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

描述(由申请人提供)：在包括阿尔兹迈尔病、帕金森病和糖尿病神经病变在内的几种神经疾病中，轴突病变对发病率和疾病进展有显著影响。轴突变性是一种主动的自毁过程，受损的轴突经历了由一个鲜为人知的信号级联引发的快速碎裂。为了更好地了解这一级联反应，我们开发了一种筛选试验，以在体外延迟小鼠横断感觉神经轴突的碎裂。我们利用这个屏幕确定了两个可能的轴突变性调节因子IKK和GSK3。拟议的研究从这一屏幕上合乎逻辑地进行，旨在证明每一种激酶与轴突细胞骨架元件的机械性分解之间的联系，这是轴突自我碎裂所必需的步骤。这项提案中概述的实验将增加我们对轴突如何致力于自我毁灭的有限理解，并因此可能为减轻神经疾病和损伤负担的治疗进展提供信息。在目标1中，我们将根据初步的基因敲除和药理学研究，验证IKK调节受损轴突中神经丝破裂的假说。首先，将研究从受损轴突中分离出的蛋白质中IKK的激活动力学。我们将使用已经确定的JNK和GSK3的抑制剂来评估IKK的激活是否发生在JNK和GSK3的活性之后。最后，我们将直接询问损伤轴突中的神经丝蛋白是否需要IKK来分解，以及神经丝的去除是否涉及依赖IKK的泛素化。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正常轴突变性所必需的，正如药理学数据所表明的那样。接下来，由于tau上的关键磷酸化位点Thr231介导了tau-微管相互作用的GSK3破坏，我们将询问该位点在受损轴突中是否被磷酸化，以及GSK3抑制是否阻止其磷酸化。最后，我们将询问，与野生型tau相比，在GSK3激活的情况下，非磷酸化突变体tau的表达是否可以稳定微管-是否延迟轴突退化。 与公共卫生相关：许多神经系统疾病和损伤都会损害轴突--神经细胞之间的微妙连接。由于尚不清楚的原因，受损的轴突经历了一个自我破坏的过程，这可能会导致疾病的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导致自我毁灭的，以便这一过程可能成为神经系统疾病新疗法的靶点。

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.