Neuropeptides and Carboxypeptidase E/ Neurotrophic Factor-1 in Neural and Cognitive Functions

神经肽和羧肽酶 E/神经营养因子 1 在神经和认知功能中的作用

• 批准号: 10001285
• 负责人: Yoke Peng Loh
• 金额: $ 128.97万
• 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/10001285
• 关键词: A kinase anchoring protein; Adrenergic Agents; Adult; Agonist; Alzheimer' s Disease; Amino Acids; Amphibia; Anterior Pituitary Gland; Antidepressive Agents; Antidiabetic Drugs; Apoptosis; Astrocytes; BCL2 gene; Behavior; Binding; Biogenesis; Brain; C-terminal; Calcineurin; Cell Death; Cell Line; Cell Proliferation; Cell membrane; Cell secretion; Cells; Cessation of life; Chromogranin A; Collaborations; Complex; Corticotropin; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Cytoplasmic Tail; Depressed mood; Diabetes Mellitus; Disease; Down-Regulation; Dynein ATPase; Ear; Embryo; Embryonic Development; Endocrine; Endocrine system; Endoplasmic Reticulum; Energy Metabolism; Enzymes; Exhibits; FGF2 gene; Gene Mutation; Genes; Glial Fibrillary Acidic Protein; Golgi Apparatus; Heart; Hippocampus (Brain); Human; IGFBP2 gene; Impaired cognition; In Vitro; Inbred SHR Rats; Inbred WKY Rats; Infertility; Ischemia; Kinesin; Knock-in Mouse; Knockout Mice; Laboratories; Lead; Learning Disabilities; Left ventricular structure; Medial; Mediating; Memory impairment; Mental Depression; Messenger RNA; Microtubules; Molecular; Motor; Movement; Mus; Mutant Strains Mice; Mutation; Myocardial; N-terminal; Names; Neocortex; Nerve Degeneration; Nervous system structure; Neurites; Neurodegenerative Disorders; Neuroendocrine Cell; Neurologic Deficit; Neurons; Neuropeptides; Neurophysiology - biologic function; Obesity; Oligodendroglia; Organelles; Oxidative Stress; PPAR gamma; Pathway interactions; Patients; Peptides; Performance; Phosphotransferases; Physiological; Play; Population; Prefrontal Cortex; Pro-Opiomelanocortin; Process; Proprotein Convertase 1; Proprotein Convertase 2; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-akt; Recovery of Function; Reperfusion Injury; Reporting; Research; Role; SNAPIN gene; Scaffolding Protein; Secretory Vesicles; Signal Transduction; Site; Sorting - Cell Movement; Stem cells; Stress; Tail; Testing; Time; Transgenic Mice; Transgenic Organisms; Transport Vesicles; Universities; Vesicle; Vesicle Transport Pathway; Weaning; Wild Type Mouse; beta catenin; carboxypeptidase H; cardioprotection; cognitive function; conditioning; dentate gyrus; depressive symptoms; dynactin; embryonic stem cell; endoplasmic reticulum stress; extracellular; forced swim test; heart function; heart pharmacology; improved; in vivo; knock-down; learning ability; maternal separation; migration; morris water maze; mouse model; multicatalytic endopeptidase complex; mutant; nerve stem cell; nervous system development; neurodevelopment; neurogenesis; neuron loss; neuroprotection; neurotoxic; neurotrophic factor; novel; null mutation; overexpression; peptide hormone; postnatal; preconditioning; prevent; prohormone; restraint stress; rosiglitazone; social; stem cell differentiation; tau Proteins; teleost; therapy development; trafficking

