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: 9348217
• 负责人: Yoke p Loh
• 金额: $ 91.36万
• 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/9348217
• 关键词: A kinase anchoring protein; Affect; Agonist; Alzheimer' s Disease; Amino Acids; Anterior Pituitary Gland; Antidiabetic Drugs; Astrocytes; BCL2 gene; Behavior; Binding; Binding Sites; C-terminal; Calcineurin; Calnexin; Cardiac; Cell Culture Techniques; Cell Death; Cell secretion; Cells; Chromogranin A; Collaborations; Complex; Contracture; Corticotropin; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Cytoplasmic Tail; Defect; Diabetes Mellitus; Disease; Down-Regulation; Dynein ATPase; Embryo; Embryonic Development; Endocrine; Endocrine system; Enzymes; Exhibits; FGF2 gene; Genes; Glial Fibrillary Acidic Protein; Golgi Apparatus; Hippocampus (Brain); Hormones; Human; Hydrogen Peroxide; Inbred SHR Rats; Inbred WKY Rats; Infarction; Ischemia; Kinesin; Knockout Mice; Laboratories; Lead; Left ventricular structure; Medial; Mediating; Membrane; Memory impairment; Mental Depression; Microtubules; Mitochondrial Proteins; Modeling; Molecular; Motor; Movement; Mus; Mutant Strains Mice; Mutation; Neocortex; Nerve Degeneration; Nervous system structure; Neurites; Neurodegenerative Disorders; Neurons; Neuropeptides; Neurophysiology - biologic function; Obesity; Oligodendroglia; Organelles; Oxidative Stress; PPAR gamma; Parahippocampal Gyrus; Pathway interactions; Patients; Peptides; Phosphotransferases; Physiological; Pituitary Gland; Play; Population; Prefrontal Cortex; Pro-Opiomelanocortin; Process; Proprotein Convertase 1; Proprotein Convertase 2; Proteins; RNA Interference; Recovery of Function; Regulation; Reperfusion Injury; Role; SNAPIN gene; Scaffolding Protein; Secretory Vesicles; Signal Transduction; Site; Sorting - Cell Movement; Staining method; Stains; Stem cells; Stress; Swimming; Tail; Testing; Time; Transgenic Mice; Transport Vesicles; Up-Regulation; Vesicle; Work; beta catenin; carboxypeptidase H; cognitive function; conditioning; dentate gyrus; depression prevention; depressive symptoms; dynactin; extracellular; improved; in vivo; knock-down; learning ability; morris water maze; multicatalytic endopeptidase complex; mutant; nerve stem cell; neurodevelopment; neurogenesis; neuron loss; neuroprotection; neurotrophic factor; normotensive; novel; overexpression; peptide hormone; postnatal; prevent; prohormone; promoter; receptor; restraint stress; rho; rosiglitazone; rosin; secretogranin III; stem cell differentiation; 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 post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells for secretion. 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. Studies in collaboration with Dr. Rina Rosin-Arbersfeld showed negative regulation of the Wnt-3a pathway by CPE in HEK293 cells. We have now shown in PC12cells and cortical neurons that CPE/NF-alpha1 can modulate the NGF-induced neurite outgrowth by negatively regulating beta-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. 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. We have identified a CPE mutation in the cortex of an Alzheimer Disease (AD) patient which results in a CPE mutant protein with a string of nine amino acids added in the sequence, and we call this protein, 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. Moreover, these mice also showed depressive-like behavior by the forced swim test. 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. 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. Furthermore, in vivo studies show that NF-alpha1 KO mice had 49% fewer GFAP+ astrocytes in the neocortex compared to WT-mice at postnatal day1 , the time of astrocytogenesis. Our results indicate that NF-alpha1 plays a critical and novel role as an extracellular signal to differentiate neural stem cells into astrocytes for normal neurodevelopment

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（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。 与Rina Rosin-Arbersfeld博士合作的研究表明，HEK293细胞中CPE对WNT-3A途径的负调控。 现在，我们已经在PC12细胞和皮质神经元中显示了CPE/NF-Alpha1可以通过在rho中调节beta-catenin以及Rho的RHO，以及Wnt-3a非canonical途径的效果，可以调节NGF诱导的神经突生长。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。 我们已经研究了NF-Alpha1，以防止约束压力诱导的抑郁症。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。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。 我们已经在阿尔茨海默氏病（AD）患者的皮质中确定了CPE突变，该突变导致序列中添加的九个氨基酸的CPE突变蛋白，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。此外，这些小鼠还通过强制游泳测试表现出类似抑郁症的行为。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，并鉴定出一种新基因CPE/NF-Alpha1，其突变可能引起神经变性。 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。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％，而星形胶质细胞发生时期的WT-MICE则与WT-MICE相比。 我们的结果表明，NF-Alpha1作为细胞外信号起着至关重要的新作用