A Novel cell culture model from olfactory neuroepithelium for investigating α-Synuclein aggregation in patients with Parkinson’s Disease

This thesis explores the olfactory epithelium as a valuable source for studying neurodegenerative processes in health and disease, with particular emphasis on its diagnostic potential for neurodegenerative disorders. The work focuses specifically on Parkinson's disease, the second most common neurodegenerative condition worldwide. Recent projections estimate that by 2050, over 25 million people globally will be affected by Parkinson's, a number equivalent to almost half of the entire Italian population. Given this growing burden, understanding and improving early diagnostic approaches is critically important. In the first part of this thesis, we established a protocol for isolating and co-culturing different cell populations from the olfactory epithelium using nasal swabs from healthy controls. We characterized these cells over time, analysing their morphology and dynamics, and identified sustentacular cells, neurons, and respiratory epithelial cells using Pan-CK, PGP9.5, and βIV-tubulin markers. We then examined these cultures for the presence and localization of α-synuclein, the pathological protein central to the development of Parkinson's disease. As expected from previous literature, we have detected both the total and the phosphorylated form of α-synuclein in the cytoplasm of neuronal and sustentacular cells, but with two distinct patterns. The phosphorylated alpha-synuclein in neurons is widespread distributed along axons, in the soma, and in the nucleus, whereas in sustentacular cells, it is mainly localized at the perinuclear level. This work also establishes a baseline alpha-synuclein signal in healthy individuals, paving the way for subsequent studies in pathological subjects. Altogether, these results demonstrate that cell cultures derived from nasal swabs replicate in vivo conditions, constituting a practical and accessible in vitro model for studying the cellular mechanisms underlying neurodegenerative diseases. The second part of the present thesis pursued diagnostic objectives. We applied the Seeding Amplification Assay for α-synuclein to olfactory epithelium samples from 192 subjects, including patients at various stages of Parkinson's disease and healthy controls. Samples were collected by otolaryngologists and trained non-specialist medical staff in two different recruitment rounds. Initial testing with the standard protocol showed suboptimal sensitivity, particularly in early-stage patients, on contrary results were better in advanced disease. After careful optimization of both pre-analytical and analytical steps, we achieved significant improvements. The sensitivity reached 93.3% in Parkinson's patients, 87.5% in Parkinson's with dementia, and 50% in the prodromal cohort, with a specificity of 92.3%. Notably, the diagnostic accuracy of samples collected by other medical staff was not significantly different, with respect to those collected by specialized otolaryngologists, demonstrating that minimal training is sufficient for reliable performance of the nasal swab sampling. These results highlight several key advantages of olfactory epithelium sampling by nasal swab: it is non-invasive, can be performed in non-specialized settings, and detects α-synuclein aggregates with high diagnostic accuracy across different disease stages. This makes the approach particularly promising for large-scale screening, early diagnosis, and monitoring disease progression in patients with Parkinson's disease.