Elucidating the role of UBXD4 at the axon initial segment

描述（由申请人提供）：轴突起始段（AIS）对于动作电位的产生和神经元极性的维持至关重要。由于疾病或损伤而导致的这一领域的破坏可导致神经系统功能障碍。动作电位起始的电压阈值在AIS处最低，这是由于电压门控钠通道的高密度簇通过与细胞骨架衔接蛋白ankG（ankG）的相互作用而在此处积累。AIS的功能组织取决于ankG。AIS蛋白非常稳定，半衰期可以超过两周。相比之下，许多突触蛋白的半衰期大约为几个小时。最近的研究强烈表明，AIS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蛋白水平如何响应神经元活动的变化而调节仍然未知。AIS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。新鉴定的泛素相关蛋白UBXD 4在AIS处富集。UBXD 4在结构上类似于泛素，并且被认为可以阻断其相互作用伴侣的降解结构域或将结合伴侣拴系到去泛素化酶。本项目的目的有三个：（1）确定AIS的蛋白质再循环是否是泛素蛋白酶体系统（UPS）介导的过程，（2）确定UBXD 4是否参与AIS的组装，（3）检验UBXD 4通过调节AIS蛋白半衰期有助于AIS稳定性的假设。AIS处的动作电位起始阈值可以在神经元之间变化，并且可以以活动依赖性方式进行调节。ankG转换率和Nav表达的差异可能会驱动阈值电位的变化。为了确定AIS处的活性依赖性蛋白质再循环是否是UPS依赖性事件，将对培养的海马神经元的活性进行人工操纵。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，并定量AIS蛋白被泛素化的程度。还将通过免疫细胞化学评估蛋白质水平。将蛋白酶体抑制剂给予细胞，以确定UPS引起的蛋白质降解减少是否会延长AIS蛋白质半衰期。UBXD 4在AIS组装中的作用将在培养的神经元中进行测试，并通过引入shRNA以沉默UBXD 4表达来体内测试。将通过免疫染色监测AIS组装。AIS处的UBXD 4定位取决于ankG。将使用His-ankG和GST-UBXD 4融合蛋白的免疫共沉淀和下拉实验来确定UBXD 4是否与ankG直接相互作用。最后，将在沉默UBXD 4表达和过表达UBXD 4后测量AIS蛋白稳定性。将通过免疫荧光法评估AIS蛋白转换率。该项目的结果将提高我们对稳定AIS的分子机制的理解，并有助于其响应活动变化的结构可塑性。

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.