A Solvatochromic Near Infrared Fluorophore Sensitive to the Full Amyloid Beta Aggregation Pathway

Alzheimer’s disease has long been associated with the aggregation of amyloid beta peptides (Aβ42) into macroscale plaques, although specific neurodegenerative agents have not been definitively identified. Much evidence has pointed to the soluble nanoscale oligomers that form early in the Aβ42 aggregation pathway, but there is little understanding of these structures, their mechanisms of formation, or how they grow into plaques. Here, we show that a solvatochromic fluorophore with near infrared (NIR) emission can track synthetic Aβ42 aggregation through environment-sensitive spectral shifts from the earliest timepoints through plaque formation. This azide-functionalized phosphine oxide azetidine rhodol (Phazr-N3) shows large polarity-dependent changes in fluorescence emission, with maxima shifting from 630 nm in toluene to 703 nm in aqueous buffer, and a maximum quantum yield of 62%. Upon induction of Aβ42 aggregation, we observe immediate solvatochromic changes in Phazr-N3 fluorescence, with multiple apparent phases over 24 h, and which culminate before the onset of any major fluorescence changes of conformation-specific aggregation fluorophore ThT. Solution anisotropy measurements show a low micromolar affinity of Phazr-N3 for non-aggregated Aβ42 in solution, and real-time measurements are consistent with rapid phase separation and slow dehydration of the growing aggregate. Spectral imaging of synthetic plaques stained in the presence of live cells and lipid-binding protein shows over 4000-fold Phazr-N3 fluorescence intensity above background under no-wash conditions, and over 100-fold intensity above co-plated microglial cells. This use of solvatochromic probes and kinetic anisotropy offers real-time, minimally invasive insight into Aβ42 aggregation and the molecular mechanisms of neurodegeneration.