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

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