Biosynthesis, Processing And Secretion Of Neuropeptides And Pituitary Hormones

神经肽和垂体激素的生物合成、加工和分泌

• 批准号: 7968441
• 负责人: Yoke p Loh
• 金额: $ 102.57万
• 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/7968441
• 关键词: Actins; Adult; Affect; Age; Alzheimer' s Disease; Amino Acids; Anabolism; Animals; Antisense RNA; Apoptosis; Area; Behavioral Assay; Binding; Biogenesis; Biological Assay; Birth; Body Weight decreased; Bone Density; Brain; Brain-Derived Neurotrophic Factor; C-terminal; Cell Line; Cell Nucleus; Cell membrane; Cells; Chromogranin A; Collaborations; Corticotropin; Cyclic AMP; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Tail; Cytoskeleton; Cytosol; Defect; Dense Core Vesicle; Development; Diabetes Mellitus; Disease; Docking; Dynein ATPase; Eating; Electrons; Endocrine; Endocrine system; Energy Metabolism; Enzymes; Event; Exclusion; F-Actin; Fluorescence; Gene Expression; Generations; Genes; Genetic Transcription; Glutamates; Golgi Apparatus; Hippocampus (Brain); Hormones; Hydrogen Peroxide; Hypothalamic structure; In Vitro; Infant; Insulin; Insulin Receptor; Laboratories; Lead; Learning; Life; Link; Long-Term Potentiation; Measurement; Mediating; Mediator of activation protein; Memory; Messenger RNA; Microscopic; Microtubules; Modeling; Motor; Movement; Mus; Mutation; Names; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neurons; Neuropeptides; Nuclear; Obesity; Organelles; Osteogenesis; Oxidative Stress; PC12 Cells; Pathway interactions; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Physiological; Pituitary Gland; Pituitary Hormones; Play; Population; Pro-Opiomelanocortin; Process; Proinsulin; Proprotein Convertase 2; Protease Inhibitor; Proteins; Role; Secretory Vesicles; Signal Transduction; Slice; Sorting - Cell Movement; Stimulus; Stress; Structure of nucleus infundibularis hypothalami; Study models; Synapses; Synaptic Membranes; Synaptic Vesicles; Synaptic plasticity; Synaptophysin; System; Tail; Testing; Total Internal Reflection Fluorescent; Universities; Vesicle; base; brain cell; carboxypeptidase H; cellular imaging; critical period; density; dynactin; gamma-adducin; in vivo; inhibitor/antagonist; maternal separation; membrane activity; morris water maze; neuronal cell body; neuronal survival; neuroprotection; neurotrophic factor; nexin; novel; overexpression; peptide hormone; preference; presynaptic; prevent; prohormone; protein degradation; receptor; rim 1; secretion process; transcription factor

We have investigated the mechanism of post-Golgi transport and delivery of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion which is critical for endocrine function and synaptic plasticity. We showed that the CPE cytoplasmic tail on these secretory vesicles plays an important role in their transport. Overexpression of the CPE cytoplasmic tail in the cytoplasm to compete with the endogenous tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of an endocrine cell line, AtT20; and hippocampal neurons. In live hippocampal neurons, primary pituitary and AtT20 cell images, overexpression of the CPE tail inhibited the movement of BDNF- and POMC/CPE-containing vesicles to the processes, respectively. S-tagged CPE tail pulled down endogenous microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from cytosol of AtT20 and brain cells. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. We also showed that the CPE tail interacted with C-terminus of gamma-adducin, a component of the cytoskeleton that binds and stabilizes F-actin. Overexpression of the C-terminal 38 amino acid of gamma-adducin inhibited the transport of POMC vesicles out of the cell body into the processes of AtT-20 cells. Thus these studies demonstrate that the vesicular CPE cytoplasmic tail plays a novel mechanistic role in anchoring regulated secretory pathway POMC/ACTH and BDNF vesicles to actin via gamma-adducin for movement immediately after budding from the TGN which is actin-based, and subsequently to the microtubule-based motor system for transport along the processes to the plasma membrane for activity-dependent secretion in endocrine cells and neurons. We recently demonstrated that transmembrane CPE is not only associated with large dense core vesicles, but also with glutamate-containing synaptic vesicles in mouse hypothalamus. High K+ stimulated release of glutamate from cultured hypothalamic neurons was absent in CPE-KO mice. Furthermore, electron microscopic analysis of 100 hypothalamic synaptic densities revealed that 40% of the synapses had no docked synaptic vesicles at the presynaptic density in CPE-KO mice in contrast to 15% for the WT mice, implicating that in some neurons, CPE may be involved in synaptic vesicle tethering/docking, possibly mediated by its cytoplasmic tail. Indeed, in vitro GST pulldown assays using both brain and PC12 cell cytosol, GST tagged CPEC10, the CPE cytoplasmic tail, bound Rab27A, Rab3A and Rim1, molecules necessary for synaptic vesicle tethering, but