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.

药物使用障碍在女性退伍军人中很常见，其中许多是生育年龄。 怀孕期间喝酒可能导致胎儿酒精谱系（FASD），这是 智力残疾。 FASD中涉及的新型机制的研究，这可能导致创新 因此，干预是与VA任务高度相关的主题。海马改变是相关的 患有FASD的人的学习和记忆缺陷。细胞外基质（ECM）发挥作用 在大脑发育中的作用和星形胶质细胞是脑ECM的主要调节剂。知识的关键差距 保持有关乙醇在胎儿海马中改变神经元发育的机制 阻碍了FASD疗法的开发。确实，没有关于影响的文献 人类妊娠三个月的酒精暴露于体内星形胶质细胞基因表达。 此外，由细胞外蛋白酶改变介导的脑ECM失调与 许多神经病理学条件和成瘾。但是，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的变化之间的联系 尚未研究新生儿酒精暴露后的调节剂和树突状发展。 初步结果表明，发育性酒精暴露通过调制改变了ECM 海马中的细胞外蛋白酶系统。实际上，ADAMTS5表达式编码为崩解蛋白 和金属蛋白酶蛋白酶具有血小板蛋白基序5（ADAMTS5），一种降解的蛋白酶，并降解。 可以激活ADAMTS的组织纤溶酶原激活剂（TPA）都在海马中上调 动物新生儿暴露于乙醇。此外，我们观察到硫化的拉克利亚人硫酸盐水平降低 这些动物中胶质糖胶质糖（Sgags）和蛋白水解的增加。我们也是 观察到针对基质金属蛋白酶（MMP）14的MMP14和MMP15的下调 MMP15和这些MMP的一个主要靶标的蛋白质水平增加：层粘连蛋白。所有这些变化是 与乙醇诱导的锥体海马神经元树突状树皮化的增加一致，如 固定剂是抑制剂，层粘连蛋白是树突状树博化的强大诱导剂。总体假设 建议是乙醇改变导致ECM的星形胶质细胞外蛋白酶的表达和活性 海马中的重塑和增加的树突状树博化。在AIM 1中，假设 发育性乙醇暴露增加了降解拉克斯人的表达和活性 通过增加TPA表达的一部分导致ADAMTS激活导致拉克斯降解 并将探索新生儿大脑中的树突状树皮化。在AIM 2中，乙醇的假设 暴露降低MMP14和MMP15，导致层粘连蛋白水平升高并增加树突状 将探索复杂性。目标1和2将采用QRT-PCR，Western印迹，共聚焦显微镜，高尔基-Cox 染色，然后对软件Neurolucida和AAV6病毒构建体进行形态分析 进入海马到过表达ADAMTS5和TPA，并使MMP14和MMP15沉默。在目标3中 乙醇在ALDH1L1-EGFP-RPL10A小鼠的新生儿海马星形胶质细胞中对基因表达的调节 将检查使用翻译核糖体亲和力纯化（TRAP）方法。效果 涉及ECM蛋白质和蛋白质的靶基因表达的新生儿酒精暴露 将分析ECM以及星形胶质细胞全基因表达的重塑。我们将孤立 在工程aldh1l1-egfp-rpl10a小鼠模型中，星形胶质细胞mRNA表达了修饰的核糖体 具有EGFP标签（EGFP-RPL10A）的蛋白质RPL10A在表达ALDH1L1的细胞中（高度特异性星形胶质细胞 标记）使用陷阱方法。这项研究将公布新型的星形胶质细胞介导的乙醇对 细胞外蛋白酶导致ECM组成和神经元发育的变化。