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

基本信息

批准号：
10915308
负责人：
Y. Peng Loh
金额：
$ 165.76万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10915308
关键词：
3xTg-AD mouse Age Agonist Alzheimer&apos s Disease Alzheimer&apos s disease model Alzheimer&apos s disease patient Amino Acids Amyloid beta-Protein Amyloid beta-Protein Precursor Antidepressive Agents Antidiabetic Drugs Astrocytes BCL1 Oncogene BCL2 gene Behavior Binding Biogenesis Biological Assay Biological Process Body Weight Brain Brain-Derived Neurotrophic Factor CREB1 gene Cell Cycle Cell Death Cell Survival Cells Central Nervous System Child Chromogranin A Clip Co-Immunoprecipitations Cognitive deficits Complex Corticotropin Cre lox recombination system Cyclic AMP-Dependent Protein Kinases Cytoplasmic Granules Development Diabetes Mellitus Disease Disease Progression Dissociation Down-Regulation Ear Embryo Endocrine Endocrine system Endocrinology Endoplasmic Reticulum Endorphins Energy Metabolism Enzymes Exhibits FGF2 gene Functional disorder G-Protein-Coupled Receptors Genes Glial Fibrillary Acidic Protein Glucose Glutamates Growth Factor Hippocampus Human Hydrogen Bonding Hydrogen Peroxide Immunohistochemistry Impaired cognition Impairment In Vitro Infertility Inflammatory Knock-in Mouse Knock-out Knockout Mice Laboratories Learning Learning Disabilities Libraries LoxP-flanked allele Medial Mediating Membrane Memory Memory impairment Mental Depression Mitochondria Mus Mutation N-terminal NF1 gene NTF3 gene Names Neocortex Nerve Degeneration Nervous System Neurites Neurodegenerative Disorders Neuroendocrine Cell Neurologic Deficit Neuronal Dysfunction Neurons Neuropeptides Neurophysiology - biologic function Neuroprotective Agents Neurosphere Neurotrophic Tyrosine Kinase Receptor Type 2 Obesity Oligodendroglia Organelles Oxidative Stress PPAR gamma Parahippocampal Gyrus Pathway interactions Peptides Physiological Play Population Prefrontal Cortex Pro-Opiomelanocortin Proliferating Proprotein Convertase 1 Proprotein Convertase 2 Protein Isoforms Proteins Proto-Oncogene Proteins c-akt Reporting Role Secretory Vesicles Serotonin Signal Transduction Site Sodium Chloride Sorting Stress Surface Tail Testing Therapeutic Agents Transgenic Mice Transgenic Organisms Viral Weaning behavior test beta catenin beta-arrestin carboxypeptidase H cognitive function conditioned fear cytotoxicity dentate gyrus depressive symptoms early childhood embryonic stem cell endoplasmic reticulum stress extracellular forced swim test gene therapy glial cell-line derived neurotrophic factor heart function hyperphosphorylated tau in vivo inhibitor learning ability maternal separation molecular dynamics morris water maze mouse model multicatalytic endopeptidase complex mutant neocortical nerve stem cell neurodevelopment neurogenesis neuron loss neuronal survival neuroprotection neurotoxic neurotoxicity neurotrophic factor novel novel therapeutics object recognition overexpression peptide hormone prevent prohormone protein expression receptor recruit restraint stress rosiglitazone screening serotonin receptor social stem cell differentiation stem cells stress reduction targeted treatment tau-1 therapy development vesicle transport