not munc18 required for docking to the synaptic membrane. Furthermore TIRF microscopy showed that expression of GFP-tagged CPE tail in PC12 cells reduced the steady-state level of the total number of dynamically mobile synaptophysin-mRFP synaptic-like vesicles in the sub-plasma membrane area. Taken together these findings have uncovered a new mediator, the CPE cytoplasmic tail, in synaptic vesicle tethering at the plasma membrane in a sub-population of hypothalamic neurons and in PC12 cells, through interaction with Rim 1, Rab27A and Rab3A. The mechanism regulating biogenesis of hormone/neuropeptide containing large dense-core secretory granule (LDCG) to replenish these organelles after stimulus-coupled secretion was investigated. We previously showed that chromogranin A (CgA) plays a critical role in the control of LDCG formation and that the mechanism was through the control of granule protein degradation within the Golgi by regulating the level of a protease inhibitor. We have identified the inhibitor as protease nexin-1 (PN-1) which expression was up-regulated transcriptionally by CgA (or a fragment of CgA). PN-1 prevented granule protein degradation and increased LDCG formation when up-regulated, but when reduced in its expression by PN-1 antisense-RNA, the proteins were degraded and LDCGs were not made. We recently identified a C-terminal fragment of CgA, which we named serpinin that was able to enhance PN-1 transcription and granule biogenesis in 6T3 cells, an endocrine cell line that normally lacks LDCGs. Serpinin was elevated in the medium after high K+ stimulation of AtT20 cells. We further demonstrated that serpinin increases cAMP presumably by binding to a cognate receptor on the plasma membrane to cause signaling to the nucleus to enhance PN-1 mRNA transcription. Thus we have discovered a new CgA-derived peptide, serpinin, which is co-secreted with POMC-derived hormones upon stimulation of pituitary corticotrophs. Serpinin signals replenishment of LDCGs by transcriptionally up-regulating the expression of the protease inhibitor, PN-1 which then stabilizes granule proteins, resulting in increased LDCG biogenesis. Recently, we have investigated the role of CPE in the nervous system in vivo. We showed that CPE KO mice were not able to process pro-CART to CART and therefore lacked this anorexigenic neuropeptide, in the hypothalamus. These animals over-eat, thus providing further evidence linking decrease of this neuropeptide to the cause of obesity. Additionally, in collaboration with the Accili group at Columbia University, it was found that the transcription factor FoxO1 negatively regulates CPE gene expression. Normally insulin binds to insulin receptors in the POMC neurons and that leads to nuclear signaling, nuclear exclusion and inactivation of FoxO1. To model this physiological event, FoxO1 was deleted in the POMC neurons in the arcuate nucleus of the hypothalamus in mice and that resulted in increased CPE levels, increased -MSH, an anorexigenic neuropeptide derived from POMC, and reduced food intake without change in energy expenditure. These findings raise the possibility of targeting CPE to develop weight loss medications. Also we showed that the extremely obese CPE KO mice have low bone mineral density, contrary to dogma that obesity is always associated with high bone mineral density. We further concluded that the lack of CART which promotes bone formation, is an important player responsible for poor bone density in these mice. Also we demonstrated deficiencies in several behavioral assays including the Morris water maze and object preference tests indicating a problem with learning and memory in CPE-KO mice. We showed that in 6-14 week old CPE-KO mice, dendritic pruning was inhibited in cortical and hippocampal neurons which would affect synaptogeneis. Additionally electrophysiological measurements showed a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. A major cause for this defect was due to the total degeneration of neurons in the CA3 region of the hippocampus. Interestingly, this was evident only in 4 week and older CPE-KO mice. Three week old KO animals were normal, suggesting that CPE is important in maintaining the survival of CA3 neurons which are enriched in this enzyme, after 3 weeks of age. These results have therefore uncovered a critical period between 3 and 4 weeks after birth when the CA3 neurons are highly sensitive to stress such as maternal separation, and that CPE is required to maintain survival of these neurons after 3 weeks of age. Indeed, we showed that when CPE was overexpressed in hippocampal neurons in culture, they were protected from apoptosis after induced oxidative stress using hydrogen peroxide. Thus, CPE has a novel neuroprotective role in adult hippocampal neurons. The CPE-KO mouse is therefore an excellent model for studying the critical period of CA3 neuronal survival and the effects of early maternal separation on infant brain development.

