS-PAGE and molecular-
mass estimations of polypeptides can be inaccurate if the
elution profiles are sensitive to the protein conformations.
SEC analysis of A? assemblies does not resolve LMW oli-
gomers but can distinguish between PF and small oligomers
. At this resolution, SEC may be useful for studying the
kinetics involved in conversion of LMW A? to PF (or disso-
ciation of PF into LMW A?). In addition to its use as an ana-
lytical method , SEC has been used extensively to purify
fractions of particular A? assemblies [30, 33, 38, 74-76].
Description of the basic instrumentation and utilization of
SEC for preparation of aggregate-free A? was published
Analytical Ultracentrifugation (AU)
AU is a versatile technique used to characterize the hy-
drodynamic and thermodynamic properties of proteins or
macromolecules. AU combines an ultracentrifuge and an
optical detection system capable of measuring the sample
concentration inside the centrifuge cell during sedimentation.
Coupled with data-analysis software, AU can determine
sample purity and molecular mass in the native state, meas-
ure sedimentation and diffusion coefficients, characterize
assembly–disassembly mechanisms of complex analytes,
determine subunit stoichiometry, detect and characterize
macromolecular conformational changes, and measure equi-
librium constants and thermodynamic parameters for self-
and hetero-associating assemblies. Two types of experiments
are commonly performed using ultracentrifugation—sedi-
mentation-velocity [78, 79] and sedimentation-equilibrium
. Sedimentation-equilibrium experiments can analyze a
mixture of moieties of various molecular masses. After each
analyte reaches its equilibrium, high-molecular-mass species
locate towards the bottom of the cell, whereas low-
molecular-mass species dominate at the top. The equilibrium
data can be fitted to calculated models for the distribution of
the solutes. Using this type of analysis, Huang et al. have
reported that A?40 existed as an equilibrium mixture of
monomers, dimers and tetramers at neutral pH . How-
ever, other equilibria, including monomer–dimer, monomer–
trimer, or monomer–tetramer, produced equivalent residuals
 hindering precise determination of the oligomerization
state of the peptide.
Dynamic Light Scattering (DLS) Spectroscopy
DLS, also known as quasielastic light scattering or pho-
ton-correlation spectroscopy, is a non-invasive analytical
method for determination of diffusion coefficients of parti-
cles undergoing Brownian motion in solution. DLS measures
the temporal dependence of light scattering emanated from
an analyte in solution over 10?7–1 s. Fluctuations in the in-
tensity of the scattered light relate to the rate of the
Brownian motion which is correlated to the diffusion coeffi-
cients of the particles. In a mixture of analytes, a distribution
of diffusion coefficients is obtained. The data are processed
to determine the particle hydrodynamic radii which relate to
the diffusion coefficients using the Stokes-Einstein equation.
DLS has an intrinsic bias for large aggregates because the
intensity of the scattered light is proportional to the square of
the particle size . Therefore, DLS is well suited to meas-
ure minute amounts of aggregated proteins (<0.01% by
weight) on the background of monomers and small oli-
gomers. Because DLS allows monitoring protein assembly
without manipulation or consumption of the analyte, it has
been used widely to study A? aggregation and assembly
processes [33, 75, 83-87].
For proteins larger than ~500 kDa or for extended pro-
teins (rod-like/unfolded), scattering varies significantly with
angle. Determining scattering at additional angles (multi-
angle laser light scattering or MALLS) allows direct meas-
ures of mass (?MDa) and radius (a measure of geometric
size). Because the light-scattering signal is directly propor-
tional to protein concentration and molecular mass, a combi-
nation of the DLS signal and concentration measurements
using refractive index or absorbance, allows calculation of
the molecular mass of each component when proteins are
fractionated chromatographically. DLS can resolve the
monomeric or dimeric state of a protein, but it cannot distin-
guish among small oligomers when their hydrodynamic radii
differ by less than a factor of 2 . Consequently, DLS is
less useful for analyzing individual small oligomers than
SEC-MALLS, PICUP coupled with SDS-PAGE, or sedi-
mentation velocity. Detailed accounts of the theory and prac-
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Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 323
tice of DLS and its application to study of A? were given by
Lomakin et al. [82, 89, 90]. Solution state and size distribu-
tion of ADDLs has been assessed recently by Hepler et al.
using SEC coupled with MALLS .
Ion-Mobility Spectrometry-Mass Spectrometry (IMS-
IMS-MS is a mass-spectrometric method that can resolve
molecules of identical mass-to-charge (m/z) values which
differ by assembly state or conformation. In IMS-MS, ions
are carried by a weak, uniform electric field through a drift
cell in which they collide at low velocity with a low-pressure
inert gas (typically helium). The analyte ions quickly reach
equilibrium resulting in a constant drift velocity. At equilib-
rium, the mobility of the ions is inversely proportional to
their collisional cross-section. Thus, ions with compact
structures drift fast through the cell, whereas ions with large
cross-sections move more slowly. The ions exit the drift cell,
pass through a mass filter, and are detected as a function of
time, producing an "arrival-time" distribution. Protein oli-
gomers often have identical m/z ratio (i.e., a singly-charged
monomer would have the same m/z as a doubly-charged
dimer, triply-charged trimer, etc.). IMS-MS analysis can
resolve these species yielding an oligomer size distribution.
IMS-MS studies of A? have shown that freshly prepared
LMW A?40 contained monomers, dimers, trimers, and
tetramers, whereas similarly prepared solutions of A?42
comprised oligomers up to a dodecamer . These results
accord with earlier observations of distinct oligomer size
distributions of A?40 and A?42 by PICUP  and may
explain differences in neurotoxic effects of the two A? allo-
ANALYSIS OF SECONDARY STRUCTURE
Circular Dichroism (CD) Spectroscopy
CD is the change in the absorption of circularly polarized
light as a function of wavelength exhibited by optically ac-
tive molecules. Because secondary structural elements such
as ?-helices, ?-strands, ?-turns and disordered regions dis-
play characteristic wavelength-dependent dichroism, CD is a
useful method to determine protein secondary structure. Sec-
ondary structure analysis by CD spectroscopy uses "far-UV"
spectra (190–250 nm), in which the chromophores are pep-
tide bonds. The CD signal reflects an average of the entire
molecular population. Therefore, CD can only determine the
overall proportion of secondary structural elements, but not
the amino acid residues involved or the fraction of molecules
that have a particular conformation. CD has been used exten-
sively to investigate secondary structure of A? peptides and
to monitor structural transitions of A? during its oligomeri-
zation and aggregation [31, 92-95].
Fourier-Transform Infrared (FTIR) Spectroscopy
Complementary to CD, FTIR enables determination of
the secondary structure of protein samples as thin films, as
solids, or in solution. Characteristic bands in IR spectra of
proteins and polypeptides include predominantly amide I and
amide II. The amide I band corresponds to the absorption in
the vibrational spectrum of the C=O component of the amide
bond, whereas the amide II band corresponds to the absorp-
tion of the N–H bond. Because C=O and N–H bonds are
involved in hydrogen bonding, the absorption wavelength of
both the amide I and amide II bands are sensitive to the sec-
ondary structure content of proteins. In many cases, instead
of a series of well-resolved peaks for each type of secondary
structure, one broad peak is observed. However, for proteins
that cannot be studied by high-resolution methods, FTIR
provides useful structural information. It is thought that
FTIR is more sensitive to ?-sheet content whereas CD meas-
urements generally tend to underestimate ?-sheet relative to
?-helix content . FTIR has been used extensively to
study the conformational changes of A? during assembly
Transmission Electron Microscopy (TEM)
TEM uses a cathode ray which emits a high-voltage elec-
tron beam focused by electrostatic and electromagnetic
lenses. When the electron beam passes through a thin, elec-
tron-transparent specimen, it carries information about the
inner structure of the sample as it reaches the TEM imaging
system. There, the spatial variation in this information,
which creates the image, is magnified by a series of electro-
magnetic lenses and detected by a fluorescent screen, photo-
graphic plate, or a light-sensitive sensor, e.g., a camera. TEM
has been used extensively to examine the morphology of
pre-fibrillar A? assemblies, including LMW A? [33, 34],
small oligomers [102-105], paranuclei [33, 34], PF [31, 75,
76, 87] and spheroids [36, 39].
Scanning Transmission Electron Microscopy (STEM)
In STEM, an electron beam scans a specimen and scat-
tered electrons are collected by detectors behind the speci-
men. In a thin proteinaceous specimen, the image intensity is
directly proportional to the mass of the irradiated region.
Therefore, following background subtraction and calibration,
the protein mass and mass-per-length unit (MPL) can be
determined quantitatively . STEM has been used to
characterize the MPL ratios of A? fibrils and PF [107-109].