We have studied the role of the CPE-cytoplasmic tail in trafficking of secretory vesicles to the plasma membrane for secretion. In collaboration with Dr. Josh Park, University of Toledo, we showed that snapin binds directly to the short cytoplasmic tail of CPE on POMC vesicles and to dynactin which in turn binds to microtubule motors consisting of kinesin-2, or cytoplasmic dynein to mediate their transport wwin anterior pituitary AtT-20 cells. 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 post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells for secretion. With Dr. Bruno Tota (Univ. of Calabria), we investigated the effect of pGlu-serpinin, a CgA-derived peptide, on cardio-protection. pGlu-serpinin mimicked pre-conditioning and post-conditioning-induced cardioprotection in both WKY and SHR rats, as well as improved left ventricle function recovery after ischemia. In pGlu-serpinin mediated post-conditioning pharmacological cardiac protection, the mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. pGlu-serpinin also depressed myocardial performance in teleost and amphibian hearts, thus supporting an evolutionary role of serpinins in sympatho-adrenergic control of the vertebrate heart. Currently, the major focus of our research is on the novel, neurotrophic functions of CPE/NF-alpha1. A human with a null mutation of CPE has been reported to have severe learning disability besides obesity, diabetes and infertility due to lack of CPE. We have also identified a CPE mutation in an Alzheimer Disease (AD) patient which results in a CPE mutant protein with an additional nine amino acids that we named CPE-QQ. When expressed in Neuro2a cells, CPE-QQ was not secreted but degraded by proteosomes. Immunocytochemical studies showed CPE-QQ localized to the endoplasmic reticulum (ER) and overexpression in hippocampal neurons increased ER stress and decreased levels of pro-survival protein, BCL-2, resulting in increased neuronal cell death. Transgenic mice overexpressing CPE-QQ exhibited memory deficits in the Morris water maze test but their spatial learning ability was unimpaired. Moreover, these mice showed depressive-like behavior by the forced swim test. These mutant mice had fewer neurites in the hippocampal CA3 region and the dentate gyrus, and the medial prefrontal cortex, indicative of neurodegeneration. They showed diminished neurogenesis in the subgranular zone and hyperphosphorylation of tau at ser395, a hallmark of AD. These studies indicate that this human mutation in CPE/NF-alpha1 is neurotoxic, and lead to neurodegeneration and cognitive decline. In addition we have found another human mutation, W235R in CPE/NF-alpha1, that caused ER stress and neuronal cell death. Our in vitro studies showed that CPE/NF-alpha1 acts extracellularly as a neurotrophic factor, independent of its enzymatic activity to protect hippocampal neurons against oxidative stress via activation of the ERK- or AKT- pathways to