Copper(II) Can Kinetically Trap Arctic and Italian Amyloid-ß40 as Toxic Oligomers, Mimicking Cu(II) Binding to Wild-Type Amyloid-ß42: Implications for Familial Alzheimer's Disease.

The self-association of amyloid-ß (Aß) peptide into neurotoxic oligomers is believed to be central to Alzheimer's disease (AD). Copper is known to impact Aß assembly, while disrupted copper homeostasis impacts phenotype in Alzheimer's models. Here we show the presence of substoichiometric Cu(II) has very different impacts on the assembly of Aß40 and Aß42 isoforms. Globally fitting microscopic rate constants for fibril assembly indicates copper will accelerate fibril formation of Aß40 by increasing primary nucleation, while seeding experiments confirm that elongation and secondary nucleation rates are unaffected by Cu(II). In marked contrast, Cu(II) traps Aß42 as prefibrillar oligomers and curvilinear protofibrils. Remarkably, the Cu(II) addition to preformed Aß42 fibrils causes the disassembly of fibrils back to protofibrils and oligomers. The very different behaviors of the two Aß isoforms are centered around differences in their fibril structures, as highlighted by studies of C-terminally amidated Aß42. Arctic and Italian familiar mutations also support a key role for fibril structure in the interplay of Cu(II) with Aß40/42 isoforms. The Cu(II) dependent switch in behavior between nonpathogenic Aß40 wild-type and Aß40 Arctic or Italian mutants suggests heightened neurotoxicity may be linked to the impact of physiological Cu(II), which traps these familial mutants as oligomers and curvilinear protofibrils, which cause membrane permeability and Ca(II) cellular influx.

Impact of Membrane Phospholipids and Exosomes on the Kinetics of Amyloid-ß Fibril Assembly.

Alzheimer's disease (AD) is linked with the self-association of the amyloid-ß peptide (Aß) into oligomers and fibrils. The brain is a lipid rich environment for Aß to assemble, while the brain membrane composition varies in an age dependent manner, we have therefore monitored the influence of lipid bilayer composition on the kinetics of Aß40 fibril assembly. Using global-fitting models of fibril formation kinetics, we show that the microscopic rate constant for primary nucleation is influenced by variations in phospholipid composition. Anionic phospholipids and particularly those with smaller headgroups shorten fibril formation lag-times, while zwitterionic phospholipids tend to extend them. Using a physiological vesicle model, we show cellular derived exosomes accelerate Aß40 and Aß42 fibril formation. Two distinct effects are observed, the presence of even small amounts of any phospholipid will impact the slope of the fibril growth curve. While subsequent additions of phospholipids only affect primary nucleation with the associated change in lag-times. Heightened anionic phospholipids and cholesterol levels are associated with aging and AD respectively, both these membrane components strongly accelerate primary nucleation during Aß assembly, making a link between disrupted lipid metabolism and Alzheimer's disease.

Imaging Amyloid-ß Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease.

The accumulation of the amyloid-ß peptides (Aß) is central to the development of Alzheimer's disease. The mechanism by which Aß triggers a cascade of events that leads to dementia is a topic of intense investigation. Aß self-associates into a series of complex assemblies with different structural and biophysical properties. It is the interaction of these oligomeric, protofibril and fibrillar assemblies with lipid membranes, or with membrane receptors, that results in membrane permeability and loss of cellular homeostasis, a key event in Alzheimer's disease pathology. Aß can have an array of impacts on lipid membranes, reports have included: a carpeting effect; a detergent effect; and Aß ion-channel pore formation. Recent advances imaging these interactions are providing a clearer picture of Aß induced membrane disruption. Understanding the relationship between different Aß structures and membrane permeability will inform therapeutics targeting Aß cytotoxicity.

Imaging Amyloid-ß Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease.