Atomic-Force Microscopy (AFM)
AFM images high-resolution (?1 nm) topography of a
sample, adsorbed on an atomically flat smooth surface, typi-
cally mica. A cantilever tip samples the surface and when the
tip contacts a spot with adsorbed sample, an ionic repulsive
force bends the cantilever upwards. The extent of bending,
measured by a laser reflected onto a split photo detector, is
translated to force units. By keeping the force constant while
scanning across the surface, the vertical movement of the tip
generates the surface contour which is recorded as the topog-
raphy of the sample. AFM has been modified for specific
applications and can be used in different modes. In "tapping
mode" (commonly referred to as "intermittent-contact" or
"dynamic-force mode"), a stiff cantilever oscillates close to
the sample. Part of the oscillation extends into the repulsive
regime so that the tip intermittently touches, or "taps," the
surface. This mode provides good resolution on soft samples
and therefore is useful for investigation of pre-fibrillar A?
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324 Current Alzheimer Research, 2008, Vol. 5, No. 3 Rahimi et al.
tinuous monitoring of the growth of oligomers in solution
. Multiple studies on the structure of soluble oligomers
have used conventional tapping-mode AFM [26, 30, 31, 111-
114]. The smallest size of individual oligomers that could be
observed by AFM corresponded to a height of ~1–4 nm
An advantage of AFM over TEM is that it allows con-
Scanning Tunneling Microscopy (STM)
STM is a non-optical microscopic technique which em-
ploys principles of quantum mechanics. The electron cloud
of atoms on the surface of a sample extends a minute dis-
tance above the surface. When a probe, with a tip as sharp as
a single atom, is brought sufficiently close to such a surface
and a small voltage is applied, a strong interaction occurs
between the electron cloud on the surface and the tip leading
to an electric tunneling current. The magnitude of the current
depends exponentially on the distance between the probe and
the surface. The tunneling current rapidly increases as the
distance between the tip and the surface decreases. This
rapid alteration in the current due to changes in distance re-
sults in construction of an atomically resolved image when
the tip scans the structure. The feedback signal, applied to a
piezoelectric element provides a measure of molecular sur-
face contour. STM was used to examine the structure of
A?40 monomers, dimers and oligomers on a surface of
atomically flat gold . At low concentrations (0.5 ?M)
small globular structures were observed. High-resolution
STM measurements of A? samples, both immediately fol-
lowing preparation and after 24 h aging, found structures of
~3–4 nm in diameter corresponding to oligomeric A?. These
results suggested that oligomer formation could potentially
proceed through a mechanism involving linear association of
STRUCTURAL AND BIOLOGICAL STUDIES OF
PRE-FIBRILLAR A? ASSEMBLIES
The use of proper terminology to describe soluble pre-
fibrillar A? assemblies is crucial to forming consensus in the
literature. However, achieving this goal is difficult because
various oligomeric forms of A? have been described struc-
turally, functionally, or both and the relationship amongst
these is unclear. Although all these structures are oligomeric,
the use of the term "oligomer" to describe all the assemblies
may be misleading for at least three reasons, as discussed
before: 1) the structure of each assembly is unique; 2) the
pathways leading to the formation of the assemblies or the
ultimate path they may take towards fibrillization may differ;
and 3) the biological activities of each assembly may differ
or similar activities may be mediated through different
mechanisms . In the following section, we describe pre-
fibrillar A? species ranging from monomers to PF (Table
(1)). In most cases, we begin with structural characterization
of each species followed by discussion of its biological ac-
"ACTIVATED MONOMERIC CONFORMER" OF A?
Monomer activation denotes a conformational change
preceding A? self-assembly that may render monomers
toxic, or cause them to nucleate further aggregation, or both.
Based on concepts taken from actin polymerization 
and a kinetic model of A? aggregation induced by constant
rotary shaking, Taylor et al. introduced the idea of "activated
monomeric conformers" of A?40 , also called "interme-
diate aggregated species" . It was postulated that this
moiety was an oxidative or hydrolytic derivative, or a
slowly-folding conformer of intact A?40 [19, 117]. It was
proposed that the "inactive" monomer slowly converted into
the "activated" monomeric conformer, several of which
might cooperate to form a growing nidus for oligomerization
and progression of aggregation . In these studies, the
presence of the active monomeric A? conformer was tested
by HPLC using acetonitrile and trifluoroacetic acid, which
might have caused structural alterations in A? sequence by
increasing its ?-sheet content . Lee et al. have provided
evidence that A? aggregation intermediates and final struc-
tures formed under slowly agitated or quiescent conditions at
37°C differed in their toxicity, stability to denaturant, and
apparent morphologies , emphasizing that parametrical
consideration of methodologies used to prepare A? for struc-
tural or biological studies and proper methods to assess the
assembly state of the resulting preparation are paramount
. Similarly, NMR studies emphasize the importance of
performing structural studies under physiological conditions
 rather than "structure-inducing" milieus as reported by
Taylor et al. .
Other studies also have shown presence of A? intermedi-
ates, however, the assembly state of these was not deter-
mined unambiguously. A study by Chimon et al. described
A?40 intermediates that contained a ?-sheet-rich character
and were thought to originate from a monomeric state, pre-
ceding PF and fibril formation . Filtration experiments
showed that these intermediates were not monomeric but
were likely larger than decamers, indicating that unambigu-
ous determination of the assembly intermediates is difficult.
By electron microscopy, these intermediates had a spherical
morphology similar to ADDLs , amylospheroids 
and ?amy balls . NMR studies showed that the interme-
diate species was well ordered in the hydrophobic core and
the C-terminal region . A?40 was used in this study at
higher concentrations than those found in biological speci-
mens and the possibility that the intermediate could have
undergone fibril formation during preparation for NMR stud-
ies was not excluded. Similarly, Lim et al. provide evidence
for presence of monomeric intermediates using CD and
NMR studies of A?40 and A?42 under both non-amyloi-
dogenic (<5°C) and amyloid-promoting conditions (>5°C) at
physiological pH . CD studies of the A? peptides sug-
gested that the initially unfolded A? peptides at low tempera-
ture gradually transformed to ?-sheet-containing monomeric
intermediates at stronger amyloidogenic conditions (higher
temperatures) . However, exclusive presence of mono-
mers after dialysis of 1,1,1,3,3,3-hexafluoro-2-propanol
(HFIP)-treated A? species against phosphate buffer was not
confirmed . Providing formal proof for the presence of
A? monomers exclusively is difficult and has not been
achieved unambiguously in the studies mentioned above.
Therefore, whether a critical conformational change in the
monomer occurs before self-assembly, or the conformational
change and the assembly occur concurrently and co-
dependently remains an open question.
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Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 325
Table 1. Summary of Structural and Biological Characteristics of Pre-Fibrillar A? Assemblies
Assembly Structural characteristics/production Biological activity
conformer" of A?
Produced during aggregation of A?40 by constant rotary
Measured by turbidity assay and HPLC
Postulated to be an oxidative or hydrolytic derivative of
A? [19, 117]
The monomeric nature of these conformers is not con-
Forms 2–3-fold more complexes with apoE4 than apoE2
or apoE3 
Enhances toxicity and apoptotic activity in neurons 
Inhibits action potential by blocking fast, inward tetrodo-
toxin-sensitive Na+ channels 
Cell–derived A? oli-
Predominantly dimers, trimers, and tetramers, produced
by CHO cells transfected with mutated or wild-type APP
Resistant to SDS denaturation and to cleavage by insulin
degrading enzyme 
Inhibit LTP in vivo 
Impair short-term memory in rats 
Cause dendritic spine loss 
Affect synaptic structure and function
Synthetic species formed in the presence of apoJ , in
F-12 media  or in PBS 
Small globules 3–8 nm in diameter measured by AFM
Polydisperse mixtures of 150–1,000 kDa determined by
Highly neurotoxic [26, 162]
Inhibit LTP 
Bind to neuronal surfaces  co-localizing with neuronal
proteins at postsynaptic punctate sites [136, 150] and NR1
and NR2B subunits of NMDAR [136, 150]
Promote oxidative stress and increased [Ca2+]i 
Induce ? phosphorylation 
Induce IL-1?, iNOS, NO and TNF-? in astrocytes 
Curvilinear fibril-like structures 6–8 nm in diameter and
?200 nm long by TEM ; a periodicity of 20 nm and
diameter of 4.3 nm were determined by AFM 
Rich in ?-sheet structure
Bind Congo red and Thioflavin T 
Increase EPSCs , and cause cell death [29, 31]
May activate NMDAR in contrast to fibrils which activate
non-NMDA glutamate receptors .
Patch-clamping using A?42 PF induces reversible, Ca2+-
dependent increase in spontaneous action potentials and
Can inhibit the A- and D-type K+ currents, but not other
outward/inward rectifying K+ channels.
PF-induced membrane activity increases [Ca2+]i spikes
A?40 containing the E22G substitution forms PF faster
and in larger quantities than wild-type A? in vitro, sug-
gesting that PF may be the main disease-causing agents in
carriers of the Arctic mutation .