我们研究了高尔基体后转运和将激素和 BDNF 囊泡递送至质膜进行活性依赖性分泌的机制，这对于内分泌功能和突触可塑性至关重要。我们发现这些分泌囊泡上的 CPE 胞质尾在它们的运输中发挥着重要作用。细胞质中 CPE 胞质尾部的过度表达与内源性尾部竞争会减少内分泌细胞系 AtT20 过程中内源性 POMC、BDNF 和荧光标记 CPE 的定位；和海马神经元。在活海马神经元、初级垂体和 AtT20 细胞图像中，CPE 尾部的过度表达分别抑制含有 BDNF 和 POMC/CPE 的囊泡向突起的运动。 S 标记的 CPE 尾部从 AtT20 和脑细胞的胞质溶胶中拉下基于内源性微管的马达、动力蛋白 (p150)、动力蛋白和 KIF1A/KIF3A。最后，CPE 尾部的过度表达抑制了 AtT20 细胞 ACTH 的调节分泌。我们还表明，CPE 尾部与γ-内收蛋白的 C 末端相互作用，γ-内收蛋白是结合并稳定 F-肌动蛋白的细胞骨架的一个组成部分。 γ-内收蛋白 C 端 38 个氨基酸的过度表达抑制 POMC 囊泡从细胞体转运到 AtT-20 细胞的过程中。因此，这些研究表明，囊泡 CPE 细胞质尾在通过 γ-内收蛋白将受调节的分泌途径 POMC/ACTH 和 BDNF 囊泡锚定到肌动蛋白中发挥着新的机制作用，以便在从基于肌动蛋白的 TGN 出芽后立即运动，并随后运动到肌动蛋白。基于微管的运动系统，用于沿着过程运输到质膜，以在内分泌细胞和神经元中进行活动依赖性分泌。 我们最近证明，跨膜 CPE 不仅与大而致密的核心囊泡相关，而且还与小鼠下丘脑中含谷氨酸的突触囊泡相关。在 CPE-KO 小鼠中，培养的下丘脑神经元不存在高 K+ 刺激的谷氨酸释放。此外，对 100 个下丘脑突触密度的电子显微镜分析显示，CPE-KO 小鼠中 40% 的突触在突触前密度处没有停靠的突触小泡，而 WT 小鼠的这一比例为 15%，这表明在某些神经元中，CPE 可能是参与突触小泡束缚/对接，可能由其细胞质尾介导。事实上，使用脑和 PC12 细胞胞浆进行体外 GST 下拉测定，GST 标记的 CPEC10（CPE 胞质尾）结合 Rab27A、Rab3A 和 Rim1（突触小泡束缚所需的分子），但不结合突触膜所需的 munc18。此外，TIRF 显微镜显示，PC12 细胞中 GFP 标记的 CPE 尾部的表达降低了质膜下区域动态移动的突触素-mRFP 突触样囊泡总数的稳态水平。总而言之，这些发现揭示了一种新的介质，即 CPE 细胞质尾，它通过与 Rim 1、Rab27A 和 Rab3A 相互作用，束缚在下丘脑神经元亚群和 PC12 细胞质膜上的突触小泡中。 研究了调节含有大致密核心分泌颗粒（LDCG）的激素/神经肽生物合成的机制，以在刺激耦合分泌后补充这些细胞器。我们之前表明，嗜铬粒蛋白 A (CgA) 在控制 LDCG 形成中起着关键作用，其机制是通过调节蛋白酶抑制剂的水平来控制高尔基体内颗粒蛋白的降解。我们已将抑制剂鉴定为蛋白酶 nexin-1 (PN-1)，其表达通过 CgA（或 CgA 片段）转录上调。