Pineal Regulation: Neural, transsynaptic and intracellular control

松果体调节：神经、突触和细胞内控制

• 批准号: 8149362
• 负责人: David Klein
• 金额: $ 53.63万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/8149362
• 关键词: neuroregulation

Analysis of global gene expression: Studies are in progress which have characterized gene expression in the pineal gland. The first stage has involved analysis of the rat pineal gland: "The rodent pineal transcriptome was investigated ..... using microarray gene expression. Comparison of midday and midnight expression profiles revealed that a global >2-fold change in the expression of 1000 genes, 2/3 of which increase at night. Among these, 400 increase >4- fold in expression; studies in organ culture reveal that in nearly all cases, the expression of the highly upregulated genes is induced by treatment with NE or cyclic nucleotide analogs. These findings are consistent with the conclusion that NE-cyclic nucleotide signaling is the primary mechanism responsible for the nocturnal increase in gene expression. However, it is also clear that other mechanisms are involved, because a small number of highly rhythmic genes are not induced or are weakly induced by NE treatment. Comparison of the level of gene expression in the pineal gland to the median expression in other tissues indicates that a set of > 300 genes are expressed >8- fold higher in the pineal gland. A significant subset of the most highly expressed genes encode proteins involved in melatonin synthesis and the control of this process, including signalling via adrenergic receptors and second messengers including cyclic nucleotides, Ca++ and phospholipids. Clusters of highly expressed genes are associated with the cellular biology of thyroid hormone, retinoid acid, glutamate biology; and, with metal ion homeostasis, membrane trafficking, and the immune response. Other highly and/or rhythmically expressed genes also encode transcription factors, ion channels, transporters, receptors, regulatory molecules and secreted products that have not previously appeared in the pineal literature. Comparison of the pineal gene expression profile to that of several other tissues adds to the evidence that the pineal gland is most similar to the retina by expanding the number of genes that are highly expressed exclusively in these two tissues. This study indicates that control of pineal biology is significantly more complex than previously thought, that the number of highly expressed genes in the pineal gland and retina is higher than previously thought, and also provides molecular evidence to suspect that the gland might function outside of the highly conserved role it plays in melatonin production." From (Bailey et al, in preparation). The work on the rodent pineal gland is being followed up with similar work on the pineal gland of the monkey and human, so as to determine the similarity of the patterns of gene expression in these three tissues. The results of the analysis of the rodent pineal gland has triggered a number of studies, some of which have been published, which have focused on genes that have been highlighted by the microarray studies. An example is detailed in HD000095-37. Control of dopamine signal transduction in the pineal gland: Dopamine plays a broad role in biology through actions mediated by specific G-protein coupled receptors. "We have discovered that the expression of the gene that encodes the dopamine D4 receptor (Drd4), can