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: 9535511
• 负责人: Shinghua Ding
• 金额: $ 32.07万
• 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/9535511
• 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; Image; In Vitro; Infarction; Ischemia; Ischemic Stroke; Knock-out; Knockout Mice; 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; excitotoxicity; functional gain; gain of function; improved; in vivo; 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; public health relevance; relating to nervous system; stem; stroke outcome; 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（由申请人提供）：局灶性缺血性中风是一种主要的神经疾病，临床治疗选择有限，因此确定可以减少神经元死亡和改善中风结果的新分子途径仍然是一个深入的研究领域，该项目的目标是阐明其作用。前 B 细胞集落增强因子 (PBEF) 在局灶性缺血性中风后发挥脑保护作用的机制 PBEF 是烟酰胺腺苷挽救途径中的限速酶。我们发现，PBEF 在哺乳动物中的二核苷酸 (NAD+) 生物合成中发挥着重要作用，在正常情况下主要在大脑中的神经元中表达。最先进的技术，包括 PBEF 条件敲除 (Pbef-/- cKO) 小鼠、病毒转导、光血栓 (PT) 缺血模型、体内双光子 (2-P)显微镜、电生理学、线粒体测定，我们提出了三个具体目标来检验我们的假设。 目标 1：检验 PBEF 通过促进 NAD+ 合成来改善缺血后急性神经元死亡和脑损伤的假设。 /- cKO 小鼠（即 Thy1-Pbef-/- cKO 小鼠）和 WT 病毒过度表达以及酶活性缺陷的突变体我们将使用体内 2-P 显微镜对活体小鼠的树突珠、谷氨酸释放和 Ca2+ 超载进行成像，并进行梗塞体积测量和神经元死亡测定，以确定 PBEF 的效果。我们将确定神经元 PBEF 是否参与 PARP-1 介导的神经元死亡（即，目标 2：检验线粒体 NAD+ 挽救途径在介导缺血后神经元保护中起主要作用的假设我们将使用 亚区室靶向分子表达来确定神经元 PBEF 在正常和缺血条件下对亚区室 NAD+ 池和线粒体功能的影响。细胞质、细胞核和线粒体中的PBEF，我们将确定线粒体NAD+挽救途径是否发挥关键作用目标 3：检验 PBEF 通过促进神经元（突触）可塑性改善长期卒中结果的假设 我们将使用多种方法评估神经元 PBEF 对长期卒中结果的影响。对 Thy1-Pbef-/- cKO 和 WT 小鼠进行从一天到一天的长期体积测量、组织损失、神经元凋亡、行为测试和神经学评估缺血后几周，我们将使用长期重复的体内 2-P 成像和电生理学来确定神经元 PBEF 对梗塞周围区域突触可塑性的影响，我们项目的结果将为潜在的治疗靶点提供新的见解。中风治疗的转化研究。