Studying the molecular consequences of the t(1;11) balanced translocation using iPSCs derived from carriers and within family controls
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Schizophrenia is a major psychiatric disorder that affects 1% of the world population and is among the 10 leading worldwide causes of disability. Disrupted-In- Schizophrenia (DISC1) is one of the most studied risk genes for mental illness and is disrupted by a balanced translocation between chromosomes 1 and 11 that co-segregates with major mental illness in a single large Scottish family. DISC1 is a scaffold protein with numerous interactors and has been shown to hold key roles in neuronal progenitor proliferation, migration, cells signalling and synapse formation and maintenance. The studies herein provide the platform in order to investigate the molecular and cellular consequences of the t(1;11) translocation using induced pluripotent stem cells (iPSCs)-derived neural precursor cells and neurons from within-family carriers and controls. Towards this end, several iPSC lines have been converted into neural progenitor cells (NPCs) and differentiated into physiologically active forebrain neurons following well-characterised protocols. These cells were characterised in terms of basic marker expression at each developmental stage. Inter-line variation was observed in all subsequent experiments but overall t(1;11) lines did not generate less neuronal or less proliferating cells compared to control lines. Furthermore, the expression pattern of genes disrupted by the t(1;11) translocation was investigated by RT-qPCR. DISC1 was reduced by ~50% in the translocation lines, both neural precursors and neurons. This observation corresponds to previous findings in lymphoblastoid cell lines (LBCs) derived from members of the same family. Moreover, DISC1 expression was found to increase as neural precursors differentiation to neurons. Two other genes are disrupted by the t(1;11) translocation;DISC2 and DISC1FP1. Their expression was detectable, but below the threshold of quantification. Similarly, DISC1/DISC1FP1 chimeric transcripts corresponding to such transcripts previously identifies in LBCs from the family were detectable, but not quantifiable. A fourth gene, TSNAX, was also investigated because it is located in close proximity to, and undergoes intergenic splicing with, DISC1. Interestingly, TSNAX was found to be altered in some but not all time points studied, in the translocation carriers compared to control lines. In addition to breakpoint gene expression profiling, iPSC-derived material was used to investigate neuronal differentiation. There seemed to be attenuation in BIII-TUBULIN expression at two weeks post-differentiation, while NESTIN, MAP2 and GFAP expression was similar between translocation carrier and control lines at all time points studied. I also had access to targeted mice designed to mimic the derived chromosome 1 of the t(1;11) balanced translocation. Using RT-qPCR Disc1 expression was found to be 50% lower in heterozygous mice compared to wild types, and I detected a similar profile of chimeric transcript expression as detected in translocation carrier-derived LBCs. These observations support my gene expression studies of the human cells and indicate that the iPSC-derived neural precursors and neurons can be studied in parallel with the genome edited mice to obtain meaningful insights into the mechanism by which the t(1;11) translocation confers substantially elevated risk of major mental illness. In conclusion, the studies described in this thesis provide an experimental platform for investigation of the effects of the t(1;11) translocation upon function and gene and protein expression in material derived from translocation carriers and in brain tissue from a corresponding mouse model.