Glia-neuron interaction in fetal alcohol spectrum disorders

胎儿酒精谱系障碍中神经胶质细胞与神经元的相互作用

• 批准号: 10200642
• 负责人: Marina Guizzetti
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200642
• 关键词: ADAMTS; Affinity Chromatography; Age; Alcohol abuse; Alteplase; Animals; Astrocytes; Behavior; Behavioral; Brain; CSPG3 gene; Cells; Cognitive; Computer software; Confocal Microscopy; Development; Disintegrins; Down-Regulation; Engineering; Enrollment; Ethanol; Extracellular Matrix; Extracellular Matrix Proteins; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; Gene Expression; Genes; Goals; Golgi Apparatus; Healthcare Systems; Hippocampus (Brain); Human; Individual; Injections; Intellectual functioning disability; Intervention; Knowledge; Laminin; Lead; Learning; Link; Literature; MMP14 gene; Matrix Metalloproteinases; Mediating; Memory; Messenger RNA; Metalloproteases; Methodology; Methods; Mission; Molecular Target; Mus; Neonatal; Neonatal Alcohol Exposure; Neuroglia; Neurons; Newborn Animals; Peptide Hydrolases; Play; Pregnancy; Proteins; Proteolysis; Publishing; Quantitative Reverse Transcriptase PCR; Rattus; Reporting; Research; Ribosomal Proteins; Ribosomes; Role; Stains; Stromelysin 1; Substance Use Disorder; System; Therapeutic Intervention; Third Pregnancy Trimester; Thrombospondins; Translating; Up-Regulation; Veterans; Viral; Western Blotting; Woman; addiction; aggrecan; alcohol consumption during pregnancy; alcohol effect; alcohol exposure; alcohol misuse; alcohol use disorder; brevican; cellular targeting; child bearing; disability; drinking; extracellular; fetal; hippocampal pyramidal neuron; in vivo; inhibitor/antagonist; innovation; military women; mouse model; neonatal brain; neuron development; novel; overexpression; polysulfated glycosaminoglycan; reproductive; therapy development; transcriptome; versican

Substance use disorders are common among women veterans, many of which are of childbearing age. Drinking during pregnancy may lead to Fetal Alcohol Spectrum Disorders (FASD), a leading cause of intellectual disability. Research on novel mechanisms involved in FASD, which may lead to innovative interventions, is therefore a topic highly relevant to the VA mission. Hippocampal alterations are associated with deficits in learning and memory in individuals with FASD. The extracellular matrix (ECM) plays a major role in brain development and astrocytes are major regulators of the brain ECM. Critical gaps in knowledge remain concerning the mechanisms by which ethanol alters neuronal development in the fetal hippocampus hampering the development of therapies for FASD. Indeed, there is no published literature on the effects of alcohol exposure during the third trimester of human gestation-equivalent on astrocyte gene expression in vivo. Furthermore, dysregulation of the brain ECM mediated by alterations in extracellular proteases is involved in many neuropathological conditions and in addiction. However, very little is known about the role of the ECM and extracellular proteases in FASD in vivo. Finally, the link between changes in astrocyte-released ECM modulators and dendritic development following neonatal alcohol exposure has not been investigated. Preliminary results suggest that developmental alcohol exposure alters the ECM through the modulation of extracellular protease systems in the hippocampus. Indeed, Adamts5 expression, encoding for a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), a protease that degrades lecticans, and tissue plasminogen activator (tPA), which can activate ADAMTSs, are both upregulated in the hippocampus of animals neonatally exposed to ethanol. Furthermore, we observed decreased levels of lectican sulfated glycosaminoglycans (sGAGs) and increased proteolysis of the lectican brevican in these animals. We also observed down-regulation of Mmp14 and Mmp15 encoding for matrix metalloproteinases (MMP)14 and MMP15 and increased protein levels of one major target of these MMPs: laminin. All of these changes are consistent with an ethanol-induced increase in dendritic arborization in pyramidal hippocampal neurons, as lecticans are inhibitors and laminin is a strong inducer of dendritic arborization. The overall hypothesis of this proposal is that ethanol alters the expression and activity of astrocyte extracellular proteases leading to ECM remodeling and increased dendritic arborization in the hippocampus. In aim 1, the hypothesis that developmental ethanol exposure increases the expression and activity of ADAMTSs that degrade lecticans in part via an increase in tPA expression leading to ADAMTS activation resulting in the degradation of lecticans and increased dendritic arborization in the neonatal brain will be explored. In aim 2, the hypothesis that ethanol exposure decreases MMP14 and MMP15 leading to increased laminin protein levels and increased dendritic complexity will be explored. Aims 1 and 2 will employ qRT-PCR, Western blot, confocal microscopy, Golgi-Cox staining followed by morphometric analysis with the software Neurolucida, and AAV6 viral construct injections into the hippocampus to overexpress ADAMTS5 and tPA and to silence MMP14 and MMP15. In aim 3 the modulation of gene expression by ethanol in neonatal hippocampal astrocytes of Aldh1l1-EGFP-Rpl10a mice using the translating ribosome affinity purification (TRAP) methodology will be examined. The effects of neonatal alcohol exposure on the expression of target genes encoding for ECM proteins and proteins involved in the remodeling of the ECM as well as on astrocyte global gene expression will be analyzed. We will isolate astrocyte mRNA in the engineered Aldh1l1-EGFP-Rpl10a mouse model that expresses a modified ribosomal protein Rpl10a with an eGFP tag (EGFP-Rpl10a) in cells expressing Aldh1l1 (a highly specific astrocytic marker) using the TRAP method. This study will unveil novel astrocyte-mediated effects of ethanol on extracellular proteases leading to changes in ECM composition and neuronal development.

