The functional and pathological pathways of α-Synuclein

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The aberrant aggregation of intrinsically disordered α-Synuclein (αS) is associated with Parkinson’s disease (PD) and other neurodegenerative diseases termed synucleopathies, whereby αS aggregates are a major constituent of Lewy bodies, a hallmark of these diseases. Despite the apparent link between αS and neurodegeneration, the exact physiological function as well as the events initiating pathology are still elusive. Extensive literature evidence suggests that αS is involved in regulating neurotransmission, a function attributed to its metamorphic character enabling the interaction with a number of presynaptic membranes, where the protein localises. Under physiological conditions, αS is present in a dynamic equilibrium between membrane-bound and cytosolic states. Understanding the subtle variations in the membrane binding modes in different biological contexts is crucial to elucidate the physiological behaviour of αS. Therefore, the first half of this PhD aims to characterise molecular interactions and structural conformations of αS upon association with various presynaptic membranes. The results indicate that cholesterol modulates the overall affinity of αS to synaptic-like vesicles (SL-SUV) by reducing the local membrane affinity in the region 65-97. This in return enhances vesicle-vesicle interactions via a previously reported double-anchor mechanism, which has important implication on synaptic vesicle clustering (chapter III). In addition, direct evidence highlights that the docking of SL-SUVs to the surface of the inner leaflet of the plasma membrane (IPM) via the double-anchor mechanism is driven by preferential binding modes of the protein to the different membranes. Further data reveals that changes in the PM lipid composition(s), commonly associated with neurodegenerative contexts, influence the binding affinity specifically of the non-amyloid component (NAC, residues 61-95) region (chapter IV). Overall, these results conclude that lipid compositions moderate the interactions of αS with the amyloidogenic NAC region playing a critical regulatory role in the physiological properties of the protein, ultimately dictating the intricate balance between functional and pathological behaviours of αS. The second part of this PhD investigates the aggregation of αS under quiescent conditions. At low pH, as found in the lysosomal pathway, the cholesterol in SL-SUVs significantly accelerates the aggregation of αS and changes the morphology of the end-product fibrils, whilst preserving the same secondary conformation (Chapter V). The aggregation of αS is also probed at neutral pH in presence of a relevant potassium chloride background illustrating a phosphate dependent fibril formation and stability. These fibrils display characteristics of dissociation in a phosphate-depleted environment. The kinetic mechanism driving the aggregation is proposed to be secondary nucleation which was previously believed to only occur in presence of mildly acidic pH (Chapter VI). The findings reveal new crucial insights into spontaneous αS aggregation and the conversion from its soluble monomer into toxic aggregates linked to neurodegeneration. Taken together, this PhD work significantly improves our understanding of how membrane binding influences properties of αS under physiological and pathological context as well as how disruptions in the fine-tuned equilibrium of αS result in protein dysfunction and aggregation. Comprehending these properties and events will assist in elucidating the role of αS in not only the function but also the aetiology and pathogenicity of PD and other synucleinopathies.

Vendruscolo, Michele
Dobson, Christopher Martin
Parkinson's disease, α-synuclein, Nuclear Magnetic Resonance, Protein aggregation, Thioflavin T
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge