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

dc.contributor.authorSjögren, Mikaela
dc.contributor.departmentChalmers tekniska högskola / Institutionen för biologi och biotekniksv
dc.contributor.examinerEsbjörner Winters, Elin
dc.contributor.supervisorEsbjörner Winters, Elin
dc.contributor.supervisorGhaeidamini, Marziyeh
dc.contributor.supervisorPhil, Johan
dc.date.accessioned2022-06-13T10:57:25Z
dc.date.available2022-06-13T10:57:25Z
dc.date.issued2022sv
dc.date.submitted2020
dc.description.abstractParkinson’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.sv
dc.identifier.urihttps://hdl.handle.net/20.500.12380/304666
dc.language.isoengsv
dc.setspec.uppsokLifeEarthScience
dc.subjectPrimary cell culturesv
dc.subjectin vitro modelsv
dc.subjectParkinson’s diseasesv
dc.subjectbiophysicssv
dc.subjectValidationsv
dc.subjectα-synuclein, amyloid fibrils,sv
dc.titleValidation of a cellular screening assay for α-synuclein propagation related to Parkinson’s diseasesv
dc.type.degreeExamensarbete för masterexamensv
dc.type.uppsokH
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