Cellular Mechanisms in Fetal Alcohol Spectrum Disorders

胎儿酒精谱系疾病的细胞机制

基本信息

批准号：
10061514
负责人：
Scott Parnell
金额：
$ 34.56万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-15 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10061514
关键词：
Address Attenuated Brain Cell Cycle Cell Proliferation Cell physiology Cells Child Cilia Clinical Research Confocal Microscopy Congenital Abnormality Data Defect Development Disease Dorsal Down-Regulation Dysmorphology Ethanol Event Eye Face Fetal Alcohol Exposure Fetal Alcohol Spectrum Disorder Fetus Foundations Functional disorder Future Gene Expression Profiling Genes Genetic Goals Growth Hair Holoprosencephaly Imaging Techniques Immunohistochemistry In Situ Hybridization Intervention Joubert syndrome Lead Literature Malignant Neoplasms Mediating Molecular Molecular and Cellular Biology Morphology Neural tube Orbital separation excessive Organ Organelles Pathogenesis Pathogenicity Pathology Pattern Phenocopy Post-Translational Protein Processing Pregnancy Prevention Prosencephalon Quantitative Reverse Transcriptase PCR Research Role SHH gene Signal Pathway Signal Transduction Sonic Hedgehog Pathway Structure Syndrome Teratogenic effects Testing Therapeutic Studies Time Tubulin Work addiction alcohol effect alcohol exposure alcohol research base brain abnormalities ciliopathy cilium biogenesis design experimental study gastrulation insight morphogens mouse model novel smoothened signaling pathway teratogenesis transcriptome sequencing transcriptomics

Address Attenuated Brain Cell Cycle Cell Proliferation Cell physiology Cells Child Cilia Clinical Research Confocal Microscopy Congenital Abnormality Data Defect Development Disease Dorsal Down-Regulation Dysmorphology Ethanol Event Eye Face Fetal Alcohol Exposure Fetal Alcohol Spectrum Disorder Fetus Foundations Functional disorder Future Gene Expression Profiling Genes Genetic Goals Growth Hair Holoprosencephaly Imaging Techniques Immunohistochemistry In Situ Hybridization Intervention Joubert syndrome Lead Literature Malignant Neoplasms Mediating Molecular Molecular and Cellular Biology Morphology Neural tube Orbital separation excessive Organ Organelles Pathogenesis Pathogenicity Pathology Pattern Phenocopy Post-Translational Protein Processing Pregnancy Prevention Prosencephalon Quantitative Reverse Transcriptase PCR Research Role SHH gene Signal Pathway Signal Transduction Sonic Hedgehog Pathway Structure Syndrome Teratogenic effects Testing Therapeutic Studies Time Tubulin Work addiction alcohol effect alcohol exposure alcohol research base brain abnormalities ciliopathy cilium biogenesis design experimental study gastrulation insight morphogens mouse model novel smoothened signaling pathway teratogenesis transcriptome sequencing transcriptomics

