The culprit behind amyloid beta peptide related neurotoxicity in Alzheimer's disease: oligomer size or conformation?

Since the reformulation of the amyloid cascade hypothesis to focus on oligomeric aggregates of amyloid beta as the prime toxic species causing Alzheimer's disease, many researchers refocused on detecting a specific molecular assembly of defined size thatis the main trigger of Alzheimer's disease. The result has been the identification of a host of molecular assemblies containing from two up to a hundred molecules of the amyloid beta peptide, which were all found to impair memory formation in mice. This clearly demonstrates that size is insufficient to define toxicity and peptide conformation has to be taken into account. In this review we discuss the interplay between oligomer size and peptide conformation as the key determinants of the neurotoxicity of the amyloid beta peptide.


Introduction
Th e original amyloid cascade hypothesis for Alzheimer's disease (AD) [1] has recently been reformulated to focus on soluble aggregates as the pathogenic molecular form of the amyloid beta peptide (Aβ) [2] (Figure 1). Aβ is naturally present in the brain and cerebrospinal fl uid of humans throughout life [3]. Its role is currently unknown. Th e mere presence of Aβ in the brain is not suffi cient to cause symptoms of neurodegeneration. It has been recog nized previously that neuronal injury is rather the result of ordered Aβ self-association [4]. Th e amyloid plaques found in AD patient brains, which serve as a hallmark for AD, have been found to contain vast amounts of Aβ organized into amyloid fi brils. Th ere is no clear correlation, however, between the presence of the Aβ containing plaques in the brain and the severity of the neurodegenerative symptoms observed in AD patients [5]. Th erefore, the focus of research in this area has shifted from senile plaques toward soluble oligomeric conformations of Aβ as the toxic species as these strongly correlate with the severity of dementia [2,6,7]. Th is oligomeric form of Aβ is highly toxic to the brain and is the trigger for loss of synapses and neuronal damage [8,9]. Because of this, many laboratories have been hunting for a specifi c molecular assembly of defi ned size that is the main trigger of AD. Th e result has been the identifi cation of a host of molecular species of Aβ, ranging from dimers [10][11][12], trimers [13] and Aβ species with a molecular weight of 56 kDa [14] to Aβ-derived diff usible ligands (ADDLs) [15,16] and protofi brils [17] in potent neurotoxic fractions. All are capable of impairing memory formation in mice and their formation and signifi cant accumulation in the brain should thus be considered a potential cause of AD.
Recent in vitro studies of the amyloid formation of Aβ demonstrate that the species mentioned all occur but that they are only transiently populated [18][19][20][21]. Moreover, their isolation and characterization are hampered by solvent extraction procedures and detection methods [22,23], making it diffi cult to study them in detail. However, looking at the studies available, a hypothesis can be formed that the key determinant for the neurotoxicity of Aβ not only involves the degree of oligomerization, but also the specifi c structural conformation of peptides in the assembly. Th is concept reconciles the apparently contradictory results that widely diff ering preparations of Aβ exert similar cytotoxic eff ects, and off ers the therapeutic potential for targeting the key conformation with small molecules or mono clonal antibodies. Th is review will discuss the degree to which a specifi c conformation rather than a specifi c oligomer size may act as the key determinant of development of AD.
identifi cation of specifi c Aβ species in the brain that could be related to AD. In spite of this, it remains unclear exactly what happens to Aβ in vivo after it is cleaved from the amyloid precursor protein by γ-secretase. It is known that the carboxy-terminal heterogeneity generated by γ-secretase may be an important contributing factor since in vitro preparations of the two major peptide fragments generated, Aβ 1-42 and Aβ 1-40 , display a marked diff erence in neurotoxicity by a range of biophysical assays and this correlates with a clear diff erence in aggregation behavior [24]. For example, samples of synthetic Aβ 1-40 primarily exist in vitro as a monomer/dimer mixture, whilst from the time of preparation (time zero) samples of Aβ 1-42 also contain a range of other low-order oligomeric species [7].
Th e assembly of mature Aβ amyloid fi brils is generally described as a nucleation-dependent polymerization reac tion. Like any chemical process, the characterization of this assembly process requires the description of the order in which the relevant molecular species occur along the reaction pathway. However, as aggregation is a stochastic process, molecules will not synchronize during the reaction and, as a result, the reaction mixture will be highly complex and composed of several species at any given time. Homogeneity of the sample is not thought to occur until after the polymerization reaction is complete and even then it is possible that the formed mature fi brils or plaques are not eternally stable. To make matters worse, the composition of the reaction will be significantly modulated by peptide concentration and physicochemical parameters, such as temperature, ionic strength and pH. It is thus not surprising that indepen dent studies of this highly dynamic reaction mixture have yielded a plethora of transient molecular species that have been claimed to occupy an essential position along this pathway. Th e transient nature of the intermediate oligomers is equally challenging for the characterization of the toxic potential of these species [25], and thus a number of diff erently sized oligomers have been suggested as the cause of AD (reviewed in [26][27][28]).
