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

自闭症 FI 和 ACC 细胞和网络中基因表达的 RNA 测序研究

• 批准号: 7844761
• 负责人: JOHN M ALLMAN
• 金额: $ 56.43万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7844761
• 关键词: Affect; Age; Anatomy; Anterior; Area; Autistic Disorder; Autopsy; Awareness; Binding; Brain; Cells; Clinical; Collaborations; Complement; Complex; Conscious; Control Groups; Copy Number Polymorphism; Coupled; Cytokine Receptors; DNA; DNA Methylation; Data; Development; Dissection; Empathy; Epigenetic Process; Family; Formaldehyde; GADD45B; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic; Genotype; Human; Hypothalamic structure; IL4R gene; IL6 gene; Immune; Individual; Inflammation; Interleukin-4; Interleukin-6; Investigation; Laboratories; Lasers; Left; Maps; Measures; Messenger RNA; Microglia; Motivation; National Institute of Child Health and Human Development; Nervous system structure; Neurons; Pattern; Phenotype; Pongidae; Population; Presynaptic Terminals; Protein Isoforms; Provider; RNA Sequences; RNA Splicing; Records; Regulation; Relative (related person); Reporting; Sampling; Self Perception; Signal Transduction; Small RNA; Social Interaction; Source; Specificity; Spottings; Structure; Synapses; Synaptic Membranes; System; Techniques; Testing; Tissue Banking; Tissue Banks; Transcript; Validation; Variant; base; brain tissue; cell type; cingulate cortex; developmental disease; hippocampal pyramidal neuron; interest; member; mind control; new technology; public health relevance; selective expression; sex; social reciprocity; synaptic function; tissue fixing

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.

描述（由申请人提供）：该提案是为了支持约翰·奥尔曼（John Allman）和芭芭拉·沃尔德（Barbara Wold）的实验室之间的合作，以研究激光微删除的细胞群体中的基因表达，该基因表达使用最近开发的Techniques，RNA-SESEQ。 RNA-seq将在SNP和拷贝数变化（CNV）基因分型的背景下进行解释。 FI（额叶岛的皮层）和ACC（前扣带回皮层）在功能上与社会相互作用和互惠，同理心以及身体功能的认识和调节有关。这些功能在自闭症中至关重要。与其他体内稳态系统（包括下丘脑中的系统）不同，FI/ACC系统似乎可以直接访问意识和动机。对FI样品的初始RNA-SEQ的分析产生了两个相干基因网络，它们在自闭症和神经型对照之间有所不同。几个网络成员的免疫染色已经开始显示网络如何映射到蜂窝电路上。具体而言，在细胞水平上，FI和ACC包含大型双极细胞（von经济神经元，Vens），它们是猿类和人类中这些结构的独特特征。我们发现，在正常和自闭症受试者中，Vens的细胞因子白细胞因子4（IL4R）和白介素6（IL6R）的Express受体，RNA-SEQ确定了与炎症相关的突出基因网络，在我们的自闭症病例的子集中，该网络强烈上调（自闭症-A）。该网络以IL6为中心，包括ATF3，SOCS3和GADD45B，它们以Vens为选择性。小胶质细胞是神经系统的免疫细胞，数量众多，并且在自闭症A中处于活化状态，可能是信号源。我们剩余的自闭症病例包括一个自闭症-B组，其特征是包括NRXN1（NEUREXIN 1）（NEUREXIN 1）在突触前末端的表达增加，该基因在后突触后膜中提供了与神经素的结合。 NRXN1是与自闭症最强烈，最始终如一的基因之一。 NRXN1具有许多剪接变体，可以提供突触连接的形成或强度的特异性。更深入地探究这些网络；将基因表达和剪接同工型分配给其适当的细胞；为了发现自闭症的剩余差异，我们建议在激光驱动的细胞上使用2代RNA-Seq。因此，Vens和其他关键细胞类型很少见（FI中的细胞<5％）。这降低了它们的转录组完整性：在自闭症中相对于对照组的基因将受到特别影响。成功的激光捕获可以克服这一障碍。我们还提出了可能被发现的NRXN1同工型和其他复杂家族的深RNA测序。 公共卫生相关性：我们试图通过使用新技术RNA-Seq来了解自闭症的细胞基础，以确定具有良好描述的自闭症与年龄和性别匹配的神经型个体的受试者的尸检大脑中基因表达的差异。我们已经调查了自闭症异常异常的两个特定的皮质区域，它们在自闭症A组中与炎症相关的基因网络中的表达增加增加，而其余的自闭症B组B则在与突触有关的基因网络中的表达增加。我们建议使用激光微分截止性，以研究自闭症A，自闭症-B和对照组的皮质群体中特定神经元和非神经元种群中的基因表达。