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Alzheimer's disease and Zinc: the missing link ?

Periodic Reporting for period 3 - aLzINK (Alzheimer's disease and Zinc: the missing link ?)

Reporting period: 2018-03-01 to 2019-08-31

The issue addressed within the framework of the aLzINK project is how metal ions, mainly copper and zinc, affect the aetiology of Alzheimer’s disease (AD). AD is a neurodegenerative disorder responsible for more than 60% of all dementia cases. Besides the constantly growing number of AD cases due to the increase of life expectancy, AD more than many other diseases has important and long-lasting fallouts on the caregivers. Hence, consequences for the society are of utmost importance.
In the aLzINK project, the project is double: (i) we aim at understanding how metal ions, which are crucial for many biological processes, could trigger the disease and (ii) at defining and probing new therapeutic strategies.
On the fundamental side, the overall objectives are to disentangle (i) the intricacy of metal ions (copper or/and zinc) binding to the peptide involved in AD, the amyloid-β (Aβ) peptide; (ii) the role played by the metal ions in Aβ aggregation that is the catalytic self-assembly of the peptide, and in the production of reactive oxygen species (ROS) that contributes to oxidative stress, where Aβ aggregation and oxidative stress are two hallmarks of AD; (iii) how cell toxicity is mediated by metal ions and the Aβ peptide. On the therapeutic side, the overall objectives are to (i) design and probe in vitro and on cells culture new molecules (named here drug candidates or ligands) able to remove the copper ions form the Aβ peptide and stop aberrant interaction responsible for Aβ aggregation and ROS production, the two deleterious events linked to the aetiology of the disease; (ii) evaluate how zinc can interfere in copper ions detoxification and counteract the hampering effect of zinc ions and (iii) ultimately, trigger copper removal using zinc ions.
The project began on the 1st March 2015. The working force hired to perform the project is: one PhD fellow (Valentina Borghesani, hired in March 2015), one post-doctoral fellow (Elena Atrian Blasco, hired in March 2016), both of them are performing research tasks; one high-level assistant (Laurent Sabater, September 2016-August 2017) who helped to settle technical, administrative and research items. In addition, two PhD fellows paid by the French Ministry of Research were involved in the project (Amandine Conte-Daban, October 2014-September 2017 and to a lesser extent Clémence Cheignon, October 2013-September 2016). Two new post-doctoral fellows will complete the research team (the first one in November 2017 and the second one in February-March 2018).
The doctoral project mainly corresponds to WP1 and aims at defining the coordination sphere of Zn within the Aβ peptide and biologically modified counterparts as well as impact in aggregation. Several results have been obtained and corresponding manuscripts are in preparation. As a side project, new methodologies to address the Aβ aggregation have been sought and studied (by electrochemistry for instance or using other fluorophores than the Thioflavine T, considered as the gold standard to monitor the aggregation of the peptide by fluorescence). In addition, the study of Reactive Oxygen Species (ROS) production by N-terminally truncated forms of the Aβ peptides have been studied. This corresponds to WP1 and WP3 of the project because those form are redox silent in contrast to the full length peptide which is redox competent and thus can be foreseen as drug candidates.
The first post-doctoral project is straddle between two WPs: WP2 and WP3. It aims at studying the mutual interference of Zn and Cu regarding their binding to the Aβ peptide and the resulting impact on ROS formation and alteration of the Aβ peptide aggregation.
The one-year technical, scientific and administrative position was opened to help the PI. Principal tasks were (i) on a technical level: to implement the fluorimeters, UV-Vis spectrometers, fplc (fast protein liquid chromatography) and the peptide synthetiser, set up the stop-flow experiments; (ii) on a scientific level: to perform preliminary tests on potential therapeutic molecules provided by external collaborators and participate in the acquisition of X-ray absorption spectroscopy (XAS) data on ESRF synchrotron ; (iii) on an administrative level: to help the PI in the management of reporting activities and expenses, welcoming of group members, interfacing the interaction with the Host institution and dealing with the open access issues.

