Excitation-energy-dependent molecular beacon detects early stage neurotoxic Aβ aggregates in the presence of cortical neurons

There is now crucial medical importance placed on understanding the role of early stage, subvisible protein aggregation, particularly in neurodegenerative disease. While there are strategies for detecting such aggregates in vitro, there is no approach at present that can detect these toxic species associated with cells and specific subcellular compartments. We have exploited excitation-energy-dependent fluorescence edge-shift of recombinant protein labeled with a molecular beacon, to provide a sensitive read out for the presence of subvisible protein aggregates. To demonstrate the potential utility of the approach, we examine the major peptide associated with the initiation of Alzheimer's disease, amyloid β-protein (Aβ) at a patho-physiologically relevant concentration in mouse cortical neurons. Using our approach, we find preliminary evidence that subvisible Aβ aggregates are detected at specific subcellular regions and that neurons drive the formation of specific Aβ aggregate conformations. These findings therefore demonstrate the potential of a novel fluorescence-based approach for detecting and imaging protein aggregates in a cellular context, which can be used to sensitively probe the association of early stage toxic protein aggregates within subcellular compartments.