Abstract
Decelerated degradation of beta-amyloid (Abeta) and its interaction with synaptic copper may be pathogenic in Alzheimer disease. Recently, Co(III)-cyclen tagged to an aromatic recognition motif was shown to degrade Abeta in vitro. Here, we report that apocyclen attached to selective Abeta recognition motifs (KLVFF or curcumin) can capture copper bound to Abeta and use the Cu(II) in place of Co(III) to become proteolytically active. The resultant complexes interfere with Abeta aggregation, degrade Abeta into fragments, preventing H2O2 formation and toxicity in neuronal cell culture. Because Abeta binds Cu in amyloid plaques, apocyclen-tagged targeting molecules may be a promising approach to the selective degradation of Abeta in Alzheimer disease. The principle of copper capture could generalize to other amyloidoses where copper is implicated.
Highlights
Dence that A neurotoxicity involves forming pathological assemblies of the peptide and evoking the generation of free radicals by binding and reducing redox-active metals such as copper (Cu(II)) [3,4,5]
We show that apocyclen ligated to an A-targeting molecule can capture Cu bound to A, become activated, and prevent both A oligomerization and toxicity as well as cleave the peptide
We used the generation of H2O2 by A-Cu(II) complexes as an index of copper interaction mixed with KLVFF peptide, cyc
Summary
Preparation of Cyc(Cu(II))-KLVFF and Other Complexes— Cyclen-hybrid cleavage agents were synthesized by routine synthetic methods, as detailed in supplemental “Experimental Procedures” and supplemental Fig. S1. Cherny) were solubilized to 2 mg/ml in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP; Acros), incubated overnight at room temperature, and stored at Ϫ80 °C in HFIP Aliquots of this solution were evaporated off under vacuum and dissolved in 20 mM NaOH sonicated for 15 min. Samples were placed in a four-sided quartz fluorescence cuvette, and data were recorded at room temperature. TEM—The TEM samples were prepared by placing 8 l of the incubated solution monitored by ThT assay on 300-mesh formvar-coated copper grids for 2 min before removing excess solution. Fresh A(1– 42) (40 M) was incubated with or without cyclen-hybrid cleavage agents (40 M) for 3 days at 37 °C in PBS (pH 7.4) and diluted 1:4 with culture medium and applied to neurons. After a 2-day incubation, cells were prepared for microscopy as described [24]
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