Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells

The Amyloid-β (Aβ) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Aβ are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Aβ using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Aβ, we identified an scFv that bound oligomeric Aβ specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Aβ aggregation and toxicity, and reduces the toxicity of preformed oligomeric Aβ towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Aβ scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Aβ in AD brains.     

Anti-oligomeric single chain variable domain antibody differentially affects huntingtin and alpha-synuclein aggregates

Huntington's and Parkinson's diseases are both neurodegenerative disorders caused at least in part by misfolding and aggregation of huntingtin (htt) and alpha-synuclein, respectively. Here we use a single chain antibody fragment (scFv) isolated against oligomeric alpha-synuclein to probe similarities and differences between the aggregation and toxic mechanisms of htt and alpha-synuclein. When incubated with htt, the scFv both blocks formation of and promotes dissociation of fibrillar aggregates, but stabilizes formation of cytotoxic oligomeric aggregates. Previous studies with monomeric alpha-synuclein showed the scFv prevented fibrillar aggregation, but blocked toxicity of oligomeric aggregates. These divergent effects suggest the toxic mechanisms of oligomeric aggregates differ among amyloidogenic protein species.     

Can size alone explain some of the differences in toxicity between β‐amyloid oligomers and fibrils?

β‐Amyloid (Aβ) peptide is believed to play a key role in the mechanism of Alzheimer's disease (AD). Aβ tends to aggregate to form amyloid fibrils. A variety of evidence indicates that Aβ aggregates are toxic in vitro and in vivo. An early “Aβ hypothesis” postulated that AD was the consequence of neuron death induced by insoluble deposits of large Aβ fibrils. Newer findings indicate that small soluble Aβ oligomers are the neurotoxic species, yet their structure is still unknown. Many researchers have tried to probe the differences in molecular structure between Aβ oligomers, protofibrils, and fibrils that give rise to their unique toxicities, but with limited success. In this report, we examine the hypothesis that differences in the toxicity of different aggregated Aβ species are the result of differences in species concentration and diffusivity. Using a simple mathematical analysis based on the assumption of a diffusion‐limited reaction, we demonstrate that near 10‐fold differences in toxicity between spherical oligomers and fibrils can be explained from size and concentration arguments. While this work does not suggest that Aβ oligomers and fibrils have identical molecular structures, it highlights the possibility that simple physical phenomena may contribute to the biological processes induced by Aβ. Biotechnol. Bioeng. 2010;106: 333–337. © 2010 Wiley Periodicals, Inc.     

Heat shock protein 70 prevents both tau aggregation and the inhibitory effects of preexisting tau aggregates on fast axonal transport

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.     

Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers

Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Aβ Oligomers and Fibrillar Aggregates Induce Different Apoptotic Pathways in LAN5 Neuroblastoma Cell Cultures

Fibril deposit formation of amyloid β-protein (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Aβ small oligomers and fibrillar aggregates to simulate the action of diffusible Aβ oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Aβ. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Aβ peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Crocetin inhibits beta-amyloid fibrillization and stabilizes beta-amyloid oligomers

Aggregation of a peptide, beta-amyloid (Aβ), is a hallmark molecular process found in Alzheimer’s disease (AD). During Aβ aggregation, oligomeric and fibrillar Aβ are formed, and these molecular self-assembly steps are implicated in generation of toxic effects in AD. Crocetin is a natural carotenoid dicarboxyl acid displaying various pharmaceutical effects and may be co-localized with Aβ mediated by human serum albumin. In the study presented here, we examined the effects of crocetin on Aβ aggregation in three different molecular pathways. Our results demonstrate that crocetin inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils. Moreover, crocetin caused stabilization of Aβ oligomers and prevented their conversion into Aβ fibrils. Our study reveals potential pathological and pharmaceutical implication of crocetin in AD and suggests possible application of crocetin for currently limited structural studies on unstable Aβ oligomers.     

Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer's disease animal models

An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ) play a key role in Alzheimer's disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet.     

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Aβ (amyloid-β peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimer's disease). The major constituent, a 42-residue Aβ, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aβ42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aβ. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aβ binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aβ, but not preformed fibrillar Aβ. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aβ and this may be mediated by mechanical damage to the membranes by Aβ aggregation. This membrane disruption may play a key role in the mechanism of Aβ-related cell toxicity in AD.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.     

Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide

Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.     

