Understanding the mechanism of alpha-synuclein proteotoxicity and development of sensor for detection of alpha-synuclein conformers
Creators: name: Bagree, Gayatri
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310607
subject: Nanobiotechnology
classificationCode: 340108
subject: Sensor technology (incl. chemical aspects)

description: Neurodegenerative disorders like Parkinson’s disease affect a large proportion of the global elderly population. They are characterized by the deposits of aggregated proteins that disrupt cellular protein homeostasis. Pathological deposits of alpha-synuclein (α-syn) protein are found in Parkinson’s and other related disorders, collectively known as synucleinopathies. α-Syn aggregates of various conformations manifest their toxicity through numerous interconnected pathways such as mitochondrial disfunction, protein aggregation, impaired protein degradation, oxidative stress, inflammation, apoptosis and cell death. In addition to its role in disease development, α-syn is the most studied candidate biomarker for synucleinopathies, where the level of total α-syn in the body as well as those of individual conformers have been found to differ in patients of synucleinopathies compared to healthy individuals. 

The aim of this research is to advance the existing knowledge about the mechanism of how α-syn exerts its toxicity on proteins. Further, this work also aims to utilize α-syn as a candidate biomarker for the development of a sensor.  
To address these aims, three primary objectives were identified:
• Understanding the mechanism of α-synuclein mediated proteotoxicity
• Exploring the interaction of aptamer-nanoparticles and aptamer-α-synuclein conformers for sensor development
• Optimization and fabrication of the sensor for detection of α-synuclein conformers.

The aggregation of α-syn gives rise to various protein conformers including monomers, oligomers and fibrils, where each conformer has unique structural and pathogenic characteristics. Studying the effect of α-syn conformers on proteins using firefly luciferase as a model protein provided information about how these conformers affect the native protein folding and its activity (Chapter 2). Of the different conformers, α-syn monomers showed protective and chaperone-like activity, where a delay was observed in the inactivation of luciferase under thermal stress conditions. Additionally, the monomeric conformers enhanced the spontaneous refolding of luciferase. In contrast, the oligomers and fibrils adversely affected luciferase activity and its refolding, where oligomers had a pronounced effect on the spontaneous refolding. This shows that oligomers and fibrils exert their toxicities by interfering with the folding of other proteins. This comparative study reveals the propensities of various protein conformers in causing misfolding and inactivation of other proteins, consequently resulting in altered protein homeostasis.

The second part of the thesis focused on the role of α-syn conformers in disease diagnosis. Here, the concept of a fluorescence based aptasensor was proposed, which used fluorescent labelled α-syn specific aptamers as molecular recognition elements. The working principle of the sensor was based on non-covalent binding and desorption of aptamers from the surface of gold nanoparticles. The start point for the sensor operation was the quenching of fluorescence by gold nanoparticles. Exposing the gold nanoparticle-aptamer conjugates to α-syn resulted in the aptamers desorbing the surface of the gold nanoparticles, which leads to a fluorescence response. This response was unique for the different α-syn conformers. The fundamental understanding of how aptamers bind to gold nanoparticles vs. α-syn conformers provided in depth fundamental understanding of the governing forces during these interactions (Chapter 3). The work further describes the complex interplay of the different interactions and the physical characteristics of the aptamers in sensor operation (Chapter 4). Considering that more than one aptamer was used for the development of the sensor, it showed capability to accurately identify the different conformers.