Investigations on the structure-toxicity relationship of different alpha-synuclein aggregates associated with Parkinson's disease

Parkinson’s disease is characterized by the disruption of motor functions as a consequence of the degeneration of the dopaminergic neurons of the substantia nigra pars compacta. This neuronal degeneration is preceded by the formation of alpha-synuclein aggregates denoted Lewy bodies and neurites. Due to their potential value for the understanding of Parkinson’s disease (PD) and the development of effective treatments, a number of mouse models of PD have been developed. A particular PD model, referred to as the preformed fibrils (PFF) PD mouse model, has been shown to reproduce many features of PD, although a great variability of phenotypes has been reported. Here, we studied how the preparation and storage of different alpha-synuclein conformers can influence this model. We concluded that only freshly prepared short alpha-synuclein fibrils were able to induce a strong phenotype in mice. After this, the difference in toxicity between kinetically trapped oligomers derived from different alpha-synuclein variants was explored. From these experiments it was concluded that WT and G51D oligomers induce a stronger toxicity than other alpha-synuclein mutational variants. It was also possible to determine that different alpha-synuclein fibrillar species are able to recruit endogenous alpha-synuclein at different rates. Finally, it was intended to explore the cellular toxicity of these different fibrillar alpha-synuclein aggregates through the widely used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. While performing these experiments it was observed that, alpha-synuclein fibrils and other unrelated amyloid aggregates were able to induce the formation of formazan crystals that resulted in a false-positive result. The nature of this phenomena, the time scale in which it happens, and which species were able to induce it were explored. It was concluded that amyloid fibrils, but not monomers or kinetically trapped oligomers, were able to induce the formation of these crystals at nanomolar concentrations and in a timescale of hours after the initial exposure. By exploring how structural characteristics of amyloid aggregates influence cell toxicity, I have been able to identify key attributes that can be used in improving in vivo models of Parkinson’s disease, find new secondary structural markers that correlate with cellular toxicity and to provide an insight of how to measure cell viability in a reliably way