In general terms, the kinetics of fi bril formation by Aβ, as well as many other disease-related and synthetic amyloidogenic peptides, consists of a lag phase, during which a thermodynamically stable nucleus needs to accumulate, which is then followed by a rapid elongation phase that sees the formation of mature amyloid fi brils [29]. However, on a structural level there is signifi cant debate over which conformational changes are essential for the timing of the reaction. Most proposed pathways for the initial stages of Aβ amyloid fi bril formation amount to a sequence of events that can be summarized as follows: unordered monomeric Aβ in solution converts into an 'activated' monomer that then recruits other Aβ molecules to form low-n oligomers [30].
Several studies proposed that monomeric Aβ in solution exists in an equilibrium between an α-helical and a β-sheet conformation (step I in Figure 2) and from this mixture only the β-sheet conformer can accommodate the formation of low molecular weight, β-sheetenriched oligomers (step II in Figure 2) [31,32]. In sharp contrast, other reports suggest that the conformation of Aβ in the activated monomeric state involves α-helical components [33]. Interestingly, the ambiguity between αhelical and β-sheet propensities is a suffi ciently frequently occurring feature of amyloid forming sequences that it can be employed to identify such sequences [34,35]. Moreover, helix-helix associations have been proposed to constitute the major mode of early associations between proteins en route to amyloid formation. Apart from Aβ, other well-known examples displaying similar charac teristics include islet amyloid polypeptide, α-synuclein and calcitonin [33]. Th is helix association pathway would require an as yet undefi ned rearrangement of the initial helical species into a β-sheet conformation, but is supported by a range of nuclear magnetic resonance (NMR) studies of the solution structure of Aβ that report an αhelical structure for the monomeric peptide.
It has to be noted that most of these studies were done in the presence of organic solvents and detergents that were intended to reduce the peptide's aggregation rate suffi ciently to allow the recording of NMR spectra.  [2] to include oligomeric species. This hypothesis suggests a sequence of pathological events leading to Alzheimer's disease. It further includes caspase 3 as a potential link between amyloid beta peptide (Aβ) and tau [129]. Interestingly, several studies of the conformations of Aβ in the presence of lipid membranes have reported increases in both α-helical [36] and β-sheet structures [37]. It is generally known that some detergents and organic solvents can induce secondary structure in proteins, particularly α-helical structure (reviewed in [38]). Yet, two carefully conducted studies employing solution NMR and molecular dynamics without the use of organic co-solvents and detergents failed to detect signifi cant α-helical stabilization for Aβ [39,40]. Whether signifi cant stabilization and population of such an intermediate is indeed required to trigger toxic oligomer formation can be debated. To illustrate this, it has been shown for both HypF-N and human lysozyme that aggregation can be initiated by a population of less than 1% of a specifi c partially folded conformation [41,42]. Small and only transiently populated confor ma tions hence could provide the key to toxic oligomer formation. Nevertheless, the confl icting nature of pub lished reports on the conformational bias in monomeric Aβ suggests several aggregation-prone conformations may coexist and that environment heavily infl uences the route taken by most of these molecules. Moreover, post-translational modifi cations and the terminal hetero geneity that characterizes Aβ in vivo may play a dominant role.
Whatever the conformations that drive the initial associations of Aβ peptides, the resulting low-n oligomers seem to be consistently enriched in β-sheet structures, although the topology of the strands remains unresolved: some groups report that oligomers contain exclusively antiparallel β-sheet structure (step III in Figure 2) [43], whereas others suggest a mixture of antiparallel and parallel β-sheets [44]. Structural investigation of the Aβ aggregation pathway has suggested that antiparallel structures need to convert to a parallel topology in order to allow formation of the so-called protofi brillar state of Aβ (step IV in Figure 2), which is the individual building block of the amyloid fi brils and is thought to consist of a single array of peptides in a parallel β-sheet like conformation [11].
Th e length-wise association of individual protofi brils produces the mature amyloid fi brils, whose structure has been studied in most detail due to their high stability under a wide range of physicochemical conditions (step The arrow at the bottom shows the toxic timeframe derived from publications. The scheme has been adapted from Bartolini and colleagues [31]. Step I describes the equilibrium of the amyloid beta peptide (Aβ) monomer between random coil, α-helix and β-sheet. The β-sheet structured Aβ molecule has been adapted from Lührs and colleagues [45] (PDB structure 2BEG) and the α-helix-containing Aβ molecule has been modifi ed from Sticht and colleagues [130] (PDB structure 1AML) using molecular dynamics with Yasara to obtain the given presentation [131]. Toxic V in Figure 2). For example, a model of Aβ amyloid fi brils derived using solution NMR with hydrogen-deuterium exchange and mutagenesis suggests that residues 18 to 42 stack as β-hairpin-like structures along the fi ber axis [45].
Although there is an ongoing debate on the exact involvement of particular residues in this arrangement (in particular the location of the loop), there seems to be general agreement that the amyloid fi brils that accumulate in plaques found in the brains of patients with AD are stabilized by a backbone of two intermolecular parallel β-sheets connected by a loop region [46]. In Figure 2, we show a schematic outline of the current known aspects of the fi brillation mechanism of Aβ, based on a recently published scheme by Bartolini and colleagues [31]. It remains unclear how oligomer size corresponds to conformational reorganization or how oligomer size and conformation combine to yield a toxicity response.

