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、帕金森病和糖尿病神经病在内的多种神经系统疾病中，轴突病对发病和疾病进展有显着影响。轴突变性是一种主动的自毁过程，受损的轴突经历由知之甚少的信号级联引发的快速断裂。为了更好地理解这种级联反应，我们开发了一种筛选试验，以筛选能够在体外延迟横切小鼠感觉轴突断裂的化合物。我们使用此筛选来鉴定两种激酶：IKK 和 GSK3，它们可能是轴突变性的调节因子。拟议的研究从逻辑上遵循该筛选，旨在证明每种激酶与轴突细胞骨架元件的机械分解之间的联系，这是轴突自我断裂的必要步骤。该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，因此可能为减轻神经系统疾病和损伤负担的治疗进展提供信息。在目标 1 中，我们将检验 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表明的那样。首先，将研究从受损轴突中分离出的蛋白质中 IKK 激活的动态。我们将使用已建立的抑制剂来评估 IKK 激活是否发生在 JNK 和 GSK3 活性之后。最后，我们将直接询问受损轴突中神经丝蛋白的分解是否需要 IKK，以及神经丝去除是否涉及 IKK 依赖性泛素化。在目标 2 中，我们将询问 GSK3 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 GSK3 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 上的关键磷酸化位点 Thr231 介导 GSK3 对 tau 微管相互作用的破坏，我们将询问该位点是否在受伤的轴突中被磷酸化，以及 GSK3 抑制是否会阻止其磷酸化。最后，我们将询问与野生型 tau 相比，不可磷酸化突变 tau 的表达（假设可以在 GSK3 激活时稳定微管）是否会延迟轴突变性。 公共卫生相关性：许多神经系统疾病和损伤会导致轴突（神经细胞之间的微妙连接）受损。由于尚不清楚的原因，受损的轴突会经历自毁过程，这可能会导致疾病进展和更糟糕的临床结果。该项目将帮助我们了解受损的轴突如何进行自我毁灭，以便神经系统疾病的新疗法可以针对这一过程。