Stem cell derived neurons and inherited epilepsy

描述（由申请人提供）：本申请针对挑战领域14，干细胞和特定挑战主题14- NS-101，逆向工程人类神经系统疾病：从对照和患者群体中产生干细胞。诱导多能干细胞的最新发展为研究一种严重形式的儿童癫痫的细胞发病机制提供了一个令人兴奋的机会，SMEI是由神经元特异性钠通道基因SCN1A突变引起的。SMEI是一种难治性疾病，发病时间为6个月至2岁。我们的实验室在2001年参与了SCN1A突变的初步鉴定。从那时起，在散发性和家族性癫痫中发现了500多个该基因突变。然而，由于神经元特异性表达模式和患者神经元的不可获取性，不可能获得关于这些SCN1A突变对受影响细胞电生理活动和放电模式影响的可靠信息。确定发病机制的特定神经元，以及了解SCN1A突变导致的细胞异常，对于开发有效的治疗方法至关重要。在过去的几个月里，使用iPSC方法，我们已经能够使用从SMEI患者的皮肤活检中制备的成纤维细胞培养物产生含有SCN1A剪接位点突变的神经元。我们已经能够记录双极和锥体神经元的动作电位。使用特定的抗体，我们已经在我们的培养中识别出gaba能和其他类型的神经元。为了继续这项工作，我们已经获得了对照和患者成纤维细胞的集合。我们建议对患者神经元的放电模式和电生理特性进行详细表征，并将其与来自对照个体的诱导神经元进行比较，以确定表达致病性SCN1A突变的神经元的独特特性。我们还将研究发育调节的选择性外显子的神经元特异性剪接模式。我们将研究携带三类SCN1A突变的神经元：剪接位点突变、蛋白质截断突变和氨基酸取代。这些研究可以在2年内完成，并将为开发筛选试验提供基础，以确定能够逆转SCN1A突变在培养细胞中的影响并将突变细胞表型转化为正常对照的药物。在这个为期两年的项目中产生的细胞系和数据将与其他研究人员共享。我们开发的方法将适用于许多其他神经系统疾病。

DESCRIPTION (provided by applicant): This application is directed to Challenge Area 14, Stem cells, and Specific Challenge Topic 14- NS-101, Reverse engineering human neurological disease: generation of stem cells from control and patient populations. The recent development of induced pluripotent stem cells offers an exciting opportunity to examine the cellular pathogenesis of a severe form of childhood epilepsy, SMEI, which is caused by mutations in a neuron-specific sodium channel gene, SCN1A. SMEI is an intractable disorder with onset between 6 months and 2 years of age. Our lab was involved in the initial identification of SCN1A mutations in 2001. Since then more than 500 mutations in this gene have been identified in sporadic and familial epilepsies. However, because of the neuron-specific expression pattern and the inaccessibility of neurons from patients, it has not been possible to obtain reliable information about the effects of these SCN1A mutations on the electrophysiological activity and firing patterns of affected cells. Identification of the specific neurons targeted for pathogenesis, and understanding of the cellular abnormalities that result from SCN1A mutations, are essential for the development of effect treatments. During the past few months, using the iPSC approach, we have been able to generate neurons containing a splice site mutation of SCN1A using fibroblast cultures prepared from a skin biopsy of an SMEI patient. We have been able to record action potentials from bipolar and pyramidal neurons. Using specific antibodies, we have identified GABAergic and other types of neurons in our culture. To continue this work, we have obtained access to a collection of control and patient fibroblasts. We propose to carry out a detailed characterization of firing patterns and electrophysiological properties of patient neurons and compare them with induced neurons from control individuals, to identify the unique properties of neurons expressing pathogenic SCN1A mutations. We will also investigate neuronal-specific splicing patterns of developmentally regulated alternative exons. We will study neurons carrying three classes of SCN1A mutations: splice site mutations, protein truncation mutations, and amino acid substitutions. These studies can be completed within 2 years and will provide the groundwork for developing a screening assay to identify pharmacological agents that can reverse the effects of SCN1A mutations in cultured cells and convert the mutant cellular phenotypes to those of normal controls. Cell lines and data generated during the course of this two year project will be shared with other investigators. The methods we develop will be applicable to many other neurological disorders. PUBLIC HEALTH RELEVANCE: Epilepsy is a common and debilitating neurological disorder, with a frequency of approximately 1 in 1,000 individuals. Many cases are resistant to currently available therapies. The severe genetic disorder SMEI has childhood onset and rapid progression and does not respond to treatment. Until now, it has not been possible to study the effects of SMEI mutations on nerve cells, because brain tissue from patients is not available for study. However, the newly developed technique of deriving neurons from skin biopsies means that SMEI and other neurological disorders can now be studied in molecular detail. We have succeeded in generating neurons from a patient with SMEI. These cells will be analyzed to determine the effects of the underlying mutation. After the molecular features of the abnormality are characterized, it may become possible to use these cultured neurons to screen for drugs which can prevent the progression of this disabling disease.