项目摘要

Our current focus is to study the neurotrophic functions of CPE/NF-alpha1. Humans with null and mis-sense CPE mutations have been reported to have severe learning disability in early childhood, 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, 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 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 hippocampus and medial prefrontal cortex, indicative of neurodegeneration. They showed diminished neurogenesis in the sub-granular zone and hyperphosphorylation of tau, a hallmark of AD. Thus, this human mutation in CPE/NF-alpha1 is neurotoxic, leading to neurodegeneration and cognitive decline. 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 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. Recently, we made a conditional CPE knock-out mouse using the Cam2a-cre system to specifically knock out expression of CPE in brain. Compared to WT mice, the CPEflox/flox mice showed impaired learning and memory using several behavioral tests including object recognition, Y-Maze and fear conditioning. Body weight and glucose were normal in these CPEflox/flox mice. However, they exhibited complete degeneration of the subiculum region, unlike the CPE full knockout mice, which exhibit hippocampal CA3 region neurodegeneration. In addition, reduced doublecortin immunostaining in the dentate gyrus of the hippocampus suggested decreased neurogenesis in CPEflox/flox mice. Thus, specific neuronal CPE knockout leads to central nervous system dysfunction in mice (1). To understand the mechanism of the trophic action of CPE/NF-a1, we searched for a membrane receptor for this factor. Screening a human G-protein coupled receptor (GPCR) library, we found a serotonin receptor, HTR1E with no known function that interacted with CPE/NF-alpha1. This interaction was confirmed by co-immunoprecipitation and pulldown assays. Binding studies revealed a Kd=13.82nM. Molecular dynamics studies indicated that CPE/NF-a1 interacts with HTR1E via 4 salt-bridges stabilized by several hydrogen bonds and is independent of the serotonin binding pocket. Immunohistochemistry revealed co-localization of HTR1E and CPE/NF-a1 on the surface of hippocampal neurons. Signal transduction studies showed that HTR1E-CPE/NF-alpha1 interaction activated the Erk1/2-CREB pathway via recruitment of beta-arrestin. This in turn activated the BCL2 pro-survival pathway. We showed that HTR1E-CPE/NF-alpha1 interaction mediated neuroprotection of human primary neurons against H2O2 -induced cytotoxicity and glutamate-induced neurotoxicity. These findings indicate that CPE/NF-alpha1 interacts with HTR1E to promote neuronal survival. Unlike CPE/NF-1, serotonin binds to a different site on 5-HTR1E and the serotonin-5-HTR1E complex activates ERK pathway via classical Gi and PKA to regulate specific biological functions, such as cell cycle and survival (2, 3). We have examined the role of CPE/NF-a1 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-a1, FGF2 and doublecortin, a marker for immature neurons, suggesting increased neurogenesis. NF-a1 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. Interestingly, rosiglitazone, a PPAR-gamma agonist and anti-diabetic drug which has anti-depression and neuroprotective activities, induced CPE/NF-alpha1 and doublecortin expression when fed to mice. Thus PPAR-gamma agonists can be potentially useful as anti-depressant and neuroprotective drugs (4). Given CPE-NF-alpha1's role in preventing cell death, we investigated its use as a therapeutic agent for Alzheimer Disease (AD). We delivered viral-(AAV)-NF-al/CPE) gene into the hippocampus of 3xTg-AD mice , an AD model, at an early pre-symptomatic age and found that it prevented later development of cognitive dysfunction as assessed by Morris water maze and novel object recognition assays. Neurodegeneration and tau hyperphosphorylation were prevented in AAV-NF-a1/CPE treated 3xTg-AD mice. Additionally, amyloid precursor protein (APP) expression was reduced to near non-AD levels, and insoluble Abeta1-42 was reduced significantly. Pro-survival proteins: mitochondrial Bcl2 and Serpina3g were increased; and mitophagy inhibitor Plin4 and pro-inflammatory protein Card14 were decreased in AAV-NF-a1/CPE treated AD mice. Thus NF-a1/CPE gene therapy targets many regulatory components to prevent cognitive deficits in 3xTg-AD mice and has implications as a new therapy to prevent AD progression by promoting cell survival, inhibiting APP expression and tau phosphorylation(5). We also investigated the role of CPE/NFa-1 in embryonic neurodevelopment. Addition of CPE/NF-a1 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-a1 treated neurospheres showed down-regulation of the wnt-beta catenin pathway known to promote proliferation. Differentiation studies using neurospheres dissociated into single cells showed an increase in astrocytes in the presence of NF-a1, without altering the percentage of neuronal and oligodendrocyte populations. Interestingly, dissociated cells from neurospheres derived from NF-a1-KO mouse embryos showed decreased astrocytes and increased neurons. NF-alpha1 KO mice had 49% fewer GFAP+ astrocytes in P1 brain. Thus NF-a1 plays a critical role in differentiating neural stem cells into astrocytes. We will continue to explore CPE/NF-a1's function in neurodevelopment.

