DEVELOPMENT OF SCAFFOLD-FREE SPHEROIDS OVEREXPRESSING ALPHA-SYNUCLEIN IN HUMAN NEUROBLASTOMA SH-SY5Y AS A MODEL OF PARKINSON'S DISEASE.

Abstract

<h3>Background</h3> Parkinson‘s disease (PD) is a chronic and progressive neurodegenerative disease, characterized by loss of dopaminergic neurons of the substantia nigra (SN) and abnormal deposits of the alpha-synuclein protein (alpha-syn). Patients with PD present characteristic motor and non-motor symptom. Spheroids came as a promising alternative in the 3D culture because the scaffold-free agarose micromolded technique allows cells to produce extracellular matrix, permitting greater cell-cell interaction, recreating the microenvironment and recapitulating tissue morphogenesis. In this study, we use the SY5Y (SH-WT) cell to develop the spheroids, they express the dopaminergic machinery. As a control we will use α-syn-A53T (SH-A53T) cells, which overexpress the alpha syn. <h3>Aim</h3> Produce the spheroids with the cell lines SH-SY5Y (SH-WT) and mutated alpha-synuclein (SH-A53T), using them as a model of Parkinson's disease. <h3>Methods</h3> Both cells lines were grown in DMEM Medium (Dulbecco‘s Modified Eagle Medium) supplemented with 100U/mL of penicillin, 100 U/ mL of streptomycin and 10% fetal bovine serum (SBF) at 37°C and 5% CO₂ atmosphere. spheroids were produced with 1 × 10⁶ cells seeded on the agarose micromolded, producing spheres with 400 µm approximately. Once a week, pictures were obtained, and the images were used for volume calculation and necrotic center evaluation. Cells viability were analyzed by Trypan Blue. <h3>Results</h3> Spheroids were successfully produced in both groups (SH-A53T and SH-WT). The volume on the first day was 2,9±1 × 10⁷ (SH-WT) and 4,0±1,3 × 10⁷ (SH-A53T). Both groups showed 95% cell viability with no changes over the time. There was significant increase in the volume of the SH-WT spheroids over time, but not in the A53T group. On day 14, SH-WT spheroids were 3X bigger than day 1 (<i>p</i><0,001) and 1,5X bigger than SH-A53T (<i>p</i><0,001). The necrotic center was first observed in SH-A53T group on day 7 and in SH-WT it become visible only on day 14. <h3>Conclusion</h3> Here we show for the first time that cells overexpressing alpha-syn in were able to form spheroids. Using this 3D model, we identified significant changes between SH-A53T and SH-WT spheroids such as: growth pattern, necrotic center formation and volume. These differences can be explained by the aggregation potential caused by the A53T mutation. Our spheroids could be used to study the disease in another perspective opening opportunities for innovations, making a model close to what is found in vivo.