Several results were obtained and published since the beginning of the project. There are reported here by chronological order.
(i) Elucidation of the zinc (Zn(II)) coordination site within the Aβ peptide. It was solved using a combination of spectroscopic methods (Nuclear Magnetic Resonance, X-ray Absorption Spectroscopy, affinity measurements by UV-Vis) and the use of a series of modified peptides. This corresponds to Part B2-Section b. §A-1(i) of the DoA and to the article doi: 10.1021/acs.inorgchem.6b01733.
(ii) Importance of the level of reductant as oxidative stress mediator. Cu-Aβ species are able to generate ROS form dioxygen and physiological reductant such as ascorbate. We showed that the initial rate of hydroxyl radical release from the Cu-Aβ / dioxygen / reductant system as a function of ascorbate concentration follows a bell-like curve. This corresponds to Part B2-Section b. §A-3(ii) of the DoA and to the communication http://dx.doi.org/10.1039/C6DT01979J.
(iii) Importance of the reorganization of Copper (Cu(I) and Cu(II)) sites after peptide oxidation. Because the production of Reactive Oxygen Species (ROS) are intimately linked to the nature of Copper binding sites in the Aβ peptide and because ROS produced can oxidized the peptide itself, we questioned the interdependency of the two events. This corresponds to Part B2-Section b. §A-3(ii) of the DoA and to the article http://dx.doi.org/10.1039/C6MT00150E selected for the themed collection: Recent HOT articles.
(iv) Illustration of the key role played by Zn(II) in the removal of Cu(II) from the Aβ peptide (thermodynamical approach). As stated in the description of the issue addressed within the framework of the aLzINK project, Zn can impede the detoxification of Cu(II) by drug candidates. We highlighted and illustrated this process using two different and prototypical drug candidates via a multi-technical study. This corresponds to Part B2-Section b. §B-1-3 of the DoA and to the article http://dx.doi.org/10.1039/C6DT02308H.
(v) Description of a new method for the determination of the affinity of Copper (Cu(II)) for the Aβ peptide. Because the affinity of Cu(II) for the Aβ peptide is a crucial parameter both for (i) the biological relevance of the Cu(II) – Aβ interaction and the design of Cu(II) ligands, we implemented a new easy-to-handle method based on UV-Vis measurements, to evaluate the affinity of Cu(II) – Aβ. This corresponds to Part B2-Section b. §B-1 of the DoA and to the article doi: 10.1021/acs.analchem.6b04979.
(vi) Identification of key structural features regarding the Cu-Aβ species responsible for the ROS production. The production of ROS by the Cu-Aβ species relies on a very specific mechanism that we thoroughly identified for the first time using a combination of spectroscopic methods and measurements of ROS production as function of several parameters, mainly variation in the peptidic sequence. This corresponds to Part B2-Section b. §A-3(ii) of the DoA and to the article http://dx.doi.org/10.1039/C7SC00809K.
(vii) Review of the reciprocal impact of Zn and Cu in their respective interaction with the Aβ peptide. This article reviews the main elements behind the aLzINK project, thus partly corresponding to Part B2-Section a. §A-2 and B-1 of the DoA and is reported in on invitation, front inside cover.
(viii) Review of the biological importance of N-terminally truncated forms of the Aβ peptide. In the meanwhile of the progression of the aLzINK project, a revival of interest for such modified forms of the Aβ peptide appears. Hence we update our view of the field through this review. A good knowledge of these new peptides is crucial for the design of appropriate Cu(II) ligands. This corresponds to Part B2-Section b. §A-1 and B-1 of the DoA and to the article DOI: 10.1002/ejic.201700776 (on invitation, VIP, front cover, and to the corresponding interview in ChemistryViews of the PI, DOI: 10.1002/chemv.201700081.)
(ix) Identification of a new strategy to remove Copper from Aβ and stop the associated ROS production. Because the valence state of Copper ions within the synaptic cleft is not well-known, we reported a chelating approach where the two redox states of the copper ions are targeted. This is a more pragmatic approach than targeting only Cu(II) or Cu(I) and differs from the current view in the field where only Cu(II) is considered as the therapeutic target. This corresponds to Part B2-Section b. §B-1-3 of the DoA and to the article http://dx.doi.org/10.1002/chem.201703429.
(x) Illustration of a prodrug strategy to remove Cu(II) from Aβ and stop associated ROS production and Cu-altered aggregation of the Ab peptide. The Cu-Aβ species is able to produce ROS, hence a beneficial impact can be foreseen for the use of Mn-based SuperOxide Dismutase (SOD) mimics. We described such a Mn(II) complex that has a SOD-like activity and is able to retrieve Cu from the Aβ peptide and precludes associated deleterious events (ROS, aggregation). We had thus studied the first inorganic pro-drug targeting Cu(II) in the AD context, which in addition has the ability to reduce the content in superoxide. This corresponds to Part B2-Section b. §B-1-3 of the DoA and has recently been submitted for publication.
(xi) Reviews of the role of metallothionein (MTs) in AD and literature update regarding copper and zinc binding to Aβ peptides and familial mutants. As parts of two articles, we reviewed (i) the cross-talk between metal ions, metallothioneins (MTs), a key protein in metal trafficking in the brain, and the Aβ peptide and (ii) update the coordination chemistry of copper and zinc with the Aβ peptide and familial mutants involved in the early onset of the disease. The two reviews were timely for us, because we plan to study the therapeutic potential of MTs and because, as for the N-terminally truncated forms, copper and zinc binding to mutated Aβ can influence the design of ligands. This corresponds to Part B2-Section b. §B-1-3 of the DoA and to DOI: 10.1039/C7CS00448F.
(xii) Illustration of the importance of kinetic parameters in Cu(II) removal from Aβ and on associated ROS production. Until now, we (and others) focus on the thermodynamic parameters regarding the ability of a ligand to retrieve Cu from the Aβ peptide. In this study, we proved that kinetic issues are of utmost importance as well. We challenged a family of tetra-azamacrocycles of various sizes and substitutions for their ability to remove Cu(II) from Aβ and redox silence it during the production of ROS. Characterizations were made using a combination of spectroscopic methods (EPR, UV-Vis, XANES…). This corresponds to Part B2-Section b. §B-1-3 of the DoA. The corresponding manuscript is in preparation.
(xiii) Importance of Aβ and oxidative stress in AD. As a consequence of the works we recently published on the impact of ROS produced by Cu-Aβ in the oxidative stress involved in AD, we proposed a general review on this topic. This corresponds Part B2-Section b. §A-3(ii) of the DoA and to the article https://doi.org/10.1016/j.redox.2017.10.014.

