Identification of TSC cellular phenotypes using patient-derived iPSCs

 描述（由申请人提供）：脑硬化症（TSC）是一种发育障碍，其特征为多器官肿瘤易感性、脑畸形和神经学表现。尽管在理解疾病的遗传和信号机制方面取得了相当大的进展，但仍然缺乏有效的治疗方法，特别是在控制神经症状方面。在这个提议中，我们计划从患者和未受影响的兄弟姐妹中开发诱导多能干细胞（iPSC）系，并使这些细胞分化以产生神经元培养物，作为一种新的体外系统来鉴定疾病的细胞和分子表型。由于患者在TSC 2基因中携带经鉴定的从头杂合突变，因此我们还将尝试使用TALEN技术纠正iPSC中的遗传TSC突变。有了这些iPSC模型，我们将确定杂合TSC 2神经元是否表现出形态学或突触表型。其次，我们将确定它们是否表现出通常由TSC调节的信号转导复合物的局部改变，如mTORC 1和mTORC 2。我们假设，杂合子TSC 2神经元表达一个微妙的形态表型，结果从局部失调的mTOR含有信号复合物。这种表型可能是TSC患者认知功能障碍和自闭症病因学的关键。TSC是一种相对罕见的疾病，大约每6,000人中就有1人受到影响。然而，它与许多皮质畸形综合征有着共同的机制基础，并且经常与癫痫（>90%），智力残疾和自闭症（40-50%）有关。因此，我们的研究结果与所有这些发育性脑疾病的治疗相关，将我们工作的潜在影响扩展到TSC领域之外。

 DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is a developmental disorder characterized by tumor susceptibility in multiple organs, brain malformations, and neurological manifestations. Despite considerable progress in understanding the genetic and signaling mechanisms underlying the disease, effective treatments are still lacking, particularly with regard to the control of neurological symptoms. In this proposal, we plan to develop induced pluripotent stem cell (iPSC) lines from patient and unaffected siblings, and differentiate these cells to generate neuronal cultures as a novel in vitr system to identify cellular and molecular phenotypes of the disease. Since the patients carry identified, de novo, heterozygous mutations in the TSC2 gene, we will also attempt to correct the genetic TSC mutations in iPSCs using the TALEN technology. With these iPSC models at hand, we will determine whether heterozygous TSC2 neurons exhibit a morphological or synaptic phenotype. Second, we will determine whether they exhibit localized alterations in signal transduction complexes that are normally regulated by the TSC, such as mTORC1 and mTORC2. We hypothesize that heterozygous TSC2 neurons express a subtle morphological phenotype that results from the localized de-regulation of mTOR-containing signaling complexes. This phenotype may be key to the etiology of cognitive dysfunction and autism in TSC patients. TSC is a relatively rare disorder affecting approximately 1 in 6,000 individuals. However, it shares mechanistic underpinnings with a number of cortical malformation syndromes, and it is frequently associated with epilepsy (>90%), intellectual disability and autism (40-50%). Thus, our findings are relevant to the treatment of all these developmental brain disorders, extending the potential impact of our work beyond the field of TSC.