Insights into the function and dysfunction of α-synuclein in cells

This thesis sheds light on the function and dysfunction of the protein α-synuclein (α-S) in the test tube and in cells and ultimately its possible involvement in Parkinson’s disease (PD). Following the introduction in Chapter 1, Chapters 2 and 3 concentrate on the investigation of the interaction between the proteins α-S and actin. In Chapter 2 we determine their dissociation constants with microscale thermophoresis and reveal that α-S has a high affinity for G-actin. The determined interaction strength was even comparable to other G-actin binding proteins. Despite this high binding affinity and the postulated function of α-S as a G-actin regulating protein, the binding of α-S to G-actin has no effect on the actin polymerization kinetics. Instead, α-S seems to bind to and stabilize F-actin structures. Chapter 3 reports on how the formation of G-actin/αS complexes attenuates the lag time of α-S aggregation depending on the actin concentration. Whereas low actin concentrations inhibit the onset of α-S aggregation this effect disappears at higher concentrations. However, although the presence of actin can inhibit the onset of aggregation it cannot prevent it. Chapter 4 draws attention to the intra-cellular occurrence of α-S spots, smaller than the diffraction limit, consisting of many proteins. To study and visualize these diffraction-limited α-S spots, we made use of stimulated emission depletion (STED) microscopy. The colocalization of these α-S clusters with a membrane marker and the appearance of α-S on the surface of giant intracellular vesicles suggest that that the spots are small vesicles and α-S functions as membrane binding protein. Chapter 5 focuses on cell models in which α-S inclusion formation is induced in cells. We describe the resemblance of induced α-S inclusion to LBs. Moreover, we not only observe different morphologies, but also observe that α-S aggregates even occur in functionally different inclusions both in vitro and in vivo. Finally, in chapter 6, we investigate the effect of exogenous α-S on the viability and electrophysiology of neuronal networks. Whereas the viability was unaffected, we noted development of α-S aggregates in cells and a progressive decrease in neuronal connectivity and excitability.