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: 9150040
• 负责人: Yoke p Loh
• 金额: $ 93.42万
• 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/9150040
• 关键词: A kinase anchoring protein; Adult; Affect; Agonist; Alzheimer' s Disease; Anabolism; Anterior Pituitary Gland; Antidiabetic Drugs; Astrocytes; BCL2 gene; Behavior; Binding; Binding Sites; Brain; C-terminal; Calcineurin; Calnexin; Cardiac; Cell Culture Techniques; Cell Death; Cells; Chromogranin A; Collaborations; Complex; Contracture; Corticotropin; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Cytoplasmic Tail; Defect; Dementia; Development; Diabetes Mellitus; Disease; Down-Regulation; Dynein ATPase; Embryo; Embryonic Development; Endocrine; Endocrine system; Enzymes; Exhibits; Fibroblast Growth Factor 2; Genes; Glutamates; Golgi Apparatus; Hippocampus (Brain); Hormones; Human; Hydrogen Peroxide; In Situ Hybridization; Inbred SHR Rats; Inbred WKY Rats; Infarction; Investigation; Ischemia; Kinesin; Laboratories; Lead; Left ventricular structure; Link; Medial; Mediating; Membrane; Memory impairment; Mental Depression; Messenger RNA; Microtubules; Mitochondrial Proteins; Modeling; Molecular; Motor; Movement; Mus; Mutant Strains Mice; Mutation; Neocortex; Nerve Degeneration; Nervous system structure; Neurites; Neurodegenerative Disorders; Neurons; Neuropeptides; Obesity; Oligodendroglia; Organelles; Oxidative Stress; PC12 Cells; PPAR gamma; Parahippocampal Gyrus; Pathway interactions; Patients; Peptides; Phosphotransferases; Physiological; Pituitary Gland; Pituitary Hormones; Play; Population; Prefrontal Cortex; Pro-Opiomelanocortin; Process; Proprotein Convertase 1; Proprotein Convertase 2; Prosencephalon; Protein Isoforms; Proteins; RNA Interference; RNA Splicing; Recovery of Function; Regulation; Reperfusion Injury; Role; SNAPIN gene; Scaffolding Protein; Secretory Vesicles; Signal Transduction; Site; Somites; Sorting - Cell Movement; Staining method; Stains; Stem cells; Stress; Tail; Testing; Transgenic Mice; Transport Vesicles; Up-Regulation; Variant; Vesicle; Work; beta catenin; carboxypeptidase H; conditioning; dentate gyrus; depression prevention; depressive symptoms; dynactin; extracellular; falls; improved; knock-down; learning ability; mRNA Expression; morris water maze; multicatalytic endopeptidase complex; mutant; nerve stem cell; neurodevelopment; neurogenesis; neuron loss; neuroprotection; neurotoxicity; neurotrophic factor; normotensive; novel; overexpression; peptide hormone; prevent; prohormone; promoter; receptor; restraint stress; rho; rosiglitazone; rosin; secretogranin III; tau Proteins; trafficking

We continue to investigate the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway (RSP). Both proteins have been shown to be capable of trafficking POMC into the RSP, a mechanism that may reflect multiple components working together to accomplish the very important task of an endocrine cell, that being the controlled regulated secretion of bioactive peptide hormones. Using RNA interference to knock down SgIII and CPE, we showed that both proteins affect the normal secretion of POMC in AtT20 cells, i.e. POMC was secreted at an elevated rate through the constitutive secretory pathway when either CPE or SgIII are reduced. When both are