Ruthenium Red Colorimetric and Birefringent Staining of Amyloid-β Aggregates in Vitro and in Tg2576 Mice

Abstract

Alzheimer's disease (AD) is a devastating neurodegenerative disease most notably characterized by the misfolding of amyloid-β (Aβ) into fibrils and its accumulation into plaques. In this Article, we utilize the affinity of Aβ fibrils to bind metal cations and subsequently imprint their chirality to bound molecules to develop novel imaging compounds for staining Aβ aggregates. Here, we investigate the cationic dye ruthenium red (ammoniated ruthenium oxychloride) that binds calcium-binding proteins, as a labeling agent for Aβ deposits. Ruthenium red stained amyloid plaques red under light microscopy, and exhibited birefringence under crossed polarizers when bound to Aβ plaques in brain tissue sections from the Tg2576 mouse model of AD. Staining of Aβ plaques was confirmed via staining of the same sections with the fluorescent amyloid binding dye Thioflavin S. In addition, it was confirmed that divalent cations such as calcium displace ruthenium red, consistent with a mechanism of binding by electrostatic interaction. We further characterized the interaction of ruthenium red with synthetic Aβ fibrils using independent biophysical techniques. Ruthenium red exhibited birefringence and induced circular dichroic bands at 540 nm upon binding to Aβ fibrils due to induced chirality. Thus, the chirality and cation binding properties of Aβ aggregates could be capitalized for the development of novel amyloid labeling methods, adding to the arsenal of AD imaging techniques and diagnostic tools.

Probing Amyloid Nanostructures Using Photoluminescent Metal Complexes

“…The existence of a hydrophobic cleft between Phe 20 and Val 18, that forms as a consequence of the assembly of Aβ monomers to form the fibril, has been previously discussed. Computational, biophysical and photochemical methods have been used to validate this hydrophobic cleft, which is exposed to the aqueous interphase, and can accommodate this hydrophobic extended systems [66,123] . Therefore, it can be extrapolated that hydrophobic aromatic molecules in general will bind (and could be employed as binding elements) to Aβ fibrils.…”

Recent progress in the development of metal complexes as β-amyloid imaging probes in the brain

“…16) as an Aβ labeling agent. 101 Under light microscopy, Aβ plaques in brain sections of Tg2576 mice were dyed red by 93; under cross-polarization, birefringence was observed when 93 bound to Aβ plaques. However, in addition to Aβ plaques, neurons in the dentate cellular layer of the hippocampus and the Purkinje layer of the cerebellum were also stained by 93, which is undesirable for Aβ-specific dyes.…”

Chirality at the Nanoparticle Surface: Functionalization and Applications

“…Consequently, one of the approaches for the treatment of this pathology is to inhibit Aβ fibrillogenesis. With this aim, Sun et al prepared chiral penicillamine-capped selenium (L(D)-Pen@Se) NPs that could perform as Aβ inhibitors [69]. They observed that D-Pen@Se NPs were more efficient inhibitors and they improved both understanding and memory impairments.…”

“…A characteristic of Alzheimer's disease (AD) is the misfolding of amyloid-β (Aβ) into fibrils and its accumulation into plaques [69]. Consequently, one of the approaches for the treatment of this pathology is to inhibit Aβ fibrillogenesis.…”