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

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几种神经系统疾病中，轴突病对发病率和疾病进展有重要影响。轴突退化是一种主动的自毁过程，受损的轴突经历由一个鲜为人知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，我们开发了一种体外延缓小鼠感觉轴突断裂的化合物筛选试验。我们使用这个筛选来鉴定两种激酶，IKK和GSK3，作为轴突退化的可能调节因子。拟议的研究从这个屏幕逻辑上遵循，旨在证明每个激酶和轴突细胞骨架元件的机械拆除之间的联系，轴突自裂的必要步骤。本提案中概述的实验将增加我们对轴突如何进行自我毁灭的有限理解，并可能因此为减轻神经系统疾病和损伤负担的治疗进展提供信息。在Aim 1中，我们将验证IKK调节受损轴突神经丝断裂的假设，正如初步的敲除和药理学研究所建议的那样。首先，IKK激活的动力学将研究从受损轴突分离的蛋白质。我们将评估IKK激活是否发生在JNK和GSK3活性之后。最后，我们将直接询问IKK是否需要损伤轴突中神经丝蛋白的分解，以及神经丝的去除是否涉及IKK依赖的泛素化。在Aim 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退化。首先，我们将使用GSK3的基因消融来确定是否需要根据药理学数据来治疗正常的轴突变性。接下来，由于tau蛋白上的关键磷酸化位点Thr231介导了GSK3对tau-微管相互作用的破坏，我们将询问该位点是否在受损轴突中被磷酸化，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，非磷酸化突变型tau的表达是否会延迟轴突退化。

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.