Uncovering primary cilium defects in Joubert Syndrome using iPSCs derived-Neural Stem Cells

Background: Joubert Syndrome is a rare neurodevelopmental disease belonging to the group of ciliopathies, characterized by defects on the cerebellar vermis and malformation of the brain stem. To date, all mutations responsible for the onset of the pathology cause defects in the primary cilium structure or function. This organelle participates in several processes, and its function is also fundamental for many pathways involved with neurogenesis. However, very little is known about the role of primary cilium in the onset of Joubert Syndrome, and how its aberrant mechanism determines cerebellar malformations. Aim: To uncover the connection between primary cilium and Joubert syndrome, we propose to use patient-specific iPSCs and differentiate them in Neural Stem Cells. Thus, we will obtain a cellular model for the JS useful for characterizing the primary cilium and how it influences neural differentiation. Results: We collected biopsies from two JS patients carrying different mutations in the AHI1 gene. First, primary fibroblast cultures were used as a model for the primary cilium characterization. Immunofluorescence analysis revealed a decrease in the number of ciliated cells with increased axoneme length. Both cell lines were reprogrammed into iPSCs, and differentiated in Neural Stem Cells. Here, we grew cells in a starving condition to promote PC ciliogenesis. However, most JS NSCs didn’t present the cilium when analyzed with immunofluorescence. To further investigate this phenotype we also differentiated cells in the three neural populations and confirmed alterations in the percentage of ciliated cells. Conclusion: The reduction and delay of ciliogenesis in Neural Stem Cells and Neuronal/glial precursors may highlight a key role of the primary cilium during the first stages of neurogenesis, by impacting the timing of cell proliferation and cell differentiation in Joubert Syndrome patients.