On the molecular basis of α-synuclein aggregation on phospholipid membranes in the presence and absence of anle138b

Abstract

A physiologic hallmark of synucleinopathies such as Parkinson’s Disease (PD) or Multiple System Atrophy (MSA) are deposits in patients’ brains, which consist of the α-synuclein. Growing evidence suggests, that oligomeric protein intermediates, formed during the aggregation process, constitute the major toxic species. The 3,5-diphenyl-pyrazole derivative anle138b has been designed and synthesized to interfere with these intermediates and showed significant efficacy in mouse models of Parkinson’s Disease. The success of anle138b in vivo calls for an elucidation of the underlying mechanism in vitro. In this thesis a combination of NMR-, fluorescence- and CD-spectroscopy was used to thoroughly characterize the kinetics of α-synuclein (αS) in the presence of phospholipid membranes under Protein Misfolding Cyclic Amplification (PMCA) conditions. The exact knowledge of the kinetics in return allowed the enrichment of oligomeric αS intermediates. Morphological information on these intermediates was obtained by AFM-microscopy and Transmission Electron Microscopy. Solid-sate NMR revealed that formation of two loop regions in these pre-organized intermediate initiates αS fibril formation. This species then rapidly rearranges with parts of the NAC region adopting the final fibril conformation. The structural changes observed during aggregation are each accompanied by a distinct response to ThT, allowing identification of key time points for the isolation of intermediates. Using DNP-enhanced solid-state-NMR in combination with photoaffinity studies an interaction of membrane-embedded anle138b with αS oligomers and fibrils was characterized. Site specific information was refined by MD-simulations for αS fibrils. In conclusion, the findings in this work add to the understanding of both the mechanism of a αS aggregation and the interaction of aggregates with small molecules.