The interwoven fibril-like structure of amyloid-beta plaques in mouse brain tissue visualized using super-resolution STED microscopy.

BACKGROUND: Standard neuropathologic analysis of Alzheimer's brain relies on traditional fluorescence microscopy, which suffers from limited spatial resolution due to light diffraction. As a result, it fails to reveal intricate details of amyloid plaques. While electron microscopy (EM) offers higher resolution, its extensive sample preparation, involving fixation, dehydration, embedding, and sectioning, can introduce artifacts and distortions in the complex brain tissue. Moreover, EM lacks molecular specificity and has limited field of view and imaging depth. RESULTS: In our study, we employed super-resolution Stimulated Emission Depletion (STED) microscopy in conjunction with the anti-human APP recombinant antibody 1C3 fluorescently labelled with DyLightTM633 (1C3-DyLight633). This combination allowed us to visualize amyloidogenic aggregates in vitro and in brain sections from a 17-month-old 3×Tg-AD mouse with sub-diffraction limited spatial resolution. Remarkably, we achieved a spatial resolution of 29 nm in vitro and 62 nm in brain tissue sections, surpassing the capabilities of conventional confocal microscopy by 5-10 times. Consequently, we could discern individual fibrils within plaques, an achievement previously only possible with EM. CONCLUSIONS: The utilization of STED microscopy represents a groundbreaking advancement in the field, enabling researchers to delve into the characterization of local mechanisms that underlie Amyloid (Aß) deposition into plaques and their subsequent clearance. This unprecedented level of detail is especially crucial for comprehending the etiology of Alzheimer's disease and developing the next generation of anti-amyloid treatments. By facilitating the evaluation of drug candidates and non-pharmacological interventions aiming to reduce amyloid burden, STED microscopy emerges as an indispensable tool for driving scientific progress in Alzheimer's research.

Small Molecule Decoys of Aggregation for Elimination of Aß-Peptide Toxicity.

Several lines of evidence suggest that a characteristic of the neuropathology of Alzheimer's disease (AD) is the aggregation of the amyloid beta peptides (Aß), fragments of the human amyloid precursor protein (hAPP). The dominating species are the Aß40 and Aß42 fragments with 40 and 42 amino acids, respectively. Aß initially forms soluble oligomers that continue to expand to protofibrils, suggestively the neurotoxic intermediates, and thereafter turn into insoluble fibrils that are markers of the disease. Using the powerful tool of pharmacophore simulation, we selected small molecules not known to possess central nervous system (CNS) activity but that might interact with Aß aggregation, from the NCI Chemotherapeutic Agents Repository, Bethesda, MD. We assessed the activity of these compounds on Aß aggregation using the thioflavin T fluorescence correlation spectroscopy (ThT-FCS) assay. Förster resonance energy transfer-based fluorescence correlation spectroscopy (FRET-FCS) was used to characterize the dose-dependent activity of selected compounds at an early stage of Aß aggregation. Transmission electron microscopy (TEM) confirmed that the interfering substances block fibril formation and identified the macrostructures of Aß aggregates formed in their presence. We first found three compounds generating protofibrils with branching and budding never observed in the control. One compound generated a two-dimensional sheet structure and another generated a double-stranded filament. Importantly, these compounds generating protofibrils with altered macrostructure protected against Aß-induced toxicity in a cell model while showing no toxicity in a model of cognition in normal mice. The data suggest that the active compounds act as decoys turning the aggregation into nontoxic trajectories and pointing toward novel approaches to therapy.

Serum Amyloidogenic Nanoplaques and Cytokines in Alzheimer's Disease: Pilot Study in a Small Naturalistic Memory Clinic Cohort.

