Cu-Aβ Complex Research Articles

A cocktail of Cu2+- and Zn2+-peptoid-based chelators can stop ROS formation for Alzheimer's disease therapy.

The formation of reactive oxygen species (ROS) in the brain is a major cause of neuropathologic degradation associated with Alzheimer's Disease (AD). It has been suggested that the copper (Cu)-amyloid-β (Aβ) peptide complex can lead to ROS formation in the brain. An external chelator for Cu that can extract Cu from the CuAβ complex should inhibit the formation of ROS, making Cu chelation an excellent therapeutic approach for AD. Such a chelator should possess high selectivity for Cu over zinc (Zn), which is also present within the synaptic cleft. However, such selectivity is generally hard to achieve in one molecule due to the similarities in the binding preferences of these two metal ions. As an alternative to monotherapy (where Cu extraction is performed using a single chelator), herein we describe a variation of combination therapy - a novel cocktail approach, which is based on the co-administration of two structurally different peptidomimetic chelators, aiming to target both Cu2+ and Zn2+ ions simultaneously but independently from each other. Based on rigorous spectroscopic experiments, we demonstrate that our peptidomimetic cocktail allows, for the first time, the complete and immediate inhibition of ROS production by the CuAβ complex in the presence of Zn2+. In addition, we further demonstrate the high stability of the cocktail under simulated physiological conditions and its resistance to proteolytic degradation by trypsin and report the water/n-octanol partition coefficient, initially assessing the blood-brain barrier (BBB) permeability potential of the chelators.

Active Site Environment and Reactivity of Copper-Aβ in Membrane Mimetic SDS Micellar Environment.

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid β (Aβ) peptide in extracellular deposits generated upon proteolysis of Amyloid Precursor Protein (APP). While copper (Cu(II)) binds to Aβ in soluble oligomeric and aggregated forms, its interaction with membrane-bound Aβ remains elusive. Investigating these interactions is crucial for understanding AD pathogenesis. Here, utilizing SDS micelles as a simplified membrane mimic, we focus on elucidating the interplay between membrane-anchored Aβ and copper, given their pivotal roles in AD. We employed spectroscopic techniques including UV, CD, and EPR to characterize the active site of Cu-Aβ complexes. Our findings demonstrate that copper interacts with Aβ peptides in membrane-mimicking micellar environments similarly to aqueous buffer solutions. Cu-Aβ complexes in this medium also induce higher hydrogen peroxide (H2O2) production, potentially contributing to AD-related oxidative stress. Moreover, we observe an increased oxidation rate of neurotransmitter such as dopamine by Cu-Aβ complexes. These results enhance our understanding of Cu-Aβ interactions in AD pathology and offer insights into potential therapeutic interventions targeting this interaction.

Redox behaviour of Cu-Aβ(4-16) complexes related to Alzheimer's Disease

The development of Alzheimer's Disease (AD) has been linked to abnormal quantities of β-amyloid peptides in the brain. The majority of studies have focussed on Aβ(1-40/42) amyloids and their Cu(II)-Aβ(1-40/42) complexes which are responsible for production of reactive oxygen and nitrogen species (ROS and RNS), which are highly toxic to neurons. According to recent studies on amyloid plaques, Aβ(4–42), which is an N-truncated version of Aβ(1–42), is as prevalent as Aβ(1–42) in the brain. Although Cu(II) ions, bounded by Aβ(4–42), can be oxidized to highly reactive Cu(III) ions, its Cu(II) complexes do not appear to contribute to ROS/RNS formation. In this paper, the pH-dependent voltammetric response of Aβ(4–16)-Cu(II) complexes is investigated to understand the influence of the deprotonation of tyrosine within the complex towards the Cu(II)/Cu(III) reaction. The results will help to better understand the scavenging role of tyrosine in quenching highly reactive Cu(III) ions not only in Aβ(4-x)-Cu(II) complexes but also provide clues to the reactive properties of other tyrosine-containing amyloid-metal complexes.

Hybrid Bis-Histidine Phenanthroline-Based Ligands to Lessen Aβ-Bound Cu ROS Production: An Illustration of Cu(I) Significance.

