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，因为它是影响~2%的活产儿发育精神障碍的主要原因(实际上，它是美国智力低下的主要原因)，它为其他(通常是并存的)精神健康障碍(例如，注意力缺陷多动障碍和自闭症)的病因提供了洞察力。大脑的组成和大小是由神经干细胞的增殖以及衍生细胞的数量和谱系决定的。不适当的神经元亚群数量或平衡可能导致精神功能障碍。因此，我们将验证乙醇影响发育中的中枢神经系统产生的细胞的循环行为和命运的假设。大脑皮质由两类神经元组成：兴奋性投射神经元(PNS)和抑制性局部环路神经元(LCNs)。这些胎盘起源于两个不同的增殖区。三叉神经节来自端脑背侧，它包括两个区：脑室区(VZ)及其衍生的脑室下区(SZ)。大多数皮质LCNs产生于端脑腹侧，位于内侧神经节隆起(MGE)。VZ/SZ和MGe电池的命运在两步过程中确定。(1)确定细胞是否留在循环种群中，以及(2)定义表型(例如，神经元的类型)。在之前的支持期间，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。本项目将探索乙醇对细胞增殖动力学和对细胞命运的定义的影响。特定的AIMS 1和2将使用体内模型来确定乙醇对皮质增殖区神经干细胞的循环活动(细胞周期动力学和退出)以及对TGF2受体表达/激活的影响。在发育过程中，神经干细胞表达一种同源异型盒基因产物(S)，即Pax6和/或Tbr2。这些蛋白质定义了从VZ的(Pax6+)干细胞到(Tbr2+)中间祖细胞阶段的过渡。这一转变对于胎儿期暴露于乙醇的目标--浅层皮质的发育至关重要(例如，穹隆突起的起源)。具体目标3将使用两种类型的培养(保留体内类似组织的器官型切片和从VZ/SZ或MGE获得的神经干细胞系)来探索乙醇对TGF21调控的细胞增殖和命运决定的影响。此外，我们将识别那些被乙醇和/或TGF21上调和下调并沉默(甲基化)的基因。在具体目标4中，神经干细胞将被移植(同位或异位)，以确定乙醇和/或TGF21对遗传和环境的影响，从而决定细胞的周期行为和决定细胞的命运。同时，新的AIMS将使用三个互补的模型来关键地洞察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。与公共卫生相关：在美国，估计有2%的活产婴儿患有胎儿酒精谱系障碍。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。本研究将探索酒精导致缺陷的机制--通过改变(A)增殖的神经干细胞的命运和(B)它们对这种增殖的关键调节因子-转化生长因子的反应。

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.