DETECTAMYLOID · Detection of membrane-interacting cytotoxic amyloid intermediates with novel fluorescent probes

Formation and deposition of fibrillar protein aggregates (amyloids) is a fundamental feature of neurodegenerative diseases. It has been suggested that neurotoxicity in Parkinson’s disease originates from the binding of α-synuclein (AS) oligomers to cellular membranes, resulting in bilayer disruption and leakage. Membrane binding is also presumed to be involved in the (as yet unknown) physiological function(s) of AS. Thus, it is essential to assess: (i) the diverse conformational states of AS bound to membranes; and (ii) whether in-situ (on-membrane) formation and/or binding of the “toxic” (pre)amyloid-AS leads to specific membrane damage and ultimate neuronal death. Studies addressing these issues have been hampered by the lack of probes able to monitor conformational states, the binding of different protein forms to membranes, and the early stages of aggregation. The aim is to identify and characterize aggregation intermediates of AS having the highest membrane-disruptive ability potentially responsible for toxicity. The focus will be on the binding of monomeric and pre-aggregated forms of AS labeled with a newly developed class of ratiometric Excited State Intramolecular Proton Transfer (ESIPT) probes. These dyes, 3 hydroxychromones, exhibit a dual emission exquisitely sensitive to the molecular microenvironment and will thus discriminate changes in lipid environment and protein states (conformation, association) as a function of (different) lipid composition and conditions. The host lab has already shown that ESIPT probes are very effective in solution studies of AS aggregation. In the project, I intend to monitor AS interactions with synthetic and cellular membranes by expression probes based on ESIPT and other environment sensitive dyes, which I will develop, optimize, and employ for multiparametric fluorescence microscopy and rapid kinetics. The effort should lead to efficient methods for screening compounds antagonizing AS toxicity.