Function and Structure Adaptations in Forebrain Development

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。第二，确定MET基因调控和前脑发育改变之间的关系，这可能导致自闭症谱系障碍（ASD）的功能障碍特征。MET及其配体肝细胞生长因子（HGF）与体外神经回路的发育和成熟有关。在体内，HGF-Met信号的间接遗传操作导致小鼠皮质中间神经元发育、间歇性癫痫发作、焦虑增加和社会行为减少的改变。我们的初步数据从条件性删除的Met在新皮层提供了直接的证据，Met信号参与突触的结构和生化成熟。此外，我们发现，在人类MET基因的52个转录调控区的单核苷酸多态性（SNP rs 1858830）与ASD密切相关（P= 5 × 10 -6）。这种变体是功能性的，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，因为我们最近已经表明，与对照相比，患有ASD的受试者的颞叶皮层中MET蛋白表达降低至对照水平的50%。在ASD和基础发育神经生物学中的人类遗传和生物化学研究的融合表明，MET信号传导对于前脑回路的正确组装是重要的，失调导致模型系统和人类的功能中断。在这一更新申请中，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在新皮层发育中的作用，促进MET基因调控的因素，以及ASD相关的人类遗传调控变体对MET相关前脑个体发育的影响。目的1将使用Emx 1Cre有条件地从背侧软腭中删除Met的小鼠来确定直接消除大脑皮层中Met信号传导的影响。这些研究的目标是确定树突和突触结构以及突触信号系统的变化。目的2将确定和实验操作，在细胞系中，转录因子和组装的复合物，调节人类MET基因转录。将在基因的52个调控区中的广泛CpG岛的甲基化研究中检查ASD病例的尸检脑和患者外周细胞中表观遗传机制对MET的调控。在目标3中，将工程化新的“人源化”小鼠系，其含有人52调节序列，所述人52调节序列具有“G”或“C”rs 1858830等位基因和CpG岛。该目标的目的是确定ASD相关的“C”等位基因如何影响MET基因转录和体内模型系统中的脑发育，并确定随时间推移基因表达的表观遗传调节的影响。公共卫生相关性：CDC指出，ASD影响美国每150名儿童中就有1名。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ASD的风险基因。该项目包括基础和临床转化研究，将提供深入了解基因-基因和基因-环境相互作用，可能是ASD非典型脑功能的基础。

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.