Elucidating the role of UBXD4 at the axon initial segment

描述(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的这一领域的破坏可能会导致神经系统功能障碍。动作电位启动的电压阈值在AIS处最低，因为高密度的电压门控钠通道聚集在这里，通过与细胞骨架适配器蛋白ankyrinG(AnkG)的相互作用在这里聚集。AIS的职能组织依赖于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在几个小时左右。最近的研究有力地表明，人工智能也是可塑性的，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蛋白水平是如何随着神经元活动的变化而调节的，仍然是未知的。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中得到了丰富。UBXD4在结构上类似于泛素，被认为要么阻断其相互作用伙伴的降解结构域，要么将结合伙伴拴在脱泛素化酶上。该项目的目标有三个：(1)确定AIS的蛋白质循环是否是泛素蛋白酶体系统(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蛋白半衰期而促进AIS稳定性的假设。AIS处的动作电位起始阈值在不同神经元之间可以不同，并且可以以活动依赖的方式进行调节。AnkG转换率和NAV表达的差异可能会导致阈值电位的变化。为了确定AIS中活性依赖的蛋白质循环是否是UPS依赖的事件，将对培养的海马神经元的活动进行药物操纵。然后，细胞裂解产物将被免疫印迹，以检测AIS蛋白水平的变化，并量化AIS蛋白泛素化的程度。蛋白质水平也将通过免疫细胞化学进行评估。蛋白酶体抑制剂将被应用于细胞，以确定UPS减少的蛋白质降解是否增加了AIS蛋白质的半衰期。通过引入shRNA来沉默UBXD4的表达，将在培养的神经元和体内测试UBXD4在AIS组装中的作用。人工免疫系统的组装将通过免疫染色进行监测。UBXD4在AIS的本地化取决于ankG。His-ankG和GST-UBXD4融合蛋白的免疫共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，检测沉默UBXD4表达和过表达UBXD4后AIS蛋白的稳定性。AIS蛋白质周转率将通过免疫荧光法进行评估。该项目的结果将提高我们对稳定AIS的分子机制的理解，并有助于其结构可塑性对活性变化的响应。

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.