Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ₄₀ peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ₄₂ peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide.     

General Amyloid Inhibitors? A Critical Examination of the Inhibition of IAPP Amyloid Formation by Inositol Stereoisomers

Islet amyloid polypeptide (IAPP or amylin) forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer''s disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibit Aβ amyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design.     

Formation of Dynamic Soluble Surfactant-induced Amyloid β Peptide Aggregation Intermediates

Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k_ex ∼1100 s⁻¹) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.     

Transthyretin Interferes with Aβ Amyloid Formation by Redirecting Oligomeric Nuclei into Non-Amyloid Aggregates

The pathological Aβ aggregates associated with Alzheimer's disease follow a nucleation-dependent path of formation. A nucleus represents an oligomeric assembly of Aβ peptides that acts as a template for subsequent incorporation of monomers to form a fibrillar structure. Nuclei can form de novo or via surface-catalyzed secondary nucleation, and the combined rates of elongation and nucleation control the overall rate of fibril formation. Transthyretin (TTR) obstructs Aβ fibril formation in favor of alternative non-fibrillar assemblies, but the mechanism behind this activity is not fully understood. This study shows that TTR does not significantly disturb fibril elongation; rather, it effectively interferes with the formation of oligomeric nuclei. We demonstrate that this interference can be modulated by altering the relative contribution of elongation and nucleation, and we show how TTR's effects can range from being essentially ineffective to almost complete inhibition of fibril formation without changing the concentration of TTR or monomeric Aβ.     

Binding Modes of Phthalocyanines to Amyloid β Peptide and Their Effects on Amyloid Fibril Formation

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer’s and Parkinson’s diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid β-peptide (Aβ) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aβ₄₀ revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aβ₄₀-PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aβ₄₀-PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aβ₄₀ and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of pathological aggregates.     

Curcumin Decreases Amyloid-β Peptide Levels by Attenuating the Maturation of Amyloid-β Precursor Protein

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-β (Aβ), the principal component of senile plaques. Aβ is an ∼4-kDa peptide generated via cleavage of the amyloid-β precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Aβ-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Aβ levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Aβ levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-β pathology.     

Racemization of the Aspartic Acid Residue of Amyloid-β Peptide by a Radical Reaction

An E. coli cell extract containing thermostable α-amylase and maltogenic amylase expressed from the clone pTMA322 was used to produce an anomalously linked oligosaccharides (Alo) mixture from starch. The liquefaction and saccharification of starch were done by one-step procedure using the above cell extract. The resulted Alo mixture contained over 40% oligosaccharides having DP 3 or more including branched forms.     

Delineating the Mechanism of Alzheimer’s Disease Aβ Peptide Neurotoxicity

The Alzheimer’s disease neurotoxic amyloid-β (Aβ) peptide is derived from the larger amyloid precursor protein (APP) and is the principal component of the senile plaques in Alzheimer’s disease (AD) brains. This mechanism by which Aβ mediates neurotoxicity or neuronal dysfunction is not fully resolved. This review will outline some of the key determinants that modulate Aβ’s activity and the cellular pathways and mechanisms involved.     

Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.     

Selection of Peptide Inhibitors of Soluble Aβ1-42 Oligomer Formation by Phage Display

There have been many reports suggesting that soluble oligomers of amyloid β (Aβ) are neurotoxins causing Alzheimer’s disease (AD). Although inhibition of the soluble oligomerization of Aβ is considered to be effective in the treatment of AD, almost all peptide inhibitors have been designed from the β-sheet structure (H14-D23) of Aβ_1-42. To obtain more potent peptides than the known inhibitors of the soluble-oligomer formation of Aβ_1-42, we performed random screening by phage display. After fifth-round panning of a hepta-peptide library against soluble Aβ_1-42, novel peptides containing arginine residues were enriched. These peptides were found to suppress specifically 37/48 kDa oligomer formation and to keep the monomeric form of Aβ_1-42 even after 24 h of incubation, as disclosed by SDS–PAGE and size-exclusion chromatography. Thus we succeeded in acquiring novel efficient peptides for inhibition of soluble 37/48 kDa oligomer formation of Aβ_1-42.     

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Aβ) peptide by using a small engineered binding protein (Z_Aβ3) that binds with nanomolar affinity to Aβ, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z_Aβ3 in the brains of Drosophila melanogaster expressing either Aβ₄₂ or the aggressive familial associated E22G variant of Aβ₄₂ abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Aβ binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Aβ aggregation and reveal that Z_Aβ3 not only inhibits the initial association of Aβ monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.     