knocked-down, the affect is augmented, suggesting that POMC trafficking is dependent on both proteins for efficient trafficking to the RSP for subsequent processing to ACTH. With Dr. Josh Park, Uni. of Toledo, we showed that snapin connects a microtubule motor complex consisting of kinesin-2, cytoplasmic dynein, and dynactin to the cytoplasmic tail of CPE on POMC vesicles to mediate their transport in anterior pituitary AtT-20 cells. Snapin directly binds to CPE cytoplasmic tail and interacts with microtubule motors. Overexpression of snapin reduced process-localization, processivity and velocity of movement of ACTH/POMC vesicles, similar to overexpression of CPE C-terminal tail. Knockdown of snapin decreased stimulated ACTH secretion. Moreover, A kinase anchor protein 150 (AKAP150), a scaffold for protein kinase A and calcineurin, associate with snapin-microtubule motor complex to facilitate the process-localization of ACTH/POMC vesicles. Thus, our study uncovered a new molecular complex that mediates and possibly regulates post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells. With Dr. Bruno Tota (Uni. of Calabria), we have investigated the effect of pGlu-serpinin, a CgA-derived peptide, on cardio-protection. Using normotensive (WKY) and hypertensive (SHR) rats as models; we showed that pGlu-serpinin mimicked pre-conditioning and post-conditioning-induced cardioprotection. In both WKY and SHR rats, pGlu-serpinin improved left ventricle function recovery after ischemia. Moreover, it reduced ischemic induced contracture state and decreased infarct size. In pGlu-serpinin mediated post-conditioning pharmacological cardiac protection, the mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. Our recent work showing the negative regulation of Wnt-3a by CPE in collaboration with Dr. Rina Rosin-Arbersfeld spurred the investigation of neurite outgrowth in PC12 cells and cortical neurons by Wnt-3a and Wnt-5a and the role CPE/NF-alpha1 may play. We have shown that CPE/NF-alpha1 can modulate the NGF-induced neurite outgrowth by reducing B-catenin in the canonical Wnt-3a pathway as well as Rho, an effector of the Wnt-3a non-canonical pathway. Interestingly, we showed that Wnt-5a can complex with CPE and induce neurite outgrowth which can be enhanced by NGF. Recently we have investigated NF-alpha1 in preventing restraint stress-induced depression. Prolonged (6h/d for 21 days), but not short-term (1h/d for 7d) restraint stress reduced fibroblast growth factor 2 (FGF2) in the hippocampus, leading to depressive-like behavior in mice. We found that mice after short-term restraint stress increased hippocampal NF-alpha1, FGF2 and doublecortin, a marker for immature neurons, suggesting increased neurogenesis. Indeed we showed that in cultured hippocampal neurons, exogenous NF-alpha1 could increase FGF2 expression. Moreover, NF-alpha1-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the subgranular zone. These mice displayed depressive-like behavior that was rescued by FGF2 administration. Thus, NF-alpha1 prevents stress-induced depression by up-regulating hippocampal FGF2 expression which leads to enhanced neurogenesis and anti-depressant activity. Analysis of the CPE promoter identified potential PPARgamma binding sites that could induce its