BACKGROUND: Neuroinflammation is a central component of Alzheimer's disease (AD) and correlates closely with amyloid pathology. Markers of inflammation such as cytokines, and amyloidogenic aggregates, so-called nanoplaques, are both promising biomarker candidates for AD. We have previously shown that there is a relationship between the levels of nanoplaques and cytokines in cerebrospinal fluid, but it is unknown whether this association extends to serum. OBJECTIVE: Investigate in a naturalistic memory clinic cohort whether the associations between nanoplaques and cytokines in the cerebrospinal fluid extends to serum. METHODS: We collected serum from 49 patients assessed for cognitive complaints at the Oslo University Hospital Memory Clinic (15 with clinical AD). We assessed the levels of serum nanoplaques with the novel Thioflavin-T fluorescence correlation spectroscopy (ThT-FCS) assay. Serum levels of nine cytokines (eotaxin-1, granulocyte colony-stimulating factor [G-CSF], interleukin [IL]-6, IL-7, IL-8, monocyte chemoattractant protein-1 (MCP-1), gamma induced protein 10 (IP-10), macrophage inflammatory protein [MIP]-1α, and MIP-1ß) were quantified with a multiplex assay and read on a Luminex IS 200 instrument. RESULTS: Serum nanoplaques were not increased in clinical AD patients compared to non-AD memory clinic patients and nanoplaques were not associated with any cytokines. The cytokines IL-8 and G-CSF were increased in patients with clinical AD compared to non-AD patients. CONCLUSION: In this small pilot study, serum nanoplaques were not associated with serum cytokines. Nanoplaque levels could not be used to separate clinical AD patients from non-AD patients in this unselected memory clinic cohort.

Associations of cerebrospinal fluid amyloidogenic nanoplaques with cytokines in Alzheimer's disease.

BACKGROUND: The aggregation of amyloid ß (Aß) is central in the pathogenesis of Alzheimer's disease (AD). Recently it has been shown that specifically, larger, Thioflavin T-binding Aß aggregates are associated with increased neuroinflammation and cytokine release. This study was aimed to quantify fibrillary amyloid aggregates, so-called nanoplaques, and investigate their relationship with cytokines in the cerebrospinal fluid (CSF). METHODS: CSF was collected from 111 patients assessed for cognitive complaints at the Oslo University Hospital Memory Clinic. The patients were grouped based on their amyloid status. The CSF nanoplaque concentration was quantified with the Thioflavin T-fluorescence correlation spectroscopy (ThT-FCS) assay. The levels of nine cytokines (eotaxin-1, granulocyte stimulating factor, interleukin [IL]-6, IL-7, IL-8, monocyte chemoattractant protein-1, gamma-induced protein 10, macrophage inflammatory protein [MIP]-1α, and MIP-1ß) were quantified with a magnetic bead-based multiplex assay and read on a Luminex IS 200 instrument. RESULTS: There were 49 amyloid-negative and 62 amyloid-positive patients in the cohort; none of the cytokines differed significantly between the amyloid groups. The increased nanoplaque levels were associated with levels of MIP-1ß below the lower limit of quantification, and with decreased levels of MIP-1α and IL-8. The associations remained significant when adjusted for age, sex, cognitive function, apolipoprotein ε4 status and CSF core biomarker levels. CONCLUSION: The cytokine levels were not associated with amyloid status in this cohort. The nanoplaque levels were negatively associated with MIP-1ß, MIP-1α and IL-8, which is in line with recent findings suggesting that the upregulation of some cytokine markers has a protective role and is negatively associated with AD progression.

Smallest Secondary Nucleation Competent Aß Aggregates Probed by an ATP-Independent Molecular Chaperone Domain.

Protein oligomerization is a commonly encountered strategy by which the functional repertoire of proteins is increased. This, however, is a double-edged sword strategy because protein oligomerization is notoriously difficult to control. Living organisms have therefore developed a number of chaperones that prevent protein aggregation. The small ATP-independent molecular chaperone domain proSP-C BRICHOS, which is mainly trimeric, specifically inhibits fibril surface-catalyzed nucleation reactions that give rise to toxic oligomers during the aggregation of the Alzheimer's disease-related amyloid-ß peptide (Aß42). Here, we have created a stable proSP-C BRICHOS monomer mutant and show that it does not bind to monomeric Aß42 but has a high affinity for Aß42 fibrils, using surface plasmon resonance. Kinetic analysis of Aß42 aggregation profiles, measured by thioflavin T fluorescence, reveals that the proSP-C BRICHOS monomer mutant strongly inhibits secondary nucleation reactions and thereby reduces the level of catalytic formation of toxic Aß42 oligomers. To study binding between the proSP-C BRICHOS monomer mutant and small soluble Aß42 aggregates, we analyzed fluorescence cross-correlation spectroscopy measurements with the maximum entropy method for fluorescence correlation spectroscopy. We found that the proSP-C BRICHOS monomer mutant binds to the smallest emerging Aß42 aggregates that are comprised of eight or fewer Aß42 molecules, which are already secondary nucleation competent. Our approach can be used to provide molecular-level insights into the mechanisms of action of substances that interfere with protein aggregation.