Does the size of Aβ oligomers determine neurotoxicity?
Th e search for an Aβ species that is capable of causing cognitive disorder as observed in AD patients led to several reports of animal-cell derived [13,47,48], humanderived [10] and synthetically prepared [9,[49][50][51][52] oligomers that have been found to induce disruptions of synaptic activity in vitro or impair cognitive function in animal models. For extensive reviews on these species see [26,27,53].

Monomeric and fi brillar Aβ
Monomeric forms of Aβ have frequently been proposed as toxic modulators in the development of AD. For example, Taylor and colleagues [30] reported that maximum cell damage observed in SH-EP1 cells and hippocampal neurons using a SYTOX Green assay coincides with the accumulation of a monomeric Aβ species able to multimerize into higher-n Aβ species, also called 'activated monomer' . Similarly, mature Aβ fi brils have been suggested as potent neurotoxic AD-inducers, although with similar inconsistent fi ndings as for mono meric Aβ. Th e hypothesis that not all fi bril morphologies are equally toxic, leading to variable results with regard to cytotoxicity, was successfully challenged by Yoshiike and colleagues [54]. Th ey reported that, using point mutations and chemical modifi cation, both a β-sheet fi brillar structure as well as the surface physicochemical composition of the fi bril defi ne the toxic potency of Aβ. One year earlier, Puzzo and Arancio [55] had shown that synthetically derived fi brillar Aβ can impair the late phase of long-term potentiation. As it is very diffi cult to ensure the purity of a monomeric or fi brillar Aβ solution, without contamination of either preseeds or protofi brillar material, it can not be excluded that the toxicity observed for monomeric and fi brillar Aβ is actually the result of contamination. Moreover, increasing evidence suggests that the toxicity of Aβ originates instead from oligomeric Aβ, for which reason this review will further focus on the role of oligomeric Aβ in the development of AD.

The synaptotoxic SDS-stable dimer
In 2008, the Walsh lab identifi ed an enrichment of sodium dodecyl sulfate (SDS)-stable Aβ dimers in both human AD patients and rat cerebrospinal fl uid (CSF) [10] that activate glial cells and can lead to nerve cell death in cultures containing astrocytes [56]. Injection of human CSF containing Aβ dimers but not higher-n Aβ oligomers into animals showed a complete abolishment of long-term potentiation ( Figure 3e); this adverse eff ect could be reversed by the systemic infusion of the synthetically derived anti-Aβ 1-40 polyclonal antibody R1282. CSF samples that contained only Aβ monomer and no detectable dimer did not inhibit long-term potentiation. At this time it was also recognized that the isolation of large quantities of the SDS-stable dimer from human CSF was diffi cult, and a synthetic, disulfi de stabilized Aβ dimer (Aβ 1-40 Ser26Cys) was prepared [57] and used to further explore any detrimental eff ects of Aβ dimers on synaptic activity [11]. Later studies [12] used a combined approach of immunoblotting and western blotting tech niques to study the Aβ population in J20 mice carrying Swedish and Indiana mutations in amyloid precursor protein. Th ese studies showed that before SDS-stable dimers can be detected, Tris-buff ered saline and triton-insoluble Aβ aggregates are present, suggesting that the assembly of Aβ species throughout life is dynamic and heterogeneous. Th e authors further concluded that it would be diffi cult to attribute synaptotoxicity to one single Aβ species.

Toxic Aβ*56
In 2006, the Ashe lab reported that the presence of an extracellular, soluble Aβ 1-42 species with a molecular weight of 56 kDa (Aβ*56) coincides with memory loss of Tg2576 mice and that administration of an isolated fraction of Aβ of this molecular weight induced similar memory loss in young rats [14] (Figure 3c). Interestingly, this much larger 56 kDa species results in a similar ADlike phenotype to that occurring with the dimer described by the Walsh lab, suggesting that AD-related toxicity is extended over a very wide range of Aβ oligomer sizes. A recently published work systematically com paring the eff ects of brain-or cell-derived Aβ assemblies with synthetic preparations further corro borated a concentration-dependent detrimental eff ect of Aβ*56 oligomers on cognition in rats [58].

