THE ROLE AND MECHANISMS OF PBEF IN ACUTE BRAIN INJURY AND LONG-TERM STROKE OUTCOMES AFTER FOCAL ISCHEMIC STROKE

PBEF 在急性脑损伤和局灶性缺血性卒中后长期卒中结局中的作用和机制

• 批准号: 9147010
• 负责人: Shinghua Ding
• 金额: $ 31.74万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9147010
• 关键词: Acute; Acute Brain Injuries; Adenosine; Affect; Anabolism; Apoptotic; Area; Behavioral; Biological Assay; Brain; Brain Injuries; Cell Nucleus; Cessation of life; Clinical Treatment; Cytoplasm; Data; Dendrites; Dinucleoside Phosphates; Disease; Electrophysiology (science); Enzymes; Evaluation; Glutamates; Goals; Health; Image; In Vitro; Infarction; Ischemia; Ischemic Stroke; Knock-out; Knockout Mice; Life; Mammals; Measurement; Mediating; Membrane Potentials; Microscopy; Mitochondria; Modeling; Molecular; Molecular Biology; Molecular Target; Mus; Neurologic; Neuronal Injury; Neurons; Niacinamide; Nicotinamide adenine dinucleotide; Outcome; PBEF Gene; Pathway interactions; Phase; Play; Production; Public Health; Recombinant adeno-associated virus (rAAV); Recovery; Recovery of Function; Research; Respiration; Role; Stroke; Synaptic plasticity; System; Technology; Testing; Time; Tissues; Translational Research; Vertebral column; Viral; Wild Type Mouse; behavior test; brain cell; dentate gyrus; functional gain; gain of function; improved; in vivo; in vivo Model; insight; knock-down; loss of function; mitochondrial membrane; mouse model; mutant; nervous system disorder; neuron loss; neuroprotection; new therapeutic target; nicotinamide phosphoribosyltransferase; novel; overexpression; pre-B-cell colony-enhancing factor protein; progenitor; promoter; relating to nervous system; stem; stroke treatment; subventricular zone; therapeutic target; two-photon

 DESCRIPTION (provided by applicant): Focal ischemic stroke is a leading neural disorder with limited choices for clinical treatment, thus identifying new molecular pathways that can reduce neuronal death and improve stroke outcomes remains an intensive research area. The project goal is to elucidate the role and mechanisms by which Pre-B-Cell Colony- Enhancing Factor (PBEF) exerts brain protection following focal ischemic stroke. PBEF is a rate limiting enzyme in the salvage pathway of nicotinamide adenosine dinucleotide (NAD+) biosynthesis in mammals and is mainly expressed in neurons in the brain under normal conditions. We hypothesize that PBEF plays a critical role in ameliorating neuronal death and brain damage and promoting behavioral recovery following focal ischemic stroke. Using a combination of state-of-the art technologies including PBEF conditional knockout (Pbef-/- cKO) mice, viral transduction, photothrombosis (PT) ischemia models, in vivo two-photon (2-P) microscopy, electrophysiology, mitochondrial assay, we propose three SPECIFIC aims to test our hypothesis. Aim 1: To test the hypothesis that PBEF ameliorates acute neuronal death and brain damage after ischemia by facilitating NAD+ synthesis. We will generate inducible and neuron-specific Pbef-/- cKO mice (i.e., Thy1-Pbef-/- cKO mice) and viral overexpression of WT and mutant enzymatic activity-deficient mutant PBEF for loss- and gain-of functional studies. We will image dendrite beading, glutamate release and Ca2+ overloading in live mice with in vivo 2-P microscopy, and conduct infarct volume measurement and, neuronal death assay to determine the effect of PBEF on neuronal injury and death in the acute phase of ischemia. We will determine whether neuronal PBEF is involved in PARP-1-mediated neuronal death (i.e., parthanatos) after ischemia. Aim 2: To test the hypothesis that mitochondrial NAD+ salvage pathway plays a predominant role in mediating neuronal protection after ischemia We will determine the effect of neuronal PBEF on subcompartment NAD+ pools and mitochondrial function under normal and ischemic conditions. Using subcompartment-targeting molecular expression of PBEF in cytoplasm, nuclei, and mitochondria, we will determine whether the mitochondrial NAD+ salvage pathway plays a critical role in neuroprotection after ischemia. Aim 3: To test the hypothesis that PBEF improves long-term stroke outcomes through promoting neuronal (synaptic) plasticity. We will evaluate the effect of neuronal PBEF on long-term stroke outcomes using multiple approaches. We will conduct infarct volume measurements, tissue loss, apoptotic neuronal death, behavior tests, and neurological evaluations on Thy1-Pbef-/- cKO and WT mice over a long- term period ranging from one day to a few weeks following ischemia. We will determine the effect of neuronal PBEF on synaptic plasticity in the peri-infarct region using long-term repeated in vivo 2-P imaging and electrophysiology. Results from our project will provide new insights into potential therapeutic targets for translational research on stroke treatment.

 描述（由适用提供）：局灶性缺血性中风是一种领先的神经元疾病，其临床治疗选择有限，因此可以确定可以减少神经元死亡并改善中风结果的新分子途径仍然是一个密集的研究领域。项目的目标是阐明局灶性缺血性中风后B细胞菌落增强因子（PBEF）施加脑部保护的作用和机制。 PBEF是一种限制杀菌剂腺苷二核苷酸（NAD+）生物合成的速率酶，在正常条件下在大脑的神经元中主要表达。我们假设PBEF在改善神经元死亡和脑损伤并促进局灶性缺血性中风后的行为恢复中起着至关重要的作用。结合包括PBEF有条件敲除（PBEF - / - CKO）小鼠，病毒转导，光疗（PT）缺血模型，体内双光子（2-P）显微镜，电物质学，电动体生理学，我们提出三个特定目标的三个特定目标。目的1：检验以下假设：PBEF通过支持NAD+合成来缓解缺血后急性神经元死亡和脑损伤。我们将生成可诱导和神经元特异性的PBEF - / - CKO小鼠（即Thy1-PBEF - / - CKO小鼠）以及WT和突变体酶活性突变突变体PBEF的病毒过表达，用于损失和获得功能研究。我们将在具有体内2-P显微镜的活小鼠中对树突珠，谷氨酸释放和Ca2+过载，并进行梗塞体积测量，并进行神经元死亡评估，以确定PBEF对缺血性急性相的神经元损伤和死亡的影响。我们将确定缺血后的神经元PBEF是否参与PARP-1介导的神经元死亡（即Parthanatos）。目的2：为了测试线粒体NAD+挽救途径在局部介导神经元保护中起主要作用的假设，我们将确定神经元PBEF在正常和缺血性条件下神经元PBEF对子分会NAD+池和线粒体功能的影响。使用PBEF在细胞质，核和线粒体中的子分末期分子表达，我们将确定线粒体NAD+分裂途径在缺血后的神经保护中是否起关键作用。 AIM 3：测试PBEF通过促进神经元（突触）可塑性改善长期中风结果的假设。我们将使用多种方法评估神经元PBEF对长期中风结果的影响。我们将对THY1-PBEF - / - CKO和WT小鼠进行梗塞体积测量，组织丢失，凋亡神经元死亡，行为测试以及神经系统评估，范围从一天到几周之后的几周不等。我们将使用长期重复体内2-P成像和电生理学重复进行神经元PBEF对突触可塑性的影响。我们项目的结果将为中风治疗的转化研究提供新的见解。