Validation of a cellular screening assay for α-synuclein propagation related to Parkinson’s disease
Typ
Examensarbete för masterexamen
Program
Publicerad
2022
Författare
Sjögren, Mikaela
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
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.
Beskrivning
Ämne/nyckelord
Primary cell culture , in vitro model , Parkinson’s disease , biophysics , Validation , α-synuclein, amyloid fibrils,