change rapidly. Drd4 mRNA increases 20-fold at night in the pineal gland and retina to levels that are >10-fold higher than those in other tissues The abundance of pineal Drd4 transcripts is controlled by the well described circadian regulatory system that controls pineal function. In vitro studies indicate that Drd4 is induced by an And gate mechanism which is activated by adrenergic /cyclic AMP signaling and is dependent on thyroid hormone (T3). These findings point to an important role of dopamine/Drd4 signalling in the pineal gland and retina. On a more general level, it appears reasonable to consider that dopamine/D4Rsignaling in other tissues could reflect the interaction of cyclic AMP and T3." (From Kim et al,) Localization and regulation of dopamine receptor D4 expression in the adult and developing rat retina: "Levels of dopamine and melatonin exhibit diurnal rhythms in the rat retina. Dopamine ishigh during daytime adapting the retina to light, whereas melatonin is high during nighttimeparticipating in the adaptation of the retina to low light intensities. Dopamine inhibits the synthesis of melatonin in the photoreceptors via Drd4-receptors located on the cell membrane of these cells. In this study, we show by semiquantitative in situ hybridization a prominent day/night variation in Drd4 expression in the retina of the Sprague Dawley rat with a peak during the nighttime. Drd4 expression is seen in all retinal layers but the nocturnal increase is confined to the photoreceptors. Retinal Drd4 expression is not affected by removal of the sympathetic input to the eye, but triiodothyronine treatment induces Drd4 the expression in the photoreceptors. In a developmental series, we show that the expression of Drd4 is restricted to postnatal stages with a peak at postnatal day 12. The high Drd4 expression in the rat retinal photoreceptors during the night supports physiological and pharmacologic evidence that the Drd4 receptor is involved in the dopaminergic inhibition of melatonin synthesis upon light stimulation. The sharp increase of Drd4 expression at a specific postnatal time suggests that dopamine is involved in retinal development." Control of cyclic AMP degradation: "The pineal gland is a photoneuroendocrine transducer that influences circadian and circannual dynamics of many physiological functions via the daily rhythm in melatonin production and release. Melatonin synthesis is stimulated at night by a photoneural system through which pineal adenylate cyclase is adrenergically activated, resulting in an elevation of cAMP. cAMP enhances melatonin synthesis through actions on several elements of the biosynthetic pathway. cAMP degradation also appears to increase at night due to an increase in phosphodiesterase (PDE) activity, which peaks in the middle of the night. Here, it was found that this nocturnal increase in PDE activity results from an increase in the abundance of PDE4B2 mRNA (approximately 5-fold; doubling time, approximately 2 h). The resulting level is notably higher (>6-fold) than in all other tissues examined, none of which exhibit a robust daily rhythm. The increase in PDE4B2 mRNA is followed by increases in PDE4B2 protein and PDE4 enzyme activity. Results from in vivo and in vitro studies indicate that these changes are due to activation of adrenergic receptors and a cAMP-dependent protein kinase A mechanism. Inhibition of PDE4 activity during the late phase of adrenergic stimulation