Lipid-dissociated mature fi brils
In 2008, work by our own group suggested that mature fi brils may not be the inert end-products of a pathway Passive avoidance test of mice injected with backward protofi brils. Light-dark step through test showed latency of entrance during the training accompanied with electrical shock (white bars) and during the testing 24 hours later (black bars). Injection of Aβ fi brils/brain total extract (BTE) mixture-soluble fractions 1.5 hours before the shock impaired memory in contrast to groups injected with control vehicle. From Martins and colleagues [59]. (b). Soluble Aβ extracted from Alzheimer's disease (AD) brain alters hippocampal synapse physiology and learned behaviour. Rats receiving AD Tris-buff ered saline (TBS; dashed line) had a signifi cantly shorter mean escape latency than animals receiving immunodepleted AD TBS (continuous line) at 48 hours after training. From Shankar and colleagues [11]. (c) Eff ects of purifi ed brain Aβ*56 (soluble Aβ species with an apparent molecular weight of 56 kDa) on memory of young rats. Aβ*56 impairs spatial memory. Rats that received vehicle but not Aβ*56 injections showed a signifi cant spatial bias for the escape location 24 hours after training [14]. (d) Vulnerability of NT2 cells to soluble oligomeric Aβ in vitro. The ability of cells to oxidize MTT was used as a measurement of cell viability after treatment with Aβ. Cells were incubated in the presence of diff erent concentrations of either Aβ or equivalent amounts of dimethyl sulfoxide (DMSO) control for 20 hours. The x-axis represents the concentration of soluble oligomeric Aβ. The y-axis represents the percentage of viability of cells compared with the DMSO control. From Kim and colleagues [68]. (e) Human cerebrospinal fl uid (huCSF) containing clearly detectable Aβ dimers disrupts synaptic plasticity in vivo. Samples of huCSF containing Aβ dimers (huCSF D) completely inhibited long-term potentiation, and this inhibition was prevented by previous immunodepletion of Aβ. Untreated huCSF (open circles), and immunodepleted samples (fi lled circles) were injected 10 minutes before high-frequency stimulation (arrow). From Klyubin and colleagues [10]. ADDL, Aβ-derived diff usible ligand; EPSP, excitatory postsynaptic potential. involving the formation of a heterogeneous population of transient toxic Aβ oligomeric species [59]. We found that co-incubation of mature Aβ fi brils with biomimetic mem brane particles results in the release of toxic Aβ oligomers, suggesting a fi bril to oligomer pathway. Incubation of released Aβ oligomers from fi brils on hippocampal primary neuronal cell cultures resulted in profound cytotoxicity, and animals injected with these oligomers showed signifi cant cognitive decline compared with control animals (Figure 3a). Th e main question arising from these observations is whether this fi bril to oligomer pathway might occur in vivo. Nevertheless, it has been reported that secondary lipid metabolic disorders, such as hypercholesterolemia [60,61] or deregulation of sphingolipid metabolism [62], frequently cooccur with a diagnosis of AD. An interesting link with regard to this is the established fact that the ε4 allele of the gene encoding apolipoprotein, a cholesterol-carrying protein, has been defi ned as the major risk factor for AD, while the ε2 allele is protective [63,64]. An extensive review on the role of the apolipoprotein E allele type on progress of AD has appeared recently [65]. Th e relevance of such an apolipoprotein E allele type in the lipid-induced dissociation mechanism leading to sporadic forms of AD as well as the precise contribution of lipid-induced Aβ fi bril dissociation and the link with apolipoprotein E phenotype in vitro remain to be confi rmed.

Aβ-derived diff usible ligands
In 1994 Oda and colleagues [66,67] fi rst mentioned that incorporation of clusterin (apoJ) into an Aβ 1-42 solution inhibited mature fi bril formation but stabilized a slowly sedimenting Aβ 1-42 aggregate. Aggregates formed according to this protocol are resistant to low concentrations of SDS (prepared in the presence of clusterin (apoJ)) and enhance oxidative stress in PC12 cells. A similar aggregating species can also be formed in the absence of clusterin by employing cold-induced aggregation [9]. Th ese so-called ADDLs potently disrupt long-term potentiation in hippocampal slices from young adult rats at very low concentrations [9], reduce cell viability in a range of diff erent cell lines [68] (Figure 3d) and were found to alter cell viability by aff ecting membrane thickness and inducing overall ionic leakiness [69]. Structural characterization revealed that ADDLs are small (approximately 4.8 to 5.7 nm), soluble globular structures [9,70] with an estimated mass of 17 to 42 kDa (derived using atomic force microscopy (AFM)) [9] that migrate during SDS gel electrophoresis to 17 kDa and 27 kDa, with the latter being the predominant species [9]. Th e very low concentration required for a toxic response led to the hypothesis that ADDL-induced toxicity may be specifi c and it was postulated that such toxicity might involve specifi c cell-surface receptors [9,68].

Protofi brils
Aggregation kinetics of Aβ 1-40 and Aβ 1-42 were investigated by Harper and colleagues [18] and Walsh and colleagues [21] using AFM. Th ese studies detected curvilinear, soluble assemblies, termed protofi brillar aggregates, which appeared to be intermediates on the pathway to amyloid fi bril formation. Th eir transient and intermediate nature was confi rmed by the fi nding that these aggregates grow slowly at fi rst and then rapidly disappear in favor of the formation of mature amyloid fi brils [18]. Th eir involvement in disease was derived from size exclusion chromatography experiments showing that Aβ 1-42 and Aβ 1-40 E22Q (later called the 'Dutch' mutation), accumulate more protofi brils compared with wild-type Aβ 1-40 [13], and they were found to inhibit memory formation in animals [11] (Figure 3b). Morphological characterization of these protofi brillar structures by AFM, transmission electron microscopy, quasielastic light scattering and size exclusion chromatography revealed that these curvilinear, soluble assemblies have an apparent mass of >100 kDa, a diameter of 6 to 8 nm, and a length of ≤200 nm [21].

