Elucidating the role of UBXD4 at the axon initial segment

描述（由申请人提供）：轴突初始段（AIS）对动作电位的产生和神经元极性的维持至关重要。由于疾病或损伤导致该区域的破坏可导致神经系统功能障碍。由于通过与细胞骨架接头蛋白ankyrinG （ankG）的相互作用，在AIS处积累了高密度的电压门控钠通道簇，因此动作电位启动的电压阈值在AIS处最低。AIS的功能组织依赖于ankG。AIS蛋白非常稳定，半衰期可超过两周。相比之下，许多突触蛋白的半衰期只有几个小时。最近的研究强烈表明，AIS也是可塑的，可以适应活动水平的变化。然而，AIS蛋白稳定性的分子机制以及AIS蛋白水平如何响应神经元活动的变化而被调节仍是未知的。AIS的稳定性可能取决于干扰蛋白质降解的事件。实现这种稳定性的一种方法可能是防止AIS蛋白的泛素化。新发现的泛素相关蛋白UBXD4在AIS富集。UBXD4在结构上与泛素相似，被认为可以阻断其相互作用伙伴的降解结构域，或者将结合伙伴束缚在去泛素化酶上。该项目的目的有三个方面：(1)确定AIS的蛋白质回收是否是一个泛素蛋白酶体系统（UPS）介导的过程，(2)确定UBXD4是否参与AIS的组装，(3)检验UBXD4通过调节AIS蛋白质半衰期有助于AIS稳定性的假设。AIS的动作电位起始阈值在神经元之间可能不同，并可能以活动依赖的方式进行调节。ankG周转率和Nav表达的差异可能会导致阈值电位的变化。为了确定AIS的活性依赖蛋白再循环是否是ups依赖事件，将对培养的海马神经元的活性进行药理学操作。然后对细胞裂解物进行免疫印迹，以检测AIS蛋白水平的变化，并量化AIS蛋白泛素化的程度。蛋白水平也将通过免疫细胞化学进行评估。将给细胞施用蛋白酶体抑制剂，以确定UPS降低的蛋白质降解是否会增加AIS蛋白质的半衰期。通过引入shRNA沉默UBXD4的表达，我们将在培养的神经元和体内测试UBXD4在AIS组装中的作用。AIS组装将通过免疫染色监测。AIS的UBXD4定位取决于ankG。His-ankG和GST-UBXD4融合蛋白的共免疫沉淀和下拉实验将用于确定UBXD4是否直接与ankG相互作用。最后，在沉默UBXD4表达和过表达UBXD4后，检测AIS蛋白的稳定性。免疫荧光法评估AIS蛋白周转率。该项目的结果将提高我们对稳定AIS的分子机制的理解，并有助于其响应活性变化的结构可塑性。

DESCRIPTION (provided by applicant): The axon initial segment (AIS) is critical for the generation of action potentials and the maintenance of neuronal polarity. Disruption of this domain due to disease or injury can lead to nervous system dysfunction. The voltage threshold for action potential initiation is lowest at the AIS due to the high- density clusters of voltage-gated sodium channels accumulated here through interaction with the cytoskeletal adaptor protein ankyrinG (ankG). The functional organization of the AIS depends on ankG. AIS proteins are remarkably stable with half-lives that can exceed two weeks. In contrast, the half-lives of many synaptic proteins are on the order of hours. Recent work strongly suggests that the AIS is also plastic and can adapt in response to changes in activity levels. The molecular mechanisms underlying AIS protein stability and how AIS protein levels are modulated in response to changes in neuronal activity, however, remain unknown. AIS stability may depend on events that interfere with protein degradation. One way to achieve such stability could be to prevent ubiquitination of AIS proteins. The newly identified ubiquitin-related protein UBXD4 is enriched at the AIS. UBXD4 is structurally similar to ubiquitin and is thought to either block the degradation domains of its interacting partners or to tether binding partners to a deubiquitinating enzyme. The objective of this project is three-fold: To (1) determine if protein recycling at the AIS is a ubiquitin proteasome system (UPS) mediated process, (2) determine if UBXD4 is involved in the assembly of the AIS, and (3) test the hypothesis that UBXD4 contributes to AIS stability by modulating AIS protein half-life. Action potential initiation threshold at the AIS can vary between neurons and may be modulated in an activity-dependent manner. Differences in ankG turnover rates and Nav expression could drive shifts in threshold potential. To determine if activity-dependent protein recycling at the AIS is a UPS-dependent event, the activity of cultured hippocampal neurons will be pharmacologically manipulated. Cell lysates will then be immunoblotted to detect changes in AIS protein levels and to quantify the extent to which AIS proteins are ubiquitinated. Protein levels will also be assessed by immunocytochemistry. Proteasome inhibitors will be administered to cells to determine if decreased protein degradation by the UPS increases AIS protein half-life. The role of UBXD4 in AIS assembly will be tested in cultured neurons and in vivo by introducing shRNA to silence UBXD4 expression. AIS assembly will be monitored by immunostaining. UBXD4 localization at the AIS depends on ankG. Co-immunoprecipitation and pull-down experiments with His-ankG and GST-UBXD4 fusion proteins will be used to determine if UBXD4 directly interacts with ankG. Finally, AIS protein stability will be measured after the silencing UBXD4 expression and overexpressing UBXD4. AIS protein turnover rates will be assessed by immunofluorescence. The results of this project will improve our understanding of the molecular mechanisms that stabilize the AIS and contribute to its structural plasticity in response to changes in activity.