The development of therapies and diagnostic methods for major neurodegenerative diseases (ND), including Alzheimer’s (AD) and Parkinson’s disease (PD), remains challenging due to our limited understanding of their pathology. Since more and more reports have revealed the pathogenic significance of protein aggregates in ND, this PhD thesis aims to answer 2 key questions in the field: How to characterise these protein species in the complex human samples and How to find the key species associated with these diseases.

To address these questions, this thesis begins by establishing an improved single-molecule pull-down (SiMPull) assay, which was used to characterise soluble α-Synuclein (αS) and Amyloid-β (Aβ) aggregates in serum from patients with PD and controls. Our findings reveal that the ratio of detected aggregates (αS/(Aβ+αS)) and morphological information of αS aggregates can be differentiated between PD samples and controls. We further improved the assay's surface coating using Rain-X, a common household chemical, for simpler and more specific detections. With this improved assay, we explored the diagnostic potential of an inflammasome-related protein, apoptosis-associated speck-like protein containing a CARD (ASC specks) protein, characterised αS and Aβ aggregates in saliva, assessed the binding affinity of rationally designed anti-Islet amyloid polypeptide (IAPP) antibodies, and tested ultra-sensitive single-molecule detection of protein aggregates in human serum. Additionally, for the first time, we demonstrated that small soluble αS can co-aggregate with Aβ and tau in human serum, highlighting that protein aggregation can occur between different protein species in a soluble state, while previous reports mainly focus on larger insoluble aggregates.

In summary, we established a single-molecule approach to characterise protein aggregates in human samples and identified several key protein aggregates in samples from patients with PD. This research platform can be extended to other systems and samples, enabling the acquisition of more detailed single-molecule information on target species. As such, it serves as a fundamental research tool in the study of neurodegenerative diseases.