Neuropeptides and Carboxypeptidase E/ Neurotrophic Factor-alpha1 in Neural and Cognitive Functions

神经肽和羧肽酶 E/神经营养因子-α1 在神经和认知功能中的作用

• 批准号: 10461671
• 负责人: Yoke Peng Loh
• 金额: $ 146.41万
• 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/10461671
• 关键词: Adrenergic Agents; Agonist; Alzheimer' s Disease; Alzheimer' s disease patient; Amino Acids; Amphibia; Anterior Pituitary Gland; Antidepressive Agents; Antidiabetic Drugs; Astrocytes; BCL2 gene; Behavior; Binding; Biogenesis; Brain; Brain-Derived Neurotrophic Factor; Cell Death; Cell membrane; Cell secretion; Cells; Chromogranin A; Collaborations; Complex; Corticotropin; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Cytoplasmic Tail; Dependence; Depressed mood; Diabetes Mellitus; Disease; Down-Regulation; Ear; Embryo; Embryonic Development; Endocrine; Endocrine system; Endoplasmic Reticulum; Energy Metabolism; Enzymes; Exhibits; FGF2 gene; Fatty Acids; Forskolin; Future; Genes; Glial Fibrillary Acidic Protein; Golgi Apparatus; Growth Factor; Heart; Hippocampus (Brain); Human; Impaired cognition; In Vitro; Infertility; Ischemia; Kinesin; Knock-in Mouse; Knockout Mice; Laboratories; Lead; Learning Disabilities; Left ventricular structure; Lipids; Medial; Mediating; Memory impairment; Mental Depression; Microtubules; Missense Mutation; Molecular; Motor; Movement; Mus; Mutation; Myocardial; N-terminal; NTF3 gene; Names; Neocortex; Nerve Degeneration; Nervous system structure; Neurites; Neurodegenerative Disorders; Neuroendocrine Cell; Neurologic Deficit; Neurons; Neuropeptides; Neurophysiology - biologic function; Neurotrophic Tyrosine Kinase Receptor Type 2; Obesity; Oligodendroglia; Organelles; Oxidative Phosphorylation; Oxidative Stress; PPAR gamma; Pathway interactions; Peptides; Performance; Phosphorylation; Phosphotransferases; Physiological; Play; Population; Prefrontal Cortex; Pro-Opiomelanocortin; Process; Production; Proprotein Convertase 1; Proprotein Convertase 2; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-akt; Rattus; Recovery of Function; Reperfusion Injury; Reporting; Role; SNAPIN gene; Secretory Vesicles; Signal Transduction; Site; Sorting - Cell Movement; Stress; Tail; Testing; Time; Transgenic Mice; Transgenic Organisms; Universities; Vesicle; Weaning; Work; anterograde transport; beta catenin; bone mass; carboxypeptidase H; cardioprotection; cognitive function; conditioning; dentate gyrus; depressive symptoms; embryonic stem cell; endoplasmic reticulum stress; extracellular; fatty acid metabolism; forced swim test; glial cell-line derived neurotrophic factor; heart function; heart pharmacology; improved; in vivo; inhibitor/antagonist; knock-down; learning ability; maternal separation; morris water maze; mouse model; multicatalytic endopeptidase complex; mutant; nerve stem cell; nervous system development; neurodevelopment; neurogenesis; neuron loss; neuroprotection; neurotoxic; neurotrophic factor; normotensive; novel; null mutation; osteoblast differentiation; overexpression; peptide hormone; postnatal; preconditioning; prevent; prohormone; restraint stress; rosiglitazone; skeletal stem cell; social; stem cell differentiation; stem cells; tau Proteins; teleost; therapy development; trafficking; vesicle transport