Cytotoxicity and neurotoxicity as parameters for development of AD -practical considerations Extraction procedures
Th e wide variety of transient and intermediate Aβ species on the pathway to fi bril formation (dimers, trimers, higher n-mers, protofi brils) that have been detected by several studies over the years have all been found to exert toxicity on cells in a manner that supposedly causes AD ( Figure 3). Interestingly, Bernstein and colleagues [71] showed by using ion mobility spectroscopy coupled with nanoelectrospray mass spectrometry that Aβ 1-42 can spon taneously co-exist as monomeric or as large oligomeric aggregated species. Th ese large species rapidly developed into large aggregates. However, fi ltration employed to remove large species resulted in the formation of small oligomers that did not convert into large aggregates rapidly. Various subfi brillar Aβ-derived toxins have been detected in vivo [72][73][74] and these all correlate well with brain dysfunction and degeneration observed in both transgenic mice and humans [7,75]. Th e extraction and analysis procedures used to study these Aβ fractions involve methods that have been established to dissociate or, at least, destabilize fi brillar proteins and include homogenization, addition of SDS, β-mercaptoethanol or urea and boiling [7,75]. Th erefore, it is not clear whether any of these species really occur in the diseased brain.
Another approach, involving the preparation of oligomeric Aβ from a synthetic source and subsequent evaluation of their impact on synaptic activity or cytotoxicity has proven a valuable approach to circumvent the use of harsh extraction procedures for analysis of in vivo occurring Aβ. A recent publication by Reed and colleagues [58] compares the eff ects of Aβ derived from synthetic sources, transfected cells and mouse or human brains. Cognitive eff ects were studied using the Alter nating Lever Cyclic Ratio cognitive assay upon intraventricular injection with Aβ from various sources into rat brains and it was found that Aβ oligomers from all sources are potently able to induce cognitive defects.

The detection of toxic Aβ assemblies by A11 antibody
Th e mature fi brillar form and monomeric Aβ have both been confi rmed on many occasions as the only non-toxic species. Moreover, co-incubation with the toxic-conformation-recognizing antibody A11 suppresses the toxic eff ect of Aβ oligomers, suggesting that, even though these Aβ species vary widely in size and shape, there must be a common denominator to their toxic origin. Even more interestingly, many proteins, either disease-related or nondisease-related, have been found to undergo a specifi c conformational transition that is toxic to cell cultures and is A11-reactive [19,76]. However, A11 reactivity as such does not always correlate well with the potency of Aβ assemblies to induce toxicity, as was reported recently by Noguchi and colleagues [77]. Th ese researchers isolated an A11-negative fraction of Aβ from brains infl icted by AD or dementia with Lewy Bodies morphologically appearing as a 10-to 15-nm spherical Aβ species and reproduced these structures using synthetic Aβ. Both assemblies induced damage to human neuronal cells and mature rat hippocampal neurons, and these Aβ species were assigned a role in disturbing presynaptic signaling mechanisms [77]. It appears that even though it is potentially useful for preliminary screening for toxic potency, A11-negativity does not exclude the possibility of Aβ assemblies playing a major role in the disease mechanism of AD. It is hence advisable to combine studies using the A11 antibody with complementary results on the neurotoxicity of detected or isolated Aβ species.

The complex cellular environment
An important discussion that tightly links into the fi eld of defi ning the most toxic species of Aβ is that on defi ning to what extent cytotoxic and neurotoxic results actually represent in vivo pathophysiological mechanisms leading to AD. A wide range of results are used to assay for the eff ects of Aβ on (neuronal) cells, which can be divided into experiments probing for general cytotoxic, and more specifi c neurotoxic deviations. For example, assays that exclusively evaluate the cytotoxic eff ects of Aβ species largely ignore subtle changes that have been shown to occur in the metabolism and functional output of neuronal cells prior to overt cell death (reviewed in [78]).
Aβ is known to be a dynamic molecule, converting from monomeric through a number of lesser-defi ned oligomeric stages to mature plaques; as a consequence, time points at which cytotoxicity is observed do not refl ect the initial stages of the damage Aβ infl icts.
A further complication with regard to neurotoxic results is that the actual initial stages of the disease are not known and have been reported to involve, for example, microglia cells of other than neuronal origin (reviewed in [79]). An elaborative review on the toxic eff ects of Cu/Zn superoxide dismutase (SOD1), implicated in amyotrophic lateral sclerosis, illustrates how the protein can aff ect multiple cell types in a highly complex manner which, judging from the complexity of the brain anatomy as well as the diffi culty in developing a treatment and pinpointing the actual triggers for onset of AD, may well be applicable to AD as well [80].
Th ese fi ndings suggest that one single result does not necessarily embrace all the facets involved in the development of AD and underlines the necessity to employ a multidisciplinary approach, including assays to report on the early eff ects (for example, long-term depression (LDP) or synaptic activity read-outs) and late eff ects (cytotoxic eff ects) of Aβ on a cellular level as well as, for example, cognition assays to evaluate the progress of the condition overall. It further implies the importance of investigating the continuity of the disease in terms of time lines of appearance of specifi c symptoms and cellular eff ects rather than a single time point or Aβ species. A publication that illustrates the dynamic eff ects of Aβ toxicity rather well shows that Aβ-mediated neuronal cell death is a dynamic process, depending on the progress of the aggregation pathway of Aβ, rather than a stable situation [81].