We here report the synthesis of three new hybrid ligands built around the phenanthroline scaffold and encompassing two histidine-like moieties: phenHH, phenHGH and H’phenH’, where H correspond to histidine and H’ to histamine. These ligands were designed to capture Cu(I/II) from the amyloid-β peptide and to prevent the formation of reactive oxygen species produced by amyloid-β bound copper in presence of physiological reductant (e.g., ascorbate) and dioxygen. The amyloid-β peptide is a well-known key player in Alzheimer’s disease, a debilitating and devasting neurological disorder the mankind has to fight against. The Cu-Aβ complex does participate in the oxidative stress observed in the disease, due to the redox ability of the Cu(I/II) ions. The complete characterization of the copper complexes made with phenHH, phenHGH and H’phenH’ is reported, along with the ability of ligands to remove Cu from Aβ, and to prevent the formation of reactive oxygen species catalyzed by Cu and Cu-Aβ, including in presence of zinc, the second metal ions important in the etiology of Alzheimer’s disease. The importance of the reduced state of copper, Cu(I), in the prevention and arrest of ROS is mechanistically described with the help of cyclic voltammetry experiments.

Copper Toxicity Links to Pathogenesis of Alzheimer's Disease and Therapeutics Approaches.

Alzheimer’s disease (AD) is an irreversible, age-related progressive neurological disorder, and the most common type of dementia in aged people. Neuropathological lesions of AD are neurofibrillary tangles (NFTs), and senile plaques comprise the accumulated amyloid-beta (Aβ), loaded with metal ions including Cu, Fe, or Zn. Some reports have identified metal dyshomeostasis as a neurotoxic factor of AD, among which Cu ions seem to be a central cationic metal in the formation of plaque and soluble oligomers, and have an essential role in the AD pathology. Cu-Aβ complex catalyzes the generation of reactive oxygen species (ROS) and results in oxidative damage. Several studies have indicated that oxidative stress plays a crucial role in the pathogenesis of AD. The connection of copper levels in AD is still ambiguous, as some researches indicate a Cu deficiency, while others show its higher content in AD, and therefore there is a need to increase and decrease its levels in animal models, respectively, to study which one is the cause. For more than twenty years, many in vitro studies have been devoted to identifying metals’ roles in Aβ accumulation, oxidative damage, and neurotoxicity. Towards the end, a short review of the modern therapeutic approach in chelation therapy, with the main focus on Cu ions, is discussed. Despite the lack of strong proofs of clinical advantage so far, the conjecture that using a therapeutic metal chelator is an effective strategy for AD remains popular. However, some recent reports of genetic-regulating copper transporters in AD models have shed light on treating this refractory disease. This review aims to succinctly present a better understanding of Cu ions’ current status in several AD features, and some conflicting reports are present herein.

Salen-based bifunctional chemosensor for copper (II) ions: Inhibition of copper-induced amyloid-β aggregation

Disruption of copper homeostasis is associated with a number of severe diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), Wilson’s disease, and Menkes syndrome. Given this association, the detection and capture of Cu2+ in biological fluids and tissues may provide a new direction for the diagnosis and treatment of related disorders. The current analytical approaches, however, are challenging due to the high cost, complexity, and long time required to prepare and analyze samples. Here, we report a novel salen ligand, namely N,N’-(1,2-phenylene)bis(1-(1H-imidazol-4-yl)methanimine) (pimi), which can readily detect and concurrently capture Cu2+ from aqueous as well as biological mediums. Pimi can selectively and specifically detect Cu2+ from biofluid and cellular samples with rapid ccresponse time (<3 s) and an ultra-sensitive detecting limit (2.7 nM). More importantly, pimi showed excellent environmental tolerance and had a very wide pH range for detecting Cu2+ in a variety of biological samples. Attributed to the strong binding affinity and selectivity towards Cu2+, pimi was found to capture Cu2+ ions from Cu-Aβ complexes, thus inhibiting copper-induced aggregation of Aβ and protecting neuronal cells from the toxicity of aggregated Aβ. These results provide a compelling starting point for further fine-tuning of salen-based chemosensor for the diagnosis and treatment of diseases associated with the hyperaccumulation of copper.

A Trishistidine Pseudopeptide with Ability to Remove Both CuΙ and CuΙΙ from the Amyloid-β Peptide and to Stop the Associated ROS Formation.

The pseudopeptide L, derived from a nitrilotriacetic acid scaffold and functionalized with three histidine moieties, is reminiscent of the amino acid side chains encountered in the Alzheimer's peptide (Aβ). Its synthesis and coordination properties for CuΙ and CuΙΙ are described. L efficiently complex CuΙΙ in a square-planar geometry involving three imidazole nitrogen atoms and an amidate-Cu bond. By contrast, CuΙ is coordinated in a tetrahedral environment. The redox behavior is irreversible and follows an ECEC mechanism in accordance with the very different environments of the two redox states of the Cu center. This is in line with the observed resistance of the CuΙ complex to oxidation by oxygen and the CuΙΙ complex reduction by ascorbate. The affinities of L for CuΙΙ and CuΙ at physiological pH are larger than that reported for the Aβ peptide. Therefore, due to its peculiar Cu coordination properties, the ligand L is able to target both redox states of Cu, redox silence them and prevent reactive oxygen species production by the CuAβ complex. Because reactive oxygen species contribute to the oxidative stress, a key issue in Alzheimer's disease, this ligand thus represents a new strategy in the long route of finding molecular concepts for fighting Alzheimer's disease.