The accumulation of the amyloid-ß peptides (Aß) is central to the development of Alzheimer's disease. The mechanism by which Aß triggers a cascade of events that leads to dementia is a topic of intense investigation. Aß self-associates into a series of complex assemblies with different structural and biophysical properties. It is the interaction of these oligomeric, protofibril and fibrillar assemblies with lipid membranes, or with membrane receptors, that results in membrane permeability and loss of cellular homeostasis, a key event in Alzheimer's disease pathology. Aß can have an array of impacts on lipid membranes, reports have included: a carpeting effect; a detergent effect; and Aß ion-channel pore formation. Recent advances imaging these interactions are providing a clearer picture of Aß induced membrane disruption. Understanding the relationship between different Aß structures and membrane permeability will inform therapeutics targeting Aß cytotoxicity.

pH Dependence of Amyloid-ß Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes.

Central to Alzheimer's disease (AD) is the assembly of the amyloid-beta peptide (Aß) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aß's isoelectric point (pI). Using global fitting of fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual fibril assembly microscopic rate constants. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid-point or pKa of 7.0, close to the pKa of Aß histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aß, in particular histidine protonation, has little impact on this stage of Aß assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aß into toxic oligomers and fibrils.

Therapeutic potential for amyloid surface inhibitor: only amyloid-ß oligomers formed by secondary nucleation disrupt lipid membrane integrity.

Inhibition of amyloid-ß peptide (Aß) aggregation is a promising therapeutic strategy for Alzheimer's disease (AD), as Aß aggregation is generally believed to trigger AD pathology. Pre-fibril Aß-oligomers induce membrane disruption and are crucial to neurotoxicity. We have previously designed a short peptide called cyclic helical amyloid surface inhibitor (cHASI) that can selectively bind to the Aß fibril surface. Here, we use cHASI to efficiently inhibit the surface-catalysed secondary nucleation process of Aß in a lipid membrane environment. By incubating Aß monomers with lipid vesicles, we show that during the assembly of Aß into amyloid fibrils, oligomers are formed that markedly disrupt the lipid bilayer. Remarkably, when Aß monomers are incubated with cHASI, although Aß forms amyloid fibrils via primary nucleation and elongation, this pathway to fibrils does not damage the lipid bilayer. This indicates that only oligomers produced during secondary surface nucleation disrupt membrane integrity. The protective effect of cHASI is confirmed by cytotoxicity assays. Our study highlights the therapeutic potential for inhibiting the secondary nucleation process in Aß aggregation, rather than inhibiting all pathways to fibril formation.

Cross-seeding of WT amyloid-ß with Arctic but not Italian familial mutants accelerates fibril formation in Alzheimer's disease.

Alzheimer's disease (AD) involves the neurotoxic self-assembly of a 40 and 42 residue peptide, Amyloid-ß (Aß). Inherited early-onset AD can be caused by single point mutations within the Aß sequence, including Arctic (E22G) and Italian (E22K) familial mutants. These mutations are heterozygous, resulting in an equal proportion of the WT and mutant Aß isoform expression. It is therefore important to understand how these mixtures of Aß isoforms interact with each other and influence the kinetics and morphology of their assembly into oligomers and fibrils. Using small amounts of nucleating fibril seeds, here, we systematically monitored the kinetics of fibril formation, comparing self-seeding with cross-seeding behavior of a range of isoform mixtures of Aß42 and Aß40. We confirm that Aß40(WT) does not readily cross-seed Aß42(WT) fibril formation. In contrast, fibril formation of Aß40(Arctic) is hugely accelerated by Aß42(WT) fibrils, causing an eight-fold reduction in the lag-time to fibrillization. We propose that cross-seeding between the more abundant Aß40(Arctic) and Aß42(WT) may be important for driving early-onset AD and will propagate fibril morphology as indicated by fibril twist periodicity. This kinetic behavior is not emulated by the Italian mutant, where minimal cross-seeding is observed. In addition, we studied the cross-seeding behavior of a C-terminal-amidated Aß42 analog to probe the coulombic charge interplay between Glu22/Asp23/Lys28 and the C-terminal carboxylate. Overall, these studies highlight the role of cross-seeding between WT and mutant Aß40/42 isoforms, which can impact the rate and structure of fibril assembly.

pH Dependence of Amyloid-ß Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes.

