Copper Transfer from Cu–Aβ to Human Serum Albumin Inhibits Aggregation,Radical Production and Reduces Aβ Toxicity

Amyloid‐β peptides (Aβ) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Aβ and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Aβ aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper‐exchange reaction between Aβ and HSA or the tetrapeptide DAHK (N‐terminal Cu‐binding domain of HSA) and the consequence of this exchange on Aβ‐induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu^II from Aβ rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu‐induced aggregation of Aβ, 3) HSA and DAHK suppressed the catalytic HO^. production in vitro and ROS production in neuroblastoma cells generated by Cu–Aβ and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu–Aβ with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu^II–HSA than Cu–DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu^II–Aβ interaction. The repercussion of the competition between HSA and Aβ to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.     

Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s‐IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co‐localization with proteins reported to accumulate in s‐IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.     

Perspectives on Inhibiting β‐Amyloid Aggregation through Structure‐Based Drug Design

Targeting β‐amyloid (Aβ) remains the most desired strategy in Alzheimer’s disease (AD) drug discovery research. Many peptides that specifically target Aβ aggregates are known, encompassing efforts from both industrial and academic research settings. However, in clinical terms, not much success has been gained with peptide research; in turn, small drug‐like molecules are already globally recognized as showing promise as an alternate approach. Aβ aggregation inhibitors are the most important part of the multifunctional drug design regimen for treating AD. Unfortunately, rational drug design approaches with small molecules are still in the initial stages. Herein we highlight, update, and elaborate on the structural anatomy of Aβ and known Aβ aggregation inhibitors in hopes of helping to optimize their use in structure‐based drug design approaches toward inhibitors with greater specificity. Furthermore, we present the first review of efforts to target a previously uncharacterized region of acetylcholinesterase: the N‐terminal 7–20 sub‐region, which was experimentally elucidated to participate in Aβ aggregation and deposition.     

Copper(I/II), α/β‐Synuclein and Amyloid‐β: Menage à Trois?

Copper binding to α‐synuclein (aS) and to amyloid‐β (Ab) has been connected to Parkinson's and Alzheimer's disease (AD), respectively, because Cu ions can modulate the peptide aggregation, and these Cu ? peptide complexes can catalyse the production of reactive oxygen species (ROS). In a significant proportion of AD brains, aggregation of aS and Ab has been detected, and it was proposed that Ab and aS interact with each other. Thus, we investigated the potential interactions of Ab and aS through their binding of copper(I) and copper(II). Additionally, β‐synuclein (bS) was investigated, due to its additional methionine residue, a potential Cu^I ligand. We found that: 1) the peptides containing the Cu‐binding domains Ab1–16, aS1–15 and bS1–15 have similar affinities towards Cu^II and towards Cu^I, with Ab1–16 being slightly stronger, 2) in the case of Cu^I, the additional Met residue in bS1–15 increased the affinity slightly, 3) the exchange of Cu^I/II between the two peptides is rapid (≤ms), 4) a/bS1–15 and Ab1–16 form a heterodimeric complex with Cu^II, 5) Cu^I probably promotes a transient ternary complex, 6) the different Cu^I/II coordination of Ab1–16, aS1–15 and bS1–15 impacts the capacity to produce ROS and to oxidise catechol, and 7) when Ab1–16, aS1–15 and Cu are present, the ROS production more closely resembles that by Ab1–16. The work gives insights into the coordination chemistry of these related peptides, and the relevance of coordination differences, the ternary complex and ROS production are discussed.     

Aggregation‐Prone Amyloid‐β⋅CuII Species Formed on the Millisecond Timescale under Mildly Acidic Conditions

Metal ions and their interaction with the amyloid beta (Aβ) peptide might be key elements in the development of Alzheimer's disease. In this work the effect of Cu^II on the aggregation of Aβ is explored on a timescale from milliseconds to days, both at physiological pH and under mildly acidic conditions, by using stopped‐flow kinetic measurements (fluorescence and light‐scattering), ¹H NMR relaxation and ThT fluorescence. A minimal reaction model that relates the initial Cu^II binding and Aβ folding with downstream aggregation is presented. We demonstrate that a highly aggregation prone Aβ ? Cu^II species is formed on the sub‐second timescale at mildly acidic pH. This observation might be central to the molecular origin of the known detrimental effect of acidosis in Alzheimer's disease.     

Modulation of Aβ42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.     

The effect of red pigment on the amyloidization of yeast proteins

The intensity of amyloid-bound thioflavine T fluorescence was studied in crude lysates of yeast strains carrying mutations in the ADE1 or ADE2 genes and accumulating the red pigment (a result of polymerization of aminoimidazoleribotide), and in white isogenic strains--either adenine prototrophs or carrying mutations at the first stages of purine biosynthesis. We found that the red pigment leads to a drop of amyloid content. This result, along with the data on separation of protein polymers of white and red strains in PAGE, suggests that the red pigment inhibits amyloid fibril formation. The differences in transmission of the thioflavine T fluorescence pattern by cytoduction and in blot-hybridization of pellet proteins of red and white [PSI(+) ] strains with Sup35p antibodies confirmed this conclusion. Purified red pigment treatment also led to a decrease of fluorescence intensity of thioflavine T bound to insulin fibrils and to yeast pellet protein aggregates from [PSI(+) ] strains. This suggests red pigment interaction with amyloid fibrils. Comparison of pellet proteins from red and white isogenic strains separated by 2D-electrophoresis followed by MALDI analysis has allowed us to identify 48 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, closely corresponding to prion-dependent proteins that we characterized previously. Also present were some proteins involved in stress response and proteolysis. We suppose that the red pigment acts by blocking certain sites on amyloid fibrils that, in some cases, can lead in vivo to interfere with their contacts with chaperones and the generation of prion seeds.