Analyses for AD-linked pathophysiology: the MTT assay
It is worth mentioning some issues with regard to the actual measurement of the potency of Aβ species to induce potentially pathophysiological eff ects in AD models. A frequently used assay for this purpose is the MTT assay, which is based on the conversion of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (a tetrazole) into purple insoluble formazan. Th e formazan compound is subsequently solubilized by a solubilization solution and the absorbance of soluble formazan can be measured spectrophotometrically [82]. Th e reduction reaction to formazan can, however, be aff ected by Aβ in the absence of cell death [83][84][85]. A number of potential reasons for this eff ect have been discussed by Wogulis and colleagues [81] and range from variations in formazan crystal morphology, exocytosis of MTT formazan, MTT-induced interaction of Aβ with intracellular targets and eff ects of Aβ on the neuronal membrane.

The concentration debate
A further discrepancy that is frequently highlighted in the literature is the observation that often non-physiologically related and excessive concentrations of in vitro prepared Aβ are required to observe detrimental eff ects. For example, in cell cultures Aβ usually needs to be added in the micromolar range for eff ects to be observed, while physiological concentrations of Aβ in vivo are estimated to be in the nanomolar range. One recent publication provides a potential answer to this question by showing that Aβ can be taken up by neuronal-like cells but not neuron-unrelated kidney cells. Upon uptake, Aβ can accumulate in late endosomes or lysosomes, where Aβ concentrations of up to 2.5 μM can be reached. At such elevated concentrations Aβ was found to form high molecular weight Aβ assemblies and these assemblies can, in turn, seed amyloid fi bril formation [86].

Toxic species or building block?
It is entirely possible that all the preparations mentioned in the previous paragraphs are unifi ed by a highly potent neurotoxic Aβ assembly of well defi ned size that is present at concentrations that preclude direct detection but mediate toxicity eff ectively, and that this elusive species is the cause of AD. However, it is more likely that a wide range of Aβ oligomers are toxic. Given that the reaction mixture is highly heterogeneous, the purifi ed species is an almost arbitrary result of the conditions chosen to enrich the fraction and the method employed to establish a size estimate. Th e apparent molecular weight of a particular fraction is usually estimated from its elution profi le from matrix-assisted molecular sieving techniques, such as gel electrophoresis and size exclusion chromatography, often carried out in the presence of low concentrations of SDS (0.1%). In order to arrive at a molecular weight estimate, the mobility of the oligomeric fraction needs to be compared to those of other proteins and peptides that act as molecular weight standards. Th ere are several problems with this approach. First, in the absence of even these low doses of detergent, the elution profi les show almost exclusively higher molecular weight assemblies, raising the question of whether the reported molecular size corresponds to the neurotoxin itself or merely to a stable fragment [22,23,87]. Second, comparison with migration profi les of reference proteins is not reliable for aggregating proteins that have a notorious feature that is systematically underestimated in the AD fi eld: amyloid fi brils and their precursors are employed in nature for their mechanical strength and high adhesiveness on a range of surfaces. Bacteria, for example, employ curli amyloids for adhesion to host organism tissues or to colonize a synthetic surface [88]. When such preparations are subjected to molecular sieving, they will hence be sorted by their affi nity for the matrix and resistance to the employed fl ow rate, thereby obliterating the main assumption required to use molecu lar sieving profi les to estimate size. When sample fraction ation is achieved in detergent free buff ers, and the molecular weight is measured using an absolute technique, such as static light scattering, that does not require comparison of the migration rate through some chemical matrix, signifi cantly higher molecular weight species have been observed, both by us [59] and by others [89].
In conclusion, it remains unclear which of the species discussed above occurs as quasi-independent units in the brain of AD patients or if they are usually part of larger molecular assemblies. After all, the extraction procedures used may importantly aff ect the Aβ species obtained, which might not necessarily refl ect those involved in the development of AD. One apparent conclusion that can be drawn is that the toxic form is neither monomeric nor fi brillar, and that the toxic species is a soluble form of Aβ. As oligomer size does not correlate tightly with the progression of AD, it seems likely that the toxic behavior can be induced by factors other than oligomer size alone.

