PhD Student University Sao Paulo (USP) São Paulo, Sao Paulo, Brazil
Abstract: Alzheimer's Disease (AD) has been extensively studied, utilizing models like post-mortem brain samples, cell lines, and animal models. However, it is vital to acknowledge the limitations of these models, as they can impact research outcomes and its translational potential. In this regard, the emergence of iPSC has revolutionized AD research by generating patient-specific neurons for more specific investigations into molecular and cellular mechanisms. Differentiating iPSC into cholinergic neurons, crucial for cognition and affected in AD, remains a challenge, and further research is warranted. Given the above, the aim of this study is to optimize and direct an existing differentiation protocol towards the generation of cholinergic neurons, seeking to develop a specialized cellular model for investigating AD and contributing to a more comprehensive understanding of the initial molecular aspects that underlie this disease. To achieve this goal, our model directs the differentiation towards cholinergic neurons by adding neurotrophins BDNF, GDNF, and NT3 (10 ng/ml) during the final 15 days of differentiation. To model AD, we edited iPSC by introducing a PSEN1 mutation in control lines through CRISPR-CAS9 system. This step allowed us to generate isogenic lines harboring the PSEN1 mutation in both heterozygous and homozygous states, which not only complements our cholinergic neuron differentiation protocol but also offers an opportunity to investigate the impact of this mutation on neural development and function. To assess the effectiveness of our approach, the main analysis was performed using Fiji software and consisted of cell counting. Additionally, neurons were characterized through immunofluorescence staining for choline acetyltransferase (ChoAC) and MAP-2 (neuron marker); which showed a 78.2% efficiency in iPSC differentiation into cholinergic neurons. CRISPR-CAS9 was also performed, and clones were validated through Sanger sequencing. Based on these findings, we conclude that these improvements to our cellular model, combining advanced differentiation techniques with precise genome editing, provide a robust platform for studying the early molecular events of AD and exploring potential therapeutic strategies.
