Exploring the Neuronal Phenotype of Autism Spectrum Disorders Using Induced Pluri

使用诱导 Pluri 探索自闭症谱系障碍的神经元表型

• 批准号: 8321078
• 负责人: Ricardo E. Dolmetsch
• 金额: $ 36.65万
• 依托单位: STANFORD UNIVERSITY
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321078
• 关键词: 16p11.2; 22q13.3; Address; Affect; Apoptosis; Autistic Disorder; Behavior; Behavioral; Biochemical; Biological; Biological Assay; Biopsy; Brain; Calcium; Calcium Channel; Calcium Signaling; Cell Line; Cell Survival; Cells; Chemicals; Child; Chromosome Deletion; Chromosomes; Clinical Data; Cluster Analysis; Complex; Copy Number Polymorphism; Data; Defect; Development; Developmental Disabilities; Diagnosis; Disease; Electric Stimulation; Excitatory Synapse; Fibroblasts; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Expression Profile; Gene Mutation; Generations; Genes; Genetic; Genetic Polymorphism; Goals; Harvest; Human; Image; Impairment; In Vitro; Individual; Inhibitory Synapse; Laboratories; Language; Lead; Measures; Mental Retardation; Methodology; Methods; Methylation; Microarray Analysis; Microscopy; Modeling; Morphology; Mutation; National Institute of Mental Health; Neurobiology; Neurodevelopmental Disorder; Neuroglia; Neuronal Differentiation; Neurons; Oligonucleotide Microarrays; Outcome; Patients; Phase; Phenotype; Phospho-Specific Antibodies; Plasmids; Play; Population; Predisposition; Property; Protocols documentation; Recruitment Activity; Reproducibility; Research Personnel; Resources; Retroviridae; Role; Signal Pathway; Signal Transduction; Skin; Social Behavior; Social Interaction; Somatic Cell; Speed; Staging; Staining method; Stains; Stem cells; Stereotyping; Syndrome; Techniques; Teratoma; Timothy syndrome; Transfection; assay development; autism spectrum disorder; cellular imaging; cost; disorder control; dopaminergic neuron; human embryonic stem cell; improved; in vitro Assay; induced pluripotent stem cell; inhibitory neuron; mRNA Expression; method development; mouse model; neuron development; neuronal excitability; novel; progenitor; promoter; public health relevance; repository; research study; stem; synaptogenesis

DESCRIPTION (provided by applicant): Autism spectrum disorders (ASD) are highly heritable complex neurodevelopmental disorders of the brain, which cannot be explained by mutation or mutations in any single gene. In the last couple of years linkage and association studies have led to the identification of several mutations that confer susceptibility to ASDs. Studying the functional effects of these mutations offers a unique window to a better understanding of the underlying neurobiology. One of the major obstacles is the difficulty in obtaining neurons and glial cells from patients with an ASD. The goal of this project is to develop the methods to convert skin cells from patients with ASDs into neurons and to characterize these neurons using high content screens. To achieve this goal we will convert fibroblasts into pluripotent progenitor (iPS) cells. In the next step we will differentiate these iPS cells into neurons in vitro. Finally we will study the specific cell- intrinsic aspects of neuronal function that are likely to be disrupted in ASDs including synapse formation, axonal and dendritic morphology and calcium signaling. We have already established all of these techniques in our laboratory. Before we can apply these techniques on a larger scale we need to first address some of their limitations. The focus of R21 phase of the proposal is to improve and standardize the methodology. We will first generate and characterize iPS cells from human fibroblasts harvested from healthy controls and ASD patients with mutations in the CACNA1C and SHANK3 gene, mutations known to affect neuronal development, and optimize and characterize the differentiation of iPS cells (Specific Aim 1). We will develop standardized protocols for differentiating iPS cells into mixed populations of cortical, dopaminergic, and inhibitory neurons (Specific Aim 2). We will then characterize the cellular phenotypes of neurons from ASD and from controls, focusing on calcium signaling, dendritic arborization, and cell survival (Specific Aim 3). In the R33 phase of the project we will target a larger number of individuals with ASD a known to have a mutation in others gene/s affecting neuronal development (Specific Aim 4 and 5). PUBLIC HEALTH RELEVANCE: Autism is considered to be among the most common of the serious developmental disabilities, second only to mental retardation. The lifetime per capita incremental societal cost is estimated at $3.2 million. Because the deficits in autism affect human- specific social behaviors, the mechanisms underlying autism will need to be studied in human patients and in cells. The goal of this project is to develop the methods to convert skin cells from patients with autism into neurons and to characterize these neurons using high content screens. These experiments will allow researchers to study the neurons of individuals diagnosed with autism and will lead to a better understanding of the development and differentiation of neurons.

描述(申请人提供)：自闭症谱系障碍(ASD)是高度遗传性的复杂的大脑神经发育障碍，不能用任何单一基因的突变或突变来解释。在过去的几年里，连锁和关联研究导致了几个使人对自闭症易感性的突变的鉴定。研究这些突变的功能影响为更好地理解潜在的神经生物学提供了一个独特的窗口。主要障碍之一是难以从ASD患者那里获得神经元和神经胶质细胞。该项目的目标是开发将自闭症患者的皮肤细胞转化为神经元的方法，并使用高含量筛选来表征这些神经元。为了实现这个目标，我们将把成纤维细胞转化为多能祖细胞(IPS)。下一步，我们将在体外将这些iPS细胞分化为神经元。最后，我们将研究ASDS中可能被破坏的神经元功能的特定细胞固有方面，包括突触形成、轴突和树突形态以及钙信号。我们已经在实验室里建立了所有这些技术。在我们能够在更大范围内应用这些技术之前，我们需要首先解决它们的一些限制。R21阶段提案的重点是改进和规范方法。我们将首先从健康对照组和ASD患者的人成纤维细胞中提取CACNA1C和SHANK3基因突变(已知影响神经元发育的突变)来培养和鉴定iPS细胞，并优化和鉴定iPS细胞的分化(特定目标1)。我们将制定标准化的方案，将iPS细胞分化为皮质、多巴胺能和抑制性神经元的混合群体(特定目标2)。然后，我们将描述ASD和对照组神经元的细胞表型，重点是钙信号、树突分枝和细胞存活(特定目标3)。在项目的R33阶段，我们将针对更多患有自闭症的人，已知其他基因/S有突变影响神经元发育(具体目标4和5)。 公共卫生相关性：自闭症被认为是最常见的严重发育障碍之一，仅次于精神发育迟滞。终身人均增加的社会成本估计为320万美元。由于自闭症的缺陷影响人类特有的社会行为，自闭症的机制需要在人类患者和细胞中进行研究。该项目的目标是开发将自闭症患者的皮肤细胞转化为神经元的方法，并使用高含量屏幕来表征这些神经元。这些实验将使研究人员能够研究被诊断为自闭症患者的神经元，并将有助于更好地了解神经元的发育和分化。