Optimising diagnostic strategies for the early detection of Parkinson’s disease, using seed amplification assays

Abstract

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease, and it is linked with aggregation of α-synuclein protein. PD can be diagnosed using real-time quaking induced conversion (RT-QuIC) assay. RT-QuIC, despite its effectiveness, is not really understood in terms of the mechanisms driving it.
Aim(s): The aim of the project was to reproduce RT-QuIC protocol developed in prof. Piero Parchi’s lab and to understand the effect of different parameters in its efficiency and reproducibility.
Methods: CSF samples from healthy and sick individuals were analysed in RT-QuIC setup with varying conditions.
Results: We reproduced the RT-QuIC protocol and compared the impact of monomer preparation, the protein’s sequence, and shaking on the outcome.
Conclusion:
Our data show that RT-QuIC can be successfully used to diagnose PD and can be reproduced. Additionally, we introduce the half-time of aggregation as an additional criterion that could be used for differentiating samples.

Popular Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide. It impacts not only 6 million patients but also their relatives and society as a whole. Therefore, understanding the molecular basis of PD means a great deal for both patients and taxpayers.
A molecular outcome of Parkinson’s disease is the appearance of fibrillar plaques in the brain made of an amyloid protein – α-synuclein. The middle part of it is folded into a helical structure that is responsible for forming the core of fibrils that then pile into a plaque. Two terminal regions of α-synuclein are intrinsically disordered which means that they don’t fold in any specific structure but rather float freely. One of the ends (C-terminus) creates a fuzzy coat around a fibril and the other (N-terminus) provides a binding site for lipid particles. That function is crucial as α-synuclein can be transported outside the brain by being bound to extracellular vesicles, small lipid bobbles that cross the brain-blood barrier. Thanks to that it has been possible to find α-synuclein in various body fluids such as saliva, blood, cerebrospinal fluid (CSF) but also skin and olfactory mucosa.
Diagnosis of synucleinopathies is still a challenge. Usually, doctors recognize PD based on the clinical symptoms such as tremors, slowness of movement, and rigidity but also non-motor symptoms such as dementia, fatigue, and cognitive and mood disorders. However, once the symptoms have occurred, the disease is already highly developed, medications slowing down the symptoms are less effective and differentiating from different diseases harder. Therefore, a diagnostic tool that could catch PD before its clinical onset would dramatically improve patients’ quality of life as well as it could help develop new therapeutics. So far, few methods have been developed to do so. One of them is positron emission tomography (PET) which allows us to directly image specific disease markers in the brain. Even though it is very powerful it has also its flaws such as high costs, radiation, and low availability. Hence, methods based on biomarkers from body fluids have recently gained the interest of researchers. Blood tests could be the most convenient way to detect PD. However, despite recent very promising findings, they haven’t been so far developed in a way that they could be clinically used. CSF-based tests have dominated so far. One of those tests, real-time quaking-induced conversion (RT-QuIC), is the focus of this thesis. RT-QuIC uses α-synuclein ability to aggregate differently depending on the conditions. We can follow fibrils appearing using a fluorescent dye called thioflavin T (ThT). CSF obtained from patients contains so-called seeds, fibrils that can accelerate aggregation of α-synuclein monomers. As it turns out, monomeric α-synuclein seeded with CSF from sick patients aggregate giving much higher fluorescence signal than when seeded with CSF of a healthy person. The measurement takes place in a shaking fluorescence-detecting reader.
Despite its relative simplicity, a few challenges arise in the RT-QuIC protocol. Mainly, reproducing results across different laboratories. Here, we tried to reproduce RT-QuIC from a pioneering lab in Bologna and to understand the mechanisms driving the method.

• author: Matulewska, Karolina
• supervisor: Sara Linse; Oskar Hansson
• organization: Department of Chemistry
• alternative title: Shake it till you make it
• course: KEMR30 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: RT-QuIC, biomarkers, alpha-synuclein, polymorphism, amyloids, biochemistry
• language: English
• id: 9165576
• date added to LUP: 2024-06-20 14:44:06
• date last changed: 2024-06-20 14:44:06