Poly (ADP-ribose) Mediates Cell Death in Stroke by Inhibiting Glucose Metabolism

 描述（由申请人提供）：本研究将确定聚（ADP-核糖）聚合物（PAR）依赖性病理过程在卒中中的作用，我们认为该病理过程是诱导卒中中生物能量缺陷、氧化应激和线粒体通透性转换的中心途径。中风仍然是美国和世界范围内死亡和发病的主要原因，缺乏有效的治疗神经支配。迫切需要鉴定中风中细胞死亡的分子途径，以便可以鉴定新的治疗靶点。 聚（ADP-核糖）聚合酶-1（PARP-1）的过度激活与卒中中诱导细胞死亡密切相关。研究表明，PAR是PARP-1激活中的死亡信号分子，PAR与己糖激酶（HK-1）结合可能导致生物能量崩溃。然而，这种PAR/HK-1相互作用在中风中的作用尚不清楚。HK-1是脑中通过糖酵解和线粒体功能调节细胞生物能量的必需酶，并通过磷酸戊糖途径维持氧化还原稳态。此外，保护线粒体免受通透性转换是HK-1在脑中的重要功能。本申请的初步数据支持以下观点：在暴露于OGD（体外缺血/中风模型）的皮质神经元中，PAR结合HK-1并改变其功能。HK-1依赖的细胞存活功能，即生物能量平衡、氧化还原稳态和线粒体膜保护的崩溃是众所周知的，可诱导中风中的细胞死亡。因此，可以想象PAR与HK-1的结合是中风中诱导细胞死亡的中心病理途径，因此，可能是治疗干预的可靠靶点。 中风为了直接和严格地确定这种新途径在中风中的作用，我们将使用病毒介导的内源性HK-1的敲低和替换与PAR结合突变形式的HK-1（pbmHK-1）结合OGD在神经元培养物和MCAO中风模型在成年小鼠。我们提出了以下4个目标：目标#1：PAR依赖性抑制HK-1介导OGD处理的神经元中的生物能量缺陷吗？目的#2：OGD神经元中的氧化应激和氧化还原失衡是否是PAR/HK-1相互作用的结果？目的#3：PAR与HK-1结合是否导致神经元中OGD后线粒体通透性的转变？目的4：PAR与HK-1的结合在体内卒中中对细胞死亡的作用是什么？ 每个提出的目标都得到了一组重要的初步数据的支持，这些数据强烈支持了以下假设：PAR对HK-1是中风中诱导细胞死亡的中心病理过程。在中风的实验模型中将病毒介导的pbmHK-1表达与Seahorse Flux分析、代谢示踪剂研究、活细胞成像和生化分析相结合代表了更深入地理解中风中PAR依赖性细胞死亡信号传导的新方法。这些研究对于进一步揭示这种新的HK-1/PAR通路在卒中中的作用以及确定这种重要的病理通路是否是卒中治疗干预的可靠靶点至关重要。

 DESCRIPTION (provided by applicant): This study will identify the role of a Poly (ADP-ribose) polymer (PAR)-dependent pathological process in stroke that we believe is a central pathway to induce bioenergetic defects, oxidative stress and mitochondrial permeability transition in stroke. Stroke remains a major cause of mortality and morbidity in United States and worldwide and lacks effective therapeutic innervations. There is a critical need to identify the molecular pathways of cell death in stroke so that novel therapeutic targets can be identified. Excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) activation is strongly implicated to induce cell death in stroke. Studies have shown that PAR is a death signaling molecule in PARP-1 activation and that binding of PAR to hexokinase (HK-1) may cause bioenergetic collapse. However, the role of this PAR/HK-1 interaction in stroke is not known. HK-1 is an essential enzyme in the brain to regulate cellular bioenergetics via glycolysis and mitochondrial function, and maintains redox homeostasis via pentose phosphate pathway. In addition, protection of mitochondria against permeability transition is an important function of HK-1 in the brain. Preliminary data in this application supports the view that in cortical neurons exposed to OGD (an in vitro ischemia / stroke model), PAR binds HK-1 and alters its functions. Collapse of the HK-1 dependent cell survival functions namely bioenergetic balance, redox homeostasis and mitochondrial membrane protection are well known to induce cell death in stroke. Therefore, it is conceivable that binding of PAR to HK-1 is a central pathological pathway to induce cell death in stroke and therefore, may be a credible target for therapeutic intervention in stroke. To directly and rigorously identify the role of this novel pathway in stroke, we will use viral-mediated knockdown of endogenous HK-1 and replacement with PAR-binding mutant form of HK-1 (pbmHK-1) in combination with OGD in neuronal cultures and an MCAO model of stroke in adult mice. We propose the following 4 aims: Aim #1: Does PAR-dependent inhibition of HK-1 mediate bioenergetic defects in OGD-treated neurons? Aim #2: Are oxidative stress and redox imbalance in OGD-subjected neurons the consequence of PAR/HK-1 interaction? Aim # 3: Does PAR-binding to HK-1 lead to mitochondrial permeability transition after OGD in neurons? Aim # 4: What is the role of PAR-binding to HK-1 on cell death in stroke in vivo? Each of the proposed aims are supported by a set of important preliminary data, which strongly support the hypothesis that PAR to HK-1 is a central pathological process to induce cell death in stroke. Combining viral-mediated expression of pbmHK-1 with Seahorse Flux analysis, metabolic tracer studies, live-cell imaging and biochemical analysis in experimental models of stroke represent a novel approach to understand PAR- dependent cell death signaling in stroke in greater depth. These studies are crucial to further reveal the role of this novel HK-1/PAR pathway in stroke and to determine whether this important pathological pathway is a credible target for therapeutic intervention in stroke.