Central to Alzheimer's disease (AD) is the assembly of the amyloid-beta peptide (Aß) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aß's isoelectric point (pI). Using global fitting of fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual fibril assembly microscopic rate constants. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid-point or pK a of 7.0, close to the pK a of Aß histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aß, in particular histidine protonation, has little impact on this stage of Aß assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aß into toxic oligomers and fibrils.

3D-visualization of amyloid-ß oligomer interactions with lipid membranes by cryo-electron tomography.

Amyloid-ß (Aß) assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer's disease. However, molecular mechanisms of Aß cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aß assembly types and their interaction with liposomes. Aß oligomers and curvilinear protofibrils bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Aß oligomers concentrate at the interface of vesicles and form a network of Aß-linked liposomes, while crucially, monomeric and fibrillar Aß have relatively little impact on the membrane. Changes to lipid membrane composition highlight a significant role for GM1-ganglioside in promoting Aß-membrane interactions. The different effects of Aß assembly forms observed align with the highlighted cytotoxicity reported for Aß oligomers. The wide-scale incorporation of Aß oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

Copper2+ Binding to α-Synuclein. Histidine50 Can Form a Ternary Complex with Cu2+ at the N-Terminus but Not a Macrochelate.

α-Synuclein (αSyn) forms amyloid fibrils in the neurons of Parkinson's disease (PD) patients'. Despite a role for Cu2+ in accelerating αSyn fibril formation, coupled with reports of copper dis-homeostasis in PD, there remain controversies surrounding the coordination geometry of Cu2+ with αSyn. Here we compare visible circular dichroism (CD) spectra of Cu2+ loaded on to full-length αSyn together with four peptides that model aspects of Cu2+ binding to the N-terminus and histidine50 of αSyn. With glycine as a competitive ligand, the affinity of Cu2+ for full-length αSyn is determined to have a conditional dissociation constant, at pH 7.4, of 0.1 nM. A similar affinity of 0.3 nM is determined for the tripeptide Met-Asp-Val(MDV) that mimics the N-terminus of αSyn, while the incorporation of a putative histidine side chain in the N-terminal complex facilitates the formation of a macrochelate with the histidine, which results in an increase in the affinity for Cu2+ to 0.03 nM at pH 7.4. Comparisons of the visible absorbance and CD spectra over a range of pH values also indicates that the MDV tripeptide closely models Cu2+ binding to full-length αSyn and rules out a role for His50 in the primary Cu2+ binding complex of monomeric αSyn. However, there are reports that suggest His50 does form a macrochelate with the N-terminal Cu2+ complex; we reconcile these conflicting observations by identifying a concentration dependence of the interaction. Only at the higher concentrations can the imidazole nitrogen bind to the N-terminal Cu2+ to form a ternary complex rather than via a macrochelate. This work shows even for this intrinsically disordered protein a large macrochelate with Cu2+ is not favored. Understanding Cu2+ coordination to αSyn gives a more complete picture of its place in amyloid assembly and cytotoxicity.

Amyloid-ß oligomers have a profound detergent-like effect on lipid membrane bilayers, imaged by atomic force and electron microscopy.

The ability of amyloid-ß peptide (Aß) to disrupt membrane integrity and cellular homeostasis is believed to be central to Alzheimer's disease pathology. Aß is reported to have various impacts on the lipid bilayer, but a clearer picture of Aß influence on membranes is required. Here, we use atomic force and transmission electron microscopies to image the impact of different isolated Aß assembly types on lipid bilayers. We show that only oligomeric Aß can profoundly disrupt the bilayer, visualized as widespread lipid extraction and subsequent deposition, which can be likened to an effect expected from the action of a detergent. We further show that Aß oligomers cause widespread curvature and discontinuities within lipid vesicle membranes. In contrast, this detergent-like effect was not observed for Aß monomers and fibers, although Aß fibers did laterally associate and embed into the upper leaflet of the bilayer. The marked impact of Aß oligomers on membrane integrity identified here reveals a mechanism by which these oligomers may be cytotoxic.

Copper Redox Cycling Inhibits Aß Fibre Formation and Promotes Fibre Fragmentation, while Generating a Dityrosine Aß Dimer.