up-regulate BCL-2 expression. The neuroprotective effect of CPE/NF-alpha1 was demonstrated in vivo using a transgenic knock-in mouse model expressing a non-enzymatic form of CPE/NF-alpha1, CPE-E342Q, but no WT form. Knock-out mice lacking CPE/NF-alpha1showed complete degeneration of the hippocampal CA3 region and cognitive dysfunction after social (maternal separation) and physical stress (ear tagging and tail clipping) associated the weaning paradigm. However, the CPE-E342Q mice, although lacking WT-CPE showed no degeneration or cognitive dysfunction after the weaning paradigm, similar to WT mice, indicating that CPE/NF-alpha1could prevent stress-induced hippocampal neurodegeneration and cognitive dysfunction, independent of its enzymatic activity. We also investigated CPE/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. Mice after short-term restraint stress increased hippocampal NF-alpha1, FGF2 and doublecortin, a marker for immature neurons, suggesting increased neurogenesis. NF-alpha1 added to cultured hippocampal neurons, increased 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, CPE/NF-alpha1 prevents stress-induced depression by up-regulating hippocampal FGF2 expression which leads to enhanced neurogenesis and anti-depressant activity. We found that rosiglitazone, a PPARgamma agonist and anti-diabetic drug which has anti-depression activities, induced the expression of CPE/NF-alpha1 and doublecortin expression when fed to mice. Thus PPAR-gamma agonists can be potentially useful anti-depressant drugs. We also studied the role of CPE/NF-alpha1 in embryonic development of the nervous system. We showed that addition of CPE/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. These CPE/NF-alpha1 treated neurospheres showed down-regulation of the wnt pathway protein, beta-catenin, which is known to promote proliferation. Differentiation studies using neurospheres in culture that were dissociated into single cells 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. Furthermore, in vivo studies show that NF-alpha1 KO mice had 49% fewer GFAP+ astrocytes in the neocortex compared to WT-mice at postnatal day 1, the time of astrocytogenesis. Thus, NF-alpha1 plays a critical and novel role as an extracellular signal to differentiate neural stem cells into astrocytes for normal neurodevelopment. Recently, we have cloned three CPE mRNAs in embryonic mouse brain. They were 2.1, 1.9 and 1.73 kb in size, encoding 53kD, 47kD and 40kD proteins representing wild-type(WT) and two N-terminal truncated isoforms (CPE-deltaN), respectively. Interestingly, all these isoforms showed a surge of expression at embryonic E10.5 and at postnatal day 1. While CPE-WT continued to be expressed in adult mouse brain, the CPE-deltaN forms were not. Furthermore, we showed that overexpression of 40kD CPE-deltaN in a mouse hippocampal neuronal cell line and primary cortical neurons up-regulated the expression of Insulin-like growth factor binding protein 2, death associated protein 1 and ephrinA1 which mediate cell proliferation, programmed cell death and neuronal migration, respectively, during neurodevelopment. Our findings emphasize the importance of the CPE-NF-alpha1 in regulating embryonic neurodevelopment and mutations of the gene could lead to various neurological deficits.

