Identification of TSC cellular phenotypes using patient-derived iPSCs

 描述(申请人提供)：结节性硬化症(TSC)是一种以多器官肿瘤易感性、脑畸形和神经表现为特征的发育障碍。尽管在了解疾病背后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。在这项计划中，我们计划从患者和未受影响的同胞中开发诱导多能干细胞(IPSC)系，并将这些细胞分化为神经元培养物，作为VITR系统中鉴定疾病细胞和分子表型的新方法。由于患者携带已识别的TSC2基因杂合突变，我们还将尝试使用TALEN技术纠正IPSCs中的TSC基因突变。有了这些IPSC模型，我们将确定杂合子TSC2神经元是否表现出形态或突触表型。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体的局部变化，如mTORC1和mTORC2。我们假设杂合的TSC2神经元表达一种微妙的形态表型，这是含有mTOR的信号复合体局部去调节的结果。这种表型可能是TSC患者认知功能障碍和自闭症病因的关键。TSC是一种相对罕见的疾病，大约每6000人中就有一人受到影响。然而，它与许多皮质畸形综合征有共同的机制基础，而且它经常与癫痫(&gt；90%)、智力残疾和自闭症(40%-50%)有关。因此，我们的发现与所有这些发育性大脑疾病的治疗相关，将我们的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.