The Pathogenic A2V Mutant Exhibits Distinct Aggregation Kinetics, Metal Site Structure, and Metal Exchange of the Cu2+ -Aβ Complex.

A prominent current hypothesis is that impaired metal ion homeostasis may contribute to Alzheimer's disease (AD). We elucidate the interaction of Cu2+ with wild-type (WT) Aβ1-40 and the genetic variants A2T and A2V which display increasing pathogenicity as A2T<WT<A2V. Cu2+ significantly extends the lag phase in aggregation kinetics, in particular for the pathogenic A2V variant. Additionally, a rapid, initial, low intensity ThT response is observed, possibly reflecting formation of Cu2+ induced amorphous aggregates, as supported by atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy, again most notably for the A2V variant. Electron paramagnetic resonance (EPR) spectroscopy gives pKa values for transition between two Cu2+ coordination geometries (component I and II) of 7.4 (A2T), 7.9 (WT), and 8.4 (A2V), that is, component I is stabilized at physiological pH in the order A2T<WT<A2V. 1 H NMR relaxation exhibits the same trend for the non-coordinating aromatic residues (A2T<WT<A2V), and implies markedly faster inter-peptide Cu2+ exchange for the A2V variant than for WT and A2T. We therefore hypothesize that component I of the Cu-Aβ complex is related to pathogenicity, accounting for both the pathogenic nature of the A2V variant and the protective nature of the A2T variant.

Identification of key structural features of the elusive Cu-Aβ complex that generates ROS in Alzheimer's disease.

Oxidative stress is linked to the etiology of Alzheimer's disease (AD), the most common cause of dementia in the elderly. Redox active metal ions such as copper catalyze the production of Reactive Oxygen Species (ROS) when bound to the amyloid-β (Aβ) peptide encountered in AD. We propose that this reaction proceeds through a low-populated Cu-Aβ state, denoted the "catalytic in-between state" (CIBS), which is in equilibrium with the resting state (RS) of both Cu(i)-Aβ and Cu(ii)-Aβ. The nature of this CIBS is investigated in the present work. We report the use of complementary spectroscopic methods (X-ray absorption spectroscopy, EPR and NMR) to characterize the binding of Cu to a wide series of modified peptides in the RS. ROS production by the resulting Cu-peptide complexes was evaluated using fluorescence and UV-vis based methods and led to the identification of the amino acid residues involved in the Cu-Aβ CIBS species. In addition, a possible mechanism by which the ROS are produced is also proposed. These two main results are expected to affect the current vision of the ROS production mechanism by Cu-Aβ but also in other diseases involving amyloidogenic peptides with weakly structured copper binding sites.

Modeling Copper Binding to the Amyloid-β Peptide at Different pH: Toward a Molecular Mechanism for Cu Reduction

Oxidative stress, including the production of reactive oxygen species (ROS), has been reported to be a key event in the etiology of Alzheimer's disease (AD). Cu has been found in high concentrations in amyloid plaques, a hallmark of AD, where it is bound to the main constituent amyloid-β (Aβ) peptide. Whereas it has been proposed that Cu-Aβ complexes catalyze the production of ROS via redox-cycling between the Cu(I) and Cu(II) state, the redox chemistry of Cu-Aβ and the precise mechanism of redox reactions are still unclear. Because experiments indicate different coordination environments for Cu(II) and Cu(I), it is expected that the electron is not transferred between Cu-Aβ and reactants in a straightforward manner but involves structural rearrangement. In this work the structures indicated by experimental data are modeled at the level of modern density-functional theory approximations. Possible pathways for Cu(II) reduction in different coordination sites are investigated by means of first-principles molecular dynamics simulations in the water solvent and at room temperature. The models of the ligand reorganization around Cu allow the proposal of a preferential mechanism for Cu-Aβ complex reduction at physiological pH. Models reveal that for efficient reduction the deprotonated amide N in the Ala 2-Glu 3 peptide bond has to be protonated and that interactions in the second coordination sphere make important contributions to the reductive pathway, in particular the interaction between COO(-) and NH(2) groups of Asp 1. The proposed mechanism is an important step forward to a clear understanding of the redox chemistry of Cu-Aβ, a difficult task for spectroscopic approaches as the Cu-peptide interactions are weak and dynamical in nature.