Redox reactivities of membrane-bound Amyloid-β-Cu complexes and their targeting by metallothionein-3.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β peptide (Aβ ) in the central nervous system (CNS) and its aggregation in senile amyloid plaques. Copper coordination to Aβ triggers Aβ aggregation and, in the presence of biological reducing agents, it promotes the catalytic generation of reactive oxygen species (ROS) via Fenton-type and Haber-Weiss reactions. Due to its amphiphilic nature, Aβ can interact with cell membranes and compromise their integrity by thinning the lipid bilayer and forming channel-like structures potentially leading to cell death. In this work, by applying biophysical and biochemical approaches, we characterized the insertion of Aβ into an artificial lipid bilayer system mimicking cell membranes and demonstrate that the Aβ -lipid interaction does not prevent the Cu coordination to Aβ . We performed a comparative analysis of the redox reactivities of membrane-bound Aβ (memAβ -Cu ) species with soluble Aβ -Cu establishing that membrane insertion leads to memAβ -Cu complexes featuring an enhanced detrimental catechol oxidase activity towards the neurotransmitter dopamine. Moreover, memAβ -Cu efficiently catalyzes Aβ di-tyrosine crosslinking and hydroxyl radical production in the presence of ascorbate. In addition, we establish that memAβ -Cu redox reactivity catalyze lipid peroxidation in membranes containing polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), leading to the generation of malondialdehyde (MDA) toxic end-products. This reactivity compromises the structural integrity of the lipid bilayers resulting in membrane leakage, further substantiating how important is to control aberrant Aβ -Cu interactions in AD. Metallothioneins (MTs) are key metalloproteins central to neuronal and astrocytic transition metal homeostasis and buffering. These cysteine-rich proteins bind with high affinity d metals (Cu and Zn ) forming two metal thiolate clusters in their N-terminal β-domain and C-terminal α-domain. The metallothionein-3 (MT-3) isoform is central to metal homeostasis in the CNS, but it is downregulated in AD patients, possessing a neuroprotective role in AD. MT-3 can control aberrant protein-Cu interactions and the Cu-centered redox reactivities of amyloidogenic protein-Cu complexes such as α-synuclein (Parkinson's disease), PrP (prion disease), and soluble and aggregated Aβ (AD). In this work, we unravel that the detrimental memAβ -Cu catechol oxidase and redox reactivities can be efficiently silenced by MT-3 via metal swap reactions, effectively scavenging and reducing Cu to Cu in its β-domain using thiolates as electron source, forming the redox-inert species Cu Zn MT-3. Consequently, MT-3 can efficiently prevent lipid peroxidation and protect membrane structural integrity. New strategies targeting membrane-bound Aβ -Cu complexes as key players of the AD etiology could be envisioned.