Function and Structure Adaptations in Forebrain Development

前脑发育中的功能和结构适应

• 批准号: 7578564
• 负责人: PAT LEVITT
• 金额: $ 56.88万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7578564
• 关键词: Address; Affect; Alleles; Anxiety; Apical; Architecture; Area; Autopsy; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Blood Cells; Brain; Cell Line; Cells; Cellular Morphology; Centers for Disease Control and Prevention (U.S.); Cerebral cortex; Characteristics; Child; Clinical; Clinical Research; Complex; CpG Islands; Data; Dendrites; Development; Disease; Dorsal; Electrophoretic Mobility Shift Assay; Engineering; Epigenetic Process; Excitatory Synapse; Forebrain Development; Fragile X Syndrome; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Growth; Hepatocyte Growth Factor; Hippocampus (Brain); Human; Human Genetics; Immunoprecipitation; In Situ Hybridization; In Vitro; Individual; Injection of therapeutic agent; Interneurons; Knock-in Mouse; Knockout Mice; Lead; Ligands; MET gene; Methylation; Molecular; Mus; Mutate; Neocortex; Neuraxis; Neurons; Nuclear Extract; Nucleic Acid Regulatory Sequences; Patients; Peripheral; Phosphorylation; Process; Prosencephalon; Pyramidal Cells; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Regulation; Reporting; Research; Research Proposals; Risk; Role; SP1 gene; Seizures; Signal Transduction; Signaling Protein; Single Nucleotide Polymorphism; Small Interfering RNA; Social Behavior; Structure; Synapses; System; Temporal Lobe; Time; Tissues; Transcript; Transcription Initiation Site; Transcriptional Regulation; Translating; United States; Urokinase Plasminogen Activator Receptor; Variant; Western Blotting; autism spectrum disorder; brain tissue; calmodulin-dependent protein kinase II; developmental neurobiology; disorder control; functional disability; gene environment interaction; genetic manipulation; hippocampal pyramidal neuron; in vivo; in vivo Model; insight; lucifer yellow; meetings; mouse model; neocortical; nervous system development; neuron development; overexpression; postnatal; prenatal; pro-hepatocyte growth factor; promoter; protein expression; public health relevance; social; synaptic function; transcription factor; translational study

DESCRIPTION (provided by applicant): There are two related long-term goals of the proposed research. First, to determine the mechanisms through which the receptor tyrosine kinase Met signaling system influences forebrain development. Second, to define the relationship between MET gene regulation and altered forebrain development that may lead to functional impairments characteristic of autism spectrum disorder (ASD). MET and its ligand, hepatocyte growth factor (HGF), have been implicated in the development and maturation of neuronal circuits in vitro. In vivo, indirect genetic manipulation of HGF-Met signaling results in alterations in cortical interneuron development, intermittent seizures, increased anxiety and reduced social behavior in mice. Our preliminary data from the conditional deletion of Met in the neocortex provides direct evidence that Met signaling is involved in the structural and biochemical maturation of synapses. Moreover, we discovered that a single nucleotide polymorphism (SNP rs1858830) in the 52 transcriptional regulatory region of the human MET gene is strongly associated with ASD (P=5X10-6). This variant is functional, as it reduces gene transcription by interfering with transcription factor binding. This has clinical validity, as we have shown recently that MET protein expression is reduced to 50% of control levels in the temporal cortex of subjects with ASD compared to controls. The convergence of the human genetic and biochemical studies in ASD and basic developmental neurobiology suggests that MET signaling is important for the proper assembly of forebrain circuits, with dysregulation leading to functional disruptions in both model systems and in humans. In this renewal application, we propose to take advantage of the convergence of basic and clinical research data, organized around three specific aims to address the role of MET in neocortical development, the factors that contribute to MET gene regulation, and the influence of the ASD-associated human genetic regulatory variant on MET-related forebrain ontogeny. Aim 1 will determine the impact of direct elimination of Met signaling in the cerebral cortex using mice in which Emx1Cre conditionally deletes Met from the dorsal pallium. The goal of these studies is to define changes in dendritic and synaptic architecture, and in synaptic signaling systems. Aim 2 will define and experimentally manipulate, in cell lines, the transcription factors and assembled complex that regulate human MET gene transcription. The regulation of MET by epigenetic mechanisms in ASD cases of postmortem brains and peripheral cells from patients will be examined in methylation studies of the extensive CpG island in the 52 regulatory region of the gene. In Aim 3, new `humanized' mouse lines will be engineered that contain the human 52 regulatory sequence that has either the `G' or `C' rs1858830 allele and the CpG island. The goals of this aim are to determine how the ASD-associated `C' allele influences MET gene transcription and brain development in an in vivo model system, and to define the influence of epigenetic regulation of gene expression over time. PUBLIC HEALTH RELEVANCE: The CDC notes that ASD affects 1 in every 150 children in the United States. The research proposal will directly investigate directly a risk gene for ASD by determining how alterations in gene expression impact brain development. This project includes basic and clinical translational studies that will provide insight into gene- gene and gene-environment interactions that may underlie atypical brain functions in ASD.

