Fluorescent Probes for Detection of Misfolded Protein Oligomers in Alzheimer's Disease and Related Disorder There is significant evidence that the clinical symptoms of Alzheimer’s Disease (AD) and related disorders are closely linked to the formation and proliferation of small oligomers that precede the emergence of the prominent late-stage fibrils and plaques. Therefore, amyloid beta oligomers (AβOs) are the most direct biomarkers for monitoring the onset and progression of AD. Attempts at utilizing this biomarker, however, have been severely hampered by the dearth of techniques for the reliable detection of AβOs in biological samples and tissues. While oligomer-selective antibodies have provided important insights into AβOs, their use doesn't extend to detecting oligomer populations in vivo, let alone monitoring their temporal evolution. The overall objectives of this proposal are therefore to identify small oligomer-selective dyes for the detection of AβOs and to validate their specificity for AβOs, and potentially related oligomers, in tissues of animal models of AD and patient tissue. Multiple laboratories have observed that in vivo Aβ assembly displays not only purely sigmoidal but also biphasic ThT kinetics. We have shown that the onset of biphasic ThT kinetics directly correlates with the onset and rapid increase in prefibrillar oligomer populations with increases in monomer concentrations. Here we propose to use this transition from essentially oligomer-free sigmoidal to oligomer-dominated biphasic kinetics to screen a selection of readily available fluorescent dyes for their selectivity for AβOs over AβFs and monomers. An initial test of this approach already yielded a highly promising dye candidate. Our preliminary data also indicate that this dye specifically stains oligomer deposits in animal models of AD. While very encouraging, the utility of our current oligomer-selective dye requires further validation. In addition, we seek to identify multiple chemically and structurally distinct oligomer-selective dyes to improve the chances to develop one of them into a PET probe for in vivo imaging of oligomers. We will therefore extend our current screen for AβO-selective dyes to a larger set of fluorescent dyes selected from different dye categories. In parallel, we will scrutinize whether the current dye reliably detects AβOs at various stages of the disease, and does so in animal models as well as patient tissues. Promising novel AβO-selective dyes identified through our screening assay will be subjected to the same ex vivo validation of their specificity in tissues. We anticipate that these experiments will yield multiple promising AβO-selective dyes with application for fundamental studies of oligomer formation, for the development of new assays for detecting AβOs ex vivo, and, most importantly, as the detection moiety for a future oligomer-selective PET probe for antemortem in vivo oligomer imaging in patients.

Relevance to Public Health The lack of reliable detection methods of small amyloid oligomers in vivo has hampered elucidating their contributions to Alzheimer's Disease and related disorders. This projects aims to identify and validate new fluorescent probes for reliable detection of the elusive and transient amyloid oligomers. Such oligomer-selective dyes are highly promising candidates for developing in vivo PET imaging probes for oligomer detection in patients.