Extracted from brains of Tg2576 mice and isolated by
immunoaffinity and SEC 
Concentration correlates with degree of cognitive deficits
Cause defects in long-term spatial memory in rats 
Channel-like structures of synthetic A?, having outer
diameter of 8–12 nm and inner diameter of 2–2.5 nm, as-
sociated with artificial membrane bilayers [18, 37]
Pore- and channel-forming capacity that may lead to
membrane leakage and increased [Ca2+]i [18, 37]
In cell biological studies, toxicity and apoptotic activity
were enhanced when the concentration of the "activated
monomeric conformer" described by Taylor et al. was
maximal during the aggregation continuum . Similarly,
in electrophysiological experiments, A? species described as
monomeric conformers obtained between ~60–120 min dur-
ing rotary-shaken aggregation inhibited neuronal action po-
tentials . This neurotoxicity was attributed only to the
"active" monomeric conformers detected at 60 min after the
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326 Current Alzheimer Research, 2008, Vol. 5, No. 3 Rahimi et al.
initiation of aggregation but not to inactive monomers or
fully formed aggregates [19, 117, 122]. In these studies, the
kinetics of aggregation was monitored by turbidity and the
loss of the low-molecular-mass starting material was moni-
tored by HPLC . The sensitivity of turbidity measure-
ments for detection of small particles is substantially lower
than that of DLS and it is therefore unlikely that the method
can distinguish monomers from small oligomers. Also of
note is that biological activities similar to those obtained by
Taylor et al.  have been described for A? oligomers ,
thus it is unclear that these toxicity data can be ascribed to
BRAIN- AND CELL-DERIVED LOW-ORDER A? OLI-
Dimeric and trimeric assemblies of A? have been iso-
lated from amyloid plaque cores , cerebrospinal fluid
(CSF) , and human cortical homogenates , and pro-
duced by cells transfected with amyloid precursor protein
(APP) . Dimeric and trimeric A? (apparent SDS-PAGE
mobility corresponding to ~9 and ~13.5 kDa) purified from
neuritic plaques and leptomeningeal mural amyloid, were
characterized by SEC, AFM, and electron microscopy .
Matrix-assisted laser-desorption ionization (MALDI) mass
spectrometry was used to determine the mass of the "di-
meric" components . However, to extract A? from tis-
sue sources, SDS was included in extraction buffers and the
possibility that these A? species could form during the ex-
traction process was not excluded. Immunoaffinity purifica-
tion, HPLC, and MALDI analyses of CSF from patients suf-
fering from meningitides and other neurological conditions
including dementia, revealed A? species of various length,
including two truncated trimer species, (Asp1–Met35)3 and
(His6–Ala42)3 and an A?40 dimer, (Asp1–Val40)2 . A? in
CSF was shown to be associated with high-density lipopro-
tein particles in an apparent monomeric form detected by
SDS-PAGE [123, 124]. Similarly, 24 and 27% of brain A?40
and A?42, respectively, were shown to be concentrated in
lipid-raft extracts as dimers determined by SDS-PAGE in the
Tg2576 murine model of AD . However, as discussed
above, SDS-PAGE is not a reliable method for A? size de-
Walsh et al. have reported "natural" A? oligomers that
are "SDS-stable" low-order oligomers (dimers, trimers, and
tetramers) detected in the conditioned media and/or lysates
of cells [23, 24, 126-128]. These oligomers are produced by
Chinese hamster ovarian cells that express human mutant
(V717F, V717I, or E693Q, cells referred to as 7PA2), or
wild-type APP [23, 129]. Low-abundance species of masses
~10, 14 and sometimes 16 kDa, which were reactive with
antibodies against A? also were detected in the culture media
of these cells . The nature of these low-order oligomers
is not fully understood but the observations that they are not
disassembled by several types of denaturants suggests that
they may be covalently linked .
Initially, biological effects of oligomeric A? fractions
purified from neuritic plaques and leptomeningeal mural
deposits were investigated in an astroglial-neuronal co-
culture system believed to approximate in vivo conditions
. Neuronal viability was compromised by AD-derived
A? only when microglial cells were co-cultured, indicating
that toxicity was mediated through a microglia-dependent
Ease of maintenance and fast growth-rate of 7PA2 cells
facilitated investigations of the biological activities of "natu-
ral", cell-derived A? oligomers. Intracerebroventricular mi-
croinjection of small volumes (~1.5 ?L containing ~3 ng/mL
A?) of SEC-fractionated cell-culture media to anesthetized
wild-type rats was shown to inhibit hippocampal long-term
potentiation (LTP) [25, 130]. This inhibition was predomi-
nantly mediated by A? trimers in wild-type murine hippo-
campal brain slices, whereas dimers and tetramers had in-
termediate potencies, and monomers were apparently inef-
fective . Cell-derived oligomers were shown to inter-
fere primarily with the induction of LTP, but not its expres-
sion, once the signaling cascades responsible for LTP were
initiated . These results suggested that cell-derived A?
oligomeric assemblies altered certain aspects of hippocampal
synaptic plasticity both in vivo and in vitro (reviewed in
The validity of LTP as an electrophysiological paradigm
for learning and memory composition has been debated
. To test the effect of the "natural A? oligomers" in a
non-LTP paradigm, Cleary et al. used an in vivo behavioral
model in rats that were injected with cell-conditioned media
containing A? assemblies into the dorsal lateral cerebral ven-
tricles. The treatment was found to cause a transient interrup-
tion of pre-learned behaviors . This was attributed to
A? oligomers because SEC fractions containing oligomers
caused the deficits, whereas monomer-containing fractions
were ineffective .
Actin-based cytoskeletal network dynamics is critical for
the regulation of neuronal spine morphology and function
. Alterations in spine morphology and actin-regulatory
mechanisms recently have emerged as a sensitive measure of
early neuronal functional deficits and neurotoxicity [135-
137]. Because loss of synaptic termini strongly correlates
with the severity of dementia, Shankar et al. assessed the
effect of cell-derived soluble oligomers on synapses .
They showed that the density of dendritic spines decreased
substantially in neurons treated with sub-nanomolar levels of
cell-derived A? oligomers . This effect was shown to
be A?-oligomer-specific, i.e., SEC monomer fractions alone
were ineffective. The decrease in spine density was reverted
by monoclonal immunodepletion of A?, by transfer of cells
back into the control media, and by scyllo-inositol , a
molecule thought to stabilize synthetic A? as nontoxic spe-
cies, possibly preventing A? interaction with neuronal target
proteins [139, 140].
In cultures of dissociated cortical neurons, synthetic A?
was shown to activate nicotinic acetylcholine receptors
(nAchRs) and trigger internalization of N-methyl-D-
aspartate (NMDA) receptors (NMDARs) . However,
Shankar et al. found that an irreversible nAchR antagonist
did not affect A?-oligomer-mediated spine loss, indicating
that nAchR activity was unnecessary for this effect .
Signaling cascades involving NMDARs, cofilin, and cal-
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Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 327
cineurin were found to be involved in A?-induced spine loss
as determined by inhibition studies . Together these
data demonstrated that cell-derived low-order A? oligomers
could cause reduction of synapse density and loss of electro-
physiologically active synapses in hippocampal pyramidal
neurons, suggesting that their deleterious effects may be im-
portant mechanistic contributors to synaptic dysfunction in
AD in vivo .
A?-DERIVED DIFFUSIBLE LIGANDS (ADDLS)
ADDLs are exclusively A?42-derived soluble pre-
fibrillar assemblies that morphologically appear as 3–8-nm
globules by AFM [26, 121] and have estimated masses be-
tween 17–42 kDa  (reviewed in ). It was first ob-
served that apolipoprotein J (apoJ, also called clusterin, is a
ubiquitous multifunctional glycoprotein co-localizing with
fibrillar deposits in systemic/localized amyloid disorders
) partially inhibited A? aggregation and caused forma-
tion of "slowly-precipitating" A?42 complexes of >200 kDa
. Follow-up studies showed that ADDLs with the same
biochemical and neurotoxic characteristics could be pro-
duced in clusterin-free solutions by incubating aggregate-
free A?42 in phenol-red-free F-12 medium at 4–8°C for 24
h, in clusterin-free brain-slice culture media at 37°C for 24 h
[26, 142], or even in phosphate-buffered saline .
By SEC, ADDL preparations contained two distinct
peaks–an early-eluting high-mass component, which exhib-
ited punctate binding to primary neurons, and a late-eluting
low-mass component (13 kDa), which lacked this property
. These peaks are similar to those reported by Walsh et
al. for protofibrillar and LMW preparations of A?, respec-
tively (see below) . However, Walsh et al. showed by
TEM that the early-eluting peak contained abundant PF
whereas the late-eluting peak was reported later to contain a
mixture of monomer and small oligomers detected using
PICUP . In a recent study of ADDLs, when SEC coupled
with MALLS and AU was used to determine the size distri-
bution of ADDLs, the SEC peaks corresponding to 75 and
13 kDa showed oligomer masses ranging from 150–1,000
kDa, and a monomeric component of 4.5 kDa by MALLS
. This study suggested that previous reports identifying
low-molecular-mass components as a composite of low-
number oligomers were misrepresentations of what may ac-
tually be a monomeric A?42 fraction . ADDLs found in
the early-eluting peak were shown to be in a dynamic equi-
librium comprising a polydisperse population of oligomers
. Multiple parameters such as peptide concentration,
temperature, pH, storage duration, and excipient addition
were shown to affect this equilibrium dramatically .
Importantly, A?40 does not form ADDLs . NMR
studies have shown that the C-terminus of A?42 is more
rigid compared to that of A?40 [60-62] potentially due to the
extended hydrophobic C-terminus of A?42. This C-terminal
difference and potentially the different monomeric A?42
conformers generated due to this structural difference may
account for the increased toxicity and plaque competence of
A?42 compared to A?40 [60, 61]. Indeed, some oligomeric
moieties (ADDLs, paranuclei, and globulomers, see below)
were shown to form by A?42 only. In fact, the exclusive
A?42 derivation of ADDLs and paranuclei, and the fact that
ADDLs and paranuclei are indistinguishable morphologi-
cally (compare [26, 121] with ), suggests that ADDLs
and paranuclei may be related to each other or even be the
same species obtained under different conditions [26, 33].
ADDLs have been shown to resist dissociation by low
SDS concentrations (0.01%) . However, when su-
pramicellar SDS concentrations were used, ADDLs and fi-
brils migrated with the same electrophoretic profile yielding
monomeric, trimeric and tetrameric moieties . A similar
profile was observed for LMW A?42  produced by SEC
or filtration . At submicellar concentrations of SDS, oli-
gomers were detected both by denaturing electrophoresis and
SEC . When A?42 was electrophoresed in the presence
of submicellar or supramicellar concentrations of SDS, high-
molecular-mass aggregates and intermediate-sized assem-
blies formed, respectively . During SDS-PAGE, these
aggregates may partially dissociate as diffuse A?42
trimer/tetramer components  as observed for ADDLs
. These observations re-emphasize that visualization of
SDS-stable oligomeric A? may be misrepresenting the actual
Initial ApoJ-induced ADDL preparations (0.34 mg/mL)
were neurotoxic . Later studies showed that ADDLs
selectively targeted the principal neurons in the hippocampal
strata pyramidale and granulosum in organotypic murine
brain slices  and inhibited LTP in rat hippocampal brain
slices [147, 148]. ADDLs also were shown to augment the
negative synaptic plasticity of long-term depression (LTD)
. Prolonged maintenance of LTD along with LTP inhi-
bition leads to an overall synapse-inhibitory effect (reviewed
When cultured hippocampal neurons were incubated with
synthetic ADDLs, F-12-prepared soluble brain extracts, or
crude human CSF, and probed with ADDL-specific antibod-
ies a punctate binding pattern reminiscent of synaptic termini
was observed  (Fig. (2)). The antibodies used were
shown to be 100-fold more sensitive to ADDLs (fmol levels)
than to monomeric A? [27, 136, 150-153]. The ADDL-
binding sites were demonstrated to coincide with dendritic
spines at postsynaptic termini of excitatory synapses .
ADDL binding also overlapped with NMDAR subunit NR1
on highly arborized neurons positive for ? calcium-
calmodulin kinase II  which accumulates in postsynap-
tic termini of neurons involved in memory function . In
addition, ADDLs specifically bound to excitatory pyramidal,
but not GABAergic, neurons , and to neurons positive
for NMDAR subunits NR1 and NR2B . Similarly,
ADDLs did not bind astrocytes or inhibitory neurons ex-
pressing glutamic acid decarboxylase . Preferential
binding of ADDLs to excitatory synapses at postsynaptic
sites was consistent with the inhibitory impact of ADDLs on
NMDAR-dependent LTP [26, 149] and NMDAR-mediated
phosphorylation of cAMP response-element binding-protein
, demonstrating that ADDLs could impact crucial re-
ceptors involved in synaptic plasticity.
Defective neuronal actin-regulatory machinery is an un-
derlying factor in dendritic and synaptic dysfunctions in
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328 Current Alzheimer Research, 2008, Vol. 5, No. 3Rahimi et al.
many neurological disorders accompanied by cognitive defi-
cits, including AD and Down syndrome (reviewed in ).
ADDLs were shown to affect spine shape, which, like recep-
tor expression is a facet of spine cell biology with ramifica-
tions for signaling and plasticity [155, 156]. ADDL-induced
alterations of spine morphology resembled the morphology
of immature and diseased spines associated with mental re-
tardation and prionoses .
ADDL binding to neuritic spines was reported to induce
expression of the activity-regulated cytoskeleton-associated
protein (Arc), a synaptic immediate-early gene [150, 158].
Proper expression of Arc is essential for LTP but its ectopic
and aberrant expression causes failure of long-term memory
formation . ADDLs generated a rapid and sustained
increase in synaptic Arc protein expression , which
interfered with long-term memory formation  and was
hypothesized to lead to synapse failure and memory loss. De
Felice et al. showed that ADDL treatment of mature primary
hippocampal neurons led to reproducible and dose-
dependent generation of reactive oxygen species (ROS) in
the vicinity of synaptic ADDL-binding sites . It was
shown that ADDL binding to the NR1 subunit of NMDAR
and NMDA-mediated Ca2+ influx led to ROS generation,
further delineating the mechanisms of ADDL neurotoxicity
. Interestingly, ADDL treatment was shown to induce ?
hyperphosphorylation in neuroblastoma cells and rat hippo-
campal primary neurons before neuronal death occurred
. Intrahippocampal injection of an oligomer-specific
antibody was sufficient to reverse the effect of amyloid and ?
pathologies, providing an additional insight into ADDL-
mediated neurotoxicity .
Although in vitro experiments demonstrated that ADDLs
interfered specifically with memory-associated experimental
phenomena, a crucial question is whether ADDLs exist, and
cause the same toxic effects, in vivo. Conformation-specific
polyclonal  and monoclonal antibodies  shown to
discriminate between ADDLs and A? monomers have been
used to address this question. Dot-blot assays have detected
ADDL immunoreactivity in transgenic mice and in AD
brains which were extracted without detergents or harsh
chemicals precluding extraction-induced alterations of the
assembly structures [27, 153]. ADDL concentrations were
70-fold higher in AD brains compared to controls . In
the nontransgenic mouse brain, no ADDLs (detection limit
<10 fmol/?g) were detected by dot-blotting brain extracts
using an ADDL-specific antibody . However, in brains
of transgenic mice, ADDL concentrations varied from ~20–
250 fmol/?g depending on the brain region tested .
Soluble brain proteins extracted in F-12 culture medium by
ultracentrifugation contained ADDL immunoreactivity that
correlated with presence of AD [27, 150] (reviewed in ).
These findings support the hypothesis that ADDLs may be
an important component in the amyloid cascade, as opposed
to the poor correlation between insoluble amyloid deposits
and cognitive impairment .
PF are A?40- and A?42-derived curvilinear, soluble as-
semblies, which were described originally by Walsh et al.
 and Harper et al. . Walsh et al. reported studies us-
ing SEC, DLS, and TEM examining initial stages of A? oli-
gomerization and characterizing A? intermediates during
fibrillogenesis. By SEC, PF had apparent mass >100 kDa.
They predominantly comprised curved, fibril-like structures
of 6–8 nm in diameter and ?200 nm long as observed by
TEM . Harper et al. detected the same protofibrillar as-
semblies of A? during polymerization by AFM . They
noted that A?40 PF, which appeared during the first week of
incubation, had diameters of 3.1 nm and were 20–70 nm
long . The PF showed a periodic diametrical variation
every 20 nm. In contrast, A?42 PF formed within the first
day of incubation and had larger diameters (4.2 nm) than
those of A?40. A?42 PF elongated overtime with diametrical
periodicity similar to A?40 .
TEM examination of A? PF with rotary shadowing 
demonstrated flexible rods up to ~200 nm long, with larger
diameters than those observed by regular TEM or AFM. PF
appeared more beaded with a periodicity of 3–6 nm, and the
proportion of small PF (<10 nm) was higher, suggesting that
these smaller structures might have been overlooked using
TEM with routine negative staining or that the preparations
Fig. (2). Punctate ADDL-binding to neurons. ADDLs isolated from AD brain or prepared in vitro show identical punctate binding to neu-
ronal cell-surface proteins. Cultured hippocampal neurons were incubated with soluble extracts of human brain or synthetic ADDLs. Im-
munoreactivity against ADDLs was visualized by microscopy using M93 antibody. Soluble AD-brain proteins (a), soluble control-brain pro-
teins (b), synthetic ADDLs (c), and synthetic ADDLs pre-treated (1 h) with oligomer-specific antibody M71 (d) are shown. Small puncta
distributed along neurites, are evident for AD extracts and synthetic ADDLs, but not for control extracts or antibody-preadsorbed ADDLs
(Scale bar = 10 ?m). Adopted with permission from .
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Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 329
were simply different . The beaded structures were
shown to be typical of early PF, whereas at later time points,
PF appeared smoother . High-resolution AFM studies
have demonstrated that A?40 PF had a diameter of ~4.3 nm,
with periodicity of ~20 nm, and coexisted with spherical
species of the same diameter . Spheres similar to those
have been hypothesized by us and others to be precursors
which join together to form PF [33, 110].