The structural requirements of a neurotoxin
A complete overview of the pathways through which Aβ oligomers are thought to mediate toxicity falls well outside the scope of this article and these are reviewed extensively elsewhere [90,91]. However, we need to briefl y consider them here in order to understand the structural requirements for toxicity-mediating interactions. It now seems that soluble Aβ oligomers do not aff ect the lifecycle of neurons in general but that the eff ect may be more specifi cally related to the interference of synaptic function, judging from the low eff ective dosage required to induce a toxic response [9], and more specifi cally, that Aβ oligomers interfere with the mach inery responsible for synaptic vesicle release at the presynaptic terminal (for reviews see [53,92]).
Two major classes of mechanisms have been proposed to explain how Aβ oligomers interact toxically with the cell. One class is receptor-mediated interactions, such as the previously proposed interactions between receptor X and ADDLs [40], the interaction with N-methyl Daspartate receptors [93,94] or the interaction with human prion proteins by the Strittmatter group [95]. Although the conformational requirements for receptor-mediated toxicity have not been extensively studied, we know from other protein-peptide interactions that structural require ments are usually very specifi c [96]. Th e second major group of toxic mechanisms involve some form of membrane disruption, which is also a much explored potential disease mechanism for several other amyloidoses (for a review see [97]), lending support to the idea that amyloid formation by any peptide sequence goes through a toxic intermediate structure. In fact, a common mechanism for amyloid toxicity, such as was proposed by Dobson and Stefani and colleagues [98] from toxicity studies of a range of amyloid fi brils, could be equally compatible with a receptor-mediated interaction, although the focus in this fi eld has been strongly on membrane disruption. Another strong line of support for the notion of a toxic conformation comes from the conformationspecifi c antibody A11, which also recognizes several other amyloidogenic proteins and peptides that are known to form pore-compatible oligomers [19,99]. A recent review by Glabe [100] explores the concept of conformationdependent toxicity in more detail.
Th e strong focus on the sizes of Aβ assemblies that display potency in the disruption of neuronal function may falsely give the impression that other structural aspects, like peptide conformation, are being neglected. However, when considering the possible molecular mechanisms by which the Aβ peptide exerts its toxicity, the conformation of the individual peptide in the molecular assembly is clearly a critical factor that needs to be carefully considered, and both receptor-mediated interaction and membrane disruption models impose specifi c structural requirements on the oligomers. Progress in this fi eld is slow, however, since techniques for studying peptide conformations suff er from the experimental diffi culties of working with heterogeneous aggregating samples even more than molecular sizing techniques as they generally require higher peptide concentrations, further promoting aggregation.
Th e seemingly confl icting results obtained by diff erent groups on the size requirements of the toxic Aβ oligomer could be elegantly unifi ed when a model is considered in which peptide oligomerization provides the necessary intermolecular interactions required to stabilize a toxic conformation that cannot be adopted by a monomer in isolation. In such a model any oligomer could be toxic as long as its constituent peptides are maintained by the intermolecular interactions in the appropriate confor mation to mediate toxicity, rendering dimers as potently toxic as 12-mers or higher order oligomers. Th e model would further predict that oligomerization is required per se (independent of exact size) and that a specifi c conformation needs to accumulate in the Aβ oligomer fraction in correspondence with toxicity.

Structural studies detect a specifi c conformation of toxic oligomers
Substantial evidence supports the hypothesis that fi brillar Aβ is conformationally distinct from monomeric Aβ but the investigation into the specifi c structural transition states of Aβ along the pathway to mature fi brils has been hampered by the inability to arrest the transient Aβ oligomers in stable, intermediate conformations. A variety of spectroscopic techniques have shown that monomeric Aβ is disordered in aqueous solution, adopting some nonrandom, local conformations [101,102]; thus, it can be categorized as a 'natively disordered' protein, such as α-synuclein, which is implicated in Parkinson's disease (for a review on natively disordered proteins see [103]). Solid-state NMR studies have revealed that Aβ fi brils are organized in a parallel β-sheet structure (for a review see [104]). Site-directed spin labeling showed that fi brillar core regions are composed of 20 amino acids or more (for a review see [105]) and the cores of most amyloid fi brils, including those composed of Aβ, have been found to assume a typical steric zipper conformation [106].
Th e question that arises is: does unstructured Aβ directly transform into a well-organized fi bril or is/are an intermediate(s) involved? Moreover, how does such a transition in structure relate to the toxicity observed for oligomeric Aβ? A structural investigation using circular dichroism on the fi brillation pathway of Aβ 1-40 by Walsh and colleagues [107] suggests that Aβ fi rst forms a transitory α-helical conformation and then transforms into a β-sheet characteristic for fi brillar Aβ. Accumulation of early α-helical enriched intermediates has been reported before using in vitro experiments and molecular modeling [43,[107][108][109][110]. Whether α-helix formation is relevant within the AD context or is on- [108] or offpathway [111] has been an issue of debate. However, the fact that aggregation and toxic eff ects in cell culture and hippocampal slices are inhibited and that locomotor activity is improved and the lifespan prolonged in Drosophila melanogaster in the presence of ligands that bind to and stabilize a region in Aβ in an α-helical conformation [110] supports the on-pathway paradigm. Such an eff ect can be explained either by a neutralizing eff ect on toxic α-helical Aβ or, alternatively, by these inhibitors blocking earlier stages in the aggregation process that can lead to the formation of the actual toxic species. Th e occurrence of a kinetic intermediate composed of α-helical components is not limited to Aβ but is also observed for a range of other peptides, such as insulin [112,113] and a model 38-residue helix-turn-helix peptide, αtα [114].
Computational analysis of the fi brillation pathways of Aβ 1-40 and familial AD-related mutations suggests that early events involve the formation of an ordered, cross-βstructured nucleus composed of six to ten monomer chains [115]. Th is supports an earlier proposition by Grant and colleagues [116] that the aggregate seed for Aβ involves a specifi c type of collapsed structure involving exposed β-strands. Th e evolution of β-structure upon higher order oligomerization and fi brillation has been published many times using a range of spectroscopic techniques, including circular dichroism [31,107,108], fi ber X-ray crystallography [106] and Fourier transform infrared spectroscopy [32,43], and also by computational methods [109]. If both mature fi brils and growing oligo mers exhibit β-sheet structure, then what determines their diff erential toxic eff ects? Work by the group of Goormaghtigh has shown, using attenuated total refl ec tion Fourier transform infrared spectroscopy, that an anti-parallel β-sheet conformation of Aβ distinguishes the oligomeric structure from the parallel β-sheet structure of mature fi brils [43]. Th e experimental set-up used ensured careful preparation conditions for Aβ to obtain solutions enriched in either oligomeric or fi brillar Aβ. To support this suggestion, the work by Yu and colleagues [44] used NMR to show that oligomeric intermediate assemblies stabilized by the addition of detergents and fatty acids have a mixed parallel and antiparallel β-sheet structure that can alter synaptic activity. On a longer timescale this anti-parallel β-sheet rearranges into a less fl exible parallel β-sheet characteristic for fi brillar Aβ [43]. If the membrane-peptide interactions are indeed responsible for the onset of the cascade of toxic responses leading to cell death, then mutations in Aβ leading to early onset cases of AD should show considerably more pronounced interaction with membranes.

