Targeting α-synuclein oligomers: The importance of characterisation

Parkinson’s Disease (PD) is a complex disease, characterised by degeneration of neocortical, limbic and nigrostriatal neurons. It is unknown what initiates neurodegeneration, but soluble oligomers of the protein α-synuclein (αSn) seem to be particularly toxic, compared to insoluble fibrils. Although there is currently no cure for PD, αSn oligomers (αSOs) are a potential therapeutic target, but a major drawback it that little is known about the nature of PD-associated αSOs. The scientific literature describes a wide variety of protocols to generate αSOs in vitro, with a subsequent heterogeneity in reported structure and bioactivities, making it difficult to predict which species is/are most relevant. The aim of this thesis is to improve and expand current knowledge on αSOs and their role in PD, with a focus on αSOs modified by the polyunsaturated fatty acid docosahexaenoic acid (DHA), and the reactive aldehyde 4-hydroxynonenal (HNE), on which current literature is limited. We performed extended characterisation using techniques aimed at resolving morphological, structural, chemical, dynamical and functional properties. We showed that, despite their clear chemical differences, DHA- and HNE-αSOs share a remarkable amount of resemblance with unmodified αSOs: they were spherical, had an anti-parallel β-sheet character, were most protected from hydrogen/deuterium exchange in the core region of αSn, and were able to permeabilize small synthetic vesicles. Nonetheless, both species also had their own unique characteristics, e.g. they were recognized by different conformational antibodies and DHA–αSOs also formed a second elongated species in addition to the dominant spherical species. Although further functional testing is needed, this suggests that each species has its own distinct toxic mechanism, in addition to what may be a shared common contribution to pathology. Finally we showed that both species co-localised with glutamatergic synapses of hippocampal neurons upon addition to the cell culture medium, and we identified six proteins which might explain this synaptic binding of DHA-αSOs: VAMP-2B, Na+/K+ ATPase, V-type ATPase, VDAC, CaMKII and Rab-3A. The identification of these targets is a first step towards unravelling the toxic pathways which are activated upon synaptic binding of extracellularly added αSOs, and hopefully will contribute to the discovery of new disease modifying compounds, which can block the toxic interaction between the αSOs and their binding partner.

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