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 the cytoplasmic tail of transmembrane CPE on POMC vesicles binds directly to snapin, which in turn binds to microtubule motors, kinesin-2 and kinesin-3 to mediate their anterograde transport. Anterior pituitary AtT-20 cells, overexpressing snapin showed reduced process-localization and velocity of movement of ACTH/POMC vesicles, similar to CPE cytoplasmic tail overexpression. Knockdown of snapin decreased stimulated ACTH secretion. Interestingly, upon protein kinase A (PKA) activation by forskolin, the interactions of kinesin-2 and kinesin-3 with CPE and ACTH vesicle levels at the terminus of At20 cells were significantly increased. Our study has uncovered a new molecular complex consisting of the CPE cytoplasmic tail-snapin-kinesin 2 and 3 that mediates post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells for secretion in a PKA-dependent manner. 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(normotensive) and SHR(hypertensive) rats, and 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, supporting an evolutionary role of serpinins in sympatho-adrenergic control of the vertebrate heart. Currently, our major focus is on the novel, neurotrophic functions of CPE/NF-alpha1. Human null and mis-sense mutations of CPE have been reported to have severe learning disability besides obesity, diabetes and infertility due to lack of CPE. We have 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. These mice showed depressive-like behavior by the forced swim test. They had fewer neurites in the hippocampal CA3 region, dentate gyrus and medial prefrontal cortex, indicative of neurodegeneration. They showed diminished neurogenesis in the sub-granular zone and hyperphosphorylation of tau at ser395, a hallmark of AD. These studies indicate that this human mutation in CPE/NF-alpha1 is neurotoxic, leading to neurodegeneration and cognitive decline. Additionally, we 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, 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. CPE knock-out mice lacking CPE/NF-alpha1 showed complete degeneration of hippocampal CA3 neurons and cognitive dysfunction after social (maternal separation) and physical stress (ear tagging and tail clipping) associated with the weaning paradigm, despite having WT levels of other growth factors such as BDNF, GDNF, NGF and NT3. Treatment of WT mice with ANA12, an inhibitor of TrkB, the BDNF receptor did not result in neurodegeneration of the CA3 region after weaning stress. Most importantly, CPE-E342Q mice, although lacking WT-CPE and were obese and had endocrinological deficits, showed no hippocampal degeneration or cognitive dysfunction after the weaning paradigm, indicating that CPE/NF-alpha1 is critical, but not BDNF, in preventing stress-induced hippocampal cell death, 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, CPE/NF-alpha1-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the sub-granular 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 have investigated the role of CPE/NF-alpha1 in embryonic development of the nervous system. 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. With Dr. Beata Lecka-Czernik, University of Toledo, we showed that murine skeletal stem cells, treated with CPE or CPE-E342Q protein enhanced Erk phosphorylation and up-regulated expression of two wnt pathway markers, Cxn43 and Axin2, and a tendency to increase Osterix, a gene associated with osteoblastic differentiation; as well as genes associated with fatty acid metabolism and energy dissipation. These cells exhibited transient accumulation of small lipid droplets, increased oxidative phosphorylation and cellular dependence on fatty acids as fuel for energy production. Thus, CPE-NF-alpha1 acts as a trophin in brain, as well as in regulating bone mass and energy metabolism, independent of its enzymatic activity. CPE-NF-alpha1's ability to increase oxidative phosphorylation may contribute to protecting stress-induced neuronal cell death, besides up-regulating BCL2 expression. Future work will investigate CPE-NF-alpha1 on energy metabolism in neurons. Thus, CPE-NF-alpha1 may be an excellent candidate for treating neurodegenerative diseases.

