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: 8149215
• 负责人: Yoke p Loh
• 金额: $ 93.99万
• 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/8149215
• 关键词: Actins; Adenylate Cyclase; Affect; Age; Anabolism; Apoptosis; Area; Binding; Brain-Derived Neurotrophic Factor; Conditioned Culture Media; Corticotropin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Tail; Cytoskeleton; Cytosol; Defect; Diabetes Mellitus; Disease; Dynein ATPase; Eating; Endocrine system; Energy Metabolism; Enzymes; Exocytosis; F-Actin; Fluorescence; Gene Expression; Genes; Glutamates; Golgi Apparatus; Hippocampus (Brain); Hormones; Hydrogen Peroxide; Hypothalamic structure; Insulin; Knockout Mice; Link; Long-Term Potentiation; Mediator of activation protein; Motor; Neuroendocrine Cell; Neuropeptides; Pathway interactions; Pharmaceutical Preparations; Physiological; Pituitary Hormones; Process; RNA Interference; Site; Slice; Small Interfering RNA; Transport Process; Wild Type Mouse; base; dynactin; knockout animal; neurotrophic factor; nexin; secretion process; secretogranin III

We have investigated the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway(RSP). Using our CPE knockout (KO) mouse, we showed that 50% of newly synthesized POMC in primary cultures of the pituitary anterior lobe cells was degraded and suggests that in the absence of efficient sorting to the granules of the RSP due to the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII), which has been shown to bind POMC in precipitation assays. SgIII, is a member of the granins that are found in neuroendocrine cells and is involved in trafficking of chromogranin A (CgA) to the RSP. We used RNA interference (siRNA) to knock down SgIII and CPE expression in AtT20 cells and demonstrated that increased POMC was secreted via the constitutive secretory pathway in both cases. Increased constitutive secretion of CgA was only observed in the SgIII knockdown cells. In double CPE-SgIII knock down cells, increased constitutive secretion of POMC was observed and stimulated secretion of ACTH was perturbed. These results demonstrate that CPE is involved in the trafficking of POMC to the RSP; and that SgIII may play a compensatory role for CPE in the sorting of POMC to the RSP in addition to a more general role in the RSP trafficking process. Transport of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion is critical for endocrine function and synaptic plasticity. We showed that the cytoplasmic tail of a transmembrane form of CPE in hormone or BDNF-containing dense core secretory vesicles plays an important role in their transport to the release site. 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 hippocampal neurons, primary pituitary and AtT20 cells, 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 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 found that transmembrane CPE is not only associated with large dense core vesicles (LDCVs), but also with glutamate-containing synaptic vesicles (SVs) in mouse hypothalamus and synaptic-like microvesicles in PC12 cells. High K+ stimulated release of glutamate from hypothalamic neurons was diminished in CPE-KO mice. Electron microscopy revealed that the number of SVs located in the pre-active zone (within 200nm of the plasma membrane at the active zone) of synapses was significantly decreased in hypothalamic neurons of CPE-KO mice compared with wild-type mice. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells as a model showed that overexpression of the CPE cytoplasmic tail reduced the steady-state level of synaptophysin-containing synaptic-like microvesicles accumulated in the area within 200 nm from the sub-plasma membrane (TIRF zone). Our findings show that the CPE cytoplasmic tail, which interacts with gamma adduccin and actin, is a new mediator for the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells. Our recent studies in pituitary AtT-20 cells have provided evidence for an autocrine mechanism for up-regulating LDCV biogenesis to replenish LDCVs following stimulated exocytosis of these vesicles. The autocrine signal was identified as serpinin, a novel 26 amino acid CgA-derived peptide cleaved from the C-terminal of CgA. Serpinin was first isolated from AtT20 cell conditioned medium and demonstrated to be released in an activity-dependent manner from LDCVs. Subsequently, secreted serpinin was found to activate adenyl cyclase to increase cAMP levels, and protein kinase A in the cell. This then led to the translocation of the transcription factor sp1 from the cytoplasm into the nucleus and an increase in transcription of a protease inhibitor, protease nexin 1 (PN-1), which then inhibited granule protein degradation in the Golgi complex. The stabilization of those proteins increased their levels in the Golgi, resulting in significantly enhanced LDCV formation. CPE plays a significant role in obesity, and recently the gene has been coined an obesity susceptibility gene. 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 and become obese, 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 alpha-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. We also showed that extremely obese CPE-KO mice have low bone mineral density and concluded that the lack of CART which promotes bone formation, is an important player responsible for poor bone density in these mice. CPE-KO mice have deficiencies in the nervous system. Morris water maze and object preference tests indicate a problem with learning and memory. We showed that in 6-14 week old CPE-KO mice, dendritic pruning was poor 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 loss of neurons in the CA3 region of the hippocampus of CPE KO animals observed at 4 weeks of age and older. These neurons, which are normally enriched in CPE, were normal at 3 weeks of age just before the animals were weaned. Interestingly, when weaning was delayed a week, this degeneration was not observed till postnatal week 5 in the CPE KO mice. These results suggest that the degeneration is correlated with the stress of weaning and maternal separation and that CPE is important in maintaining the survival of CA3 neurons during that period. 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 hippocampal neurons.

