DEVELOPMENT AND REGULATION OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

黄体生成素释放激素系统的发育和调控

• 批准号: 5203963
• 负责人: S WRAY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5203963
• 关键词: N acetylglucosamine; carbohydrate transport; cell migration; developmental neurobiology; embryo /fetus tissue /cell culture; gamma aminobutyrate; gene expression; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; glycosylation; gonadotropin releasing factor; histochemistry /cytochemistry; laboratory mouse; messenger RNA; neural cell adhesion molecules; neuronal transport; neurons; neurotransmitters; olfactory lobe; peptide hormone biosynthesis; second messengers; secretion; tunicamycin

Luteinizing hormone releasing hormone (LHRH) neurons are derived from the olfactory placode and migrate into the brain, where they become integral members of the hypothalamic-pituitary-gonadal axis. We study the mechanisms underlying LHRH neuronal migration into the CNS in normal and transgenic animals, as well as in nasal explants. The intrinsic and transsynaptic regulation of LHRH gene expression, peptide synthesis and secretion in postnatal LHRH neurons (in the CNS) vs embryonic LHRH neurons (outside the CNS) is studied using long-term organotypic cultures and nasal explants, respectively. Working on the hypothesis that LHRH neurons migrate on peripherin positive (P+) olfactory axons via adhesion between cell surface molecules, we are examining specific carbohydrate moieties which might "highlight" these adhesive molecules. Using lectin cytochemistry, we found that: 1) olfactory axons in nasal explants express several sugar moieties; 2) the pattern of carbohydrate expression in vitro is similar to that in vivo; and 3) in vitro, D-N-acetyl-glucosamine oligomers are detected on P+ axons with which LHRH neurons are associated and are localized to LHRH neurons which migrated out of the explant. Using tunicamycin, we found that during an early time window, inhibition of N- glycosylation prevents outgrowth of P+ axons, but does not affect the association of LHRH neurons with these fibers. Using the LHRH promoter fused to a luciferase reporter, we attempted to monitor migrating LHRH neurons in situ. However, the luciferase signal was not robust enough to detect. We have now fused the LHRH promoter to the Lac Z reporter and are currently working on optimizing a fluorescent signal using this tag. Using two different transcription inhibitors, we have broadened our studies on stability and turnover rates of LHRH mRNA. Using 2nd messenger analogs, we are examining (1-24 hr after stimulation) changes in LHRH mRNA to determine a) the 2nd messenger systems active in LHRH cells; and b) an optimal timepoint to monitor changes in LHRH mRNA levels after stimulation with neurotransmitters. Currently, we are determining: 1) the mechanism by which N-glycosylation "directs" outgrowth of P+ axons; 2) the role of molecules expressing D-N- acetyl-glucosamine oligomers on LHRH neuronal movement and/or olfactory axon outgrowth; 3) whether LHRH neurons in cultures release LHRH in a pulsatile manner; 4) whether tagged-LHRH neurons can be visualized in situ to monitor movement in embryonic explants and/or determine the membrane properties of postnatal LHRH neurons in organotypic slices; and 5) the effect of GABA on LHRH gene expression in embryonic and postnatal LHRH neurons.

促黄体激素释放激素（LHRH）神经元来源于 嗅觉基板并迁移到大脑，在那里它们成为整体 下丘脑-垂体-性腺轴的成员。我们研究 LHRH神经元迁移到中枢神经系统的机制， 转基因动物以及鼻外植体中。内在和 LHRH基因表达的跨突触调节，肽合成和 出生后LHRH神经元（中枢神经系统）与胚胎LHRH分泌 使用长期器官型培养物研究神经元（CNS外） 和鼻外植体。 LHRH神经元在外周神经元上迁移的假设 阳性（P+）嗅轴突通过细胞表面之间的粘附 分子，我们正在研究特定的碳水化合物部分， "突出显示"这些粘合剂分子。使用凝集素细胞化学，我们 发现：1）鼻外植体中的嗅轴突表达多种糖 2）碳水化合物在体外的表达模式是相似的 和3）在体外，D-N-乙酰基-葡糖胺低聚物， 在与LHRH神经元相关的P+轴突上检测到， 定位于移出外植体的LHRH神经元。使用 衣霉素，我们发现，在早期的时间窗口，抑制N- 糖基化阻止了P+轴突的生长，但不影响P+轴突的生长。 LHRH神经元与这些纤维的联系。使用LHRH启动子 与荧光素酶报告基因融合，我们试图监测迁移的LHRH 原位神经元。然而，荧光素酶信号不够稳健， 检测。我们现在已经将LHRH启动子与Lac Z报告基因融合， 目前正致力于使用这种标签优化荧光信号。 使用两种不同的转录抑制剂，我们扩大了我们的研究范围。 LHRH mRNA的稳定性和周转率的研究。使用第二信使 类似物，我们正在检查（刺激后1 - 24小时）LHRH的变化 a）在LHRH细胞中有活性的第二信使系统;和 B）监测LHRH mRNA水平变化的最佳时间点， 刺激神经递质。 目前，我们正在确定：1）N-糖基化的机制， "指导" P+轴突的生长; 2）表达D-N-的分子的作用， 乙酰葡糖胺寡聚物对LHRH神经元运动和/或嗅觉的影响 轴突生长; 3）培养物中的LHRH神经元是否在一个特定的时间内释放LHRH。 4）标记的LHRH神经元是否可以在 原位监测胚胎外植体中的运动和/或确定 器官型切片中出生后LHRH神经元的膜特性;以及 5)γ-氨基丁酸对胚胎及出生后LHRH基因表达的影响 LHRH神经元