Conformational antibodies, β-sheet breaker compounds and mutation are eff ective disruptors of Aβ toxicity
Th e introduction of β-sheet breaking amino acids in the carboxyl terminus of Aβ 1-42 [117][118][119], or co-incubation with β-sheet breaking compounds [120][121][122][123] or peptides [124] have been shown to be highly eff ective inhibitors of Aβ aggregation and to reduce toxic responses to Aβ in a neuronal cell culture [117,119]. Th e relevance of β-sheet structure for toxicity has also been supported by the fi nding that many proteins respond to the conformationspecifi c antibody A11 [19], including natively folded proteins not related to disease -for example, a GroEL oligo mer complex, a bacterial chaperonin [76], heat shock proteins 27, 40, 70, and 90, yeast heat shock protein 104 and bovine heat shock cognate 70 [76], but also trans thyretin and α2-macroglobulin, both proteins associated with aggregation themselves and found to have a β-sandwich topology [125][126][127][128]. Interestingly and analogous to β-sheet breaker peptides, co-incubation of Aβ and a range of other oligomeric aggregates from αsynuclein, islet amyloid polypeptide, polyglutamine, lysozyme, human insulin, and prion peptide 106-126 with other A11-positive proteins has an anti-aggregation eff ect on Aβ [19] similar to co-incubation with A11 antibody [76] and limits toxicity in neuroblastoma SH-SY5Y cells as assessed by the 3-[4,5dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide reduction assay and lactate dehydrogenase release [19]. Moreover, recent work by Yoshiike and colleagues [76] has defi ned the epitope of the A11 antibody as the β-sheet edge of proteins. Th e A11 antibody is reactive to the Aβ oligomer conformation over a wide timeframe and recognizes pentameric Aβ up to protofi brils [19]. Th is fi nding strongly suggests a structural similarity between all these species, which could be related to toxicity. SDS-stable tetramers and dimers are not recognized by the A11 antibody [19] but still have been found to exert profound toxicity in cell culture [11,56]. However, extraction procedures to obtain dimeric and other low-n aggregated Aβ from tissue and neuronal cells that involve SDS or sonication could partly dissociate small oligomers into their basic building blocks. Th ese fi ndings show that toxic oligomeric Aβ, over a wide range of sizes, has a structure distinct from monomeric or fi brillar Aβ, which might provide the key to their toxic potential.

Conclusion
Th e studies reviewed above highlight the challenging nature of identifying the critical component of the complex and constantly evolving reaction mixture that is the ageing Aβ peptide solution. However, the potential result will be highly rewarding as it will allow us to understand how known risk factors for AD map onto the formation of toxic intermediates of amyloid formation: do well established risk factors such as carboxy-terminal heterogeneity, the apolipoprotein E allele and age act by stabilizing the toxic substructures, and if so, which therapeutic interventions are best suited for counter act ing these eff ects? Moreover, as long as we cannot accu rately model the accumulation of the neurotoxic species, we cannot be certain that therapeutic inter vention will produce the desired outcome. For example, an overall decrease in the concentration of Aβ may stabilize low-n oligomers, thereby poten tially increasing rather than decreasing toxicity.
In conclusion, a wide range of AD-related (synapto) toxic Aβ oligomeric sizes have been identifi ed. How oligo mer size precisely relates to the disease process has not been clarifi ed and recent work shows that the wide range of Aβ oligomers may have a specifi c conformation in common. Th ese fi ndings suggest that Aβ oligomer size may not be the only AD-inducing factor and we propose a new paradigm in which both oligomer size and structural arrangement act as toxic parameters in AD development. Studies targeting the toxic contributor to AD in the past have usually highlighted only one of these aspects, but we suggest that further studies should employ a multi disciplinary approach in which oligomer size, structural characteristics and synaptic activity results are recorded simultaneously over a long kinetic timeframe. Suffi cient resolution in such a kinetic study should allow access to structural information on transient intermediates, which may be further supported by employing means of stabilizing these species using cross-linking, conformation-sensitive antibodies or lipid-induced fi bril dissociation and by studying familial AD-related mutations in Aβ.

Competing interests
The authors declare that they have no competing interests.