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项目摘要

Many of the structural and functional abnormalities associated with Fetal Alcohol Spectrum Disorders (FASD) have been uncovered, yet major gaps remain in our understanding of the associated pathogenesis and mechanisms. For example, it is well known that ethanol exposure during gastrulation results in the classic hypoteloric FAS face and midline forebrain dysgenesis; yet, exposure just slightly later, during neurulation, induces expanded midline brain structures and hypertelorism. Interestingly, these abnormalities resemble (phenocopy) those of many genetic ciliopathies, such as Joubert syndrome. The central pathogenic mechanism of ciliopathies is a perturbation of the structure and/or function of primary cilia, hair-like organelles found on most cells that integrate extra- and intra-cellular signals. The proposed research tests the overall novel hypothesis that neurulation-stage ethanol exposure induces a “transient ciliopathy” (i.e., a temporary disruption of primary cilia function) that is the basic cellular mechanism for the expansion of midline brain structures and hyperteloric dysmorphologies. The proposed experiments are designed to meet the following integrated specific aims. Aim 1 will define the direct effects of early prenatal ethanol exposure on primary cilia structure and function. For this, confocal microscopy and immunohistochemistry will be used to examine the effects of ethanol on primary cilia number while gene expression assays will be used to assess cilia function. It is hypothesized that ethanol exposure causes abnormal ciliary number and/or function, reducing activation of the Shh signaling pathway. Aim 2 will characterize the secondary cellular pathogenic events in the neural tube resulting from an ethanol-induced transient ciliopathy. The experiments in this aim will test the hypothesis that the ethanol-induced transient ciliopathy and subsequent down-regulation of the Shh pathway will decrease downstream cell proliferation genes in the ventral neural tube and expand morphogen gradients that pattern the dorsal neural tube. Following ethanol exposure, genes with known roles in cell proliferation will be assessed using qRT-PCR and the gradients of ventral and dorsal morphogens will be assessed using in situ hybridization. These data will help to determine the precise mechanisms by which ethanol alters development. Aim 3 is to determine the primary cellular mechanistic events underlying an ethanol-induced transient ciliopathy. This final Aim will use RNA-seq to determine in an unbiased manner how ethanol disrupts normal ciliogenesis by examining the total transcriptomic profile at several time points immediately following ethanol exposure. We hypothesize that ethanol will alter key ciliogenesis genes; however, using this non-biased approach will aid in identifying other potential changes. Finally, we test the alternative/complementary hypothesis that ethanol alters tubulin post-translational modification, thereby disrupting normal cilia stability and function. Together, these novel experiments will provide fundamental insights into the pathogenic mechanisms underlying the effects of ethanol exposure during development, and propel alcohol research into new primary ciliary-related studies.

许多与胎儿酒精谱系障碍相关的结构和功能异常（FASD）已经发现了，但是我们对相关的发病机理的理解仍然存在主要差距机制。例如，众所周知，过口期间的乙醇暴露会导致经典低局部FAS的脸和中线前脑发病障碍；然而，在神经化过程中稍后稍后暴露，引起扩展的中线大脑结构和高血压。有趣的是，这些异常类似（表观）许多遗传性纤毛病，例如乔伯特综合征。中央病原体机制纤毛病是主要纤毛的结构和/或功能的扰动整合细胞外信号的细胞。提出的研究测试了整体新的假设神经化阶段的乙醇暴露会诱导“瞬态纤毛病”（即，原发性暂时破坏纤毛功能）是中线大脑结构和高速膨胀的基本细胞机制畸形。提出的实验旨在满足以下集成的特定目标。目的 1将定义早期产前乙醇暴露对原发性纤毛结构和功能的直接影响。为此，共聚焦显微镜和免疫组织化学将用于检查乙醇对原发性纤毛数，而基因表达分析将用于评估纤毛功能。假设乙醇暴露会导致异常睫状数和/或功能，从而减少SHH信号的激活路径。 AIM 2将表征神经管中的次级细胞致病事件，导致来自乙醇引起的瞬时纤毛病。这个目标的实验将检验以下假设。乙醇引起的瞬时纤毛病和随后的SHH途径下调将减少腹侧神经元管中的下游细胞增殖基因并扩大形态的形态梯度背神经管。暴露乙醇后，将评估在细胞增殖中具有已知作用的基因使用QRT-PCR以及通气和背形梯度的梯度将使用原位杂交评估。这些数据将有助于确定乙醇改变开发的确切机制。目标3是确定乙醇诱导的瞬时纤毛病的主要细胞机械事件。这个最终目标将使用RNA-Seq以公正的方式确定乙醇如何破坏正常的纤毛生成通过检查乙醇暴露后的几个时间点的总转录组轮廓。我们假设乙醇会改变关键的纤毛生成基因。但是，使用这种无偏见的方法将有助于确定其他潜在变化。最后，我们检验乙醇改变的替代/补充假设微管蛋白翻译后修饰，从而破坏了正常的纤毛稳定性和功能。这些小说在一起实验将提供对乙醇影响背后的致病机制的基本见解开发过程中的暴露，并将酒精研究推向新的主要睫状相关研究。