Exploring the Neuronal Phenotype of Autism Spectrum Disorders Using Induced Pluri

描述（由申请人提供）：自闭症谱系障碍（ASD）是一种高度遗传性的复杂大脑神经发育障碍，不能通过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种突变的确定，这些突变赋予了自闭症的易感性。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。其中一个主要障碍是很难从自闭症患者身上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。为了实现这一目标，我们将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细胞分化成神经元。最后，我们将研究可能在asd中被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。我们已经在实验室里建立了所有这些技术。在大规模应用这些技术之前，我们需要首先解决它们的一些局限性。提案的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具有CACNA1C和SHANK3基因突变（已知影响神经元发育的突变）的ASD患者身上采集的人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（Specific Aim 1）。我们将制定将iPS细胞分化为皮层神经元、多巴胺能神经元和抑制性神经元混合群体的标准化方案（Specific Aim 2）。然后，我们将描述来自ASD和对照的神经元的细胞表型，重点关注钙信号，树突树突化和细胞存活（Specific Aim 3）。在该项目的R33阶段，我们将针对更多已知具有影响神经元发育的其他基因突变的ASD患者（特定目标4和5）。

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.