Oxidative stress and the formation of plaques which contain amyloid-ß (Aß) peptides are two key hallmarks of Alzheimer's disease (AD). Dityrosine is found in the plaques of AD patients and Aß dimers have been linked to neurotoxicity. Here we investigate the formation of Aß dityrosine dimers promoted by Cu2+/+ Fenton reactions. Using fluorescence measurements and UV absorbance, we show that dityrosine can be formed aerobically when Aß is incubated with Cu2+ and hydrogen-peroxide, or in a Cu2+ and ascorbate redox mixture. The dityrosine cross-linking can occur for both monomeric and fibrillar forms of Aß. We show that oxidative modification of Aß impedes the ability for Aß monomer to form fibres, as indicated by the amyloid specific dye Thioflavin T (ThT). Transmission electron microscopy (TEM) indicates the limited amyloid assemblies that form have a marked reduction in fibre length for Aß(1-40). Importantly, the addition of Cu2+ and a reductant to preformed Aß(1-40) fibers causes their widespread fragmentation, reducing median fibre lengths from 800 nm to 150 nm upon oxidation. The processes of covalent cross-linking of Aß fibres, dimer formation, and fibre fragmentation within plaques are likely to have a significant impact on Aß clearance and neurotoxicity.

Prion protein stabilizes amyloid-ß (Aß) oligomers and enhances Aß neurotoxicity in a Drosophila model of Alzheimer's disease.

The cellular prion protein (PrPC) can act as a cell-surface receptor for ß-amyloid (Aß) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aß isoforms and PrPC in the Drosophila brain. We found that co-expression of Aß and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aß deposition in the fly brain. In contrast, under the same conditions, expression of Aß or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aß as oligomeric assemblies and fragment-preformed Aß fibers. The ability of membrane-anchored PrPC to trap Aß as cytotoxic oligomers at the membrane surface and fragment inert Aß fibers suggests a mechanism by which PrPC exacerbates Aß deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aß toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aß and PrPC represents a potential therapeutic strategy.

Serum Albumin's Protective Inhibition of Amyloid-ß Fiber Formation Is Suppressed by Cholesterol, Fatty Acids and Warfarin.

Central to Alzheimer's disease (AD) pathology is the assembly of monomeric amyloid-ß peptide (Aß) into oligomers and fibers. The most abundant protein in the blood plasma and cerebrospinal fluid is human serum albumin. Albumin can bind to Aß and is capable of inhibiting the fibrillization of Aß at physiological (µM) concentrations. The ability of albumin to bind Aß has recently been exploited in a phase II clinical trial, which showed a reduction in cognitive decline in AD patients undergoing albumin-plasma exchange. Here we explore the equilibrium between Aß monomer, oligomer and fiber in the presence of albumin. Using transmission electron microscopy and thioflavin-T fluorescent dye, we have shown that albumin traps Aß as oligomers, 9 nm in diameter. We show that albumin-trapped Aß oligomeric assemblies are not capable of forming ion channels, which suggests a mechanism by which albumin is protective in Aß-exposed neuronal cells. In vivo albumin binds a variety of endogenous and therapeutic exogenous hydrophobic molecules, including cholesterol, fatty acids and warfarin. We show that these molecules bind to albumin and suppress its ability to inhibit Aß fiber formation. The interplay between Aß, albumin and endogenous hydrophobic molecules impacts Aß assembly; thus, changes in cholesterol and fatty acid levels in vivo may impact Aß fibrillization, by altering the capacity of albumin to bind Aß. These observations are particularly intriguing given that high cholesterol or fatty acid diets are well-established risk factors for late-onset AD.

N-Terminally Truncated Amyloid-ß(11-40/42) Cofibrillizes with its Full-Length Counterpart: Implications for Alzheimer's Disease.

Amyloid-ß peptide (Aß) isoforms of different lengths and aggregation propensities coexist in vivo. These different isoforms are able to nucleate or frustrate the assembly of each other. N-terminally truncated Aß(11-40) and Aß(11-42) make up one fifth of plaque load yet nothing is known about their interaction with full-length Aß(1-40/42) . We show that in contrast to C-terminally truncated isoforms, which do not co-fibrillize, deletions of ten residues from the N terminus of Aß have little impact on its ability to co-fibrillize with the full-length counterpart. As a consequence, N-terminally truncated Aß will accelerate fiber formation and co-assemble into short rod-shaped fibers with its full-length Aß counterpart. This has implications for the assembly kinetics, morphology, and toxicity of all Aß isoforms.

