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（由申请人提供）：局灶性缺血性卒中是一种主要的神经疾病，临床治疗选择有限，因此确定可减少神经元死亡和改善卒中结局的新分子途径仍然是一个深入研究的领域。该项目的目标是阐明前B细胞集落增强因子（PBEF）在局灶性缺血性卒中后发挥脑保护作用的作用和机制。PBEF是哺乳动物中烟酰胺腺苷二核苷酸（NAD+）生物合成补救途径中的限速酶，并且在正常条件下主要在脑中的神经元中表达。我们假设PBEF在改善局灶性缺血性卒中后神经元死亡和脑损伤以及促进行为恢复方面起关键作用。使用最先进的技术的组合，包括PBEF条件敲除（Pbef-/- cKO）小鼠，病毒转导，光血栓（PT）缺血模型，在体内双光子（2-P）显微镜，电生理学，线粒体测定，我们提出了三个具体的目的来验证我们的假设。目的1：验证PBEF通过促进NAD+合成改善缺血后急性神经元死亡和脑损伤的假设。我们将产生诱导型和神经元特异性Pbef-/- cKO小鼠（即，Thy 1-Pbef-/- cKO小鼠）和WT和突变体酶活性缺陷突变体PBEF的病毒过表达，用于功能丧失和获得研究。我们将在活体2-P显微镜下对小鼠树突珠状、谷氨酸释放和钙超载进行成像，并进行梗死体积测量和神经元死亡测定，以确定PBEF对缺血急性期神经元损伤和死亡的影响。我们将确定神经元PBEF是否参与PARP-1介导的神经元死亡（即，parthanatos）。目标二：为了检验线粒体NAD+补救途径在缺血后介导神经元保护中起主导作用的假设，我们将确定在正常和缺血条件下神经元PBEF对亚隔室NAD+库和线粒体功能的影响。利用PBEF在细胞质、细胞核和线粒体中的亚区室靶向分子表达，我们将确定线粒体NAD+补救途径是否在缺血后的神经保护中起关键作用。目的3：验证PBEF通过促进神经元（突触）可塑性改善长期卒中结局的假设。我们将使用多种方法评估神经元PBEF对长期卒中结局的影响。我们将对Thy 1-Pbef-/- cKO和WT小鼠进行缺血后1天至数周的长期梗死体积测量、组织损失、凋亡性神经元死亡、行为测试和神经学评价。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对梗死周围区域突触可塑性的影响。我们项目的结果将为中风治疗的转化研究提供潜在治疗靶点的新见解。