Cellular and Molecular Neurobiology
Author: Sofia Mucci | Email: sofiamucci27@gmail.com
Sofia Mucci1°, María Soledad Rodríguez Varela2°,Mercedes Vautier4°, Diego García Chialva, Gustavo Emilio Sevlever, María Élida Scassa, Leonardo Romorini
1° INEU (FLENI-CONICET)
Cyclin-dependent kinase 5 (CDK5) plays a vital role in neuronal functions from embryogenesis to postnatal brain modulation. CDK5 is activated through specific binding with the protein P35. In the present study, we generated knockout (KO)-CDK5 human induced pluripotent stem cells (hiPSCs) by CRISPR/Cas9 approach and further differentiated these cells into neurons to evaluate CDK5 relevance in neuronal differentiation and the impact of its absence in the neuronal transcriptome. We differentiated WT and KO-CDK5 FN2.1 hiPSCs into neurons using a defined medium and validated their phenotype through immunostaining with the neuronal markers (TUJ-1, MAP2, MAP5) and then with the glial markers (OLIG2, S100B, GFAP) to confirm the purity of the culture. Then, we performed an RNA-seq analysis (n=3) with RNA isolated from WT and KO-CDK5 hiPSCs-derived neurons. We found 657 differentially expressed genes between WT and KO-CDK5 by using the DESeq2 package in R Studio (alpha=0.01). 4 of these genes (CBLN2, NCAM2, FUT9, NTPX) were validated by RT-qPCR. Interestingly, we observed that several up-regulated genes were synaptic components and regulators of neurite projection and cell adhesion. Remarkably, by Western blot analysis, we found that the lack of CDK5 led to decreased P35 levels and conserved the phosphorylation status of TAU at Thr205. In conclusion, the knockout of CDK5 did not impair hiPSCs neuronal differentiation despite decreasing P35 levels and altering gene expression.