Studies of the M15 β-Galactosidase Complementation Process

M15 -Galactosidase was activated by heat-denatured wild-type -galactosidase, urea, and heat-denatured wild-type -galactosidase, a peptide made up of residues 6–44 of -galactosidase and CB2, the peptide that is normally used for complementation (residues 3–92 of -galactosidase). In each case roughly equal activation levels were attained. Heat-denatured wild-type -galactosidase was present as a finely divided visible white precipitate both before and after complementation. The heat-denatured protein by itself did not migrate on native PAGE and both the protein and the activity that occurred as a result of the complementation also remained at the point of application. The N-terminal ends of the heat-denatured wild-type -galactosidase must have been available for complementation and must have been mobile enough to allow tetramer to form despite being aggregated. -Galactosidase denatured by both urea and heat resulted in a streak of interacting protein on the native PAGE. Upon activation, a streak (indicating that interaction was still occurring) was still present, but it moves more slowly. Complementation using a peptide called XP (made up of residues 6–44 plus an additional nine C-terminal amino acids) resulted in three discrete forms of active enzyme at ratios of peptide to M15 -galactosidase monomer of less than 1:1. The fastest migrating of the three bands predominated at ratios near 1:1. A single active tetrameric form of M15 -galactosidase was formed with CB2. In both of these last two cases an active slow-moving diffuse band also formed (possibly a dimer of the tetramer). A quantitation of the amount of peptide bound to M15 -galactosidase by titration with XP and with CB2 and by using gel filtration after an excess of fluorescent-labeled XP was added showed that peptide bound in a 1:1 ratio (peptide/monomer) when full activity was achieved. These fluorescent studies also showed that peptide initially bound to dimer and that the tetramer was then formed.     

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ(1-40), defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ(1-40) is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.     

Towards Multiparametric Fluorescent Imaging of Amyloid Formation: Studies of a YFP Model of α-Synuclein Aggregation

Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein α-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.     

The Monomer-Seed Interaction Mechanism in the Formation of the β2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques

Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.     

The Common Architecture of Cross-β Amyloid

Amyloid fibril deposition is central to the pathology of more than 30 unrelated diseases including Alzheimer's disease and Type 2 diabetes. It is generally accepted that amyloid fibrils share common structural features despite each disease being characterised by the deposition of an unrelated protein or peptide. The structure of amyloid fibrils has been studied using X-ray fibre diffraction and crystallography, solid-state NMR and electron paramagnetic resonance, and many different, sometimes opposing, models have been suggested. Many of these models are based on the original interpretation of the cross-β diffraction pattern for cross-β silk in which β-strands run perpendicular to the fibre axis, although alternative models include β-helices and natively structured proteins. Here, we have analysed opposing model structures and examined the necessary structural elements within the amyloid core structure, as well as producing idealised models to test the limits of the core conformation. Our work supports the view that amyloid fibrils share a number of common structural features, resulting in characteristic diffraction patterns. This pattern may be satisfied by structures in which the strands align close to perpendicular to the fibre axis and are regularly arranged to form β-sheet ribbons. Furthermore, the fibril structure contains several β-sheets that associate via side-chain packing to form the final protofilament structure.     

Selective antihypertensive dihydropyridines lower Aβ accumulation by targeting both the production and the clearance of Aβ across the blood-brain barrier

Several large population-based or clinical trial studies have suggested that certain dihydropyridine (DHP) L-type calcium channel blockers (CCBs) used for the treatment of hypertension may confer protection against the development of Alzheimer disease (AD). However, other studies with drugs of the same class have shown no beneficial clinical effects. To determine whether certain DHPs are able to impact underlying disease processes in AD (specifically the accumulation of the Alzheimer Aβ peptide), we investigated the effect of several antihypertensive DHPs and non-DHP CCBs on Aβ production. Among the antihypertensive DHPs tested, a few, including nilvadipine, nitrendipine and amlodipine inhibited Aβ production in vitro, whereas others had no effect or raised Aβ levels. In vivo, nilvadipine and nitrendipine acutely reduced brain Aβ levels in a transgenic mouse model of AD (Tg PS1/APPsw) and improved Aβ clearance across the blood-brain barrier (BBB), whereas amlodipine and nifedipine were ineffective showing that the Aβ-lowering activity of the DHPs is independent of their antihypertensive activity. Chronic oral treatment with nilvadipine decreased Aβ burden in the brains of Tg APPsw (Tg2576) and Tg PS1/APPsw mice, and also improved learning abilities and spatial memory. Our data suggest that the clinical benefit conferred by certain antihypertensive DHPs against AD is unrelated to their antihypertensive activity, but rely on their ability to lower brain Aβ accumulation by affecting both Aβ production and Aβ clearance across the BBB.     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.