Tuberous Sclerosis Complex (TSC) is a monogenic disorder with increased incidence of seizures, intellectual disability (ID), and autism spectrum disorder (ASD). Although significant progress has been achieved in understanding TSC, the ability to fully comprehend TSC as a neurodevelopmental disorder, and the shared molecular mechanisms that may explain the overlapping phenotypes between TSC and ASD is hampered by the lack of suitable human neuronal cell lines as well as challenges in establishing disease-relevant human isogenic cellular models. The capability to reprogram somatic cells into induced pluripotent stem cells (iPSCs), and the recent advances in genome editing technologies provide a timely opportunity to establish genetically matched sets of human iPSC lines that differ exclusively at the disease-causing genetic alteration. Employing CRISPR/Cas9 genome editing, we have recently generated such isogenic iPSC lines from two unrelated TSC patients, with a defined heterozygous inactivating mutation in TSC1 or TSC2, respectively. Further, we have obtained iPSC lines from three additional unrelated TSC2 individuals, which will serve as a validation cohort ensuring robustness and reproducibility of our data. These iPSC lines have allowed us to derive lineages of neural progenitor cells (NPCs), the cell of origin for the CNS manifestations of TSC, and neural crest cells (NCCs), responsible for the non-CNS aspects of TSC. Initial studies carried out with the genetically matched sets of TSC1-NPCs (Het, Null and Corrected-WT) confirm an increase in cell size and activation of mTORC1 in TSC1 Het and Null cells. We observe distinct activation of ERK1/2 signaling and an increase in MNK-eIF4E after treating NPCs with rapamycin. Interestingly, TSC1 Het and Null NPCs when compared with the matched WT control reveal an increase in NPC proliferation as well as neurite number and length, which are early-stage neurodevelopmental phenotypes linked to ASD. As mTORC1, MEK-ERK a as well s MNK-eIF4E signaling regulate translation, we propose to generate comprehensive transcriptome and translatome profiles in TSC1/2 Het, Null and Corrected-WT NPCs and NCCs, which will define the underlying molecular changes upon TSC1/2 loss. Finally, we will undertake an unbiased high-throughput single and combination drug screen in TSC1/2 isogenic sets of NPCs, in collaboration with NIH-NCATS, to identify potential drugs that exert preferential impact on TSC1/2 Het and Null cells. The top single and combination drugs will be independently validated in the Ramesh lab in multiple TSC patient-derived NPC lines. The effects of compounds that show selective bias toward TSC1/2 Het or Null cells will be further tested in secondary assays that will assess their ability to normalize transcriptome and translatome signatures. The use of patient-specific, iPSC-derived NPCs and NCCs as genetically accurate human cellular models for understanding the disease and for drug screening will provide insights into pathophysiology and novel targets for therapeutic development, thus having a direct impact on TSC research as well as patient care, and ultimately will lead to a better understanding of the shared molecular mechanisms between TSC, ASD, and ID.

Public Health Relevance

We have made tremendous progress in generating induced pluripotent stem cells (known as iPSCs) from skin cells of Tuberous Sclerosis Complex (TSC) patients, and by using the cutting-edge CRISPR/Cas9 genome editing technique, we have established genetically matched cell line sets that express TSC1/TSC2 at 1) corrected ?normal? levels, 2) half the level or 3) totally lack TSC1/2. We have successfully derived neural progenitor cells (NPCs) and neural crest cells (NCCs) from these iPSC sets, the cell types considered responsible for many of the CNS and non-CNS symptoms in TSC patients. The proposed investigation combines studies of molecular and cellular abnormalities in TSC1/2 mutant NPCs and NCCs along with a large-scale drug screen, and these results are likely to further our knowledge of the disorder as well as provide potential novel therapeutic strategies for TSC with high incidence of seizures, intellectual disability and autism spectrum disorder.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Developmental Brain Disorders Study Section (DBD)
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Mamounas, Laura
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Massachusetts General Hospital
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