Ion Channel Formation by Amyloid-ß42 Oligomers but Not Amyloid-ß40 in Cellular Membranes.

A central hallmark of Alzheimer's disease is the presence of extracellular amyloid plaques chiefly consisting of amyloid-ß (Aß) peptides in the brain interstitium. Aß largely exists in two isoforms, 40 and 42 amino acids long, but a large body of evidence points to Aß(1-42) rather than Aß(1-40) as the cytotoxic form. One proposed mechanism by which Aß exerts toxicity is the formation of ion channel pores that disrupt intracellular Ca2+ homeostasis. However, previous studies using membrane mimetics have not identified any notable difference in the channel forming properties between Aß(1-40) and Aß(1-42). Here, we tested whether a more physiological environment, membranes excised from HEK293 cells of neuronal origin, would reveal differences in the relative channel forming ability of monomeric, oligomeric, and fibrillar forms of both Aß(1-40) and Aß(1-42). Aß preparations were characterized with transmission electron microscopy and thioflavin T fluorescence. Aß was then exposed to the extracellular face of excised membranes, and transmembrane currents were monitored using patch clamp. Our data indicated that Aß(1-42) assemblies in oligomeric preparations form voltage-independent, non-selective ion channels. In contrast, Aß(1-40) oligomers, fibers, and monomers did not form channels. Ion channel conductance results suggested that Aß(1-42) oligomers, but not monomers and fibers, formed three distinct pore structures with 1.7-, 2.1-, and 2.4-nm pore diameters. Our findings demonstrate that only Aß(1-42) contains unique structural features that facilitate membrane insertion and channel formation, now aligning ion channel formation with the differential neurotoxic effect of Aß(1-40) and Aß(1-42) in Alzheimer's disease.

The Rapid Exchange of Zinc(2+) Enables Trace Levels to Profoundly Influence Amyloid-ß Misfolding and Dominates Assembly Outcomes in Cu(2+)/Zn(2+) Mixtures.

The misfolding and self-assembly of amyloid-ß (Aß) into oligomers and fibres is fundamental to Alzheimer's disease pathology. Alzheimer's disease is a multifaceted disease. One factor that is thought to have a significant role in disease aetiology is Zn(2+) homeostasis, which is disrupted in the brains of Alzheimer's disease sufferers and has been shown to modulate Alzheimer's symptoms in animal models. Here, we investigate how the kinetics of Aß fibre growth are affected at a range of Zn(2+) concentrations and we use transmission electron microscopy to characterise the aggregate assemblies formed. We demonstrate that for Aß(1-40), and Aß(1-42), as little as 0.01mol equivalent of Zn(2+) (100nM) is sufficient to greatly perturb the formation of amyloid fibres irreversibly. Instead, Aß(1-40) assembles into short, rod-like structures that pack tightly together into ordered stacks, whereas Aß(1-42) forms short, crooked assemblies that knit together to form a mesh of disordered tangles. Our data suggest that a small number of Zn(2+) ions are able to influence a great many Aß molecules through the rapid exchange of Zn(2+) between Aß peptides. Surprisingly, although Cu(2+) binds to Aß 10,000 times tighter than Zn(2+), the effect of Zn(2+) on Aß assembly dominates in Cu(2+)/Zn(2+) mixtures, suggesting that trace levels of Zn(2+) must have a profound effect on extracellular Aß accumulation. Trace Zn(2+) levels profoundly influence Aß assembly even at concentrations weaker than its affinity for Aß. These observations indicate that inhibitors of fibre assembly do not necessarily have to be at high concentration and affinity to have a profound impact.

Methionine oxidation reduces lag-times for amyloid-ß(1-40) fiber formation but generates highly fragmented fibers.

