Study of the relationship between structure and toxicity of different α-synuclein aggregates and related cellular dysfunctions

α-Synucleinopathies are a vast group of neurodegenerative disorders characterized by the abnormal accumulation of insoluble aggregates, both in neurons and in oligodendrocytes, whose major component is the protein α-synuclein (αS). Among them, Parkinson’s disease (PD) is the most widespread; it is defined by the progressive loss of dopaminergic neurons in the substantia nigra, responsible for several motor disturbances, such as bradykinesia, muscular rigidity and resting tremor. This work is focused on αS, whose abnormal self-assembly gives rise to insoluble inclusions called Lewy bodies and neurites, the most relevant neuropathological hallmarks of PD. The aggregation process of αS is extremely complex and leads to the formation of a wide range of assemblies such as oligomers, protofibrils and fibrils. To define the nature of the species responsible for neuronal damage and their mechanism of action, in the first part of this work we have evaluated the evolution in time of different readouts of cellular dysfunction in neuronal cells. We found that, at early incubation times, small oligomeric species with a rudimentary cross-β structure and high solvent exposed hydrophobicity are by far the most toxic to cells, whereas unstructured monomers and hydrophilic and disordered oligomers are unable to cause any cellular dysfunction. We also found that αS fibrils induce the same cascade of events as toxic oligomers, but more slowly and at a rate dependent on their length, despite their inability to be internalized by the cells. Thus, we associated the toxic capacity of αS fibrillar assemblies with their ability to release small hydrophobic oligomers with a cross-β architecture, particularly effective in crossing neuronal membranes and in inducing neurotoxicity. Our results indicate that oligomers are the most toxic among the analyzed αS species, but fibrillar assemblies can generate neurotoxicity through the release of small oligomeric aggregates, that can in turn contribute to the toxicity associated with their well-characterized ability to transfer from neuron-to-neuron, causing the spreading of Lewy body pathology. In the second part of this study we focused on the ability of αS species to interact with neuronal membranes and we analyzed the involvement of the different membrane components, in particular the exposed proteins, on this interaction. Our study revealed that αS oligomers accumulate on the plasma membrane in close proximity to the cellular prion protein, subsequently inducing an increase of intracellular calcium influx in cells by both channel-independent and channel-dependent mechanisms, with the N-methyl-D-aspartate receptor-channels (NMDARs) triggering a prompt and transient calcium influx, followed by a massive calcium dysregulation due to the disruption of the plasma membrane integrity. Accordingly, the pharmacological inhibition of NMDARs, as well as the blockade of the cellular prion protein and the removal of the proteins exposed on the cell membrane transiently delayed the early calcium influx, but not the sustained late one caused by αS oligomers. Furthermore, αS fibrils caused calcium dyshomeostasis with slower kinetics with respect to the oligomers, and the observed ionic alterations were not rescued by the blockade of NMDARs or by the removal of the proteins exposed in neuronal membranes. Thus, the experimental evidences accumulated in the second part of this work shed light into the interplay between αS aggregates and the plasma membrane of neuronal cells, thus expanding the range of molecular targets for the therapeutic intervention in PD. Overall, the data presented in this work provide a robust body of evidence on the prominent role of oligomeric species with high solvent exposed hydrophobicity and cross-β structure, formed either during the aggregation process of αS, or released from mature fibrils, in the neurotoxicity of αS, giving a detailed description of the toxic effects they evoke. The experimental evidences accumulated in this study also emphasize the importance of the membrane binding properties of such species for their pathological features, proposing possible strategies with therapeutic value in PD.