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。这两种蛋白质已被证明能够将 POMC 运输到 RSP 中，这种机制可能反映了多种成分协同工作以完成内分泌细胞的非常重要的任务，即生物活性肽激素的受控调​​节分泌。使用RNA干扰敲低SgIII和CPE，我们发现这两种蛋白都会影响AtT20细胞中POMC的正常分泌，即当CPE或SgIII减少时，POMC通过组成型分泌途径以升高的速率分泌。当两者都被击倒时，影响会增强，表明 POMC 运输依赖于这两种蛋白质才能有效运输到 RSP，以便随后加工成 ACTH。 与大学乔希·帕克博士一起。 Toledo 的研究表明，Snapin 将由驱动蛋白 2、细胞质动力蛋白和动力蛋白组成的微管运动复合体连接到 POMC 囊泡上 CPE 的细胞质尾部，以介导其在垂体前叶 AtT-20 细胞中的运输。 Snapin 直接结合 CPE 细胞质尾并与微管马达相互作用。 Snapin 的过度表达降低了 ACTH/POMC 囊泡的过程定位、持续性和运动速度，类似于 CPE C 末端尾部的过度表达。 Snapin 的敲低减少了刺激的 ACTH 分泌。此外，激酶锚蛋白 150 (AKAP150)（蛋白激酶 A 和钙调神经磷酸酶的支架）与管理单元-微管运动复合物结合，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，可介导高尔基体后 ACTH/POMC 囊泡转运至 AtT20 细胞的分泌过程末端。 我们与 Bruno Tota 博士（卡拉布里亚大学）一起研究了 pGlu-serpinin（一种 CgA 衍生肽）对心脏保护的作用。以正常血压（WKY）和高血压（SHR）大鼠为模型；我们发现 pGlu-serpinin 模仿预处理和后处理诱导的心脏保护作用。在 WKY 和 SHR 大鼠中，pGlu-serpinin 改善了缺血后左心室功能的恢复。此外，它还减少了缺血引起的挛缩状态并减小了梗塞面积。在 pGlu-丝氨酸蛋白酶抑制剂介导的后处理药理学心脏保护中，其机制涉及再灌注损伤挽救激酶 (RISK) 通路的激活。 与 Rina Rosin-Arbersfeld 博士合作的研究表明，HEK293 细胞中 CPE 对 Wnt-3a 通路产生负调节。 我们现在在 PC12 细胞和皮质神经元中证明，CPE/NF-α1 可以通过负向调节经典 Wnt-3a 通路中的 β-连环蛋白以及 Wnt-3a 非通路的效应子 Rho 来调节 NGF 诱导的神经突生长。 - 规范途径。有趣的是，我们发现 Wnt-5a 可以与 CPE 复合并诱导神经突生长，而 NGF 可以增强神经突生长。 我们研究了 NF-α1 在预防束缚应激诱发的抑郁症方面的作用。长时间（每天 6 小时，持续 21 天）而非短期（每天 1 小时，持续 7 天）束缚应激会降低海马中的成纤维细胞生长因子 2 (FGF2)，导致小鼠出现抑郁样行为。我们发现，短期束缚应激后的小鼠海马 NF-α1、FGF2 和双皮质素（未成熟神经元的标志物）增加，表明神经发生增加。事实上，我们发现在培养的海马神经元中，外源性 NF-α1 可以增加 FGF2 的表达。 此外，NF-α1-KO小鼠表现出海马FGF2水平严重降低和颗粒下区未成熟神经元数量。这些小鼠表现出类似抑郁的行为，但通过施用 FGF2 可以缓解这些行为。因此，NF-α1 通过上调海马 FGF2 表达来预防压力诱发的抑郁症，从而增强神经发生和抗抑郁活性。 对 CPE 启动子的分析确定了可以诱导其表达的潜在 PPARgamma 结合位点。我们发现罗格列酮（一种 PPARγ 激动剂和抗糖尿病药物，具有额外的抗抑郁活性）可以诱导 Neuro2a 细胞和海马神经元中 CPE/NF-α1 的表达。诱导的 NF-α1 通过上调 BCL-2（一种促存活线粒体蛋白）来保护细胞免受过氧化氢诱导的氧化应激。 我们在阿尔茨海默病 (AD) 患者的皮质中发现了 CPE 突变，该突变导致 CPE 突变蛋白的序列中添加了一串九个氨基酸，我们将这种蛋白称为 CPE-QQ。当在 Neuro2a 细胞中表达时，它不被分泌，而是被蛋白酶体降解。免疫细胞化学研究表明，CPE-QQ 与 ER 标记物 Calnexin 共染色，并且海马神经元中的过度表达增加了 ER 应激标记物 CHOP 的水平，降低了促生存蛋白 BCL-2 的水平，并增加了神经元细胞死亡。这表明CPE-QQ通过ER应激和BCL-2的下调诱导细胞死亡。通过莫里斯水迷宫测试，过度表达 CPE-QQ 的转基因小鼠表现出记忆缺陷，但它们的空间学习能力未受影响。此外，这些小鼠在强迫游泳测试中还表现出抑郁样行为。这些突变小鼠的 CA3 区、海马齿状回和内侧前额皮质的神经突较少，表明存在神经退行性变。此外，他们还发现颗粒下区的神经发生减少，并且 tau 蛋白 Ser395 过度磷酸化，这是 AD 的标志。这些研究证实了 CPE/NF-1 对人类的神经保护作用，并确定了一种新基因 CPE/NF-alpha1，其突变可导致神经退行性变。 我们还利用神经球研究了 NF-α1 在神经系统胚胎发育过程中的作用，以研究增殖和分化。将 NF-α1 外源添加到 E13.5 新皮质衍生的神经球（含有干细胞和神经祖细胞）中，会导致神经球增殖减少，但不会导致细胞死亡。 NF-α1 下调神经球中的 WNT 通路，导致 β-连环蛋白水平降低，而β-连环蛋白已知可增强增殖。使用来自 7 天培养物的神经球进行分化研究，这些神经球被解离成单细胞并再培养 5 天，结果表明，在 NF-α1 存在的情况下，星形胶质细胞增加，而神经元和少突胶质细胞群体的百分比没有改变。有趣的是，来自 NF-α1-KO 小鼠胚胎神经球的分离细胞显示星形胶质细胞减少和神经元增加。此外，体内研究表明，与 WT 小鼠相比，NF-α1 KO 小鼠在出生后第 1 天（星形细胞生成时间）新皮质中的 GFAP+ 星形胶质细胞减少了 49%。 我们的结果表明，NF-α1 作为细胞外信号在将神经干细胞分化为星形胶质细胞以促进正常神经发育方面发挥着关键且新颖的作用