expression. We showed that rosiglitazone, a PPARgamma agonist and anti-diabetic drug with additional anti-depression activities, could induce the expression of CPE/NF-alpha1 in Neuro2a cells and hippocampal neurons. The induced NF-alpha1 protected the cells against oxidative stress induced by hydrogen peroxide through up-regulation of BCL-2, a pro-survival mitochondrial protein. The expression of NF-alpha1 and a splice variant isoform, CPE-deltaN was examined in mouse embryos to determine if it could play a role in development and neuroprotection of embryos. We found that WT CPE and CPE-deltaN mRNA was expressed as early as day E5.5 and increased each day, peaking at E8.5, falling slightly at E9.5 prior to expression of the endocrine system. CPE mRNA expression decreased sharply at E 10.5-11.5 to below E5.5 levels and then increased sharply at E12.5 in parallel with the development of the endocrine system and continued to increase to adulthood. However, CPE-deltaN mRNA increased maximally at E10.5 followed by a decrease at E11.5-12.5, and then a small increase till PN1. In contrast to NF-alpha1, CPE-deltaN is virtually absent in the adult brain. In situ hybridization studies indicate that WT CPE and CPEdeltaN mRNA are expressed primarily in the fore brain and somites in mouse embryos. We showed that CPE-deltaN plays a role in protecting embryonic cortical and hippocampal neurons from glutamate neurotoxicity and oxidative stress through up-regulating FGF2 expression which then increased the pro-survival protein, BCl2. We also studied the role of NF-alpha1 during embryonic development of the nervous system using neurospheres to study proliferation and differentiation. Exogenous addition of NF-alpha1 to E13.5 neocortex-derived neurospheres, which contains stem cells and neuroprogenitors, resulted in reduced proliferation of the neurospheres without causing cell death. NF-alpha1 down-regulated the wnt-pathway in the neurospheres leading to reduced levels of beta-catenin which is known to enhance proliferation. Differentiation studies using neurospheres from 7d cultures that were dissociated into single cells and cultured for an additional 5d showed an increase in astrocytes in the presence of NF-alpha1, without altering the percentage of neuronal and oligodendrocyte populations. Interestingly, dissociated cells from neurospheres derived from NF-alpha1-KO mouse embryos showed decreased astrocytes and increased neurons. Our results suggest a novel role of NF-alpha1 as an extracellular signal to differentiate neural stem cells into astrocytes. We identified a mutant CPE from the cortex of an Alzheimer Disease (AD) patient which we call CPE-QQ. When expressed in Neuro2a cells it was not secreted but degraded by the proteosome. Immunocytochemical studies showed CPE-QQ co-stained with Calnexin, an ER marker and overexpression in hippocampal neurons increased levels of ER stress marker CHOP, decreased levels of pro-survival protein, BCL-2, and increased neuronal cell death. This indicates that CPE-QQ induces cell death through ER stress and down regulation of BCL-2. Transgenic mice overexpressing CPE-QQ exhibited memory deficits as tested by the Morris water maze but their spatial learning ability was unimpaired. These mutant mice showed less neurites in the CA3 region and the dentate gyrus of the hippocampus and the medial prefrontal cortex, indicative of neurodegeneration. Moreover they showed diminished neurogenesis in the subgranular zone and hyperphosphorylation of tau at ser395, a hallmark of AD. These studies have substantiated a neuroprotective role of CPE/NF-1 in humans and identified a new gene, CPE/NF-alpha1, with a mutation that can cause neurodegeneration linked to AD and other dementia.

