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

 描述(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，因此寻找能够减少神经元死亡和改善中风预后的新的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是哺乳动物体内烟酰胺腺苷二核苷酸(NAD+)生物合成回收途径中的限速酶，在正常情况下主要表达于脑神经元。我们假设，PBEF在改善局灶性脑缺血后神经元死亡和脑损伤以及促进行为恢复方面起着关键作用。结合最先进的技术，包括PBEF条件基因敲除(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、线粒体分析，我们提出了三个特定的目标来验证我们的假设。目的1：验证PBEF通过促进NAD+合成改善脑缺血后急性神经元死亡和脑损伤的假说。我们将产生可诱导的和神经元特异性的Pbef-/-CKO小鼠(即Thy1-Pbef-/-CKO小鼠)和病毒过表达WT和突变的酶活性缺陷突变体PBEF，用于功能研究的得失。我们将在活体2-P显微镜下对活体小鼠的树突状突起、谷氨酸释放和钙超载进行成像，并进行梗塞体积测量和神经元死亡实验，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是否参与了PARP-1介导的缺血后神经元死亡(即副死亡性)。目的：为了验证线粒体NAD+挽救通路在介导脑缺血后神经元保护中的主导作用的假说，我们将测定正常和缺血条件下神经元PBEF对NAD+池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室靶向分子表达，我们将确定线粒体NAD+挽救途径是否在缺血后的神经保护中发挥关键作用。目的3：验证PBEF通过促进神经元(突触)可塑性改善长期中风预后的假设。我们将使用多种方法评估神经性PBEF对长期卒中结果的影响。我们将对Thy1-Pbef-/-CKO和WT小鼠进行从缺血后一天到几周的长期脑梗塞体积测量、组织丢失、神经细胞凋亡、行为测试和神经学评估。我们将使用长期重复的活体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中风治疗的转化性研究的潜在治疗靶点提供新的见解。

 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.