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives.

In neurodegenerative diseases, a wide range of amyloid proteins or peptides such as amyloid-beta and α-synuclein fail to keep native functional conformations, followed by misfolding and self-assembling into a diverse array of aggregates. The aggregates further exert toxicity leading to the dysfunction, degeneration and loss of cells in the affected organs. Due to the disordered structure of the amyloid proteins, endogenous molecules, such as lipids, are prone to interact with amyloid proteins at a low concentration and influence amyloid cytotoxicity. The heterogeneity of amyloid proteinscomplicates the understanding of the amyloid cytotoxicity when relying only on conventional bulk and ensemble techniques. As complementary tools, single-molecule techniques (SMTs) provide novel insights into the different subpopulations of a heterogeneous amyloid mixture as well as the cytotoxicity, in particular as involved in lipid membranes. This review focuses on the recent advances of a series of SMTs, including single-molecule fluorescence imaging, single-molecule force spectroscopy and single-nanopore electrical recording, for the understanding of the amyloid molecular mechanism. The working principles, benefits and limitations of each technique are discussed and compared in amyloid protein related studies.. We also discuss why SMTs show great potential and are worthy of further investigation with feasibility studies as diagnostic tools of neurodegenerative diseases and which limitations are to be addressed.

Amyloidogenic Nanoplaques in Cerebrospinal Fluid: Relationship to Amyloid Brain Uptake and Clinical Alzheimer's Disease in a Memory Clinic Cohort.

BACKGROUND: Aggregation of amyloid-ß (Aß) is an early pathological event in Alzheimer's disease (AD). Consequently, measures of pathogenic aggregated Aß are attractive biomarkers for AD. Here, we use a recently developed Thioflavin-T-Fluorescence Correlation Spectroscopy (ThT-FCS) assay to quantify structured ThT-responsive protein aggregates, so-called nanoplaques, in the cerebrospinal fluid (CSF). OBJECTIVE: The overall aim of this work was to assess whether ThT-FCS determined CSF nanoplaque levels could predict amyloid brain uptake as determined by 18F-Flutemetamol PET analysis. Further, we assess whether nanoplaque levels could predict clinical AD. METHODS: Nanoplaque levels in the CSF from 54 memory clinic patients were compared between sub-groups classified by 18F-Flutemetamol PET as amyloid-positive or amyloid-negative, and by clinical assessment as AD or non-AD. RESULTS: Nanoplaque levels did not differ between amyloid groups and could not predict brain amyloid uptake. However, nanoplaque levels were significantly increased in patients with clinical AD, and were significant predictors for AD when adjusting for age, sex, cognitive function, and apolipoprotein E (APOE) genotype. CONCLUSION: The concentration of nanoplaques in the CSF differentiates patients with clinical AD from non-AD patients.

Comparison of Cerebrospinal Fluid Amyloidogenic Nanoplaques With Core Biomarkers of Alzheimer's Disease.