It is noteworthy that during the course of the project, new collaborations have been settled, mainly with collaborators providing Copper ligands, along the main objective to illustrate the importance of the Cu over Zn selectivity in Cu detoxification in AD.

In addition to dissemination of the results towards an academic audience via publications and presentations in international conferences, a special input has been given to dissemination to a wide general audience. The PI has, in particular, communicated through various events such as scientific coffees, European night of research, “Fête de la science”, oral presentations at the museum, in university classes... The general topic is introduced and experimental illustrations are given.
The main scientific breakthrough of the aLzINK project is to participate in making the current paradigm about Copper targeting in the context of AD move towards a more rationale view. Indeed while the impact of metal ions in AD is recognized, their role is far from being understood. Hence, first therapeutic approaches have targeted Cu(II) ions but without taking care of many crucial parameters. And because the first molecules tested failed to pass clinical trials, this has recently led to a disinterest of a part of the scientific community regarding the role of metal ions in AD. With the aLzINK project, we aim at counter-balancing such very radical thought by providing new proofs of concept regarding the targeting of Cu(II). In particular, we focus on the importance of Zn interference in Cu(II) detoxification, from thermodynamic and kinetic views. In addition, we also improve the current description of the metal ions to Aβ peptide interaction.
The end of the project will focus on moving forward to more biologically relevant conditions, that is to say studying metal ions to Aβ peptide interaction and toxicity in cellular media (and ideally in animal models). In particular, we will focus on the design of Zn-triggered drug candidates able to target and remove Copper from the Aβ peptide and stop the associated ROS and aggregation events in the test tubes and in cellular media.