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。与托莱多大学的乔什·帕克（Josh Park）博士合作，我们表明pomc囊泡上跨膜CPE的细胞质尾巴直接与Snapin结合，而Snapin又与微管电动机，Kinesin-2和Kinesin-3结合，以介导其同步运输。前表达Snapin的前垂体ATT-20细胞表现出与ACTH/POMC囊泡运动的过程 - 定位降低和速度，类似于CPE细胞质尾部的过表达。 Snapin的敲低减少了刺激的ACTH分泌。有趣的是，在Forskolin的蛋白激酶A（PKA）激活后，在AT20细胞末端，驱动蛋白-2和驱动蛋白3与CPE和ACTH囊泡水平的相互作用显着增加。 我们的研究发现了一种新的分子络合物，该复合物由CPE细胞质尾尾 - 钉蛋白2和3组成，该蛋白2和3介导了Acth/POMC囊泡后高尔基体转运到ATT20细胞的过程中以PKA依赖性方式分泌的过程。 与Univ的Bruno Tota博士在一起。在卡拉布里亚（Calabria）上，我们研究了一种CGA衍生肽PGLU-Serpinin对心脏保护的影响。 PGLU-Serpinin模仿了WKY（正常）和SHR（高血压）大鼠的前调节和调节后诱导的心脏保护作用，并改善了缺血后的左心室功能恢复。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Teleost和两栖动物心脏的心肌表现，支持Serpinins在脊椎动物心脏的交感神经控制中的进化作用。 当前，我们的主要重点是CPE/NF-Alpha1的新型神经营养功能。据报道，由于缺乏CPE而导致的肥胖症，糖尿病和不育除外，据报道，CPE的人体无效和不宽松的CPE突变具有严重的学习障碍。我们已经确定了阿尔茨海默氏病（AD）患者中的CPE突变，该突变导致CPE突变蛋白具有另外9个氨基酸，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。这些小鼠通过强制游泳测试表现出抑郁症状的行为。它们在海马CA3区域，齿状回和内侧前额叶皮质中的神经突更少，表明神经退行性。他们显示出在AD的标志的Ser395处的粒状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，导致神经退行性和认知能力下降。此外，我们在CPE/NF-Alpha1中发现了另一个人类突变，它导致了ER应激和神经元细胞死亡。 我们的体外研究表明，CPE/NF-Alpha1在细胞外起作用，独立于其酶促活性，以保护海马神经元通过激活ERK或AKT-上调BCl-2表达而通过激活ERK或AKT-途径来保护氧化应激。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CPE敲除小鼠在社交（母体分离）和身体压力（孕产妇分离）和断奶范式相关的身体压力（孕产妇分离和尾巴剪断）之后的海马CA3神经元和认知功能障碍表现出完全变性。用TRKB的抑制剂ANA12处理WT小鼠，BDNF受体在断奶应激后不会导致CA3区域的神经变性。 最重要的是，CPE-E342Q小鼠虽然缺乏WT-CPE并且肥胖并且存在内分泌缺陷，但在断奶范式后没有显示海马的退化或认知功能障碍，表明CPE/NF-Alpha1是至关重要的，但在预防势力较大的情况下，cpe/nf-alpha1是至关重要的。 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，CPE/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。 我们已经研究了CPE/NF-Alpha1在神经系统的胚胎发育中的作用。 将CPE/NF-Alpha1添加到E13.5新皮层衍生的神经球，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。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。 借助托莱多大学的Beata Lecka-Czernik博士，我们表明，用CPE或CPE-E342Q蛋白处理的鼠骨骼干细胞增强了ERK磷酸化，并上调了两个Wnt途径标记物CXN43和AXIN2的趋势，并倾向于增加与基因的相关性，而与基因相关的趋势是osteoblastic osteoblastic osteoblastic；以及与脂肪酸代谢和能量耗散有关的基因。这些细胞表现出小脂质液滴的瞬时积累，氧化磷酸化增加以及对脂肪酸作为能量产生的燃料的细胞依赖性。因此，CPE-NF-Alpha1充当大脑中的滋养蛋白，以及调节骨量和能量代谢，与其酶活性无关。 除上调BCL2表达外，CPE-NF-Alpha1增加氧化磷酸化的能力可能有助于保护应激诱导的神经元细胞死亡。未来的工作将研究CPE-NF-Alpha1关于神经元能量代谢的CPE-NF-Alpha1。因此，CPE-NF-Alpha1可能是治疗神经退行性疾病的极好候选者。