Astrocyte-neuron interactions and sulfatases in Fetal Alcohol Spectrum Disorders

 描述(由申请人提供)：胎儿酒精谱系障碍(FASD)的特征是大脑结构异常和认知和行为功能受损。发育中的酒精暴露会影响神经元的连通性和可塑性。乙醇对星形胶质细胞的暴露抑制了与乙醇处理的星形胶质细胞共培养的海马神经元的神经发生。然而，对乙醇影响发育中大脑神经元结构可塑性的机制仍然缺乏了解。填补这一空白将为确定预防或改善乙醇对发育影响的高效治疗开辟道路。凝集素是一种抑制性蛋白多糖，在大脑中，它可以防止轴突和树突的延伸。凝集素是由一个共价结合到双糖链上的核心蛋白部分组成的，它主要由软骨素-4硫酸盐(C4S)和软骨素-6硫酸酯(C6S)组成；凝集素的抑制特性取决于其核心蛋白及其SGAG侧链。芳基硫酸酯酶B(ARSB)和半乳糖-6-硫酸酯酶(GALNS)分别从C4S和C6S中去除硫酸盐基团，是降解CS-GAG所必需的。我们发现乙醇抑制了星形胶质细胞ARSB和GALNS的活性，而乙醇和ARSB沉默增加了星形胶质细胞凝集素的表达和C4S-GAG的含量。体内酒精暴露后，新生大鼠海马区ARSB活性受到抑制，神经元CaN和SGAG水平上调，锥体神经元树突分支减少。ARSB活性降低和树突分枝持续到出生后第36天，并与空间学习和记忆任务中表现的降低有关。在这项建议中，我们假设乙醇抑制发育中的海马区ARSB和GALNS的活性，导致凝集素(Neurocan，Brivican和Verscan)核心蛋白和CS-Gag水平意外增加，导致神经元结构可塑性下降；这些影响是长期的，并与学习成绩下降有关 学习和记忆任务，并由重组ARSB拯救。我们计划通过以下三个具体目标来验证我们的假设：1)研究乙醇诱导的硫酸酯酶抑制的机制及其对凝集素核心蛋白和SGAG水平以及对星形胶质细胞CS双糖水平和组成的影响。2)探讨硫酸酯酶、SUMF1、凝集素和氧化应激在乙醇处理的星形胶质细胞抑制轴突生长中的作用。3)探讨体内乙醇暴露和rARSB注射对新生大鼠海马硫酸酯酶活性、凝集素表达、SGAG和CS双糖水平、海马树突状分枝及空间学习记忆的影响。这项拟议的工作具有创新性和重要意义，因为它探索了乙醇抑制神经元可塑性的新机制，并将产生针对酒精致畸的硫酸酯酶激活的策略，从而促进FASD的研究。

 DESCRIPTION (provided by applicant): Fetal Alcohol Spectrum Disorders (FASD) are characterized by structural brain abnormalities and compromised cognitive and behavioral functions. Neuronal connectivity and plasticity are affected by developmental alcohol exposure. The exposure of astrocytes to ethanol inhibits neuritogenesis in hippocampal neurons co-cultured with ethanol-treated astrocytes. However, a lack of understanding of the mechanisms by which ethanol affects neuronal structural plasticity in the developing brain persists. Filling tis gap will open the path to the identification of highly effective treatments to prevent or ameliorat the developmental effects of ethanol. Lecticans are inhibitory proteoglycans that, in the brain, prevent the extension of axons and dendrites. Lecticans consist of a core-protein moiety covalently bound to linear chains of disaccharides, chondroitin sulfates glycosaminoglycans (CS-GAGs), which are comprised mostly of chondroitin-4 sulfate (C4S) and chondroitin-6 sulfate (C6S); the inhibitory properties of lecticans depend on their core-protein and their sGAG side-chains. Two sulfatase enzymes, arylsulfatase B (ARSB) and galactose-6-sulfatase (GALNS) remove sulfate groups from C4S and C6S respectively and are required for the degradation of CS-GAGs. We find that ethanol inhibits the activity of ARSB and GALNS and that ethanol and ARSB silencing increase the expression of the lectican neurocan and C4S-GAG content in astrocytes in vitro. ARSB activity is inhibited, neurocan and sGAG levels are upregulated, and pyramidal neuron dendritic arborization is reduced also in the hippocampus of neonatal rats after in vivo ethanol exposure. Reduced ARSB activity and dendritic arborization persist up to post-natal day 36 and is correlated with reduced performance in spatial learning and memory tasks. In this proposal we hypothesize that the inhibition of ARSB and GALNS activity by ethanol in the developing hippocampus induces an unscheduled increase in lectican (neurocan, brevican, and versican) core-protein and CS-GAG levels leading to decreased in neuronal structural plasticity; these effects are long-lasting and causally linked to reduced performance in learning and memory tasks and are rescued by recombinant (r)ARSB. We plan to test our hypothesis by pursuing the following three specific aims: 1) To investigate the mechanisms of ethanol-induced sulfatase inhibition and the consequences on lectican core-proteins and sGAG levels and on CS disaccharide levels and composition in astrocytes. 2) To investigate the role of: sulfatases, SUMF1, lecticans, and oxidative stress on ethanol-treated astrocyte inhibition of neurite outgrowth. 3) To investigate the effects of in vivo neonatal ethanol exposure and of rARSB injections on sulfatase activity, lectican expression, sGAG levels and CS disaccharide levels, and dendritic arborization in the hippocampus and on spatial learning and memory. The proposed work is innovative and significant as it explores a novel mechanism underlying ethanol-induced inhibition of neuronal plasticity and will yield strategies aimed at sulfatase enzyme activation for the treatment of alcohol teratogenesis therefore advancing FASD research.