Single-molecule studies of alpha-synuclein and abeta in Parkinson’s disease derived biofluids
Alzheimer’s and Parkinson’s disease are the two biggest neurodegenerative disorders, characterised by an irreversible loss of neurons. To date, there are still no authorised treatments that stop or reverse Parkinson’s disease (PD) progression. Age is the most significant environmental factor for the development of sporadic PD. As life expectancy has increased over the last 100 years, so has the number of patients. 3% of the world population over the age of 65 is affected by PD and by 2030, this number is expected to double. The major hallmark of neurodegenerative disorders is deposits of fibrillar protein aggregates in the human brain. However, recent evidence suggests that the small soluble aggregates of alpha-synuclein (αS) and Abeta (Aβ), often referred to as oligomers, are more neurotoxic than mature fibrils. Thus, this work aims to study the structural and biological properties of αS and Aβ aggregates involved in Parkinson’s disease. To understand the protein-specific properties of Parkinson’s disease, we established an immunoassay on a single-molecule level in chapter 4, which can detect αS and Aβ aggregates in vitro and in biofluids. With this, we identified a biomarker in a small cohort study in human blood serum by quantifying the ratio of these two proteins. Using single-molecule total internal reflection fluorescence imaging, super-resolution, and high-resolution techniques, we determined an efficient way of separating a heterogeneous protein mixture by size using a sucrose density gradient centrifugation in chapter 5. We showed that the small aggregates below 200 nm are the toxic species in two biological assays by quantifying the extent of membrane disruption and consequent Ca2+ -influx into single-liposomes and the production of a proinflammatory cytokine (tumour necrosis factor alpha) by microglia-like cells. This result supports the hypothesis that smaller protein aggregates are responsible for neurotoxicity. To gain more insights into Parkinson’s disease, we investigated the properties of aggregates obtained directly from the brain tissue of both diseased and control patients (healthy controls) through a novel soaking protocol in chapter 6. Using a variety of biophysical tools at both the bulk and single-molecule level, we provided answers regarding the structure, composition, and toxicity of human aggregates in Parkinson’s disease.