PF have a high ?-sheet content, a characteristic similar to
that of mature amyloid fibrils  and are recognized by a
conformation-sensitive antibody, WO1, which reacts with
the fibrillar form of various amyloid proteins . Thus, PF
are the latest precursors on the pathway of fibril formation
described to date. Nevertheless, apparently a substantial con-
formational rearrangement occurs upon maturation of PF
into fibrils as evidenced by the observations that PF can be
readily disassembled into LMW A?, whereas mature fibrils
do not typically disaggregate back into PF . Proline sub-
stitution experiments showed that A?40 PF were "less struc-
tured" in the region Glu22–Gly29 compared to mature fibrils
. Hydrogen–deuterium exchange data demonstrated that
the C-terminal Met35–Val40 and the N-terminal Asp1–Phe19
regions of A?40 were highly exposed to solvent both in fi-
brils and PF. In contrast, the Phe20–Leu34 segment was
highly protected from hydrogen–deuterium exchange in fi-
brils but much less so in PF . The ?-structure (?-sheet
and ?-turn) content of PF was similar to that of fibrils as as-
sessed by CD studies . Collectively, these data suggested
that the ?-sheet elements comprising the amyloid fibrils were
already present in PF. These elements could be expanded
into adjacent residues and other elements, such as lateral
association of filaments may contribute to the maturation of
Initial studies to assess the biological activity of PF were
performed in cultured primary rat cortical neurons over a
time scale of minutes to hours, presumably before PF con-
vert to fibrils . In these experiments, compromise of cell
viability by SEC-isolated PF was evaluated using the 3-(4,5-
(MTT) assay . It was found that PF and fibrils altered
the normal physiology of cultured neurons, whereas LMW
A? did not . To explore and compare the toxic effects of
LMW A?, PF and fibrils further, Hartley et al. used rat cere-
bral primary mixed cultures (containing neurons, astroglia
and microglia) and showed that PF caused neuronal injury
and altered electrophysiological activities, ultimately causing
cell death . Although LMW A? caused a rapid but tran-
sient increase in excitatory post-synaptic currents (EPSCs),
PF or fibrils (~3 ?M) invariably produced rapid and sus-
tained increases in electrical activity, six-fold greater than
that induced by LMW A? . Similarly, PF and fibrils sig-
nificantly increased the frequency of action potentials and
augmented the frequency and size of membrane depolariza-
tion compared to LMW A? preparation . Substantial
neuronal loss (80% as opposed to 10% non-treated cells) was
observed consistently using the lactate dehydrogenase assay
 and immunostaining against neuron-specific, microtu-
bule-associated protein-2, after 5 days exposure to LMW and
protofibrillar A? . It was hypothesized that LMW A?
could either convert to PF that cause the neurotoxicity or it
could induce neurotoxicity by a mechanism independent of
Important insight into the clinical relevance of PF came
from investigation of a family in Northern Sweden, members
of which carry a mutation in the app gene that leads to a sin-
gle amino acid substitution in the A? region, E22G (dubbed
the Arctic mutation) . Surprisingly, even though carri-
ers of this mutation have decreased plasma levels of A?40
and A?42 compared to non-carriers, they develop early-onset
AD . In vitro fibrillization studies showed that A? con-
taining the E22G substitution formed PF faster and in larger
quantities than wild-type A?, suggesting that PF may be the
main disease-causing agents in carriers of the Arctic muta-
tion . These findings, along with the observations of PF
formation by most other amyloidogenic proteins, have posi-
tioned PF as a likely primary pathogenic assembly state and
an important target for drug development efforts for amyloi-
Concentrations of 4-hydroxy-2-nonenal (HNE), a me-
tabolite of oxidative stress resulting from fatty-acid peroxi-
dation, and of HNE-derived lipid peroxidation products,
have been shown to increase in AD . Recently, HNE
has been shown to modify A? by 1,4 conjugation to primary
amino groups and Schiff base formation, which could result
in putative covalent intermolecular cross-linking of A?
monomers . A?40 prepared after a 285-h incubation
with HNE predominantly had a PF-like, curved morphology
when examined by AFM, whereas preparations formed in
the absence of HNE comprised straight fibrils predominantly
. Both the long, straight fibrils formed in the absence of
HNE and the curved fibrillar aggregates formed in the pres-
ence of HNE were rich in ?-sheet structure, based on their
CD spectra. In the presence of HNE, accelerated formation
of protofibrillar A?40 species was observed, whereas genera-
tion of mature straight fibrils even upon extended incuba-
tions was inhibited . These data suggested that HNE
could cause accumulation of toxic A? PF by preventing their
maturation to the less toxic fibrils . Similarly, docosa-
hexaenoic acid was shown to stabilize soluble A?42 PF and
hinder their conversion to insoluble fibrils, thus leading to a
sustained A?-induced neurotoxicity measured using cultured
PC12 cells . These observations suggest that toxic ef-
fects of PF could be promoted by molecular interactions that
prevented downstream fibril formation.
species, termed A?*56, that caused cognitive deficits in
middle-aged transgenic Tg(APPSWE)2576Kahs (Tg2576)
mice . The Tg2576 mice express high levels of a human
APP variant, which carries a familial AD-linked double mu-
tation originating in a Swedish lineage (K670N, M671L)
[172, 173]. A? concentrations in these mice increase rapidly
at 6 months of age, and abundant amyloid plaques are appar-
ent between 9–12 months . Tg2576 mice recapitulate
many other neurological features of AD, including neuroin-
flammation and oxidative stresses, dystrophic neurites, and
significant cognitive deficits (reviewed in ). However,
other important features, such as neurofibrillary tangles and
significant neuronal loss are not found in this model .
Lesné and colleagues have identified an oligomeric A?
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330 Current Alzheimer Research, 2008, Vol. 5, No. 3Rahimi et al.
stration of A?-specific antibodies has been shown to rapidly
ameliorate memory decline, and because memory deficits
were thought to precede plaques, Lesné et al. reasoned that a
particular A? species could promote cognitive decline pre-
ceding plaque maturation . They found that insoluble A?
accumulated over 7 months without noticeable spatial mem-
ory decline. In contrast, certain soluble and extracellular A?
species, which migrated as LMW bands in SDS-
PAGE/Western blots, correlated strongly with memory defi-
cits at 6 months of age, suggesting that these oligomers, par-
ticularly those with apparent gel mobility of A? dodecamers,
were important neurotoxins in this model. Based on these
data, it was proposed that similar species could be causing
AD . In support of
pathophysiologically relevant concentrations of A?*56 (8.5
pmol) were administered into the lateral cerebral ventricles
of healthy young rats pre-trained in the Morris water maze,
the rats developed defective long-term spatial memory.
These are important findings supporting the central role of
oligomeric A? in the disease mechanism of AD.
The structural characterization of A?*56 as a putative
dodecamer should be interpreted cautiously because the ap-
parent electrophoretic migration of this oligomer, corre-
sponding to 56 kDa, may not represent accurately its in vivo
mass given the artifactual effects of SDS , which was
included in the initial steps of the extraction protocol. Of
note, Jacobsen et al. have found that cognitive deficits in
Tg2756 mice occurred before the reported time of appear-
ance of A?*56 .
Recently, longitudinal water-maze spatial training was
reported to reduce A? and ? neuropathology transiently but
significantly, and improve later learning performance in a
triple transgenic (3?Tg) murine model of AD . The
3?Tg-AD mice harbor human APP containing the Swedish
mutations (KM670/671NL), human ? containing the AD-
associated mutation, P301L, and a human presenilin 1 gene
(PSEN1) containing the AD-linked mutation, PS1M146V
. The improvement in performance in 3?Tg-AD mice
occurred at 6–12 months and depended strongly on spatial
training . To achieve this effect, pre-training was re-
quired before development of overt neuropathology, pre-
sumably because it delayed A? redistribution to extracellular
plaques and reduced the concentration of A? oligomers, in-
cluding one with an apparent SDS-PAGE mobility similar to
A?*56 . These findings suggest that A?*56 is a neuro-
toxic form of A? that may be important in the etiology of
AD. Currently, the structural relationships of this species to
PF and ADDLs are unknown, although under certain condi-
tions, ADDLs also display electrophoretic migration corre-
sponding to a dodecamer .
