Experimental Fetal Alcohol Syndrome

描述（由申请人提供）：胎儿酒精谱系障碍是常见的（影响所有活产的~2%），是精神功能障碍的主要原因。在乙醇对发育中的神经系统的许多负面影响中，最严重的是神经元死亡。它是严重的，因为这种损失是永久性的;除了几个部位，CNS中的有丝分裂后神经元不会被新产生的神经元取代。事实上，在神经元生成完成后，神经系统必须决定受损的神经元是否可以修复。如果没有，就必须消除。这个决定过程中的一个关键分子是癌蛋白p53。p53对于维持神经元的完整性和弹性至关重要。我们将测试的假设，发育暴露于乙醇改变神经元的存活和DNA修复通过p53依赖的活动。 在发育中的神经系统中，神经元死亡是一个自然和关键的过程。这种死亡似乎是凋亡性的，并涉及p53。我们的初步数据和其他人对大脑皮层发育的研究表明，在自然发生的神经元死亡期间暴露于乙醇会导致活性半胱天冬酶3表达和末端尿苷酸化缺口末端标记（TUNEL）的急剧和短暂增加。另一方面，我们的新数据也表明，这种模式与皮层神经元在时间或空间上的最终损失并不一致。这意味着乙醇导致DNA片段化，但这种降解可能不是细胞凋亡所必需的。相反，它可能揭示DNA修复机制。p53是DNA修复的关键分子。三个互补的目标将使用p53缺陷小鼠和细胞解决。(1)皮质神经元对乙醇的脆弱性将在长期和短期的体内研究中得到解决。长期研究将确定乙醇诱导的缺陷小鼠皮层神经元损失。补充研究将检查皮质层中假定的"死亡标志物"（活性半胱天冬酶3免疫表达和TUNEL）表达的短期变化以及神经元损失的时间。(2)将测定p53缺陷小鼠和培养的神经干细胞对乙醇的急性基因组反应。我们将确定参与细胞凋亡和DNA修复的转录物表达的变化。此外，我们将确定乙醇通过超甲基化对基因沉默的表观遗传效应。(3)securin是一种受p53调控的蛋白质，对DNA修复至关重要。初步的微阵列和免疫细胞化学数据表明，它是深刻的影响乙醇。因此，我们将研究乙醇对缺乏securin的细胞的影响，在体外和体内移植到层后，显然是敏感和难治性乙醇。 这些研究将探索发育中的神经元对乙醇的两种长尾反应：DNA修复，以及细胞凋亡。我们将解决关键问题。例如，什么定义了年轻神经元对乙醇的敏感性？神经元可以被操纵以减少其乙醇脆弱性吗？因此，这些研究探索了乙醇的两个新靶点，p53和securin，它们对神经元的存活和完整性至关重要。 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分化细胞，特别是那些新整合到发育中大脑复杂环境中的细胞。本研究将探讨两种互补的机制，酒精诱导的缺陷导致（a）乙醇诱导的神经元死亡和（B）乙醇改变的DNA修复。

DESCRIPTION (provided by applicant): Fetal alcohol spectrum disorder is common (affects ~2% of all live births) and is a major cause of mental dysfunction. Of the many negative effects that ethanol has on the developing nervous system, the most severe is neuronal death. It is severe because this loss is permanent; with the exception of a couple of sites, post-mitotic neurons in the CNS are not replaced by newly generated ones. Indeed, after the generation of neurons is complete, the nervous system has to decide if and whether damaged neurons can be repaired. If not, they must be eliminated. A key molecule in this decision process is the oncoprotein p53. p53 is critical for maintaining neuronal integrity and resiliency. We will test the hypothesis that developmental exposure to ethanol alters neuronal survival and DNA repair through p53-dependent activities. In the developing nervous system, neuronal death is a natural and critical process. This death appears to be apoptotic and to involve p53. Our preliminary data and the work of others on developing cerebral cortex show that exposure to ethanol during the period of naturally occurring neuronal death causes a dramatic and transient increase in both active caspase 3 expression and terminal uridylated nick-end labeling (TUNEL). On the other hand, our novel data also show that this pattern does not coincide with the ultimate loss of cortical neurons either in time or space. The implication is that ethanol causes DNA fragmentation, but this degradation may not be obligatory for apoptosis. Instead, it may reveal DNA repair mechanisms. p53 is a key player in DNA repair. Three complementary aims will be addressed using p53 deficient mice and cells. (1) Vulnerability of cortical neurons to ethanol will be addressed in long- and short-term in vivo studies. Long-term studies will determine the ethanol-induced loss of neurons in cortical layers occurring in the deficient mice. Complementary studies will examine short-term changes in the expression of presumed "death markers" (active caspase3 immunoexpression and TUNEL) in cortical layers and the timing of the neuronal loss. (2) The acute genomic responses of p53 deficient mice and cultured neural stem cells to ethanol will be determined. We will identify changes in the expression of transcripts involved in apoptosis and DNA repair. In addition, we will determine the epigenetic effects of ethanol on the silencing of genes through hyper-methylation. (3) Securin is a protein that is regulated by p53 and is pivotal for DNA repair. Preliminary microarray and immunocytochemical data show that it is profoundly affected by ethanol. Thus, we will examine the effects of ethanol on cells deficient of securin in vitro and in vivo after transplantation into layers that are apparently susceptible and refractory to ethanol. These studies will explore two dovetailed responses that developing neurons have to ethanol: DNA repair, and failing that, apoptotic death. We will address critical questions. For example, what defines the susceptibility of a young neuron to ethanol? Can neurons be manipulated to reduce their ethanol vulnerability? Thus, the studies explore two new targets of ethanol, p53 and securin, that are critical for the neuronal survival and integrity. 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 differentiating cells, particularly those that are newly integrating into the complex environment of the developing brain. The present study will explore two complementary mechanisms by which alcohol-induced defects result (a) from ethanol-induced neuronal death and (b) from ethanol-altered DNA repair.