我们目前的重点是研究CPE/NF-Alpha1的神经营养功能。据报道，由于缺乏CPE，幼儿，肥胖，糖尿病和不育的人类患有无效的CPE突变的人类患有严重的学习障碍。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，在海马神经元中定位于内质网（ER）（ER）的CPE-QQ增加了ER应力和促生存性蛋白Bcl-2的水平降低，导致神经元细胞死亡。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。它们在海马和内侧前额叶皮层中的神经突较少，表明神经退行性。它们显示出AD标志的Tau的粒状区域和过度磷酸化的神经发生减少。因此，CPE/NF-Alpha1中的这种人类突变是神经毒性的，导致神经变性和认知能力下降。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且缺乏内分泌学缺陷，但在断奶范式后没有显示海马退化或认知功能障碍，表明CPE/NF-Alpha1至关重要，但在预防压力诱导的Hippocapal ins中，cpe/nf-alpha是至关重要的。最近，我们使用CAM2A-CRE系统制作了有条件的CPE敲除小鼠，以特别敲除脑中CPE的表达。与WT小鼠相比，CPEFLOX/FLOX小鼠使用多种行为测试（包括对象识别，Y-迷宫和恐惧条件）显示出学习和记忆力受损。这些CPEFLOX/FLOX小鼠中体重和葡萄糖正常。然而，与CPE全敲除小鼠不同，它们表现出了亚致区域的完全变性，后者表现出海马CA3区域神经变性。此外，在海马的齿状回中降低了双铁蛋白免疫染色，这表明CPEFLOX/FLOX小鼠的神经发生降低。因此，特定的神经元CPE敲除导致小鼠中枢神经系统功能障碍（1）。为了了解CPE/NF-A1的营养作用的机制，我们为此搜索了膜受体。筛选人类G蛋白偶联受体（GPCR）库，我们发现了一个羟色胺受体，HTR1E，没有已知功能，与CPE/NF-ALPHA1相互作用。通过共免疫沉淀和下拉分析证实了这种相互作用。结合研究显示KD = 13.82nm。分子动力学研究表明，CPE/NF-A1通过4个由几种氢键稳定的盐桥与HTR1E相互作用，并且独立于5-羟色胺结合口袋。免疫组织化学揭示了HTR1E和CPE/NF-A1在海马神经元表面的共定位。信号转导研究表明，HTR1E-CPE/NF-Alpha1相互作用通过募集β-arrestin激活了ERK1/2-CREB途径。这反过来激活了Bcl2亲生途径。我们表明，HTR1E-CPE/NF-ALPHA1相互作用介导了人类原代神经元针对H2O2诱导的细胞毒性和谷氨酸诱导的神经毒性的神经保护。这些发现表明CPE/NF-Alpha1与HTR1E相互作用以促进神经元存活。与CPE/NF-1不同，5-羟色胺在5-HTR1E上与其他位点结合，而5-羟色胺-5-HTR1E复合物通过经典GI和PKA激活ERK途径，以调节特定的生物学功能，例如细胞周期和存活（2，3）。我们已经检查了CPE/NF-A1在防止压力诱发的抑郁症中的作用。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-A1，FGF2和Doublecortin，这是未成熟神经元的标志物，表明神经发生增加。 NF-A1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。有趣的是，有抗抑郁症和神经保护活性的pPAR-gamma激动剂和抗糖尿病药物的罗格列酮会诱导CPE/NF-Alpha1和Doublecortin表达时，当给小鼠诱导CPE/NF-ALPHA1和DoubleCortin的表达。因此，PPAR-GAMMA激动剂可以作为抗抑郁药和神经保护药物有用（4）。鉴于CPE-NF-Alpha1在预防细胞死亡中的作用，我们研究了其用作阿尔茨海默氏病（AD）的治疗剂。我们将病毒（AAV）-NF-AL/CPE）基因输送到3xTG-AD小鼠的海马（一种AD模型）的海马，并在早期症状年龄的早期年龄，发现它阻止了后来的认知功能障碍的发展，该认知功能障碍通过Morris Water Maze和新颖的对象识别分析所评估。在AAV-NF-A1/CPE处理的3XTG-AD小鼠中，预防神经变性和TAU高磷酸化。另外，淀粉样蛋白前体蛋白（APP）表达降低到接近非AD水平，而不溶性ABETA1-42显着降低。促生存蛋白：线粒体BCL2和SERPINA3G增加；在AAV-NF-A1/CPE处理的AD小鼠中，线粒体抑制剂PLIN4和促炎蛋白Card14降低。因此，NF-A1/CPE基因疗法靶向许多调节成分，以防止3XTG-AD小鼠的认知缺陷，并具有一种新的疗法，可以通过促进细胞存活，抑制APP表达和tau磷酸化来防止AD进展（5）。我们还研究了CPE/NFA-1在胚胎神经发育中的作用。将CPE/NF-A1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，导致神经球的增殖降低而不会导致细胞死亡。这些CPE/NF-A1处理的神经球显示了已知促进增殖的Wnt-beta catenin途径的下调。使用神经球分离为单个细胞的神经球的分化研究表明，在存在NF-A1的情况下，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的百分比。有趣的是，来自NF-A1-KO小鼠胚胎的神经球的分离细胞显示，星形胶质细胞降低和神经元增加。 NF-Alpha1 KO小鼠在P1脑中的GFAP+星形胶质细胞少49％。因此，NF-A1在区分神经干细胞为星形胶质细胞中起关键作用。我们将继续探索CPE/NF-A1在神经发育中的功能。

项目成果

期刊论文数量（51）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Neurotrophic factor-α1, a novel tropin is critical for the prevention of stress-induced hippocampal CA3 cell death and cognitive dysfunction in mice: comparison to BDNF.

DOI：
10.1038/s41398-020-01112-w
发表时间：
2021-01-07
期刊：
Translational psychiatry
影响因子：
6.8
作者：
Xiao L;Sharma VK;Toulabi L;Yang X;Lee C;Abebe D;Peltekian A;Arnaoutova I;Lou H;Loh YP
通讯作者：
Loh YP

Carboxypeptidase E conditional knockout mice exhibit learning and memory deficits and neurodegeneration.