α-synuclein is a neuronal protein primarily found in the presynaptic terminals. It is thought to have a direct role in the cell to cell spread of the pathology underlying synucleinopathies. This spread leads to a progressive loss of dopaminergic neurons, resulting in loss of motor function and later a decline in cognitive abilities. Therefore studies of how this spread may be halted stands as an important task. Recent studies show antibodies may effectively halt this spread, thereby highlighting immunotherapy as a promising neuroprotective treatment strategy for synucleinopathies. While the efficacy of antibody treatment has been documented from multiple studies the mechanistic understanding is still limited. In this study, a cell model of extracellular α-synuclein fibrils internalization was generated and used to test the effects of α-synuclein specific antibodies. α-synuclein fibrils were generated in vitro by 5 days incubation and following sonicated to fragment the fibrils. These fragments were characterized by a laser light scatter based technique called ‘nanoparticle tracking analysis’, to assess the generated size distribution. The entrance into cells and the later processing of α-synuclein fibrils was analyzed by western blot and confocal microscopy to visualize uptake and co-localization to endosomal, lysosomal and autophagosomal compartments. A cell model of intracellular vesicle permeabilization was generated by virus mediated expression of a Gal3-GFP construct enabling the visualization of membrane permeabilization events. Together with a cell free vesicle rupture assay this could address the hypotheses stating that cytosolic entry of α-synuclein is mediated via uptake and vesicular permeabilization by extracellular α-synuclein. Furthermore it is also hypothesized that these permeabilization events could cause the observed cellular toxicity. α-synuclein fibrils were found to be internalized in a concentration and time dependent manner by pathways requiring dynamin. The cells were capable of clearing the internalized α-synuclein up to a concentration of 10 μg/ml over the course of 72 hours, possibly by involvement of the proteasome, autophagosome and lysosome. After cellular internalization in endosomes, the fibrils showed no signs of gaining cytosolic entry, but might rather follow the endo-lysosomal pathway leading to its degradation. The panel of α-synuclein antibodies analyzed in this study all exhibited inhibition of α-synuclein fibril uptake into the cells. The main effect was indicated to be blockage of entry into the cells due to extracellular complex formation with the antibodies, while a small percentage of the antibodies was taken up by co-localization with α-synuclein inside the cells. The antibodies also showed efficacy in preventing intracellular degradation of the internalized α- synuclein, preventing the formation of hypothesized toxic α-synuclein fragments. Taken together these results indicate the specific antibodies do exert effects on purely neuronal cell cultures.
|Uddannelser||Molekylærbiologi, (Bachelor/kandidatuddannelse) KandidatKemi, (Bachelor/kandidatuddannelse) Kandidat|
|Udgivelsesdato||12 maj 2015|
|Vejledere||Peter Westh, Ann-Louise Bergström & Louise Buur Vesterager|