Investigating in vitro Alpha-Synuclein Aggregation of Parkinson's Disease

Abstract

Parkinson’s Disease (PD) stands as the second most prevalent neurodegenerative disorder, associated with ageing. Its hallmark features include two key elements, the formation of Lewy pathology and the progressive loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain. The primary component of Lewy pathology is aggregated α-synuclein (α-syn), particularly involving phosphorylated forms at Serine 129. In familial PD cases, mutations in the SNCA gene encoding α-syn are involved. Despite being recognised as a key neuropathological characteristic, the role of α-syn remains not fully understood. This protein exists in a highly heterogenous array of forms, ranging from fibrils observable under histological examination, to nanoscopic oligomers. There have been a large amount of research into the fibrous forms, however significant gaps remain in our understanding of the aggregation process of α-syn. In this project, a novel phenotypically-relevant PD iPSC model was developed in 70 days, harbouring a SNCA triplication, and compared with its isogenic control and a wild-type control. This was developed to provide a model for the development of α-syn aggregation, indicative of ageing. To obtain this model, a new stress-induction was established, supplement elevated levels of cAMP to the neurons, generating cell death, alterations in cellular signalling, and evidence of the presence of cytoplasmic phosphorylated α-syn. Once this model was established, further investigation into its biological mechanism was explored, by inhibiting a major downstream effector of the cAMP pathway, PKA. It was also found that high cAMP effects could be partially restored, through the addition of the PKA inhibitor. Further analysis into the expression and characterisation of α-syn aggregates were completed, using single-molecule techniques and super-resolution imaging. These techniques were optimised to allow for nanoscopic detection of in vitro α-syn aggregates. It was revealed there was a large heterogeneity in intracellular aggregate sizes, ranging from 20-400 nm in length, with the majority of these aggregates exhibiting a small size, between 20-49 nm long and fibrillar-like in shape. Very little changes were observed between the size and shape of α-syn aggregates between genotype, time and treatment. However, it was interpreted that the PD neurons were more vulnerable to high cAMP treatment, they appeared to die rapidly, releasing their cellular contents into their environment. A hypothesis was proposed that there were only a small proportion of cultures with large number of aggregates, masked by neurons with little α-syn aggregates. Finally, the seeding potential of α-syn aggregates was assessed, showing α-syn aggregates were seeding-competent. However, it was difficult to conclude the seeding potential changes between treatment, time and genotype. Overall a cellular model of PD was developed to which novel single-molecule approaches were applied to detect and characterise the nanoscopic α-syn aggregates formed. This approach has potential to reveal the cellular and molecular mechanisms of a-syn aggregation revealing how PD pathology develops in neurons.