Validation of a cellular screening assay for α-synuclein propagation related to Parkinson’s disease

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Examensarbete för masterexamen
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2022
Författare
Sjögren, Mikaela
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Sammanfattning
Parkinson’s disease (PD) is the second most common neurodegenerative disorder, with an increasing prevalence among the global population. A pathological hallmark of PD is the presence of inclusion bodies called Lewy Bodies (LB) and Lewy Neurites (LN), composed of α-synuclein ( α-syn) amyloid fibrils. To study the disease development and formation of these inclusion bodies, cellular models are implemented. In this project, an in vitro cellular screening model utilizing primary cells was validated for α-syn propagation using pre-formed fibrils (PFFs). Both wild type (WT) and early-onset disease associated mutant A53T α-syn fibrils were included. Fibril batches were sonicated to generate various lengths and biophysical characterization measurements of the α-syn fibrils were performed, including fluorescence spectroscopy with Thioflavin T, circular dichroism (CD) and atomic force microscopy (AFM). The biophysical characteristics were aligned with the biological effects to evaluate pathological factors of the fibrils. An additional assay was included, evaluating the effect on spontaneous activation of the fibril treated neurons through monitoring intracellular calcium levels. In this thesis the functionality of the cellular screening assay was demonstrated. Aggregation of the endogenous α-syn monomers were induced upon seeding with both WT and mutant A53T α-syn fibrils. Furthermore, the seeding capacity of the fibrils was found to depend both on the concentration and the length of the α-syn fibrils, as shown for both WT and A53T α-syn variants. Moreover, A53T α-syn mutant was proven to accelerate the aggregation in comparison to WT, as reported in increased number of phosphorylated α-syn aggregates. Furthermore, the α-syn fibrils were shown to weaken the synaptic signaling in the treated cell cultures, as reported in reduced signal amplitude ratio. These results prove the functionality of the cellular screening assay for evaluation of α-syn aggregation in primary cell cultures, along with underlining the importance of the biophysical characteristics of the α-syn PFFs to induce the α-syn aggregation.
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Primary cell culture, in vitro model, Parkinson’s disease, biophysics, Validation, α-synuclein, amyloid fibrils,
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