DIFFERENTIATION OF N2A CELLS: COMPARISON BETWEEN HDAC INHIBITORS AND DIFFERENTIATING AGENTS

BERNARDI A; JUNGE S; VILLALBA S; URBANO FJ; BISAGNO V

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAB AAFE AACYTAL 2023; 2023

Sociedades SAIC SAB AAFE AACYTAL

Differentiation is a crucial process upon cell development. Epigenetic mechanisms play an important role in it and HDACs, a family of enzymes whose major function is to acetylate histones and, therefore, regulate gene expression is one the main character in cell differentiation. The aim of this work was to compare differentiating agents in the N2a cells (mouse neural crest-derived cell line) where its neuronal stem cell could differentiate into neurons. Cells were treated with differentiating drugs such as: dbcAMP (cAMP analogue), resveratrol (polyphenol) and two selective HDACi (HDAC inhibitors) MS-275 (class I) and MC-1568 (class IIa). N2a were incubated with DMEM high glucose, 1% Glutamax and 0,5% SFB at 37°C and 5% CO2. For 4 days cells were observed and counted if necessary. Finally, cells were characterized by phase-contrast or fluorescence microscopy. We first determined cell viability at day in vitro 4 (DIV4) and noticed that MS275 500 nM significantly reduced the total number of viable cells (p= 0,021) while other conditions did not affect viability. After that, we analyzed the number of differentiated cells in each condition: 24% Resveratrol 6.2 μM vs 9% ethanol, 27% dbcAMP 0.5 mM vs 3% water and 20% MS-275 50 nM vs 14% DMSO. Finally, we observed morphological changes in N2a cells comparing presence or absence of typical neuron structures such as dendrites, axons and filopodia. Here we found that HDACi induced differential transformation in cell morphology. We saw that MS-275 increased the number of axons (p=0.037) and dendrites (p=0.013) while MC-1568 showed an increase of filopodia (p=0.08) and dendrites (p=0.02). These results show differentiating agents which can convert neuronal stem cells into mature neurons as we observed. Although further work is still needed, we can assume that class I HDAcs are necessary to maintain a tumor-like conformation and, even more that HDACi stop tumoral growth and lead to N2a neuron-like differentiation.