Effect of Ethanol on Cell Proliferation

描述（由申请人提供）：了解乙醇对神经干细胞的影响对于解开与胎儿酒精谱系障碍（FASD）相关的缺陷的病因结至关重要。FASD是一个引人注目的问题，因为它是发育性精神功能障碍的主要原因，影响所有活产的约2%（事实上，它是美国精神发育迟滞的主要原因），并且它提供了对其他（通常共病）精神健康障碍（例如，注意力缺陷多动障碍和自闭症）。大脑的组成和大小是由神经干细胞的增殖和衍生物的数量和谱系确定的。神经元亚群的数量或平衡不当可能导致精神功能障碍。因此，我们将测试乙醇影响发育中的中枢神经系统中产生的细胞的循环行为和命运的假设。大脑皮层由两种类型的神经元组成：兴奋性投射神经元（PNs）和抑制性局部回路神经元（LCNs）。这些来源于产前两个不同的增殖区域。PN来自端脑背侧，端脑背侧包括两个区：脑室区（VZ）及其衍生物脑室下区（SZ）。大多数皮质LCN产生于端脑腹侧，内侧神经节隆起（MGE）。VZ/SZ和MGE细胞的命运在两步过程中定义。(1)决定细胞是否保留在循环群体中和（2）表型（例如，神经元的类型）被定义。在前期的支持中，我们发现转化生长因子（TGF）21是神经干细胞增殖的关键调节因子。本项目将探讨乙醇对细胞增殖动力学和细胞命运定义的影响。具体目标1和2将使用体内模型确定乙醇对皮质增生区神经干细胞的循环活性（细胞周期动力学和退出）和TGF 2受体表达/活化的影响。在它们的发育过程中，神经干细胞表达同源框基因产物Pax 6和/或Tbr 2。这些蛋白质定义了从VZ中的（Pax 6+）干细胞到（Tbr 2+）中间祖细胞阶段的转变。这种过渡对于表层皮层的发育至关重要（例如，胼胝体突起的起源），这是产前暴露于乙醇的目标。具体目标3将使用两种类型的培养物（保留体内样组织的器官型切片和从VZ/SZ或MGE收获的神经干细胞系）来探索乙醇对TGF 21调节的细胞增殖和命运决定的影响。此外，我们将确定基因的上调和下调和沉默（甲基化）的乙醇和/或TGF 21。在具体目标4中，将移植神经干细胞（同向或异向），以确定乙醇和/或TGF 21对遗传和环境贡献的影响，从而确定循环行为和确定细胞命运。与此同时，新的目标将使用三个互补的模型来深入了解定义细胞增殖和命运的机制以及乙醇对这些关键因素的影响。公共卫生相关性：在美国，胎儿酒精谱系障碍估计影响所有活产婴儿的2%。酒精毒性的一个常见目标是增殖细胞，特别是产生大脑的神经干细胞。本研究将探讨酒精诱导缺陷的机制--（a）增殖神经干细胞命运的变化和（B）它们对增殖关键调节因子转化生长因子的反应的变化。

DESCRIPTION (provided by applicant): Understanding the effects of ethanol on neural stem cells is critical to unraveling the etiological knots of deficits associated with fetal alcohol spectrum disorder (FASD). FASD is a compelling problem because it is a major cause of developmental mental dysfunction affecting ~2% of all live births (indeed, it is the chief cause of mental retardation in the USA), and it provides insights into the etiology of other (often co-morbid) mental health disorders (e.g., attention deficit hyperactivity disorder and autism). The composition and size of the brain are established by the proliferation of neural stem cells and by the numbers and lineage of the derivatives. Improper numbers or balance of neuronal subpopulations can underlie mental dysfunction. Thus, we will test the hypothesis that ethanol affects the cycling behavior and fates of cells generated in the developing central nervous system. Cerebral cortex is composed of two types of neurons: excitatory projection neurons (PNs) and inhibitory local circuit neurons (LCNs). These derive prenatally from two distinct proliferative regions. PNs come from the dorsal telencephalon which comprises two zones: the ventricular zone (VZ) and its derivative, the subventricular zone (SZ). Most cortical LCNs are generated in the ventral telencephalon, in the medial ganglionic eminence (MGE). The fates of VZ/SZ and MGE cells are defined in a two-step process. (1) It is decided whether the cells remain in the cycling population and (2) the phenotype (e.g., the type of neuron) is defined. During the previous period of support, we showed that transforming growth factor (TGF) 21 is a key regulator of neural stem cell proliferation. The present project will explore the effects of ethanol on dynamics of cell proliferation and on the definition of cell fate. Specific Aims 1 and 2 will use an in vivo model to determine the effects of ethanol on the cycling activity (cell cycle kinetics and exit) and on the expression/activation of TGF2 receptors by neural stem cells in the cortical proliferative zones. During their development, neural stem cells express a homeobox gene product(s), Pax6 and/or Tbr2. These proteins define the transition from (Pax6+) stem cells in the VZ to an (Tbr2+) intermediate progenitor cell stage. This transition is critical for the development of superficial cortex (e.g., the origin of callosal projections) which is a target of prenatal exposure to ethanol. Specific Aim 3 will use two types of cultures (organotypic slices that retain in vivo-like organization and lines of neural stem cells harvested from the VZ/SZ or MGE) to explore the effects of ethanol on TGF21-regulated cell proliferation and fate decisions. In addition, we will identify genes that are up- and down-regulated and silenced (methylated) by ethanol and/or TGF21. In Specific Aim 4, neural stem cells will be transplanted (homotopically or heterotopically) to determine the effects of ethanol and/or TGF21 on genetic and environmental contributions to determining cycling behavior and to defining cell fate. In concert, the novel Aims will use three complementary models to gain critical insight into mechanisms defining cell proliferation and fate and the effects of ethanol on these critical factors. PUBLIC HEALTH RELEVANCE: Fetal alcohol spectrum disorder affects an estimated 2% of all live births in the United States. One common target of alcohol toxicity is proliferating cells, particularly neural stem cells that give rise to the brain. The present study will explore a mechanism by which alcohol-induced defects result - from changes (a) in the fates of proliferating neural stem cells and (b) in their response to a key regulator of that proliferation, transforming growth factor.