enhances cAMP and melatonin levels. The evidence that PDE4B2 plays a negative feedback role in adrenergic/cAMP signaling in the pineal gland provides the first proof that cAMP control of PDE4B2 is a physiologically relevant control mechanism in cAMP signaling." From (1) Control of circadian rhythms by the Ptprn and Ptprn2. We have participated in an effort to describe the role that two synaptic vesicle proteins play in circadian biology. "We have found that synaptic vesicle proteins islet antigen 2(IA-2, Ptprn) and islet antigen 2-beta (IA-2-B, Ptprn2) are essential for circadian rhythms in activity, blood pressure, heart rate and temperature. IA-2 and IA-2-B are expressed in the suprachiasmatic nucleus (SCN), the master circadian oscillator in mammals the SCN of animals lacking these genes exhibit patterns of electrical activity which indicate that electrical activity of the SCN is not coherently rhythmic and that total activity is markedly reduced. IA-2 and IA-2-B may act in the SCN to facilitate cell-cell communication.

整体基因表达分析：有关松果体基因表达特征的研究正在进行中。 第一阶段涉及对大鼠松果体的分析：“利用微阵列基因表达对啮齿类动物的松果体转录组进行了研究。中午和午夜表达谱的比较表明，1000个基因的表达发生了2倍以上的变化，其中2/3在夜间增加。其中，400个基因的表达增加了4倍以上；器官培养研究表明，几乎在所有情况下，高度上调的基因的表达都是通过NE或NE处理诱导的。 环核苷酸类似物。 这些发现与NE-环核苷酸信号传导是夜间基因表达增加的主要机制的结论一致。 然而，也清楚的是，还涉及其他机制，因为少数高节律性基因不会被 NE 处理诱导或弱诱导。 松果体中的基因表达水平与其他组织中的中值表达水平的比较表明 一组超过 300 个基因在松果体中的表达量高出 8 倍以上。 表达最高的基因的一个重要子集编码参与褪黑激素合成和该过程控制的蛋白质，包括通过肾上腺素能受体和第二信使（包括环核苷酸、Ca ++ 和磷脂）发出的信号。 高表达基因簇与甲状腺激素的细胞生物学相关， 视黄酸、谷氨酸生物学；并且，与金属离子稳态、膜运输和免疫反应有关。 其他高度和/或有节奏表达的基因还编码以前未在松果体文献中出现的转录因子、离子通道、转运蛋白、受体、调节分子和分泌产物。 松果体基因表达谱与其他几种组织的比较进一步证明， 松果体与视网膜最相似，因为它们增加了仅在这两种组织中高度表达的基因数量。 这项研究表明，松果体生物学的控制比以前认为的要复杂得多，松果体和视网膜中高表达基因的数量比以前认为的要高，并且还提供了分子证据来怀疑松果体可能在褪黑激素产生中发挥的高度保守作用之外发挥作用。” 来自（Bailey 等人，准备中）。 对啮齿动物松果体的研究正在进行中，对猴子和人类松果体进行类似的研究，以确定这三种组织中基因表达模式的相似性。 对啮齿动物松果体的分析结果引发了一系列研究，其中一些研究已经发表，这些研究重点关注微阵列研究强调的基因。 HD000095-37 中详细介绍了一个示例。 控制松果体中的多巴胺信号转导：多巴胺通过特定 G 蛋白偶联受体介导的作用在生物学中发挥着广泛的作用。 “我们发现，编码多巴胺 D4 受体 (Drd4) 的基因的表达可以迅速变化。松果体和视网膜中的 Drd4 mRNA 在夜间增加 20 倍，达到比其他组织高 10 倍以上的水平。松果体 Drd4 转录物的丰度由控制松果体功能的昼夜节律调节系统控制。体外研究表明，Drd4 由 And 门机制诱导，该机制由肾上腺素/环 AMP 信号传导激活，并且依赖于甲状腺激素 (T3)。 这些发现表明多巴胺/Drd4 信号在松果体和视网膜中发挥着重要作用。 在更一般的层面上，认为其他组织中的多巴胺/D4R 信号传导可以反映环 AMP 和 T3 的相互作用似乎是合理的。” （来自 Kim 等人） 成年和发育中的大鼠视网膜中多巴胺受体 D4 表达的定位和调节：“多巴胺和褪黑激素的水平在大鼠视网膜中表现出昼夜节律。多巴胺在白天含量较高，使视网膜适应光线，而褪黑激素在夜间含量较高，参与视网膜对低光强度的适应。多巴胺抑制 光感受器中的褪黑激素通过位于这些细胞的细胞膜上的 Drd4 受体发挥作用。在这项研究中，我们通过半定量原位杂交显示了 Sprague Dawley 大鼠视网膜中 Drd4 表达的显着昼夜变化，并在夜间达到峰值。 Drd4 表达见于所有视网膜层，但夜间增加仅限于光感受器。视网膜Drd4 Drd4 的表达不受去除眼睛交感神经输入的影响，但三碘甲状腺原氨酸治疗会诱导 Drd4 在光感受器中的表达。在发育系列中，我们表明 Drd4 的表达仅限于出生后阶段，在出生后第 12 天达到峰值。夜间大鼠视网膜光感受器中 Drd4 的高表达支持生理学和药理学证据，即 Drd4 受体 参与光刺激下褪黑激素合成的多巴胺能抑制。 Drd4 表达在出生后特定时间急剧增加表明多巴胺参与视网膜发育。” 控制环磷酸腺苷降解：“松果体是一种光神经内分泌传感器，通过褪黑激素产生和释放的日常节律影响许多生理功能的昼夜节律和昼夜动态。褪黑激素的合成在夜间受到光神经系统的刺激，通过该系统肾上腺素能激活松果体腺苷酸环化酶，导致 cAMP 升高。cAMP 增强 通过作用于生物合成途径的几个元件来合成褪黑激素。由于磷酸二酯酶 (PDE) 活性增加（在半夜达到峰值），cAMP 降解似乎也在夜间增加。在这里，我们发现 PDE 活性的夜间增加是由于 PDE4B2 mRNA 丰度增加（约 5 倍；倍增时间，约 2 小时）所致。的 由此产生的水平明显高于（> 6倍）所有其他检查的组织，其中没有一个表现出强大的每日节律。 PDE4B2 mRNA 增加后，PDE4B2 蛋白和 PDE4 酶活性也增加。体内和体外研究的结果表明，这些变化是由于肾上腺素受体的激活和 cAMP 依赖性蛋白激酶 A 机制所致。抑制 肾上腺素能刺激后期 PDE4 活性的增加会增强 cAMP 和褪黑激素水平。 PDE4B2 在松果体肾上腺素能/cAMP 信号传导中发挥负反馈作用的证据首次证明 cAMP 对 PDE4B2 的控制是 cAMP 信号传导中生理相关的控制机制。”来自 (1) Ptprn 和 Ptprn2 控制昼夜节律。 我们参与了描述两种突触小泡蛋白在昼夜节律生物学中发挥的作用的努力。 “我们发现突触小泡蛋白胰岛抗原 2（IA-2、Ptprn）和胰岛抗原 2-β（IA-2-B、Ptprn2）对于活动、血压、心率和体温的昼夜节律至关重要。IA-2 和 IA-2-B 在视交叉上核 (SCN) 中表达，视交叉上核是哺乳动物的主要昼夜节律振荡器，即动物的 SCN 缺乏这些基因会表现出电活动模式，这表明 SCN 的电活动节奏不连贯，并且总活动显着降低。 IA-2和IA-2-B可以在SCN中起作用以促进细胞间通信。