Because in these and other APP transgenic mice admini-
this hypothesis, when
A? pores are channel-like structures believed to disrupt
cell membranes and cellular ionic homeostasis . In lipid
bilayers in vitro, A? was shown to form uniform pore-like
structures with 8–12 nm outer and 2 nm inner diameters
[177, 178]. These are thought to serve as Ca2+ channels and
thus have been hypothesized to cause excitotoxicity and me-
diate A?-induced neurotoxicity in AD [179, 180]. Reports of
various models including artificial phospholipid membrane
bilayers, excised neuronal membrane patches, whole-cell
patch-clamp experiments, and phospholipid vesicles support
a channel-forming property of A? [177, 180-192]. In these
studies, imaging techniques [177, 184, 186, 187], electro-
physiological experiments [180, 181, 183-185, 187, 188,
191, 192] or cation-sensitive dyes  were used to assess
channel-like properties of A?. However, other studies have
reported general disruption of the plasma membrane homeo-
stasis without channel formation [193-195]. In a study by
Kayed et al. the effect of spherical A?42 oligomers on mem-
brane conductivity was assessed using planar lipid bilayers
. It was found that these A?42 oligomers specifically
increased the conductance of the bilayer in a concentration-
dependent manner whereas no increase in conductance was
observed for LMW A? species (monomer or dimer) or for
A? fibrils . The increase in membrane conductance in
response to spherical oligomers occurred in the absence of
evidence for discrete ion-channel or pore formation . It
was postulated that soluble oligomers enhanced movement
of ions through the lipid bilayer by a channel-independent
mechanism . High sensitivity recording has indicated
that there was little change in the noise level of the current
trace as the current increased from 0 to ~100 pA after oli-
A?42 was reported to be more prone to forming channels
than A?40 . High-resolution examination of individual
A?42 channel-like structures revealed two subunit arrange-
ments: rectangular and hexagonal structures, putatively
comprising tetramers and hexamers, respectively . The
disease-associated mutant E22G form of A?40 was shown to
form pore-like structures akin to those formed by A?42 [37,
196]. Treatment of the hypothalamic neuronal cells GT1-7
with A?40 has led to simultaneous formation of Ca2+ chan-
nels and increased intracellular Ca2+ concentration ([Ca2+]i)
as determined by fluorometric measurements, suggesting
that A?40 also could disrupt biological and artificial mem-
branes, possibly via formation of pores [182, 197].
Using PICUP followed by SDS-PAGE analysis, A?40
and A?42 were shown to form distinct oligomer size distri-
butions, suggesting that the two A? alloforms oligomerized
through distinct pathways . In those experiments, A?42
preferentially formed pentamer/hexamer units termed
paranuclei, which self-associated into larger assemblies, in-
cluding dodecamers and octadecamers . In contrast,
A?40 formed a roughly equimolar, quasi-equilibrium mix-
ture of monomers, dimers, trimers, and tetramers .
A systematic study using PICUP assessed oligomeriza-
tion of 34 A? alloforms , including those containing fa-
milial AD-linked amino acid substitutions, N-terminal trun-
cations found in AD plaques, and modifications that altered
the charge, hydrophobicity, or conformation of A? . C-
terminal length was found to be the most important structural
determinant in early oligomerization, and the side-chain of
Ile41 in A?42 was found to be important both for effective
formation of paranuclei and for their self-association .
Thus, A?41 and longer alloforms formed abundant paranu-
clei whereas A?40 and shorter alloforms did not . The
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Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 331
side-chain of Ala42, and the C-terminal carboxyl group, af-
fected paranucleus self-association . In a related study,
oxidation of Met35 in A?42 was found to preclude paranu-
cleus formation and led to generation of oligomers indistin-
guishable from those produced by A?40 . These data
demonstrated that modification of even a single atom could
induce dramatic effects on A? paranucleus formation and
downstream assembly, providing important insights into
mechanisms of A? assembly into neurotoxic oligomers po-
tentially relevant to AD pathogenesis. As discussed above,
the difference in toxicity between A?40 and A?42  cor-
relates with observations that certain oligomeric A? forms,
such as paranuclei and ADDLs, are produced by A?42 only,
emphasizing strong correlation of the latter to the pathogenic
process of AD.
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?amy balls are A?40-derived structures that form sponta-
neously when high concentrations of A?40 (60–600 ?M) are
incubated in phosphate-buffered saline at 30°C for 8–13 days
. ?amy balls, have diameters of 20–200 ?m and were
shown to be composed of birefringent 6–10-nm diameter A?
fibrils with random orientation . Although such high A?
concentrations used to generate ?amy balls are unlikely to
occur in vivo [22, 199], it was argued that these concentra-
tions could possibly occur locally at microfoci circumscrib-
ing the amyloid plaques in AD brain . Interestingly, in
vivo extracellular retinal deposits called drusen have an ap-
parent similarity to ?amy balls. Drusen are A?-containing
macromolecular assemblies and are a pathologic sign in age-
related macular degeneration . However, these struc-
tures have larger diameters and unlike ?amy balls, which are
produced from synthetic A?40 in vitro in the absence of
other proteins, the retinal deposits contain other A?-binding
Amylospheroids, were described as A?40- and A?42-
derived assemblies with 10–15 nm diameters . A?40
amylospheroids formed by incubating 350 ?M of the peptide
in phosphate-buffered saline under slow rotation for 5–7
days at 37°C . A?42 amylospheroids were produced by
incubating 0.01–1 ?M peptide in the same buffer for 8–10 h
at 4°C . Indeed, it was observed that A?42 amylosphe-
roids, which formed faster and at substantially lower concen-
tration than those of A?40, were also more toxic than A?40
amylospheroids , correlating with the higher toxicity and
pathogenicity of A?42 in AD compared to A?40. Although
these A?42 assemblies are spherical , they are morpho-
logically distinct from ADDLs in their diameter (2.5 nm by
AFM  vs. 10 nm by TEM, respectively).
Globulomers are A?42 oligomers produced by incubating
400 ?M A?42 in phosphate-buffered saline in the presence
of 0.2% SDS at 37°C for 6 h . These species also have
been produced by incubation of A?42 with lauric, oleic, or
arachidonic acids , suggesting that they are generated
by interaction of A?42 with micelles of SDS or fatty acids.
A?42 globulomers were neurotoxic to rat brain slices and
antibodies generated against globulomers detected im-
munoreactive epitopes in tissue sections . Similar
globular structures of A?40 were reported to form after 18 h
incubation in 25 mM 2-morpholinoethanesulfonic acid
buffer (pH 4.5) in a "hanging-drop" environment .
Hanging-drop environment is used extensively for protein
crystallization and provides a static, low-convection envi-
ronment with a markedly increased hydrophobic air–buffer
interfacial area compared to that of the microfuge-tube envi-
OLIGOMERS OF DISEASE-RELATED AMYLOIDO-
GENIC PROTEINS OTHER THAN A?
Over twenty human amyloidoses are caused by aberrant
protein folding and aggregation [203-205]. As A? often is
considered an archetypal protein in studies of these diseases,
the discoveries of toxic pre-fibrillar A? assemblies and their
centrality in AD have led to a search for similar assemblies
of other amyloidosis-related proteins. To date, at least 24
different proteins have been identified as causative agents of
amyloidoses . In essentially all cases, such assemblies
have been found and had adverse biological effects similar to
those of A? oligomers [5, 7, 11, 207]. Because this review
focuses on pre-fibrillar assemblies of A?, we do not intend to
cover assemblies of other amyloidogenic proteins in detail
but rather to highlight a few examples and discuss current
features that are common to all or most of these assemblies.
The structures reported for amyloidogenic protein oli-
gomers, in general have been similar to those described for
A?, namely PF, annular (pore-like) PF, and spherical oli-
gomers. In several cases, annular PF have been the predomi-
nant structures found. However, as discussed elsewhere ,
it is important to note that in many cases the term PF has
been used even though the morphologies of the assemblies
under study were distinct from those originally defined as
One of the most studied amyloidogenic proteins is ?-
synuclein, the function of which is not clear although it is
believed to be part of the ubiquitin system that marks pro-
teins for proteasomal degradation [208, 209]. ?-synuclein is
the predominant component in Lewy bodies, the pathological
hallmarks in Parkinson's disease brain, and has been impli-
cated in other degenerative disorders (synucleinopathies),
including dementia with Lewy bodies and multiple system
atrophy . Similar to A?, ?-synuclein belongs to a grow-
ing family of "intrinsically unstructured" proteins [211, 212],
a characteristic that perhaps renders these proteins more
prone to undergoing amyloidogenic assembly. Mutant ?-
synuclein alloforms linked to familial Parkinson's disease
were found to oligomerize faster than the wild-type protein,
whereas the rate of fibril formation did not correlate with the
presence of disease-causing mutations . Pre-fibrillar
assemblies of both wild-type and mutant ?-synuclein in-
cluded spherical oligomers, protofibrillar structures, and
most abundantly, annular PF [196, 214]. The latter morphol-
ogy suggested that the mechanism by which ?-synuclein
induces toxicity is pore formation in cell membranes. In
agreement with this idea, protofibrillar ?-synuclein was
found to permeabilize synthetic vesicles . Interestingly,
this effect was increased by the familial PD-linked mutants
332 Current Alzheimer Research, 2008, Vol. 5, No. 3Rahimi et al.
A30P and A53T  but not by the mutant E46K .
Thus, although pore formation may be involved in ?-
synuclein-induced toxicity, other mechanisms also have been
implicated, but these are not well understood .