我们已经研究了CPE - 胞质尾巴在将分泌囊泡运送到质膜进行分泌方面的作用。在与托莱多大学的Josh Park博士合作，我们表明Snapin直接与POMC囊泡上的CPE的短细胞质尾巴结合，而Dynactin则与Dynactin结合，这反过来又与驱动蛋白-2或由细胞质Dynein组成的微管电动机结合，以介导其运输Wwin Wwin wwin wwin Anterior puituit puituit puituit puituity Att-et-ett-et-ett-et-ett-et-ett-ett-ett-et-20。 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），一种蛋白激酶A的支架，钙调蛋白与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络合物，该复合物介导了ACTH/POMC囊泡在ATT20细胞的过程末端的大胆后运输以进行分泌。 与Bruno Tota博士（卡拉布里亚大学）一起，我们研究了CGA衍生肽PGLU-Serpinin对心脏保护的影响。 PGLU-SERPININ模拟了WKY和SHR大鼠的前调节和调节后诱导的心脏保护，以及缺血后改善的左心室功能恢复。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。 PGLU-Serpinin还抑制了Telest和两栖动物心脏中心肌表现，从而支持Serpinins在脊椎动物心脏的交汇 - 肾上腺素能控制中的进化作用。 目前，我们研究的主要重点是CPE/NF-Alpha1的新型神经营养功能。据报道，由于缺乏CPE而导致的肥胖，糖尿病和不育除外，据报道具有无效CPE突变的人类具有严重的学习障碍。我们还鉴定了阿尔茨海默氏病（AD）患者中的CPE突变，该突变导致CPE突变蛋白，并带有另外九种氨基酸，我们将其命名为CPE-QQ。当在Neuro2a细胞中表达时，CPE-QQ不是分泌的，而是被蛋白体降解。免疫细胞化学研究表明，在海马神经元内定位于内质网（ER）的CPE-QQ会增加ER应力和促寿命蛋白Bcl-2的水平降低，导致神经元细胞死亡增加。过表达CPE-QQ的转基因小鼠在莫里斯水迷宫测试中表现出记忆缺陷，但它们的空间学习能力没有受损。此外，这些小鼠通过强制游泳测试表现出抑郁症状的行为。这些突变小鼠在海马CA3区和齿状回的神经突和内侧前额叶皮层的神经突中较少，表明神经变性。他们显示出在Ser395（AD的标志）下的晶状体区域的神经发生下降和Tau的高磷酸化。这些研究表明，CPE/NF-Alpha1中的这种人突变是神经毒性的，导致神经变性和认知能力下降。此外，我们在CPE/NF-Alpha1中发现了另一种人类突变，W235R导致ER应激和神经元细胞死亡。 我们的体外研究表明，CPE/NF-Alpha1在细胞外充当神经营养因子，与其酶促活性无关，以保护海马神经元通过激活ERK或AKT-途径上调BCL-2表达，以保护海马神经元免受氧化应激。使用表达非酶形式的CPE/NF-ALPHA1，CPE-E342Q的非酶形式的转基因敲门小鼠模型在体内证明了CPE/NF-Alpha1的神经保护作用，但没有WT形式。 缺乏CPE/NF-Alpha1的淘汰小鼠在社交（母体分离）和身体压力（耳朵标记和尾巴剪裁）之后，海马CA3区域和认知功能障碍完全变性。然而，尽管缺乏WT-CPE，但CPE-E342Q小鼠在断奶范式后没有表现出与WT小鼠相似的退化或认知功能障碍，这表明CPE/NF-ALPHA1COCOLD会阻止应激诱导的海马神经变性和认知功能疾病独立于其独立的活性。 我们还研究了CPE/NF-Alpha1，以防止约束应激诱发的抑郁症。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。短期约束应激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。 NF-Alpha1添加到培养的海马神经元中，增加了FGF2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，CPE/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。我们发现，有抗抑郁活性的ppargamma激动剂和抗糖尿病药物罗格列酮诱导喂给小鼠时诱导CPE/NF-Alpha1和Doublecortin表达的表达。因此，PPAR-GAMMA激动剂可能是潜在有用的抗抑郁药。 我们还研究了CPE/NF-Alpha1在神经系统的胚胎发育中的作用。 我们表明，将CPE/NF-Alpha1添加到E13.5新皮层衍生的神经球中，其中包含干细胞和神经生殖器，导致神经圈的增殖降低而不会导致细胞死亡。这些CPE/NF-Alpha1处理的神经球显示了Wnt途径蛋白Beta-catenin的下调，该蛋白已知会促进增殖。在NF-Alpha1存在下，使用神经球分解为单个细胞的培养物的分化研究显示，星形胶质细胞的增加，而没有改变神经元和少突胶质细胞群体的百分比。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。此外，体内研究表明，NF-Alpha1 KO小鼠在新皮层中的GFAP+星形胶质细胞少49％，而在产后第1天（星形胶质细胞发生时间）中，NF-Alpha1 KO小鼠的NF-Alpha1 KO小鼠在新皮层中的GFAP+星形胶质细胞少49％。因此，NF-Alpha1作为细胞外信号起着至关重要的新作用，将神经干细胞区分为星形胶质细胞以进行正常神经发育。 最近，我们在胚胎小鼠脑中克隆了三个CPE mRNA。它们的大小为2.1、1.9和1.73 kb，代表野生型（WT）和两个N端截短的同工型（CPE-DELTAN）的53KD，47KD和40KD蛋白。有趣的是，所有这些同工型在胚胎E10.5和产后第1天都表现出表达的激增。尽管CPE-WT继续以成年小鼠脑表示，但CPE-DELTAN形式却没有。此外，我们表明，在小鼠海马神经元细胞系中40kD CPE-deltan的过表达和原发性皮质神经元上调了胰岛素样生长因子结合蛋白2，死亡相关蛋白1和以Ephrina的表达，这些蛋白1和Ephrina1介导细胞增殖，介导编程的细胞死亡和神经元的迁移，分别介导Neurodection。我们的发现强调了CPE-NF-Alpha1在调节胚胎神经发育和基因突变中的重要性可能导致各种神经缺陷。