RNA-Seq studies of Gene Expression in Cells and Networks in FI and ACC in Autism

描述（由申请人提供）：该提案旨在支持John Allman和Barbara Wold实验室之间的合作，以研究使用最近开发的技术RNA-Seq在良好表型自闭症个体与年龄和性别匹配对照的尸检大脑中激光显微解剖细胞群中的基因表达。RNA-Seq将在SNP和拷贝数变异（CNV）基因分型的背景下进行解释。FI（额岛皮质）和ACC（前扣带回皮质）在功能上与社会互动和互惠、同理心以及身体功能的意识和调节有关。这些功能在自闭症中受到至关重要的影响。与包括下丘脑在内的其他稳态系统不同，FI/ACC系统似乎可以直接访问意识和动机。对FI样本的初始RNA-Seq分析产生了两个在自闭症患者和神经典型对照之间不同的连贯基因网络。对几个网络成员的免疫染色已经开始显示网络如何映射到细胞回路上。具体而言，在细胞水平上，FI和ACC包含大的双极细胞（Von Economo神经元，VEN），这是猿和人类这些结构的独特特征。我们发现VENs在正常和自闭症受试者中表达细胞因子白细胞介素4（IL 4 R）和白细胞介素6（IL 6 R）的受体，并且RNA-Seq鉴定了与炎症相关的突出基因网络，该网络在我们的自闭症病例（自闭症-A）的子集中强烈上调。该网络以IL 6为中心，包括ATF 3、SOCS 3和GADD 45 B，它们选择性地在VEN中表达。小胶质细胞是神经系统的免疫细胞，数量众多，在自闭症A中处于激活状态，可能是一个信号源。我们剩下的自闭症病例包括自闭症-B组，其特征在于突触前末端的基因表达增加，包括NRXN 1（neurexin 1），其提供与突触后膜中的神经连接蛋白的Velcro样结合。NRXN 1是与自闭症相关性最强和最一致的基因之一。NRXN 1具有许多剪接变体，这些变体可以在突触连接的形成或强度方面提供特异性。为了更深入地探索这些网络;将基因表达和剪接异构体分配给它们适当的细胞;并发现自闭症患者中剩余的差异，我们提出了激光切割细胞的第2代RNA-seq。因此，VEN和其他关键细胞类型是罕见的（FI中<5%的细胞）。这降低了它们的转录组完整性：相对于对照组，自闭症中表达不足的基因将受到特别的影响。成功的激光捕获可以克服这一障碍。我们还建议对NRXN 1亚型和其他可能被发现的复杂家族进行深度RNA测序。 公共卫生相关性：我们试图了解自闭症的细胞基础，通过使用一种新技术，RNA-Seq，以确定在尸检大脑中的基因表达的差异，与年龄和性别匹配的神经典型个体的自闭症受试者。我们已经研究了两个特定的皮质区域，涉及自我意识和社会互惠，这在自闭症中是异常的，并发现自闭症A组中与炎症相关的基因网络的表达增加，而剩下的病例，自闭症B组，与突触相关的基因网络的表达增加。我们建议使用激光显微解剖研究自闭症A，自闭症B和对照组的感兴趣的皮质区的特定神经元和非神经元群体的基因表达。

DESCRIPTION (provided by applicant): This proposal is to support a collaboration between the laboratories of John Allman and Barbara Wold to investigate gene expression in laser micro-dissected cell populations in autopsy brains of well-phenotyped autistic individuals versus age and sex matched controls using a recently developed techniques, RNA-Seq. RNA-Seq will be interpreted in the context of SNP and copy number variation (CNV) genotyping. FI (Fronto Insular Cortex) and ACC (Anterior Cingulate cortex) are functionally implicated in social interaction and reciprocity, in empathy, and in the awareness and regulation of bodily functions. These functions are crucially affected in autism. Unlike other homeostatic systems, including those in the hypothalamus, the FI/ACC system appears to have direct access to consciousness and motivation. Analysis of initial RNA-Seq on FI samples produced two coherent gene networks that differ between autistics and neurotypical controls. Immunostaining for several network members has begun to show how networks map onto the cellular circuitry. Specifically, at the cell level, FI and ACC contain large bipolar cells (Von Economo neurons, VENs) that are distinctive features of these structures in apes and humans. We found that VENs express receptors for the cytokines interleukin 4 (IL4R) and interleukin 6 (IL6R) in normal and autistic subjects, and RNA-Seq identified a prominent gene network related to inflammation which is strongly up-regulated in a subset of our autistic cases (autism-A). This network is centers on IL6 and includes ATF3, SOCS3, and GADD45B, which are selectively expressed in VENs. Microglia, the immune cells of the nervous system, are numerous and are in the activated state in autism-A, making a likely signal source. Our remaining autistic cases comprise an autism-B group, which is characterized by increased expression of genes in the presynaptic terminal including NRXN1 (neurexin 1), which provides Velcro-like binding to neuroligins in the post-synaptic membrane. NRXN1 is one of the genes most strongly and consistently associated with autism. NRXN1 has many splice variants which could provide specificity in formation or strength of synaptic connection. To probe these networks more deeply; to assign gene expression and splice isoforms to their proper cells; and to discover remaining differences in autistics, we propose generation-2 RNA-seq on laser-dissected cells. Thus VENs and other key cell types are rare (<5% of cells in FI). This reduces their transcriptome completeness: genes under- expressed in autism relative to controls will be especially affected. Successful laser capture can overcomes this hurdle. We also propose deep RNA sequencing for NRXN1 isoforms and other complex families that may be uncovered. PUBLIC HEALTH RELEVANCE: We seek to understand the cellular bases of autism by using a new technology, RNA-Seq, to determine differences in gene expression in autopsy brains of subjects with well described autism versus age and sex matched neurotypical individuals. We have investigated two specific cortical areas involved in self-awareness and social reciprocity which are abnormal in autism and have found increased expression in a network of genes related to inflammation in autism group A, whereas the remaining cases, autism group B, have increased expression in a network of genes related to synapses. We propose to use laser micro-dissection to investigate gene expression in specific neuronal and non-neuronal populations in the cortical areas of interest in the autism-A, autism-B and control groups.