FUNCTIONAL AND PROTEOMIC ANALYSIS OF ISOLATED EXOSOMES FROM PARKIN-MUTANT FIBROBLASTS

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder characterized by motor symptoms such as resting tremor, muscle rigidity, bradykinesia, and non-motor manifestations like depression, sleep disorders, and dementia. The disease results from the degeneration of dopaminergic neurons in the pars compacta of the substantia nigra and intracellular accumulation of Lewy bodies, filamentous inclusions mainly composed of α-synuclein protein aggregates. Systemic inflammation and mitochondrial dysfunction are key factors in neurodegeneration associated with this disease. In this context, extracellular vesicles (EVs), small membrane-bound structures released by all cells, play a significant role in intercellular communication and regulation of various biological processes. In this project, we focused on the molecular and in vitro analysis of extracellular vesicles isolated from the conditioned medium of fibroblasts derived from skin biopsies. Samples were obtained from fibroblasts of a Control subject (CTRL) and of NHDF (Normal Human Dermal Fibroblasts), both used as Controls, along with fibroblasts isolated from three Patients affected by PD (P1, P2, P3). EVs from Patient and Control samples were isolated using differential centrifugation. Following protein quantification of the EVs, fibroblast cultures from the Control subject were treated with vesicles derived from fibroblasts obtained from PD Patients and the Control subject to gather information about the progression of cell growth and any alterations in cellular and mitochondrial metabolic activity, assessed through cell counting and MTT viability assay; the presence of reactive oxygen species (ROS) and oxidative stress in fibroblasts, evaluated using fluorometric methods. Additionally, EVs’ protein extracts were characterized using proteomic techniques (LC-MS). Analysis of the data revealed 21 differentially expressed proteins between the EVs of Controls and Patients, compared with literature regarding their potential signalling roles. This thesis deepened the understanding of EVs produced by fibroblasts from Controls and Patients in intercellular communication, characterized their protein content, identified and pinpointed protein biomarkers involved in pathways related to PD pathogenesis, with potential diagnostic utility.