Cellular Mechanisms in Fetal Alcohol Spectrum Disorders

胎儿酒精谱系疾病的细胞机制

• 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
• 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

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 (FASD) 相关的结构和功能异常 已经被发现，但我们对相关发病机制的理解仍然存在重大差距 例如，众所周知，原肠胚形成过程中接触乙醇会导致经典的症状。 近端 FAS 面部和中线前脑发育不全；然而，在神经形成过程中，暴露稍晚一些， 导致中线大脑结构扩大和超距离暗示，这些异常类似于。 （表型）许多遗传性纤毛病，例如朱伯特综合征的主要致病机制。 纤毛病是对初级纤毛结构和/或功能的干扰，初级纤毛是大多数人身上发现的毛发状细胞器。 该研究测试了整合细胞外和细胞内信号的细胞。 神经溶解阶段的乙醇暴露会诱发“短暂性纤毛病”（即，原发性纤毛暂时性破坏） 纤毛功能），这是中线大脑结构和超远距扩张的基本细胞机制 所提出的实验旨在满足以下综合特定目标。 图 1 将定义早期产前乙醇暴露对初级纤毛结构和功能的直接影响。 为此，将使用共聚焦显微镜和免疫组织化学来检查乙醇对 初级纤毛数量，而检测基因表达将用于评估纤毛功能。 乙醇暴露会导致纤毛数量和/或功能异常，从而减少 Shh 信号的激活 目标 2 将描述神经管中继发性细胞致病事件的特征。 来自乙醇引起的短暂性纤毛病的实验将检验以下假设： 乙醇引起的短暂性纤毛病和随后的 Shh 通路下调将减少 腹侧神经管下游细胞增殖基因并扩大形态发生素梯度 乙醇暴露后，将评估在细胞增殖中具有已知作用的基因。 使用qRT-PCR，并使用原位杂交评估腹侧和背侧形态发生素的梯度。 这些数据将有助于确定乙醇改变发育的精确机制。 确定乙醇诱导的短暂性纤毛病的主要细胞机制事件。 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发生 通过在乙醇暴露后立即检查几个时间点的总转录组谱。 促进乙醇会改变关键的纤毛发生基因；然而，使用这种无偏见的方法将有助于 最后，我们测试乙醇改变的替代/补充假设。 微管蛋白翻译后修饰，从而破坏正常纤毛的稳定性和功能。 实验将为乙醇影响的致病机制提供基本见解 发育过程中的暴露，并将酒精研究推动到新的初级纤毛相关研究中。