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

描述（由申请人提供）：本提案旨在支持John Allman和Barbara Wold实验室之间的合作，利用最近开发的RNA-Seq技术，研究在表型良好的自闭症个体的尸检大脑中激光微解剖细胞群中的基因表达与年龄和性别匹配的对照。RNA-Seq将在SNP和拷贝数变异（CNV）基因分型的背景下进行解释。额岛皮层（FI）和前扣带皮层（ACC）在功能上与社会互动和互惠、共情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严重影响。与包括下丘脑在内的其他体内平衡系统不同，FI/ACC系统似乎与意识和动机有直接联系。对FI样本的初始RNA-Seq分析产生了两个连贯的基因网络，不同于自闭症和神经正常对照。对几个网络成员的免疫染色已经开始显示网络是如何映射到细胞电路上的。具体来说，在细胞水平上，FI和ACC包含大的双极细胞（Von Economo神经元，VENs），这是类人猿和人类这些结构的显著特征。我们发现，在正常和自闭症受试者中，VENs表达白细胞介素4 （IL4R）和白细胞介素6 （IL6R）细胞因子的受体，RNA-Seq发现了一个与炎症相关的突出基因网络，该基因网络在我们的自闭症病例（自闭症- a）中被强烈上调。该网络以IL6为中心，包括在VENs中选择性表达的ATF3、SOCS3和GADD45B。神经系统的免疫细胞小胶质细胞数量众多，在自闭症患者中处于激活状态，这可能是一个信号来源。我们剩下的自闭症病例包括自闭症b组，其特征是突触前末端基因表达增加，包括NRXN1 (neurexin 1)，它在突触后膜上与神经素提供类似尼龙扣的结合。NRXN1是与自闭症密切相关的基因之一。NRXN1具有许多剪接变异体，这些变异体可以提供突触连接形成或强度的特异性。为了更深入地探索这些网络；将基因表达和剪接同种异构体分配到合适的细胞中；为了发现自闭症患者的剩余差异，我们提出对激光解剖细胞进行第2代rna测序。因此，VENs和其他关键细胞类型是罕见的（<5%的细胞在FI）。这降低了他们转录组的完整性：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NRXN1亚型和其他可能发现的复杂家族进行深度RNA测序。

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.