我们研究了膜 CPE 和促分泌素 III 作为将 POMC 靶向调节分泌途径 (RSP) 的分选受体的作用。使用我们的 CPE 敲除 (KO) 小鼠，我们发现垂体前叶细胞原代培养物中新合成的 POMC 50% 被降解，这表明由于缺乏 CPE，无法对 RSP 颗粒进行有效分选，POMC 是降解的目标。然而，剩余的一些 POMC 被分类到 RSP 中。补偿性分选受体的候选者是 Secretogranin III (SgIII)，它已被证明在沉淀测定中结合 POMC。 SgIII 是神经内分泌细胞中颗粒蛋白的成员，参与嗜铬粒蛋白 A (CgA) 向 RSP 的运输。我们使用 RNA 干扰 (siRNA) 敲低 AtT20 细胞中的 SgIII 和 CPE 表达，并证明在两种情况下，POMC 均通过组成型分泌途径分泌增加。仅在 SgIII 敲低细胞中观察到 CgA 组成型分泌增加。在双 CPE-SgIII 敲低细胞中，观察到 POMC 的组成性分泌增加，并且 ACTH 的刺激分泌受到干扰。这些结果表明 CPE 参与了向 RSP 贩运 POMC； SgIII 除了在 RSP 贩运过程中发挥更一般的作用外，还可能在将 POMC 分类到 RSP 中对 CPE 起到补偿作用。 将激素和 BDNF 囊泡转运至质膜进行活性依赖性分泌对于内分泌功能和突触可塑性至关重要。我们发现，激素或含有 BDNF 的致密核心分泌囊泡中跨膜形式的 CPE 的细胞质尾部在其转运至释放位点的过程中发挥着重要作用。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。在海马神经元、初级垂体和 AtT20 细胞中，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。 我们最近发现跨膜CPE不仅与大致密核心囊泡（LDCV）相关，而且还与小鼠下丘脑中含谷氨酸的突触小泡（SV）和PC12细胞中的突触样微泡相关。在 CPE-KO 小鼠中，高 K+ 刺激的下丘脑神经元谷氨酸释放减少。 电镜观察显示，与野生型小鼠相比，CPE-KO小鼠下丘脑神经元中位于突触前活性区（活性区质膜200nm以内）的SV数量显着减少。以 PC12 细胞为模型的全内反射荧光 (TIRF) 显微镜显示，CPE 胞质尾部的过度表达降低了在距离亚质膜 200 nm 范围内积累的含有突触素的突触样微泡的稳态水平。 TIRF 区域）。我们的研究结果表明，与γ内收蛋白和肌动蛋白相互作用的CPE细胞质尾部是下丘脑神经元富含肌动蛋白的前活性区和PC12细胞的TIRF区中SV定位的新介质。 我们最近对垂体 AtT-20 细胞的研究为自分泌机制提供了证据，该机制可上调 LDCV 生物发生，以在刺激这些囊泡的胞吐作用后补充 LDCV。自分泌信号被鉴定为丝氨酸蛋白酶抑制剂，一种从 CgA C 末端切割的新型 26 氨基酸 CgA 衍生肽。丝氨酸蛋白酶抑制剂首先从 AtT20 细胞条件培养基中分离出来，并被证明以活性依赖性方式从 LDCV 中释放。随后，发现分泌的丝氨酸蛋白酶抑制剂可以激活腺苷酸环化酶，从而增加细胞中的 cAMP 和蛋白激酶 A 水平。这导致转录因子 sp1 从细胞质易位到细胞核，并增加蛋白酶抑制剂蛋白酶 nexin 1 (PN-1) 的转录，从而抑制高尔基复合体中颗粒蛋白的降解。这些蛋白质的稳定增加了它们在高尔基体中的水平，从而显着增强了 LDCV 的形成。 CPE在肥胖中起着重要作用，最近该基因被称为肥胖易感基因。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。这些动物吃得过多并变得肥胖，从而提供了进一步的证据，将这种神经肽的减少与肥胖的原因联系起来。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。我们还表明，极度肥胖的 CPE-KO 小鼠骨矿物质密度较低，并得出结论，缺乏促进骨形成的 CART 是导致这些小鼠骨密度低的一个重要因素。 CPE-KO 小鼠的神经系统存在缺陷。莫里斯水迷宫和物体偏好测试表明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很差，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在 4 周龄及以上观察到的 CPE KO 动物海马 CA3 区神经元丢失。这些神经元通常富含 CPE，在动物断奶前 3 周龄时表现正常。有趣的是，当断奶推迟一周时，直到出生后第 5 周，CPE KO 小鼠才观察到这种退化。这些结果表明，这种退化与断奶和母体分离的压力有关，并且 CPE 对于维持该时期 CA3 神经元的存活很重要。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 对海马神经元具有新的神经保护作用。