Stem cell derived neurons and inherited epilepsy

干细胞衍生的神经元和遗传性癫痫

• 批准号: 7935492
• 负责人: MIRIAM H MEISLER
• 金额: $ 49.98万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935492
• 关键词: 2 year old; Action Potentials; Adult; Affect; Amino Acid Substitution; Antibodies; Area; Biological Assay; Biopsy; Cell Line; Cells; Cellular biology; Childhood; Collection; Complementary DNA; Cultured Cells; Data; Development; Disease; Disease model; Epilepsy; Exons; Febrile Convulsions; Fibroblasts; Frequencies; Future; Gene Mutation; Generalized Epilepsy; Generations; Genes; Glutamates; Goals; Hereditary Disease; Human; Human Engineering; Huntington Disease; Individual; Inherited; Interneurons; Introns; Kinetics; Lead; Messenger RNA; Methods; Missense Mutation; Modeling; Molecular; Motor Neurons; Mutate; Mutation; Myoclonic Epilepsies; Neonatal; Neurons; Parkinson Disease; Pathogenesis; Patients; Pattern; Phenotype; Preclinical Drug Evaluation; Property; Prosencephalon; Protein Truncation; Pyramidal Cells; RNA; RNA Splicing; Relative (related person); Research Personnel; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Screening procedure; Site; Skin; Sodium Channel; Somatic Cell; Splice-Site Mutation; Stem cells; System; Techniques; Testing; Transcript; Work; brain tissue; disabling disease; embryonic stem cell; gamma-Aminobutyric Acid; hippocampal pyramidal neuron; human embryonic stem cell; induced pluripotent stem cell; infancy; inhibitory neuron; insight; mutant; nervous system disorder; novel strategies; null mutation; patient population; pluripotency; prevent; protein expression; public health relevance; tool; transcription factor; treatment effect; voltage

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.

描述(由申请人提供)：本申请针对挑战领域14，干细胞，以及特定挑战主题14-NS-101，反向工程人类神经疾病：从对照和患者群体中生成干细胞。诱导多能干细胞的最新发展为研究一种严重的儿童癫痫SMEI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，发病时间在6个月到2岁之间。我们的实验室在2001年参与了对SCN1A突变的初步鉴定。从那时起，在散发性和家族性癫痫中已经发现了500多个该基因的突变。然而，由于神经元的特异性表达模式和患者无法接触到的神经元，还不可能获得关于这些SCN1A突变对受影响细胞的电生理活动和放电模式的影响的可靠信息。识别特定的神经元作为发病机制的靶点，并了解由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来生成含有SCN1A剪接点突变的神经元。我们已经能够记录双极神经元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鉴定了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的表征，并将它们与来自对照个体的诱导神经元进行比较，以确定表达致病SCN1A突变的神经元的独特特性。我们还将研究发育调节的替代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替换。这些研究可以在两年内完成，并将为开发筛选试验提供基础，以确定能够逆转培养细胞中SCN1A突变的影响并将突变的细胞表型转化为正常对照的药物。在这个为期两年的项目过程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疾病。 公共卫生相关性：癫痫是一种常见的、使人衰弱的神经疾病，其频率约为每1000人中有1人。许多病例对目前可用的治疗方法具有抵抗力。严重的遗传性疾病SMEI起病于童年，进展迅速，对治疗没有反应。到目前为止，还不可能研究SMEI突变对神经细胞的影响，因为无法获得患者的脑组织进行研究。然而，新开发的从皮肤活检中提取神经元的技术意味着，现在可以对SMEI和其他神经疾病进行分子细节研究。我们已经成功地从一名SMEI患者身上培养出神经元。将对这些细胞进行分析，以确定潜在突变的影响。在表征了这种异常的分子特征后，可能会使用这些培养的神经元来筛选可以防止这种致残性疾病进展的药物。