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α-synuclein (αS) is an intrinsically disordered protein that is strongly connected with Parkinson’s disease (PD) and a number of other neurodegenerative disorders, including Parkinson’s disease with dementia, dementia with Lewy bodies and multiple system amyotrophy. Fibrillar aggregates of αS have been identified as the major constituents of proteinaceous inclusions known as Lewy bodies that form inside the neurons of patients suffering from these conditions. A number of missense mutations, as well as duplications and triplications of the gene encoding αS have also been associated with familial forms of early onset PD. Despite the association between αS aggregation and neurodegeneration is now established, the specific function of αS is still currently unclear, however, a general consensus is forming on its key role in regulating the process of neurotransmitter release, which is associated with the ability of αS to bind a variety of biological membranes. Indeed, in dopaminergic neurons, αS exists in a tightly regulated equilibrium between water-soluble disordered state and membrane-associated forms that are rich in α-helix. Characterising the nature of this binding as well as the structural and functional properties of αS at the surface of biological membranes is currently a top challenge. In particular the intrinsic limitation of current analytical techniques in studying highly heterogeneous protein states in rapid equilibrium between different physical phases demands for novel approaches to be formulated. This PhD thesis describes major achievements in developing and applying a multidisciplinary approach based on solution and solid-state NMR and extending to a number of other biophysical techniques, including cryo electron microscopy, super resolution microscopy, FRET and cellular biophysics, which enabled us to elucidate in detail the balance between structural order and disorder associated with the membrane interaction of αS in view of its physiological and pathological roles. Using this approach, we identified the key elements that govern the binding of αS to synaptic vesicles (Chapter III). In particular, three regions of αS were shown to possess distinct structural and dynamical properties at the surface of synaptic vesicles, including an N-terminal helical segment having a role of membrane anchor, an unstructured C-terminal region that is weakly associated with the membrane and a central region acting as a sensor of the lipid properties and determining the affinity of αS membrane binding. We refined the structural ensemble of the N-terminal membrane anchor at the surface of synaptic membranes, showing that the partial insertion of this region in the membrane core promotes strong but reversible binding with biological membranes in such a way to enable a fast equilibrium between membrane-bound and cytosolic forms of the protein (Chapter III). Further studies of two mutational variants of αS that are associated to early onset PD, namely A30P and E46K, revealed that two key regions of the protein, namely the N-terminal membrane-anchor (residues 1 to 25) and the central segment of the sequence (residues 65–97, having significant overlap with the non-amyloid β component - NAC - region), have independent membrane-binding properties and therefore are not only able to interact with a single SV, but can also simultaneously bind to two different vesicles thereby promoting their clustering (Chapter IV). The resulting “double-anchor” mechanism explains the biological property of αS to promote clusters of synaptic vesicles within the processes of formation of distal pools to the active zone. The double-anchor mechanism reconciles literature data showing that the deletion of the segment 71–82 in the NAC region of αS or the impairment of the membrane affinity of the N-terminal anchor region of the protein severely affect vesicle clustering in vivo. Thus our data revealed that the NAC region is not only involved in the aggregation of αS, as extensive literature evidence has previously indicated, but also has a specific role in a key molecular mechanism associated with the normal function of αS. The structural characterisation also showed that the active conformations of αS to initiate the double-anchor mechanism are particularly vulnerable to self-association leading to αS aggregation at membrane surfaces, thereby providing a new mechanistic link between functional and pathological roles of αS. In addition to studying the physiological membrane interactions by αS, we characterised the fundamental mechanism of membrane disruption by αS oligomers resulting in the generation of neuronal toxicity in PD (Chapter V). Indeed, while fibrillar aggregates of αS represent the major histopathological hallmarks of PD, small oligomeric assemblies of this protein are believed to play a crucial role in neuronal impairment. We obtained a detailed structural characterisation of toxic αS oligomers and compared these results to the study of non-toxic oligomeric species. The results reveal the fundamental structural characteristics driving the toxicity of αS oligomers, including a highly lipophilic element that promotes strong interactions with biological membranes and a structured region that inserts into lipid bilayers and disrupts their integrity. We obtained additional support for these conclusions by showing that mutations targeting the region of αS promoting such interactions with the membrane dramatically suppress the toxicity of αS aggregates in neuroblastoma cells and primary cortical neurons. Taken together our studies enabled the characterisation of a series of structural properties of the membrane-bound states of αS in both its monomeric and oligomeric forms. The results revealed the nature of the fine balance between functional and pathological membrane interactions of αS and delineated how subtle perturbations of this equilibrium can lead to the rapid evolution of processes that trigger pathological mechanisms. Understanding this balance is a top challenge for advancing the research in PD and requires innovation across different disciplines to overcome current limitations in probing the conformational transitions of this disordered and metamorphic neuronal protein. |