Two amyloidogenic proteins involved in sugar metabo-
lism are insulin and islet amyloid polypeptide (IAPP, also
called amylin). Insulin aggregation is not associated with
disease but has been studied by multiple groups as a conven-
ient in vitro model [219-222]. Biophysical investigation of
insulin fibrillogenesis has identified oligomeric populations
with conformations distinct from those of natively folded
insulin dimer and hexamer . Taking advantage of the
relative stability of insulin oligomers and using special in-
strumentation, Robinson and co-workers have provided one
of the first examples of mass spectrometric investigation of
amyloidogenic protein oligomers, and demonstrated the
power of this experimental approach for studying the effects
of pH and metal ion binding on oligomerization . In a
recent study combining structural characterization and cyto-
toxicity experiments, Grudzielanek et al. found no toxicity
for low-order insulin oligomers whereas substantial toxicity
was measured for high-order, ?-sheet-rich aggregates that
displayed either fibrillar or amorphous morphology .
In contrast to insulin, IAPP aggregation is believed to be
causative in T2D. IAPP is a 37-residue peptide hormone
produced in pancreatic ?-cells and co-secreted with insulin.
Early stages of T2D are characterized by insulin resistance
followed by increased insulin and IAPP secretion. Elevated
IAPP concentrations lead to its assembly into toxic oli-
gomers and insoluble aggregates . Oligomeric and proto-
fibrillar IAPP were shown to interact with synthetic mem-
branes , a characteristic that decreases with further ag-
gregation, providing a clue for the mechanism of IAPP toxic-
ity . The interaction with biological membranes may
induce a transient ?-helical conformation in IAPP, presuma-
bly facilitating penetration of the oligomers into the mem-
brane resulting in solute leakage across the membrane [228,
229]. Strong evidence for the neurotoxic role of IAPP oli-
gomers in T2D was given in a study in which rifampicin, an
inhibitor of IAPP fibril, but not oligomer formation, did not
protect pancreatic ?-cells against apoptosis induced by either
endogenously expressed or externally applied IAPP .
More recent data have suggested that in vivo, toxic IAPP
oligomers are formed intracellularly and therefore, oligomer-
specific antibodies do not prevent cell death in vitro and in
Numerous other examples have demonstrated the impor-
tant role of oligomers of amyloidogenic proteins as disease-
causing agents. Before the focus in the amyloid field shifted
from fibrils to oligomers, it had been known that although no
sequence similarity was found among amyloidogenic pro-
teins, the fibril structures of all were highly similar, charac-
terized by fibrillar morphology with periodic helical twist
and cross-? structures [232, 233]. The realization that the
precursor oligomers may be the proximate disease-causing
agents in the amyloidoses related to these proteins raised the
question whether oligomer structures also were similar.
High-resolution microscopic studies of oligomeric structures,
mostly by TEM and AFM, have demonstrated that in most
cases the morphologies observed were spherical, annular, or
protofibrillar (worm-like). Conformational studies of oli-
gomers have been difficult because the oligomers typically
are metastable and exist in mixtures. Structural insight has
been offered by Glabe and co-workers who developed anti-
bodies that showed specificity for oligomers of proteins with
unrelated sequences but did not bind the monomeric or fi-
brillar forms of these proteins . The first polyclonal
antibody, A11, and similar antibodies developed in follow-
up studies showed remarkable ability to bind to oligomers
formed by proteins as diverse as A?, ?-synulein, IAPP,
lysozyme, insulin, poly Q, and prion fragments . These
observations strongly suggested that a predominant mecha-
nism by which these oligomers injure cells is through per-
meabilization of the plasma membrane because toxic oli-
gomers sharing a common structure formed by both intracel-
lular and extracellular proteins [194, 195]. As discussed
above, oligomers may interact with membranes by several
mechanisms, including pore-formation and shallow penetra-
tion under the surface resulting in thinning of the membrane
and a net increase in its permeability. Further delineation of
the specific mechanisms governing these interactions re-
quires additional studies and will be highly important for
designing reagents that block them.
PRE-FIBRILLAR ASSEMBLIES OF DISEASE-UNRE-
In his 1972 Nobel-Prize acceptance speech, Anfinsen
stated that "the native conformation [of proteins] is deter-
mined by the totality of inter-atomic interactions and hence
by the amino acid sequence, in a given environment" which
does not always favor the normally functional and folded
state of proteins. Consistent with Anfinsen's theory, the con-
formational-change hypothesis postulates that one of 17
normally soluble and functional human proteins could un-
dergo structural alterations under partially denaturing condi-
tions leading to self-assembly and amyloid fibril formation
. Besides disease-associated amyloid-forming proteins,
and proteins that naturally form non-pathological, functional
amyloid-like fibrils (reviewed in ), disease-unrelated
proteins  and artificially designed peptides [238-240]
have been found to form amyloid under particular non-native
conditions. To the best of our knowledge, the ability of dis-
ease-unrelated peptides and proteins to form amyloid fibrils
was first reported by Guijarro et al.  and Litvinovich et
al. . The src-homology 3 (SH3) domain of bovine
phosphatidyl inositol 3-kinase (PI3K), an 85-residue, ?-
structured protein, was shown to aggregate slowly and form
amyloid fibrils under acidic pH . Thenceforth, the dis-
ease-unrelated SH3 domain has served as an excellent model
for systematic studies examining structural properties of
amyloid fibrils and molecular mechanisms of amyloid for-
mation [243-246]. The PI3K-SH3 was shown to adopt a
compact denatured state under acidic conditions before
formation of amyloid fibrils . Limited proteolysis
studies showed that PI3K-SH3 at low pH had a partially
folded conformation  and progressively displayed
enhanced susceptibility to proteolysis, suggesting that the
protein became more unfolded in the early stages of aggrega-
tion . In contrast, the amyloid fibrils that formed over
longer periods of time were resistant to proteolysis . It
was suggested that the protein aggregates formed initially
were relatively dynamic species and this flexibility allowed
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Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 333
dynamic species and this flexibility allowed for the particular
interactions leading to formation of the highly ordered fibrils
After Litvinovich et al. demonstrated formation of amy-
loid-like fibrils by self-association of murine fibronectin type
III module , others reported that similar conversions in
a number of disease-unrelated proteins could be induced in
vitro by a deliberate, rational choice of excipient conditions
[248, 249]. Examples (reviewed in ) include human
apolipoprotein CII, ADA2H, amphoterin, stefin B and en-
dostatin, murine V1 domain, equine acylphosphatase (AcP)
and apomyoglobin, monellin (Dioscoreophyllum camminsii),
and yeast phosphoglycerate kinase. Fezoui et al. reported de
novo design of a monomeric ?-helix-turn-?-helix peptide
(?t?) which converted to ?-sheet-rich amyloid-type, prote-
ase-resistant, 6–10-nm fibrils at 37°C in a neutral aqueous
buffer . Formation of fibrils from full-length proteins
requires solution conditions that partially or completely dis-
rupt the native structure of the protein but not completely
disturb hydrogen bonds . It was observed that proteins
with as few as four residues, and amino-acid homopolymers
that are unable to fold into stable globular structures, form
fibrils readily [237, 250, 251]. Therefore, it has been sug-
gested that the ability to form amyloid fibrils could be a ge-
neric property of polypeptide chains .
In one study of pre-fibrillar assemblies of disease-
unrelated proteins, tapping-mode AFM was used to follow
the process of HypF-N aggregation which was induced by
incubating the protein in the presence of trifluoroethanol
. HypF-N was shown to aggregate hierarchically
through a number of distinct steps with morphologically dif-
ferent intermediates . Initially, globular assemblies ap-
peared, which subsequently self-assembled into beaded
chains, similar to those found for amyloidogenic proteins
[253-255]. Subsequently, these organized into crescents,
large annular and ribbon-like structures (Fig. (3)), and even-
tually assembled into mature fibrils of different sizes .
The globule height was measured to be 2.8–3.0 nm .
Although HypF-N and AcP are similarly prone to conversion
from a predominantly ?-helical conformation to one rich in
?-sheet, HypF-N aggregation rate was found to be dramati-
cally higher (~1,000-fold) than AcP, possibly due to the
higher hydrophobicity and lower net charge of HypF-N com-
pared to AcP .
In contrast to fibrils of disease-causing amyloidogenic
proteins, those formed by disease-unrelated proteins did not
cause cytotoxicity in cell-culture experiments. For example,
fibrils formed by the aforementioned ?t? peptide displayed
no neurotoxicity, even though they were morphologically
indistinguishable from A? and IAPP fibrils, which were
toxic . It was therefore unexpected that the pre-fibrillar
assemblies of PI3K-SH3 and HypF-N were shown to be
highly toxic to PC12 cells and murine fibroblasts in vitro
. The extent of cellular injury caused by the cytotoxic
oligomers was comparable to that of A?42 oligomers,
whereas the corresponding fibrils of both PI3K-SH3 and
HypF-N were benign.
