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  • br Experimental Procedures br Author


    Experimental Procedures
    Author Contributions
    Acknowledgments P.W.B. received funding through the Alborada Trust\'s support of the Alzheimer\'s Research UK Stem Cell Research Centre. J.S. was supported by the Innovative Medicines Initiative Consortium, StemBANCC (grant no, 115439). H.Z. was supported by the Swedish Research Council (grant no: 2013-2546) and the European Research Council (grant no: 681712). F.J.L. is a Wellcome Trust Senior Investigator (grant no. 101052/2/13/2) and gratefully acknowledges the support of the Alborada Trust and Alzheimer\'s Research UK (grant no. ARUK-SCRC 2014-1). Research in the Gurdon Institute benefits from core support from the Wellcome Trust and Cancer Research UK. The authors thank Dr. Steven Moore for provision of technical support and advice.
    Introduction Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by hamartomas in multiple organs with an incidence of approximately 1 in 6,000 at birth (Orlova and Crino, 2010). It is caused by mutations in either TSC1 or TSC2, which encode the proteins hamartin and tuberin, respectively (Kandt et al., 1992; van Slegtenhorst et al., 1997). Hamartin and tuberin bind to each other to form a heterodimer that can inhibit the activation of mammalian target of rapamycin (mTOR), which regulates protein synthesis, cell growth, and proliferation (Long et al., 2005). Despite reports implying that a second-hit mutation is sufficient for the development of TSC, loss of heterozygosity is difficult to demonstrate in brain lesions and was observed in only 4% of the cortical tubers and subependymal nodules assayed in a previous study (Henske et al., 1996). Further investigation is needed to confirm whether TSC1 or TSC2 heterozygosity is sufficient for the development of neurological abnormalities. Patient-specific induced pluripotent stem myd88 pathway (iPSCs), which can model the pathology of a specific disease, represent a promising resource for studying disease mechanisms, screening for novel drug compounds, and developing new therapies (Ebert et al., 2009). The generation of expandable primitive neural stem cells (pNSCs) from iPSCs and their further differentiation into neuron and astrocyte lineages (Yan et al., 2013) enables modeling of human neurological diseases at the cellular level. During the last decade, it was demonstrated that a TSC1 defect in embryonic neural progenitor cells caused CNS malformations similar to those observed in patients with TSC by using TSC1Emx1−Cre conditional knockout animals (Carson et al., 2012). Therefore, the isolation and molecular characterization of TSC-specific pNSCs would provide a powerful model for studying the abnormal neural development that occurs in TSC and the potential underlying mechanisms. mTOR is a serine/threonine protein kinase that forms two distinct multiprotein complexes, mTORC1 and mTORC2, and stimulates protein translation by activating downstream targets, including eIF4E/4E-binding protein and p70S6 kinase/ribosomal S6 protein. mTOR regulates neuronal proliferation, survival, growth, and function, which are critical for development, and deregulation of mTOR at any stage of development can have deleterious consequences. Cell populations expressing hyperactive mTOR show many structural abnormalities that support recurrent circuit formation, including somatic and dendritic hypertrophy, aberrant basal dendrites, and axon-tract enlargement. At the functional level, mTOR hyperactivation is commonly but not always associated with enhanced synaptic transmission and plasticity (Lasarge and Danzer, 2014). The mTORC1 inhibitor everolimus (an analog of rapamycin) is now recommended for the treatment of subependymal giant cell astrocytomas and renal angiomyolipoma in tuberous sclerosis. However, the molecular mechanism underlying the development and pathologies of TSC is not yet fully understood.