当PN-1上调时，PN-1可防止颗粒蛋白降解并增加LDCG形成，但当PN-1反义RNA减少其表达时，蛋白质被降解并且不产生LDCG。我们最近鉴定了 CgA 的 C 末端片段，将其命名为丝氨酸蛋白酶抑制剂，它能够增强 6T3 细胞（一种通常缺乏 LDCG 的内分泌细胞系）中的 PN-1 转录和颗粒生物发生。高 K+ 刺激 AtT20 细胞后，培养基中的丝氨酸蛋白酶抑制剂升高。我们进一步证明，丝氨酸蛋白酶抑制剂可能通过与质膜上的同源受体结合，向细胞核发出信号，从而增强 PN-1 mRNA 转录，从而增加 cAMP。因此，我们发现了一种新的 CgA 衍生肽，丝氨酸蛋白酶抑制剂，它在刺激垂体促肾上腺皮质激素后与 POMC 衍生激素共同分泌。丝氨酸蛋白酶抑制剂通过转录上调蛋白酶抑制剂 PN-1 的表达来发出 LDCG 补充的信号，PN-1 随后稳定颗粒蛋白，从而增加 LDCG 生物发生。 最近，我们研究了CPE在体内神经系统中的作用。我们发现，CPE KO 小鼠无法将 pro-CART 加工成 CART，因此下丘脑中缺乏这种抑制食欲的神经肽。这些动物吃得过多，从而提供了进一步的证据，证明这种神经肽的减少与肥胖的原因有关。此外，与哥伦比亚大学 Accili 小组合作，发现转录因子 FoxO1 负向调节 CPE 基因表达。通常，胰岛素与 POMC 神经元中的胰岛素受体结合，导致核信号传导、核排斥和 FoxO1 失活。为了模拟这一生理事件，小鼠下丘脑弓状核的 POMC 神经元中的 FoxO1 被删除，导致 CPE 水平增加、α-MSH（一种源自 POMC 的厌食神经肽）增加，并在能量不变的情况下减少食物摄入量支出。这些发现提出了针对 CPE 开发减肥药物的可能性。我们还发现，极度肥胖的 CPE KO 小鼠骨矿物质密度较低，这与肥胖总是与高骨矿物质密度相关的教条相反。我们进一步得出结论，缺乏促进骨形成的 CART 是导致这些小鼠骨密度差的重要因素。我们还证明了一些行为测定的缺陷，包括莫里斯水迷宫和对象偏好测试，表明 CPE-KO 小鼠的学习和记忆存在问题。我们发现，在 6-14 周龄的 CPE-KO 小鼠中，皮质和海马神经元的树突修剪受到抑制，这会影响突触发生。此外，电生理学测量显示这些小鼠的海马切片中长时程增强（LTP）的产生存在缺陷。造成这种缺陷的主要原因是海马 CA3 区域神经元的完全退化。有趣的是，这一现象仅在 4 周及以上的 CPE-KO 小鼠中表现得明显。三周龄的 KO 动物是正常的，这表明 CPE 对于维持 3 周龄后富含这种酶的 CA3 神经元的存活很重要。因此，这些结果揭示了出生后 3 至 4 周之间的关键时期，此时 CA3 神经元对母体分离等压力高度敏感，并且需要 CPE 来维持 3 周龄后这些神经元的存活。事实上，我们发现，当 CPE 在培养的海马神经元中过度表达时，在使用过氧化氢诱导氧化应激后，它们可以免受细胞凋亡的影响。因此，CPE 对成人海马神经元具有新的神经保护作用。因此，CPE-KO小鼠是研究CA3神经元存活关键期以及早期母体分离对婴儿大脑发育影响的优秀模型。