Early pre-fibrillar HypF-N assemblies were shown to
permeabilize artificial phospholipid membranes more effi-
ciently than mature fibrils, indicating that this disease-
unrelated protein displayed the same toxic properties as pre-
fibrillar assemblies of pathological peptides and proteins
. Further investigation of the cellular effects of HypF-N
oligomers revealed that they entered the cytoplasm and
caused an acute rise in ROS levels and [Ca2+]i, leading to cell
death . In a study where murine fibroblasts and endo-
thelial cells were treated with pre-fibrillar HypF-N assem-
blies, the two cell types underwent two different death
mechanisms—fibroblasts exposed for 24 h to 10 ?M HypF-
N oligomers underwent necrosis, whereas endothelial cells
treated similarly sustained apoptosis . A similar study
comparing cytotoxic effects of pre-fibrillar and fibrillar
HypF-N assemblies using a panel of normal and pathological
cell-lines showed that cells were variably affected by the
same amount of pre-fibrillar aggregates, whereas mature
fibrils showed little or no toxicity . This difference in
the extent of compromise of cell viability was significantly
related to the cell-membrane cholesterol content and to dif-
ferent cellular Ca2+-buffering and antioxidant capacities of
the various cell types . Recently, it has been shown that
microinjection into rat brain nucleus basalis magnocellularis
of PI3K-SH3 or HypF-N assemblies, but not the correspond-
ing mature fibrils, compromised neuronal viability dose-
dependently . Taken together, these data clearly demon-
strate that the pre-fibrillar assemblies of disease-unrelated
proteins are highly toxic whereas the corresponding mature
fibrils are not . The toxic effect of the oligomers may arise
when these assemblies assume a "misfolded" conformation
which may expose hydrophobic residues that are natively
entombed within the core structure. Such aggregation-prone
regions may interact with membranes and other cellular
components modifying their structural/functional homeosta-
Dobson and co-workers have proposed that evolutionary
mechanisms may have been in force to ensure propagation of
proteins that resist aggregation for efficient function .
However, genetic, environmental and metabolic factors that
decrease the solubility or increase the concentration of sus-
ceptible proteins in vivo may act against those forces and
induce protein misfolding disorders including neurodegen-
erative diseases .
The fact that aggregates of some disease-unrelated pro-
teins could function similarly to those formed by amyloi-
dogenic, disease-related peptides and proteins, has profound
implications for understanding the mechanistic fundamentals
of abnormal protein deposition in amyloidoses. These obser-
vations facilitate investigation and discovery of the general
mechanistic features underlying protein misfolding and ag-
gregation  and help defining likely targets for drug de-
Since the amyloid-cascade hypothesis was formulated
, intensive research has led to an exponential accumula-
tion of data elucidating the pathogenic mechanisms of AD.
In the end of August, 2007, PubMed database searches, us-
ing the queries "Alzheimer's disease" or "amyloid" returned
53,767 and 32,576 hits, respectively. Importantly, as our
understanding of the devastating morbus Alzheimer, and
other neurodegenerative and protein misfolding diseases has
been growing, an alternative, encompassing paradigm has
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334 Current Alzheimer Research, 2008, Vol. 5, No. 3Rahimi et al.
Fig. (3). Hierarchical aggregation process of HypF-N. (a) Tap-
ping AFM images taken under liquid (height data, scan size 670
nm, Z range 20 nm) of HypF-N globular aggregates observed a few
hours after the onset of the aggregation process in the presence of
30% trifluoroethanol (TFE). Scale bar = 100 nm. Inset, STM image
(height data, scan size 42 nm, Z range 15 nm) of globular aggre-
gates obtained under the same conditions, but diluted 1:50 prior to
deposition onto the substrate; the globules are apparently asymmet-
ric and tend to form a defined mutual orientation. Scale bar = 10
nm. (b) HypF-N crescents and a ring observed after 3 days of incu-
bation in 30% TFE (scan size 5.3 ?m, Z range 45 nm). Inset, high
resolution image of a ring (scan size1.9 ?m, Z range 120 nm), re-
vealing its globular components, taken after 5 days of incubation in
30% TFE. Scale bars = 400 nm. (c) Tapping-mode AFM image
taken in air (height data) after three days of incubation in 30% TFE
showing the co-existence of annular structures with thin and wide
ribbon-like fibrils. Scan size 4.8 ?m, Z range 40 nm. Scale = 500
nm. (d) Tapping-mode AFM images taken in air (height data) of
mature fibrils obtained in 30% TFE showing supercoiled fibrils
after eight days (scan size 3.6 ?m, Z range 80 nm). Adopted with
permission from .
emerged. This paradigm postulates that pre-fibrillar protein
assemblies rather than mature amyloidogenic fibrils likely
are the key neurotoxins responsible for most of the patho-
genic mechanisms in protein-misfolding and neurodegenera-
tive diseases, including AD. Accordingly, oligomeric species
with different degrees of structural order are thought to me-
diate various pathogenic mechanisms that may lead to cyto-
toxicity and cell loss eventuating in organic and systemic
morbidity. With the progressive use of classical techniques,
and the advent of novel, sophisticated methodologies, sev-
eral different forms of pre-fibrillar assemblies of A? and
those of other amyloidogenic proteins have been described.
This has led to considerable progress in the elucidation of
structural and functional features and fundamentals of the
assembly of these proteins at the molecular level. Overall, it
is postulated that the pre-fibrillar amyloidogenic proteins are
on path to fibrillogenesis. The resulting protein fibrils are
thought to be the end-stage sinks for the toxic pre-fibrillar
species. Fibrillar assemblies accumulate progressively into
intra- and/or extracellular proteinaceous amyloid aggregates
generating the disease-specific lesions in vivo.
In AD research, various forms of A? pre-fibrillar assem-
blies, including activated monomeric conformers, ADDLs,
PF, cell-derived dimers and trimers, and annular assemblies
have been described (Table 1). In many cases, the structural
and functional interrelationships amongst these assemblies
are still elusive. Nevertheless, some have been shown to be
pathogenic through one or several common pathways, sug-
gesting that they may share structural features and possibly
mechanisms of action. Understanding these intricate struc-
ture–function correlations will decipher a complex and inter-
connected array of pathogenic mechanisms.
Global research efforts have established a framework for
understanding the fundamentals of A? assembly [7, 10, 11].
A remaining challenge is to assess how these fundamental
structural principles are linked to cellular and tissular micro-
environments during progression of AD. Many experimental
conditions have been used to study the structure and function
of pre-fibrillar assemblies, but it is difficult to regenerate and
scrutinize the actual in vivo milieus and conditions in which
protein assembly, oligomerization, fibrillization, and deposi-
tion occur. Similarly, it is extremely difficult to assess all the
possible interactions these assemblies may have with various
cellular components and organelles. A multitude of detri-
mental mechanisms, including disruption of cellular metabo-
lism, synapse structure and function deregulation, membrane
damage, ionic imbalance, oxidative/inflammatory stress,
apoptotic, and other cytotoxic effects, have been shown to be
mediated by pre-fibrillar A? assemblies, emphasizing that a
single therapeutic approach likely will be insufficient to pre-
vent or treat the progression of AD. By inference, this likely
is true also for other amyloidoses. The intricacy of the
pathogenic mechanisms in these diseases calls for rational
diagnostic and therapeutic approaches that would target not
only a single assembly or a single mechanism but a multi-
tude of assemblies and mechanisms. It is important to design
and discover therapeutic agents that could target various as-
semblies that potentially become active early or are continu-
ously active throughout the course of disease. Successful
targeting of pre-fibrillar assemblies in vitro and in animal
models could have crucial diagnostic and prognostic impli-
cations for amyloid-related diseases.
We acknowledge Mr. Sean M. Spring for conducting the
experiment described in Fig. (1). This work was supported
by grants AG027818 from NIH/NIA and 20052E from the
Larry L. Hillblom Foundation, and by a generous gift from
the Turken family.
= Alzheimer’s disease
= A?-derived diffusible ligands
Not For Distribution
Structure–Function Relationships of Pre-Fibrillar Protein Assemblies Current Alzheimer Research, 2008, Vol. 5, No. 3 335
= Atomic-force microscopy
= Apolipoprotein J
= Amyloid ?-protein precursor
= Activity-regulated cytoskeleton-associated
= Analytical ultracentrifugation
= Amyloid ?-protein
= Intracellular Ca2+ concentration
= Circular dichroism
= Cerebrospinal fluid
= Dynamic light scattering
= Excitatory post-synaptic current
= Fourier-transform infrared spectroscopy
= High-performance liquid chromatography
= Hydrogenase maturation-factor
= Ion-mobility spectrometry-mass spectrome-
= Low-molecular weight
= Long-term depression
= Long-term potentiation
= Matrix-assisted laser-desorption ionization
= Multi-angle laser light scattering
= Nicotinic acetylcholine receptor
NMDAR(s) = NMDA receptor(s)
PF = Protofibrils
PI3K = Phosphatidyl inositol 3-kinase
PICUP = Photo-induced cross-linking of unmodified
ROS = Reactive oxygen species
SDS = Sodium dodecyl sulfate
SEC = Size-exclusion chromatography
SH3 = Src-homology 3
SPPS = Solid-phase peptide synthesis
STEM = Scanning transmission electron microscopy
STM = Scanning tunneling microscopy
T2D = Type-2 diabetes mellitus
TEM = Transmission electron microscopy
TFE = Trifluoroethanol
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Received: September 15, 2007 Revised: February 29, 2008 Accepted: February 29, 2008