描述（由申请人提供）：拟议研究有两个相关的长期目标。首先，确定受体酪氨酸激酶 Met 信号系统影响前脑发育的机制。其次，确定 MET 基因调控与前脑发育改变之间的关系，前脑发育改变可能导致自闭症谱系障碍 (ASD) 的功能障碍。 MET 及其配体肝细胞生长因子 (HGF) 与体外神经元回路的发育和成熟有关。在体内，HGF-Met 信号传导的间接基因操作会导致小鼠皮质中间神经元发育的改变、间歇性癫痫发作、焦虑增加和社交行为减少。我们从新皮质中条件性删除 Met 获得的初步数据提供了直接证据，表明 Met 信号传导参与突触的结构和生化成熟。此外，我们发现人类MET基因52个转录调控区的单核苷酸多态性（SNP rs1858830）与ASD密切相关（P=5X10-6）。这种变体是有功能的，因为它通过干扰转录因子结合来减少基因转录。这具有临床有效性，正如我们最近表明的那样，与对照组相比，自闭症谱系障碍受试者的颞叶皮层中 MET 蛋白表达降低至对照水平的 50%。自闭症谱系障碍 (ASD) 和基本发育神经生物学中的人类遗传和生化研究的融合表明，MET 信号传导对于前脑回路的正确组装非常重要，失调会导致模型系统和人类的功能破坏。在此更新申请中，我们建议利用基础和临床研究数据的融合，围绕三个具体目标组织，以解决 MET 在新皮质发育中的作用、有助于 MET 基因调控的因素，以及 ASD 相关的人类遗传调控变异对 MET 相关前脑个体发育的影响。目标 1 将使用 Emx1Cre 有条件地从背侧大脑皮层删除 Met 的小鼠来确定直接消除大脑皮层中 Met 信号传导的影响。这些研究的目标是定义树突和突触结构以及突触信号系统的变化。目标 2 将在细胞系中定义并通过实验操作调节人类 MET 基因转录的转录因子和组装复合物。在自闭症谱系障碍（ASD）病例的死后大脑和患者外周细胞中，表观遗传机制对 MET 的调节将通过对该基因 52 调节区中广泛的 CpG 岛的甲基化研究进行检查。在目标 3 中，将设计新的“人源化”小鼠品系，其中包含具有“G”或“C”rs1858830 等位基因和 CpG 岛的人类 52 调控序列。该目标的目的是确定 ASD 相关的“C”等位基因如何影响体内模型系统中的 MET 基因转录和大脑发育，并确定随着时间的推移基因表达的表观遗传调控的影响。公共卫生相关性：CDC 指出，美国每 150 名儿童中就有 1 名患有自闭症谱系障碍 (ASD)。该研究计划将通过确定基因表达的改变如何影响大脑发育来直接研究自闭症谱系障碍的风险基因。该项目包括基础和临床转化研究，将深入了解可能构成自闭症谱系障碍非典型大脑功能的基因-基因和基因-环境相互作用。