物质使用障碍在女性退伍军人中很常见，其中许多人处于生育年龄。 怀孕期间饮酒可能会导致胎儿酒精谱系障碍（FASD），这是导致胎儿酒精谱系障碍的主要原因。 智力残疾。研究FASD中涉及的新机制，这可能导致创新 因此，干预是与VA使命高度相关的主题。海马结构的改变与 FASD患者的学习和记忆缺陷。细胞外基质（ECM）在肿瘤的发生发展中起着重要作用。 在脑发育中的作用，星形胶质细胞是脑ECM的主要调节剂。知识方面的重大差距 乙醇改变胎儿海马神经元发育的机制仍然是个问题 阻碍了FASD疗法的发展。事实上，没有发表文献的影响， 人类妊娠晚期酒精暴露对星形胶质细胞基因表达的影响。 此外，由细胞外蛋白酶的改变介导的脑ECM的失调参与了脑细胞外基质的形成。 许多神经病理状况和成瘾。然而，很少有人知道ECM的作用， 和细胞外蛋白酶。最后，星形胶质细胞释放的ECM的变化之间的联系 调节剂和树突发育新生儿酒精暴露后尚未研究。 初步结果表明，发育酒精暴露改变ECM通过调制 细胞外蛋白酶系统。事实上，Adamts 5表达，编码去整合素 和具有血小板反应蛋白基序的金属蛋白酶5（ADAMTS 5），一种降解凝集素的蛋白酶，和 组织纤溶酶原激活物（tPA），它可以激活ADAMTS，在海马中上调， 暴露于乙醇的动物。此外，我们观察到硫酸凝集素水平降低， 这些动物中的糖胺聚糖（sGAG）和短凝集素聚糖的蛋白水解增加。我们也 观察到编码基质金属蛋白酶（MMP）14的Mmp 14和Mmp 15的下调， MMP 15和这些MMPs的一个主要靶点：层粘连蛋白的蛋白质水平增加。所有这些变化都是 与乙醇诱导的锥体海马神经元树突分支增加一致， 凝集素是抑制剂，而层粘连蛋白是树突状树枝化的强诱导剂。这个问题的总体假设 有人认为乙醇改变了星形胶质细胞胞外蛋白酶的表达和活性，导致ECM 海马中的重塑和增加的树突状分支。在目标1中，假设 发育中的乙醇暴露增加了降解凝集素的ADAMTS的表达和活性， 部分通过tPA表达的增加导致ADAMTS激活，导致凝集素降解 以及新生儿大脑中增加的树突状分支将被探索。在目标2中，假设乙醇 暴露降低MMP 14和MMP 15，导致层粘连蛋白水平增加和树突状细胞增加。 将探索复杂性。目的1和2将采用qRT-PCR、蛋白质印迹、共聚焦显微镜、Golgi-Cox 染色，然后用Neurolucida软件进行形态测定分析，并注射AAV 6病毒构建体 使ADAMTS 5和tPA过表达，并使MMP 14和MMP 15沉默。在aim 3中， 乙醇对Aldh 1 l1-EGFP-Rpl 10a小鼠新生海马星形胶质细胞基因表达的调节 将检查使用翻译核糖体亲和纯化（TRAP）方法。的影响 新生儿酒精暴露对编码ECM蛋白和相关蛋白的靶基因表达的影响 将分析ECM重塑以及星形胶质细胞整体基因表达。我们将隔离 在表达修饰的核糖体的工程化Aldh 1 l1-EGFP-Rpl 10a小鼠模型中的星形胶质细胞mRNA 在表达Aldh 111（一种高度特异性星形胶质细胞 标记物）。这项研究将揭示新的星形胶质细胞介导的影响，乙醇对 细胞外蛋白酶导致ECM组成和神经元发育的变化。