Mechanisms of ethanol-induced neurodevelopmental effects

描述（由申请人提供）：本提案将解决乙醇发育神经毒性的一个新方面，即其对中枢神经系统胆固醇稳态的影响。虽然过多的胆固醇可能是有害的，如在动脉粥样硬化和阿尔茨海默病的情况下，太少会产生出生缺陷。不同程度的智力迟钝常见于先天性胆固醇合成错误，如Smith-Lemli-Opitz综合征。由于这些遗传缺陷，在大脑发育过程中缺乏胆固醇会导致严重的脑损伤，包括小头畸形和智力迟钝，这两种情况都是胎儿酒精综合征的标志。乙醇对发育中的大脑中胆固醇稳态的影响尚未被研究。在大脑中，胆固醇主要是内源性产生的，其稳态由内源性产生的脂蛋白和胆固醇转运蛋白调节。该提案将研究乙醇通过上调中枢神经系统细胞的胆固醇转运体和脂蛋白产生，从而增加大脑对胆固醇的清除，导致胆固醇消耗的假设。低胆固醇水平与子宫内酒精暴露引起的一些影响相一致，如抑制sonic hedgehog通路、神经元发育和存活以及细胞增殖。具体目的是：1)研究ABCA1和ABCG1在乙醇诱导的胆固醇外排中的作用。乙醇对体外皮质神经元胆固醇外排和胆固醇水平的影响将被研究；此外，ABCA1、ABCG1和磷脂酶D在乙醇诱导的星形胶质细胞和可能的神经元中胆固醇外排和胆固醇转运体诱导上调中的作用将通过选择性地从星形胶质细胞和神经元培养物中去除这些蛋白质来检测。2)乙醇对星形胶质细胞生成脂蛋白的影响及其与神经元相互作用的研究。乙醇对凝胶过滤分离的星形胶质细胞产生的脂蛋白组成的影响，以及它们在神经元胆固醇外排中的作用将被分析。此外，乙醇对脂蛋白组成和胆固醇外排的影响将在星形胶质细胞/神经元共培养系统中进行研究。3)体内给药乙醇对ABC胆固醇转运蛋白及发育中的脑内胆固醇水平影响的研究。妊娠期体内暴露于乙醇对胆固醇转运体ABCA1和ABCG1在神经元和星形胶质细胞中的转录和表达以及胆固醇水平的影响将在妊娠第21天的大鼠胎儿新皮质中进行评估。总之，这些研究的目的是描述一种可能的新机制，涉及乙醇诱导的神经发育效应，即它对大脑中胆固醇稳态的影响。由于观察到乙醇引起的几种神经发育影响与缺乏胆固醇引起的影响是一致的，因此提出了关于乙醇对发育中的大脑中胆固醇稳态影响的研究。胆固醇确实对大脑发育的各个方面都是必需的。我们假设乙醇，通过在拟议项目中研究的机制，可能影响胆固醇稳态并降低发育中的大脑中的胆固醇水平。胆固醇运输领域的最新发现将应用于研究乙醇对胆固醇转运体、脂蛋白生成以及最终对发育中的大脑中胆固醇水平的影响。由于胆固醇是日常饮食的常规组成部分，我们的研究可能会导致对高危孕妇的饮食指南进行修订。这也有助于理解为什么在南非一些非常贫困的地区，FAS的患病率远高于西方国家，因为这些地区的人口也经历着严重的营养不良。

DESCRIPTION (provided by applicant): This proposal will address a novel aspect of ethanol's developmental neurotoxicity, i.e. its effect on cholesterol homeostasis in the central nervous system. While too much cholesterol may be deleterious, as in case of atherosclerosis and Alzheimer's disease, too little can produce birth defects. Different degrees of mental retardation are often observed in inborn errors of cholesterol synthesis such as Smith-Lemli-Opitz syndrome. Lack of cholesterol during brain development as a consequence of these genetic defects leads to severe brain damage, including microcephaly and mental retardation, both of which are hallmarks of the fetal alcohol syndrome. The effects of ethanol on cholesterol homeostasis in the developing brain have not been investigated. In the brain, cholesterol is mostly produced endogenously, and its homeostasis is regulated by endogenously produced lipoproteins and cholesterol transporters. This proposal will investigate the hypothesis that ethanol, by upregulating cholesterol transporters and lipoprotein production by central nervous system cells, will increase the clearance of cholesterol from the brain, causing cholesterol depletion. Low levels of cholesterol are consistent with some of the effects caused by in utero alcohol exposure such as inhibition of the sonic hedgehog pathway, neuronal development and survival, and cell proliferation. Specific Aims of the proposal are: 1) The investigation of the role of ABCA1 and ABCG1 in ethanol-induced cholesterol efflux. The effect of ethanol on cholesterol efflux and cholesterol levels will be investigated in cortical neurons in vitro; in addition, the role of ABCA1, ABCG1, and phospholipase D in ethanol-induced upregulation of cholesterol efflux and cholesterol transporter induction in astrocytes and, possibly, in neurons will be examined by selectively removing these proteins form astrocyte and neuron cultures. 2) The investigation of the effect of ethanol on astrocyte-generated lipoproteins and their interaction with neurons. The effect of ethanol on the composition of astrocyte-produced lipoproteins separated by gel filtration, and their role on cholesterol efflux from neurons will be analyzed. Furthermore, the effect of ethanol on lipoprotein composition and cholesterol efflux will be examined in an astrocyte/neuron co- culture system. 3) The investigation of the effect of in vivo ethanol administration on ABC cholesterol transporters and on cholesterol levels in the developing brain. The effect of in vivo exposure to ethanol during gestation on the cholesterol transporters ABCA1 and ABCG1 transcription and expression in neurons and astrocytes and on cholesterol levels will be assessed in the neocortex of rat fetuses at gestational day 21. Altogether, these studies are directed at characterizing a possible novel mechanism involved in ethanol-induced neurodevelopmental effects, i.e. its effects on cholesterol homeostasis in the brain. PUBLIC HEALTH RELEVANCE The proposed studies on the effect of ethanol on cholesterol homeostasis in the developing brain were prompted by the observation that several of the neurodevelopmental effects caused by ethanol are consistent with effects caused by lack of cholesterol. Cholesterol is indeed necessary for various aspects of brain development. We hypothesized that ethanol, by mechanisms to be investigated in the proposed project, may affect cholesterol homoeostasis and reduce cholesterol levels in the developing brain. The most recent discoveries in the field of cholesterol trafficking will be applied to the investigation of the effects of ethanol on cholesterol transporters, lipoprotein generation and, ultimately, cholesterol levels in the developing brain. As cholesterol is a regular component of the diet, our studies could potentially lead to the revision of dietary guidelines for pregnant women at risk. It may also help understanding why in some very poor regions of South Africa the prevalence of FAS is much higher than in Western countries as these populations also experience severe malnutrition.