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.

药物滥用障碍在女性退伍军人中很常见，其中许多人处于育龄期。 怀孕期间饮酒可能导致胎儿酒精谱系障碍 (FASD)，这是导致胎儿酒精中毒的主要原因 智力障碍。对 FASD 涉及的新机制的研究可能会带来创新 因此，干预措施是与 VA 使命高度相关的主题。海马体的改变相关 患有 FASD 的人存在学习和记忆缺陷。细胞外基质（ECM）起着重要作用 在大脑发育中的作用和星形胶质细胞是大脑 ECM 的主要调节者。知识方面的关键差距 仍然关注乙醇改变胎儿海马神经元发育的机制 阻碍了 FASD 疗法的开发。事实上，目前还没有关于其影响的公开文献。 人类妊娠晚期酒精暴露对体内星形胶质细胞基因表达的影响。 此外，细胞外蛋白酶改变介导的大脑 ECM 失调也参与了 许多神经病理学病症和成瘾。然而，人们对 ECM 的作用知之甚少。 和体内 FASD 中的细胞外蛋白酶。最后，星形胶质细胞释放的 ECM 变化之间的联系 尚未研究新生儿酒精暴露后的调节剂和树突状发育。 初步结果表明，发育时期的酒精暴露通过调节 ECM 来改变 ECM。 海马体中的细胞外蛋白酶系统。事实上，Adamts5 表达，编码解整合素 以及具有血小板反应蛋白基序 5 (ADAMTS5) 的金属蛋白酶，这是一种降解凝集素的蛋白酶，以及 可以激活 ADAMTS 的组织纤溶酶原激活剂 (tPA) 在海马中均上调 新生儿接触乙醇的动物。此外，我们观察到硫酸凝集素水平降低 这些动物体内的糖胺聚糖（sGAG）和 lectican brevican 的蛋白水解作用增加。我们也 观察到编码基质金属蛋白酶 (MMP)14 的 Mmp14 和 Mmp15 下调 MMP15 和这些 MMP 的一个主要目标：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状树枝化的增加一致， 凝集素是抑制剂，层粘连蛋白是树突分枝的强诱导剂。本次的总体假设 建议认为乙醇会改变星形胶质细胞胞外蛋白酶的表达和活性，从而导致 ECM 海马体的重塑和树突状树枝化的增加。在目标 1 中，假设 发育期乙醇暴露会增加 ADAMTS 的表达和活性，从而降解 lecticans 部分通过 tPA 表达增加导致 ADAMTS 激活，从而导致 lecticans 降解 并将探索新生儿大脑中树突状树枝化的增加。在目标 2 中，假设乙醇 暴露会降低 MMP14 和 MMP15，导致层粘连蛋白水平增加和树突增加 将探讨复杂性。目标 1 和 2 将采用 qRT-PCR、Western blot、共聚焦显微镜、Golgi-Cox 染色，然后使用 Neurolucida 软件进行形态分析，并注射 AAV6 病毒构建体 进入海马体以过表达 ADAMTS5 和 tPA 并沉默 MMP14 和 MMP15。在目标 3 中 乙醇对 Aldh1l1-EGFP-Rpl10a 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 表达修饰核糖体的工程化 Aldh1l1-EGFP-Rpl10a 小鼠模型中的星形胶质细胞 mRNA 表达 Aldh1l1（一种高度特异性星形胶质细胞）的细胞中带有 eGFP 标签的蛋白 Rpl10a (EGFP-Rpl10a) 标记）使用TRAP方法。这项研究将揭示乙醇对星形胶质细胞介导的新作用 细胞外蛋白酶导致 ECM 成分和神经元发育的变化。