Experimental Fetal Alcohol Syndrome

描述（由申请人提供）：胎儿酒精谱系障碍是常见的（影响所有活产婴儿的约2%），是精神功能障碍的主要原因。乙醇对发育中的神经系统有许多负面影响，其中最严重的是神经元死亡。它之所以严重，是因为这种损失是永久性的；除了几个位点外，中枢神经系统中有丝分裂后的神经元不会被新生成的神经元所取代。的确，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。否则，他们必须被淘汰。在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。我们将测试发育暴露于乙醇通过p53依赖性活动改变神经元存活和DNA修复的假设。在发育中的神经系统中，神经元死亡是一个自然而关键的过程。这种死亡似乎是凋亡，并涉及p53。我们的初步数据和其他人关于大脑皮层发育的工作表明，在自然发生的神经元死亡期间暴露于乙醇会导致活性caspase 3表达和末端尿苷化镍端标记（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的最终损失在时间或空间上都不一致。这意味着乙醇导致DNA断裂，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在DNA修复中起着关键作用。使用p53缺陷小鼠和细胞将解决三个互补的目标。(1)皮质神经元对乙醇的脆弱性将在长期和短期体内研究中得到解决。长期研究将确定乙醇引起的缺陷小鼠皮质层神经元的丢失。补充研究将检查皮质层中假定的“死亡标志物”（caspase3免疫活性表达和TUNEL）表达的短期变化以及神经元丢失的时间。(2)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修复的转录本表达的变化。此外，我们将通过超甲基化确定乙醇对基因沉默的表观遗传效应。(3) Securin是一种受p53调控的蛋白，在DNA修复中起关键作用。初步的微阵列和免疫细胞化学数据表明，它受到乙醇的深刻影响。因此，我们将在体外和体内研究乙醇对缺乏安全蛋白的细胞移植后的影响，这些细胞显然对乙醇敏感且难以耐受。这些研究将探索发育中的神经元对乙醇的两种相互关联的反应：DNA修复，以及失败的凋亡性死亡。我们将解决关键问题。例如，是什么定义了一个年轻的神经元对乙醇的敏感性？神经元能否通过操纵来降低它们对乙醇的脆弱性？因此，本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。

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.