Accurate biomarkers of Alzheimer's disease (AD) are essential for early diagnosis and intervention. Available biomarkers are not sufficient to permit the monitoring of AD progression over time, and additional biomarkers are required. Measures of aggregated amyloid-ß (Aß) could be useful biomarkers for AD. Here, we investigate whether levels of Thioflavin-T (ThT) positive amyloid aggregates, i.e., nanoplaques, in cerebrospinal fluid (CSF) could serve as useful biomarkers for AD. One-hundred and eighteen memory clinic patients were AT(N) classified, and CSF nanoplaque concentrations were compared between patients on the "Alzheimer's continuum" (A+ patients) and patients with "Normal AD biomarkers" or "Non-AD pathologic change" (A- patients). CSF nanoplaque concentrations and sizes were quantified using the novel ThT-Fluorescence Correlation Spectroscopy (ThT-FCS) assay, and core biomarkers (Aß42, total tau and phosphorylated tau) were determined by enzyme-linked immunosorbent assays. We investigated the association between nanoplaque concentrations and core biomarkers, and the diagnostic value of nanoplaque levels. Nanoplaque levels were increased in A+ patients compared to A- patients. Nanoplaque concentrations were negatively associated with Aß42, but not related to total tau or phosphorylated tau measures. Quantification of nanoplaques did not improve the classification of patients on the Alzheimer's continuum compared to the core biomarkers alone. Dynamic changes in nanoplaques concentration and size throughout AD stages should be explored in longitudinal studies.

Metal binding to the amyloid-ß peptides in the presence of biomembranes: potential mechanisms of cell toxicity.

The amyloid-ß (Aß) peptides are key molecules in Alzheimer's disease (AD) pathology. They interact with cellular membranes, and can bind metal ions outside the membrane. Certain oligomeric Aß aggregates are known to induce membrane perturbations and the structure of these oligomers-and their membrane-perturbing effects-can be modulated by metal ion binding. If the bound metal ions are redox active, as e.g., Cu and Fe ions are, they will generate harmful reactive oxygen species (ROS) just outside the membrane surface. Thus, the membrane damage incurred by toxic Aß oligomers is likely aggravated when redox-active metal ions are present. The combined interactions between Aß oligomers, metal ions, and biomembranes may be responsible for at least some of the neuronal death in AD patients.

Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer's Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis.

BACKGROUND: Biomarkers are central to current research on molecular mechanisms underlying Alzheimer's disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies. OBJECTIVE: The overall aim of this work is to develop a minimally invasive method for fast, ultra-sensitive and cost-effective detection of structurally modified peptide/protein self-assemblies in the peripheral blood and in other biological fluids. Specifically, we focus here on using this method to detect structured amyloidogenic oligomeric aggregates in the blood serum of apparently healthy individuals and patients in early AD stage, and measure their concentration and size. METHODS: Time-resolved detection of Thioflavin T (ThT) fluorescence intensity fluctuations in a sub-femtoliter observation volume element was used to identify in blood serum ThT-active structured amyloidogenic oligomeric aggregates, hereafter called nanoplaques, and measure with single-particle sensitivity their concentration and size. RESULTS: The concentration and size of structured amyloidogenic nanoplaques are significantly higher in the blood serum of individuals diagnosed with AD than in control subjects. CONCLUSION: A new method with the ultimate, single-particle sensitivity was successfully developed. The proposed approach neither relies on the use of immune-based probes, nor on the use of radiotracers, signal-amplification or protein separation techniques, and provides a minimally invasive test for fast and cost-effective early determination of structurally modified peptides/proteins in the peripheral blood, as shown here, but also in other biological fluids.

Conformation-specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen.

We engineered and employed a chaperone-like amyloid-binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross-reacted with amyloid beta-peptide (Aß42) protofibrils, but not with Aß40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1-hIAPP complex cross-react with Aß42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation-specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation-sensitive and sequence-independent and can target more than one type of protofibril species.

Specific Binding of Cu(II) Ions to Amyloid-Beta Peptides Bound to Aggregation-Inhibiting Molecules or SDS Micelles Creates Complexes that Generate Radical Oxygen Species.

Aggregation of the amyloid-beta (Aß) peptide into insoluble plaques is a major factor in Alzheimer's disease (AD) pathology. Another major factor in AD is arguably metal ions, as metal dyshomeostasis is observed in AD patients, metal ions modulate Aß aggregation, and AD plaques contain numerous metals including redox-active Cu and Fe ions. In vivo, Aß is found in various cellular locations including membranes. So far, Cu(II)/Aß interactions and ROS generation have not been investigated in a membrane environment. Here, we study Cu(II) and Zn(II) interactions with Aß bound to SDS micelles or to engineered aggregation-inhibiting molecules (the cyclic peptide CP-2 and the ZAß3(12-58)Y18L Affibody molecule). In all studied systems the Aß N-terminal segment was found to be unbound, unstructured, and free to bind metal ions. In SDS micelles, Aß was found to bind Cu(II) and Zn(II) with the same ligands and the same KD as in aqueous solution. ROS was generated in all Cu(II)/Aß complexes. These results indicate that binding of Aß to membranes, drugs, and other entities that do not interact with the Aß N-terminal part, appears not to compromise the N-terminal segment's ability to bind metal ions, nor impede the capacity of N-terminally bound Cu(II) to generate ROS.

