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

基本信息

批准号：
7939753
负责人：
Ricardo E. Dolmetsch
金额：
$ 24.15万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7939753
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 Fibroblasts Fluorescence-Activated Cell Sorting Gene Expression 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 Pattern 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

项目摘要

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（由申请人提供）：自闭症谱系障碍（ASD）是高度遗传性的复杂大脑神经发育障碍，不能用任何单个基因的突变来解释。在过去的几年中，连锁和关联研究已经确定了几种导致自闭症谱系障碍易感性的突变。研究这些突变的功能效应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 患者身上获取神经元和神经胶质细胞。该项目的目标是开发将 ASD 患者的皮肤细胞转化为神经元的方法，并使用高内涵屏幕来表征这些神经元。为了实现这一目标，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，我们将研究自闭症谱系障碍中可能受到破坏的神经元功能的特定细胞内在方面，包括突触形成、轴突和树突形态以及钙信号传导。我们已经在实验室建立了所有这些技术。在更大规模地应用这些技术之前，我们需要首先解决它们的一些局限性。该提案 R21 阶段的重点是改进和标准化方法。我们将首先从健康对照和患有 CACNA1C 和 SHANK3 基因突变（已知会影响神经元发育的 ASD 患者）的人类成纤维细胞中生成和表征 iPS 细胞，并优化和表征 iPS 细胞的分化（具体目标 1）。我们将开发标准化方案，用于将 iPS 细胞分化为皮质神经元、多巴胺能神经元和抑制性神经元的混合群体（具体目标 2）。然后，我们将表征 ASD 和对照神经元的细胞表型，重点关注钙信号传导、树突分枝和细胞存活（具体目标 3）。在该项目的 R33 阶段，我们将针对大量患有 ASD 的个体，已知这些个体在影响神经元发育的其他基因中存在突变（具体目标 4 和 5）。公众健康相关性：自闭症被认为是最常见的严重发育障碍之一，仅次于智力低下。人均一生增量社会成本估计为 320 万美元。由于自闭症的缺陷会影响人类特定的社会行为，因此需要在人类患者和细胞中研究自闭症的机制。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，并使用高内涵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，并更好地了解神经元的发育和分化。