我们继续研究膜CPE和分泌蛋白III作为将POMC靶向受调节分泌途径（RSP）的分类受体的作用。两种蛋白质都已证明能够将POMC运输到RSP中，这种机制可能反映了多种组件，以完成内分泌细胞的非常重要的任务，该任务是受控调节的生物活性肽激素的分泌。利用RNA干扰击倒SGIII和CPE，我们表明两种蛋白质都会影响att20细胞中POMC的正常分泌，即当CPE或SGIII减少时，POMC通过本构分泌途径以较高的速率分泌。当两者都被击倒时，影响会增加，这表明POMC运输取决于两种蛋白质，以有效地运输到RSP，以便随后加工到ACTH。 与Uni的Josh Park博士在一起。在托莱多（Toledo），我们表明Snapin连接了由驱动蛋白-2，细胞质动力蛋白和DynActin组成的微管运动复合物与POMC囊泡上CPE的细胞质尾巴介导，以介导其在垂体putitit att-20细胞中的转运。 Snapin直接与CPE细胞质尾巴结合，并与微管电机相互作用。 Snapin的过表达降低了ACTH/POMC囊泡运动的过程 - 定位，加工性和速度，类似于CPE C末端尾巴的过表达。 Snapin的敲低减少了刺激的ACTH分泌。此外，激酶锚固蛋白150（AKAP150）是蛋白激酶A和钙调蛋白的支架，与Snapin-Microubule运动复合物相关，以促进Acth/POMC囊泡的过程 - 定位。因此，我们的研究发现了一种新的分子复合物，该复合物介导并可能调节Acth/POMC囊泡在ATT20细胞的过程末端的高尔基体后运输。 与Bruno Tota博士（Calabria的Uni。），我们研究了CGA衍生的肽PGLU-Serpinin对心脏保护的影响。使用正常的（WKY）和高血压（SHR）大鼠作为模型；我们表明，PGLU-辛替肽模仿了前调节和调节后诱导的心脏保护。在WKY和SHR大鼠中，PGLU-Serpinin在缺血后改善了左心室功能恢复。此外，它减少了缺血性诱导的染色状态并降低了梗塞大小。在PGLU-盐酸素介导的调节后药理学心脏保护中，该机制涉及重新灌注损伤刺激性激酶（风险）途径的激活。 我们最近的工作表明，CPE与Rina Rosin-Arbersfeld博士合作对WNT-3A负面调节，促使对PC12细胞中神经突的生长和Wnt-3A和WNT-5A的皮质神经元以及CPE/NF-Alpha1的作用进行了研究。我们已经表明，CPE/NF-Alpha1可以通过减少规范WNT-3A途径中的B-catenin以及Rho来调节NGF诱导的神经突生长，Rho是WNT-3A非canonical途径的效果。有趣的是，我们表明Wnt-5a可以与CPE复合并诱导神经突生长，而NGF可以增强。 最近，我们研究了NF-Alpha1在防止压力诱发的抑郁症方面。长时间（6h/d持续21天），但没有短期（7D）约束应力降低了海马的成纤维细胞生长因子2（FGF2），导致小鼠的抑郁样行为。我们发现，短期约束应力后的小鼠增加了海马NF-Alpha1，FGF2和Doublecortin，这是未成熟神经元的标志物，表明神经发生增加。确实，我们表明，在培养的海马神经元中，外源性NF-Alpha1可以增加FGF2的表达。 此外，NF-Alpha1-KO小鼠在亚颗粒区域表现出严重降低的海马FGF2水平和未成熟的神经元数。这些小鼠表现出抑郁症状的行为，这些行为是由FGF2给药救出的。因此，NF-Alpha1通过上调海马FGF2表达来阻止应力诱导的抑郁，从而导致神经发生和抗抑郁活性增强。 对CPE启动子的分析确定了可能诱导其表达的潜在ppargamma结合位点。我们表明，罗格列酮是一种具有其他抗抑郁活性的ppargamma激动剂和抗糖尿病药物，可以诱导神经2A细胞和海马神经元中CPE/NF-Alpha1的表达。诱导的NF-Alpha1通过上调Bcl-2（一种促卵巢线粒体蛋白），保护细胞免受过氧化氢诱导的氧化应激。 在小鼠胚胎中检查了NF-Alpha1和剪接变体同工型，CPE-DELTAN的表达，以确定它是否可以在胚胎的发育和神经保护中起作用。我们发现WT CPE和CPE-DELTAN mRNA早在每天E5.5时表达，并且每天增加，峰值达到E8.5，在表达内分泌系统之前略微下降到E9.5。 CPE mRNA的表达在E 10.5-11.5至E5.5以下的水平下急剧下降，然后在E12.5时与内分泌系统的发展并行急剧增加，并继续增加到成年。然而，在E10.5时，CPE-DELTAN mRNA最大增加，然后在E11.5-12.5下降，然后少量增加直至PN1。与NF-Alpha1相反，成人大脑实际上不存在CPE-Deltan。原位杂交研究表明，WT CPE和CPEDELTAN mRNA主要在小鼠胚胎中的前脑和节点中表达。我们表明，CPE-Deltan通过上调FGF2表达来保护胚胎皮质和海马神经元免受谷氨酸神经毒性和氧化应激的作用，从而增加了促卵巢蛋白Bcl2。 我们还研究了NF-Alpha1在神经系统的胚胎发育过程中使用神经球来研究增殖和分化的作用。将NF-Alpha1添加到E13.5新皮层衍生的神经圈中，其中包含干细胞和神经生殖器，导致神经球的增殖降低而不会导致细胞死亡。 NF-Alpha1在神经球中下调了WNT-Pathway，导致β-catenin水平降低，这已知会增强增殖。使用来自7D培养物的神经球进行分化研究，这些培养物分解为单个细胞并培养以额外的5D培养显示在存在NF-Alpha1的情况下，星形胶质细胞增加，而没有改变神经元和少突胶质细胞种群的百分比。有趣的是，来自NF-Alpha1-KO小鼠胚胎的神经球的解离细胞显示，星形胶质细胞降低并增加了神经元。 我们的结果表明，NF-Alpha1是将神经干细胞区分为星形胶质细胞的细胞外信号的新作用。 我们从阿尔茨海默氏病（AD）患者的皮质中鉴定了一个突变的CPE，我们称之为CPE-QQ。当在Neuro2a细胞中表达时，它不是分泌的，而是被蛋白体降解。免疫细胞化学研究表明，CPE-QQ与钙钙蛋白酶染色，ER标记和海马神经元中的过表达增加了ER应力标记章的水平，促血肠蛋白，BCL-2和神经元细胞死亡的水平降低。这表明CPE-QQ通过ER应力和BCL-2的调节诱导细胞死亡。过表达CPE-QQ的转基因小鼠表现出由莫里斯水迷宫测试的记忆缺陷，但它们的空间学习能力没有受损。这些突变小鼠在CA3区和海马和内侧前额叶皮层的齿状回中显示出较少的神经突。此外，它们显示出在Ser395（AD的标志）下晶状区域的神经发生下降和Tau的高磷酸化。这些研究证实了CPE/NF-1在人类中的神经保护作用，并鉴定了一个新基因CPE/NF-Alpha1，其突变可能会导致与AD和其他痴呆症相关的神经变性。