Oxidative stress and the formation of amyloid plaques containing amyloid-ß (Aß) peptides are two key hallmarks of Alzheimer's disease. A proportion of methionine (Met) at position 35 within Aß is oxidized to methionine sulphoxide (Met(OX)) within the Alzheimer's plaques. These oxidative processes may be the key to understanding the early stages of Alzheimer's disease. In vitro oxidation of Aß, by the physiological oxidant H2O2, was monitored using (1)H NMR and mass spectrometry. Here we investigate the effect of Aß methionine oxidation on fiber formation kinetics and morphology using the amyloid specific fluorescence dye Thioflavin T (ThT) and Transmission Electron Microscopy (TEM). Methionine oxidation reduces the total amount of fibers generated for both dominant forms of Aß, however there are marked differences in the effect of Met(OX) between Aß(1-40) and Aß(1-42). Surprisingly the presence of Met(OX) reduces lag-times for Aß(1-40) fiber formation but extends lag-times for Aß(1-42). TEM indicates a change in fiber morphology with a pronounced reduction in fiber length for both methionine oxidized Aß(1-40) and Aß(1-42). In contrast, the morphology of preformed amyloid fibers is largely unaffected by the presence of H2O2. Our studies suggest that methionine oxidation promotes highly fragmented fiber assemblies of Aß. Oxidative stress associated with Alzheimer's disease can cause oxidation of methionine within Aß and this in turn will influence the complex assembly of Aß monomer into amyloid fibers, which is likely to impact Aß toxicity.

Endocytosis of the tachykinin neuropeptide, neurokinin B, in astrocytes and its role in cellular copper uptake.

The tachykinin neuropeptide, neurokinin B (NKB), belongs to a family of peptides having diverse roles in the brain. NKB, along with several other tachykinins, has been identified as a copper-binding peptide, however the physiological relevance of the binding is unclear. Previously, NKB was shown to limit the ability of copper to enter astrocytes and disrupt calcium homeostasis and it was thought that the peptide was sequestering the metal extracellularly. Here we use a fluorescein-labelled NKB peptide (F-NKB) to show that NKB is not retained extracellularly, but is endocytosed within 10-20min after addition to the cell media. The endocytosis is not inhibited when NKB is delivered as a copper-complex, [CuII(F-NKB)2]. Endocytosis of NKB can increase intracellular copper. Comparison to cells cultured in copper-free buffer indicated that apo-NKB can facilitate uptake of copper found in normal culture media. To achieve this NKB must compete with a variety of copper proteins, and we show that NKB can successfully compete with copper-binding peptides derived from the prion protein, itself associated with Cu(II) and Zn(II) metabolism. We suggest a mechanism of receptor mediated endocytosis to account for the observations.

Truncated Amyloid-ß(11-40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly.

Alzheimer disease coincides with the formation of extracellular amyloid plaques composed of the amyloid-ß (Aß) peptide. Aß is typically 40 residues long (Aß(1-40)) but can have variable C and N termini. Naturally occurring N-terminally truncated Aß(11-40/42) is found in the cerebrospinal fluid and has a similar abundance to Aß(1-42), constituting one-fifth of the plaque load. Based on its specific N-terminal sequence we hypothesized that truncated Aß(11-40/42) would have an elevated affinity for Cu(2+). Various spectroscopic techniques, complemented with transmission electron microscopy, were used to determine the properties of the Cu(2+)-Aß(11-40/42) interaction and how Cu(2+) influences amyloid fiber formation. We show that Cu(2+)-Aß(11-40) forms a tetragonal complex with a 34 ± 5 fm dissociation constant at pH 7.4. This affinity is 3 orders of magnitude tighter than Cu(2+) binding to Aß(1-40/42) and more than an order of magnitude tighter than that of serum albumin, the extracellular Cu(2+) transport protein. Furthermore, Aß(11-40/42) forms fibers twice as fast as Aß(1-40) with a very different morphology, forming bundles of very short amyloid rods. Substoichiometric Cu(2+) drastically perturbs Aß(11-40/42) assembly, stabilizing much longer fibers. The very tight fm affinity of Cu(2+) for Aß(11-40/42) explains the high levels of Cu(2+) observed in Alzheimer disease plaques.