The role of a novel AIF-associated nuclease PAAN1 in neuronal injury

描述（由申请人提供）： 成年神经元是控制人体功能的重要而珍贵的细胞类型。中风和其他神经系统疾病后的神经元损伤导致显著的功能丧失。我的长期目标是研究神经元损伤的分子机制，并开发新的治疗策略来治疗患有神经功能障碍的患者。聚腺苷二磷酸核糖聚合酶-1（PARP-1）在谷氨酸神经毒性和脑梗死中起关键作用，脑梗死是卒中后发病率和死亡率的主要原因。PARP-1基因敲除小鼠对中风具有强大的抵抗力。因此，了解卒中中PARP-1依赖性细胞死亡（parthanatos）的分子机制至关重要。线粒体凋亡诱导因子（AIF）的释放和易位到细胞核引起染色质溶解，这是死亡的承诺点。然而，很少有人知道AIF如何诱导核转位后的染色质溶解和神经元死亡，因为AIF本身没有内切酶活性。通过无偏的17 K人蛋白质芯片筛选和第二次基于siRNA的PARP-1依赖性细胞活力高通量筛选，鉴定了13个命中，包括AIF interactor 18和蛋白质精氨酸甲基转移酶6（PRMT 6）。在这里，我们关注AIF相互作用子18，因为我们的初步数据显示，AIF相互作用子18的敲低与Parthanatos中的PARP-1敲低一样具有保护性，并且它具有迄今未鉴定的内切核酸酶活性。因此，我们将AIF相互作用子18命名为PAAN 1（Parthanatos依赖性AIF相关核酸酶1）。在指导K99阶段，我们将首先确定PAAN 1在体外和体内缺血性神经元死亡中的作用。然后，我们将确定特定的PAAN 1核酸内切酶活性，并确定其核酸内切酶活性是否是神经元损伤所必需的。组蛋白翻译后修饰可调节PAAN 1在紧密包裹的基因组DNA中的识别，从而诱导PARP-1活化后的染色质溶解和细胞死亡。PRMT 6是一种组蛋白甲基转移酶，被鉴定为参与parthanatos。在独立的R 00阶段，我将研究PRMT 6在PAAN 1介导的缺血性细胞死亡中的作用，以了解PAAN 1募集到DNA损伤位点的机制。我将使用两个高通量筛选进一步鉴定和表征parthanatos所需的功能性PAAN 1相互作用蛋白，以了解PAAN 1内切核酸酶活性如何通过其网络进行调节。该项目的成功完成将对PAAN 1内切核酸酶活性和parthanatos的细胞控制产生重要的见解，并可能为开发治疗中风患者的创新治疗方法提供新的靶点。指导方法将在约翰霍普金斯大学医学院的Valina和Ted Dawson博士以及Raymond Koehler博士的实验室进行。我可以访问道森博士和克勒实验室以及细胞工程研究所的神经再生和干细胞项目的资源。为了成功完成拟议的研究，在指导阶段，我打算参加表观遗传学课程，并获得实验中风模型和生物信息学技术的技能，以及授予写作和教学技能。这次培训将使我能够扩展我的专业知识，并帮助我过渡到一个成功的独立学术研究人员。

DESCRIPTION (provided by applicant): Adult neurons are crucial and precious cell types controlling human body functions. Neuron injury following stroke and other neurologic diseases causes a significant loss of function. My long-term goal is to investigate the molecular mechanisms of neuronal injury and develop novel therapeutic strategies to treat patients suffering neurologic dysfunction. Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in glutamate neurotoxicity and cerebral infarction, which is a primary cause of subsequent morbidity and mortality following stroke. PARP-1 knockout mice are robustly resistant to stroke. Thus, it is critically important to understand the molecular mechanisms underlying PARP-1-dependent cell death (parthanatos) in stroke. Mitochondrial apoptosis-inducing factor (AIF) release and translocation to the nucleus causes chromatinolysis, which is the commitment point for parthanatos. However, little is known how AIF induces chromatinolysis and neuronal death after its nuclear translocation, since AIF itself has no endonuclease activity. Through an unbiased 17K human protein chip screening and a second siRNA-based PARP-1-dependent cell viability high-throughput screening, thirteen hits including AIF interactor 18 and protein arginine methyltransferase 6 (PRMT6) were identified. Here we focus on AIF interactor 18 as our preliminary data showed that knockdown of AIF interactor 18 is as protective as PARP-1 knockdown in parthanatos and it possesses hitherto unidentified endonuclease activity. Therefore, we name AIF interactor 18 as PAAN1 (Parthanatos-dependent AIF-Associated Nuclease 1). In the mentored K99 phase, we will first define the role of PAAN1 in ischemic neuronal death both in vitro and in vivo. We will then determine the specific PAAN1 endonuclease activity and determine if its endonuclease activity is required for neuronal injury. Histone post-translational modifications may regulate PAAN1 recognition in tightly wrapped genomic DNA to induce chromatinolysis and cell death after PARP-1 activation. PRMT6, a histone methyltransferase, was identified to be involved in parthanatos. In the independent R00 phase, I will study the role of PRMT6 in PAAN1-mediated ischemic cell death to understand the mechanism of PAAN1 recruitment to the DNA damage sites. I will further identify and characterize the functional PAAN1-interacting proteins required for parthanatos using two high-throughput screens, in order to understand how PAAN1 endonuclease activity is regulated by its networks. The successful completion of this project will yield important insights into cellular control of PAAN1 endonuclease activity and parthanatos and may provide new targets for developing innovative therapeutic approaches to treat patients with stroke. The mentored approach will be conducted in the laboratories of Drs. Valina and Ted Dawson and Dr. Raymond Koehler at the Johns Hopkins University School of Medicine. I have access to the resources available to Drs. Dawson and Koehler laboratories and the Neuroregeneration and Stem Cell Programs in Institute for Cell Engineering. To successfully accomplish the proposed research, during the mentored phase, I intend to take epigenetics courses and acquire skills in experimental stroke models and bioinformatics technologies, as well as grant writing and teaching skills. This training will allow me to expand my expertise and help my transition into a successful independent academic researcher.