Heterogeneity and Turnover of Intermediates during Amyloid-ß (Aß) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy.

Self-assembly of amyloid ß (Aß) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among Aß molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. Aß self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding Aß aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying Aß aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of Aß peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 × 10(6) kDa and revealed the hitherto unobserved kinetic turnover of intermediates during Aß aggregation. The effect of different Aß concentrations, Aß:ThT ratios, differences between the 40 (Aß40) and 42 (Aß42) residue long variants of Aß, and the effect of stirring were also examined.

Effect of methionine-35 oxidation on the aggregation of amyloid-ß peptide.

Aggregation of Aß peptides into amyloid plaques is considered to trigger the Alzheimer's disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aß aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H2O2. The fibrillization of Aß with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aß with oxidized Met35 in the presence of H2O2, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aß-Cu(II) complex.

The hairpin conformation of the amyloid ß peptide is an important structural motif along the aggregation pathway.

The amyloid ß (Aß) peptides are 39-42 residue-long peptides found in the senile plaques in the brains of Alzheimer's disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the Aß peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aß peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of Aß complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.

Biophysical studies of the amyloid ß-peptide: interactions with metal ions and small molecules.

Alzheimer's disease is the most common of the protein misfolding ("amyloid") diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so-called amyloid ß-peptides (Aß peptides): fragments of the amyloid precursor protein of 39-43 residues in length. This review focuses on biophysical studies of the Aß peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of Aß with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high-resolution and solid-state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of Aß peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the Aß peptides and the prion protein, where the latter is involved in other protein misfolding diseases.

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid-ß peptide 1-42 but not amylin and insulin fibrils can grow under quiescent conditions.

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides--Alzheimer's amyloid-ß (Aß) 1-40 and 1-42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aß40 substantially decreased, whereas the fibrillization of Aß42 peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive.

The missing link in the amyloid cascade of Alzheimer's disease - metal ions.

Progressive deposition of amyloid beta (Aß) peptides into amyloid plaques is the pathological hallmark of Alzheimer's disease (AD). The amyloid cascade hypothesis pins this deposition as the primary cause of the disease, but the mechanisms that causes this deposition remain elusive. An increasing amount of evidence shows that biometals Zn(II) and Cu(II) can interact with Aß, thus influencing the fibrillization and toxicity. This review focuses on the role of Zn(II) and Cu(II) in AD, and revisits the amyloid cascade hypothesis demonstrating the possible roles of Zn(II) and Cu(II) in the disease pathogenesis.

Interactions of Zn(II) and Cu(II) ions with Alzheimer's amyloid-beta peptide. Metal ion binding, contribution to fibrillization and toxicity.

Amyloid-ß peptides (Aß) are key molecules in Alzheimer's disease (AD) pathology as they form amyloid plaques that are primary hallmarks of AD. There is increasing evidence demonstrating that the biometals zinc(ii) and copper(ii) interact with Aß peptides and have an influence on their fibrillization and toxicity. Zinc and copper ions are abundantly present in the synaptic areas of the brain, and it is likely that the age-related dyshomeostasis of these biometals is associated with AD pathology. In this review we summarize the knowledge of the interactions of zinc and copper ions with Aß peptides, their role in Aß fibrillization and toxicity and provide a critical analysis of the conflicting results in the field. Copper ions entrapped in Aß fibrils are electrochemically active and can generate ROS in the presence of hydrogen peroxide and reducing agents. This might provide a key for understanding the putative role of copper in Aß toxicity and AD pathology.