Exploring G-Protein-Coupled Receptors Regulation, Specificity and Controllability of Exosomes Release in the Neuronal Cell Line SH-SY5Y
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PublisherThe University of Arizona.
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AbstractParkinson's disease is a neurodegenerative disease characterized by the buildup of aggregated and spread of misfolded alpha-synuclein. How the misfolded alpha-synuclein contributing to the toxicity and death of neuronal cells has been the focal point of research. The spread of alpha-synuclein has been attributed to many mechanisms, one of which is via cell-derived vesicles called exosomes. This project aims to examine the controllability of exosome release. SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with dopamine receptor 3-green fluorescent protein, G-protein receptor 143 or green fluorescent protein and treated with either dopamine or L-DOPA. Medium was harvested and subjected to ultracentrifugation and a silver stain and western blot were performed. There was no significant difference in the total protein in the exosome fraction lanes between the treatment groups or within them. Another aim was to test the specificity of exosomes. Exosomes isolated from SH-SY5Y or MCF-7 were labeled with Exo-Red dye and introduced to wells containing SH-SY5Y, MCF-7 and CHO-K1 cells at room temperature and -4C. At room temperature, exosomes were observed intercellular in all of the cell lines, however, they did not deliver their content. At -4C exosome uptake was halted and they remained on the surface of the cells. Exo-Red labeled SH-SY5Y exosomes were treated with proteinase K and were introduced to CHO-K1 cells at -4C and room temperature. CHO-K1 did not take up exosomes, suggesting exosomes contain one or more necessary proteins needed to interact with the cellular membrane to initiate internalization. CHO-K1 cells were treated with versene to examine the involvement of integrin proteins. Exo-Red labeled SH-SY5Y exosomes were trapped on the surface of CHO-K1 after versene treatment. Lastly, Exo-Red labeled SH-SY5Y exosomes were biotinylated and magnetically captured then introduced to SH-SY5Y and MCF-7 cells and a silver stain and a biotinylated blot were performed. MCF-7 bound more Exo-Red labeled SH-SY5Y exosomes.
Degree ProgramGraduate College