Systemic vaccination with anti‐oligomeric monoclonal antibodies improves cognitive function by reducing Aβ deposition and tau pathology in 3xTg‐AD mice

Alzheimer's disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid‐beta (Aβ) peptide plays a pivotal role in the pathogenesis of AD. In AD, the conversion of Aβ from a physiological soluble monomeric form into insoluble fibrillar conformation is an important event. The most toxic form of Aβ is oligomers, which is the intermediate step during the conversion of monomeric form to fibrillar form. There are at least two types of oligomers: oligomers that are immunologically related to fibrils and those that are not. In transgenic AD animal models, both active and passive anti‐Aβ immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. In this report we studied effect of immunotherapy of two sequence‐independent non‐fibrillar oligomer specific monoclonal antibodies on the cognitive function, amyloid load and tau pathology in 3xTg‐AD mice. Anti‐oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduced tau hyperphosphorylation and improvement in cognition. These results demonstrate that systemic immunotherapy using oligomer‐specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in 3xTg‐AD mice. These findings demonstrate the potential of using oligomer specific monoclonal antibodies as a therapeutic approach to prevent and treat Alzheimer's disease.     

Characterization of prefibrillar Tau oligomers in vitro and in Alzheimer disease

Neurofibrillary tangles, composed of insoluble aggregates of the microtubule-associated protein Tau, are a pathological hallmark of Alzheimer disease (AD) and other tauopathies. However, recent evidence indicates that neuronal dysfunction precedes the formation of these insoluble fibrillar deposits, suggesting that earlier prefibrillar Tau aggregates may be neurotoxic. To determine the composition of these aggregates, we have employed a photochemical cross-linking technique to examine intermolecular interactions of full-length Tau in vitro. Using this method, we demonstrate that dimerization is an early event in the Tau aggregation process and that these dimers self-associate to form larger oligomeric aggregates. Moreover, using these stabilized Tau aggregates as immunogens, we generated a monoclonal antibody that selectively recognizes Tau dimers and higher order oligomeric aggregates but shows little reactivity to Tau filaments in vitro. Immunostaining indicates that these dimers/oligomers are markedly elevated in AD, appearing in early pathological inclusions such as neuropil threads and pretangle neurons as well as colocalizing with other early markers of Tau pathogenesis. Taken as a whole, the work presented herein demonstrates the existence of alternative Tau aggregates that precede formation of fibrillar Tau pathologies and raises the possibility that these hierarchical oligomeric forms of Tau may contribute to neurodegeneration.     

Identification of distinct physiochemical properties of toxic prefibrillar species formed by Aβ peptide variants

The formation of amyloid-β peptide (Aβ) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of Aβ. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of Aβ-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aβ aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those Aβ peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the Aβ peptide to form nontoxic versus toxic species.     

Novel curcumin derivatives as potent inhibitors of amyloid β aggregation

Modulation of abnormal amyloid β (Aβ) aggregation is considered to be a potential therapeutic target for Alzheimer’s disease (AD). Recent in vitro and in vivo experiments suggest that inhibition of Aβ aggregation by curcumin would exert favorable effects for preventing or treating AD. We have previously synthesized a series of novel curcumin derivatives. In this study, we investigated the effects of our curcumin derivatives on Aβ aggregation and the cell toxicities of Aβ aggregates. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles, 14 of 41 compounds showed a significant increase in the densities of the bands of Aβ (1–42) by incubation during the aggregation process relative to those of Aβ (1–42) prepared in the presence of the vehicle control. Of the 14 compounds, four compounds additionally reduced cell toxicity of the Aβ aggregates by incubation during the aggregation process. A significant positive correlation was observed between the cell viability and densities of the bands at ranges of 15–20, 20–37, 37–75, and 75–200 kDa in SDS-PAGE. On the basis of these results, we propose four curcumin derivatives with potential for preventing AD. These curcumin derivatives exhibited high inhibitory effects on Aβ aggregation and induced the formation of lower molecular size Aβ species that have weaker cell toxicity. These compounds may exert therapeutic effects on AD in future in vivo studies.     

Kinetic characterization of amyloid-beta 1-42 aggregation with a multimethodological approach

Extensive evidence suggests that the self-assembly of amyloid-beta peptide (Aβ) is a nucleation-dependent process that involves the formation of several oligomeric intermediates. Despite neuronal toxicity being recently related to Aβ soluble oligomers, results from aggregation studies are often controversial, mainly because of the low reproducibility of several experimental protocols. Here a multimethodological study that included atomic force microscopy (AFM), transmission electron microscopy (TEM), fluorescence microscopy (FLM), mass spectrometry techniques (matrix-assisted laser desorption/ionization time-of-flight [MALDI–TOF] and electrospray ionization quadrupole time-of-flight [ESI–QTOF]), and direct thioflavin T (ThT) fluorescence spectroscopy were enabled to set up a reliable and highly reproducible experimental protocol for the characterization of the morphology and dimension of Aβ 1–42 (Aβ42) aggregates along the self-assembly pathway. This multimethodological approach allowed elucidating the diverse assembly species formed during the Aβ aggregation process and was applied to the detailed investigation of the mechanism of Aβ42 inhibition by myricetin. In particular, a very striking result was the molecular weight determination of the initial oligomeric nuclei by MALDI–TOF, composed of up to 10 monomers, and their morphology by AFM.