Konsequenzen der Misregulation in der PP2A/mTOR Signalkaskade und der lokalen Proteinsynthese auf Funktionalität und Konnektivität der Neuronen bei Patienten mit Opitz BBB/G Syndrom
Zusammenfassung der Projektergebnisse
In this project we primarily went out to unravel the pathogenesis of Opitz BBB/G syndrome, a neurodevelopmental disorder that is caused by mutations in the X-chromosomal MID1 gene by using cerebral organoids of patients. In order to generate isogenic controls without the CRISPR/Cas9 system that has the potential to induce artificial changes to cells, we used iPSCs from a mother of a patient and heterozygous carrier of a mutation in the MID1 gene. iPSCs were generated and clones were then selected based on the expression of either the mutant or the wildtype allele from the active X- chromosome. With these clones we made the surprising observation of a re-activation of the second MID1 allele from the inactive X-chromosome induced by neuronal differentiation. Re-activation of X-chromosomal genes had not been described before but can be an important step that connects X-inactivation and variable escape of selected genes. In light of these exciting observations, we changed the scope of the project and focused on a detailed analysis of re-activation of X-chromosomal genes from the inactive X-chromosome during neuronal differentiation as a general phenomenon. While random X-chromosome inactivation in female cells of placental mammalians silences one allele of the majority of X-chromosomal genes, a considerable fraction is only incompletely and variably inactivated resulting in a tissue-specific pattern of mono- and biallelic expression. Konstitutive escapees show bi-allelic expression in any tissue and cell tested, while variable escapees are only biallelically expressed in selected tissues and cells. How tissue-specific, variable escape from X-inactivation arises, however, is so far entirely unclear. The clonal human female induced pluripotent stem cells (iPSCs) allowed us to trace the (in)activation status of the two alleles of all X-chromosomal genes carrying expressed SNPs (single nucleotide polymorphism) individually along neural differentiation trajectories in MID1 mutant cells. Expressed SNPs in X-chromosomal genes were used to generate iPSC clones of identical X-inactivation from female controls. Following differentiation trajectories in 2D and 3D cultures, we discovered X-chromosome-wide locus- and lineage-specific dynamic usage of the two X-chromosomal alleles in female cells induced by differentiation. Leveraging brain organoids generated from the iPSC clones expressing mutant MID1 from the active X-chromosome, we furthermore could demonstrate that activation of alleles on the inactive X-chromosome can exert protective effects on the manifestation of disease phenotypes in female neural cells and tissue. Taken together, our data demonstrate that alleles on the inactive X-chromosome can serve as a critical reservoir reactivated during differentiation, thereby enhancing resilience of female neural tissue.
