The Role of Ikappa-B Kinase and Glycogen Synthase Kinase 3-beta in Axon Degenerat

Ikappa-B 激酶和糖原合酶激酶 3-β 在轴突退化中的作用

• 批准号: 8262386
• 负责人: JOSIAH GERDTS
• 金额: $ 2.9万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8262386
• 关键词: Ablation; Afferent Neurons; Antibodies; Axon; Axotomy; Biological Assay; Clinical; Commit; Cytoskeleton; Data; Diabetic Neuropathies; Disease; Disease Progression; Dominant-Negative Mutation; Elements; Ensure; Event; Excision; Figs - dietary; Genetic; Glycogen Synthase Kinase 3; Image; Immunoblotting; In Vitro; Injury; Libraries; Link; Literature; MAP Kinase Gene; Mechanics; Mediating; Microtubule-Associated Proteins; Microtubules; Morbidity - disease rate; Mus; N-terminal; Neurofilament Proteins; Neurofilament-M; Neurologic; Neurons; Outcome; Parkinson Disease; Pathologic; Pathway interactions; Phase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Proteasome Inhibition; Proteasome Inhibitor; Proteins; RNA Interference; Role; Screening procedure; Sensory; Signal Pathway; Signal Transduction; Site; Subfamily lentivirinae; Testing; Therapeutic; Ubiquitination; Western Blotting; Work; axonopathy; base; design; expression vector; glycogen synthase kinase 3 beta; immunoreactivity; inhibitor/antagonist; injured; multicatalytic endopeptidase complex; mutant; nervous system disorder; neurofilament; overexpression; polymerization; prevent; public health relevance; recombinase; research study; stress-activated protein kinase 1; tau Proteins; tau mutation; tau phosphorylation; tau-microtubule interaction

DESCRIPTION (provided by applicant): In several neurologic disorders including Alzhemier disease, Parkinson disease, and diabetic neuropathy, axonopathy contributes significantly to morbidity and disease progression. Axon degeneration is an active self- destruct process by which compromised axons undergo rapid fragmentation initiated by a poorly-understood signaling cascade. To better understand this cascade, we developed a screening assay for compounds that delay fragmentation of transected mouse sensory axons in vitro. We used this screen to identify two kinases, IKK and GSK3, as probable regulators of axon degeneration. The proposed studies follow logically from this screen and are designed to demonstrate a link between each kinase and the mechanistic dismantling of axon cytoskeletal elements, a required step for axon self-fragmentation. The experiments outlined in this proposal will add to our limited understanding of how axons commit to self-destruction and may therefore inform therapeutic advances that reduce the burden of neurologic disease and injury. In Aim 1, we will test the hypothesis that IKK regulates Neurofilament breakdown in injured axons, as suggested by preliminary knockdown and pharmacologic studies. First, the dynamics of IKK activation will be studied in protein isolated from injured axons. We will assess whether IKK activation occurs subsequent to JNK and GSK3 activity using established inhibitors of each. Finally we will directly ask whether IKK is required for breakdown of Neurofilament protein in injured axons and whether Neurofilament removal involves IKK- dependent ubiquitination. In Aim 2, we will ask whether GSK3 contributes to axon degeneration by disrupting tau-microtubule interactions. First, we will use genetic ablation of GSK3 to determine whether it is required for normal axon degeneration as suggested by pharmacologic data. Next, because the critical phosphorylation site on tau, Thr231, mediates GSK3 disruption of tau-microtubule interactions, we will ask whether this site is becomes phosphorylated in injured axons and whether GSK3 inhibition blocks its phosphorylation. Finally, we will ask whether expression of non-phosphorylatable mutant tau - hypothesized to stabilize microtubules in the face of GSK3 activation - delays axon degeneration compared to wild-type tau. PUBLIC HEALTH RELEVANCE: Many nervous system diseases and injuries result in damage to axons - the delicate connections between nerve cells. For reasons not yet understood, damaged axons undergo a self-destruct process that may contribute to disease progression and worse clinical outcomes. This project will help us understand how damaged axons commit to self-destruction so that this process might be targeted by new therapies for nervous system diseases.

描述（由申请人提供）： 在包括阿尔茨海默病、帕金森病和糖尿病性神经病变在内的几种神经系统疾病中，轴突病变显著促进发病率和疾病进展。轴突变性是一种主动的自我破坏过程，受损的轴突通过该过程经历由不太清楚的信号级联启动的快速片段化。为了更好地理解这一级联反应，我们开发了一种筛选试验，用于延迟体外横断小鼠感觉轴突碎片化的化合物。我们用这个屏幕，以确定两个激酶，IKK和GSK 3，作为轴突变性的可能调节。拟议的研究遵循逻辑从这个屏幕和设计，以证明每个激酶和轴突细胞骨架元素的机械拆除，轴突自我分裂所需的步骤之间的联系。本提案中概述的实验将增加我们对轴突如何自我毁灭的有限理解，因此可能会为减少神经疾病和损伤负担的治疗进展提供信息。在目标1中，我们将测试IKK调节受损轴突中神经丝断裂的假设，正如初步敲除和药理学研究所建议的那样。首先，IKK激活的动力学将在从损伤的轴突分离的蛋白质中进行研究。我们将评估IKK激活是否发生在JNK和GSK 3活性之后，使用已建立的各自的抑制剂。最后，我们将直接询问IKK是否是受损轴突中神经丝蛋白分解所必需的，以及神经丝的去除是否涉及IKK依赖的泛素化。在目标2中，我们将询问GSK 3是否通过破坏tau-微管相互作用而导致轴突变性。首先，我们将使用GSK 3的基因消融来确定它是否是药理学数据所建议的正常轴突变性所必需的。接下来，由于tau蛋白上的关键磷酸化位点Thr 231介导GSK 3破坏tau-微管相互作用，我们将询问该位点是否在受损轴突中被磷酸化，以及GSK 3抑制是否阻断其磷酸化。最后，我们将询问与野生型tau蛋白相比，非磷酸化突变tau蛋白的表达-假设在GSK 3活化的情况下稳定微管-是否延迟轴突变性。 公共卫生关系： 许多神经系统疾病和损伤都会导致轴突（神经细胞之间的微妙连接）受损。由于尚不清楚的原因，受损的轴突经历了一个自毁过程，这可能导致疾病进展和更糟糕的临床结果。这个项目将帮助我们了解受损的轴突是如何自我毁灭的，这样这个过程就可能成为神经系统疾病新疗法的目标。