Determining the mechanisms that regulate neurofascin155 distribution in myelin

描述（由申请人提供）：Neurofascin155 （NF155）积聚在髓鞘的外侧边缘，并通过结合contactin/caspr的神经元异二聚体将鞘系在轴突上。在多发性硬化症（MS）的早期阶段，NF155簇丢失导致髓鞘-轴突粘连受损，导致脱髓鞘和破坏性运动和认知缺陷的发展。因此，维持NF155簇的稳定性对于保持神经元功能至关重要。不幸的是，调控NF155簇稳定性的机制尚不清楚。根据我实验室的初步数据，我已经确定了两种可能的NF155锚定方法，它们可能在维持NF155簇和髓磷脂稳定性方面发挥重要作用，但在时间上是不同的。我的初步研究结果表明，在髓鞘形成过程中，NF155和脂质之间的膜内相互作用调节了NF155在髓鞘中的位置。也许令人惊讶的是，在这个发育阶段，肌动蛋白细胞骨架网络似乎没有在NF155结构域稳定中发挥作用。然而，随着年龄的增长，NF155结构域的稳定性似乎依赖于肌动蛋白网络。nf155 -脂质相互作用在成熟鞘中起什么作用（如果有的话）还有待确定。为了证实这些发现，我将采用一种改进的原位提取方法来破坏脂质结构域或肌动蛋白网络。在确定具体的破坏后，我将使用各种洗涤剂来测试NF155簇的稳定性。根据我的实验室发表的工作，脂质，特别是髓磷脂糖鞘脂脂，是维持NF155簇所必需的。为了阐明脂质如何调节NF155的分布，我将生成几个荧光标记的NF155构建体，其中有或没有棕榈酰化所需的氨基酸突变，棕榈酰化是一种已知的介导蛋白质-脂质相互作用的翻译后修饰。转染这些构建体后，我将使用光漂白后的荧光恢复来量化棕榈酰化在NF155集群维持调控中的作用。公共卫生相关性：神经fascin155聚集在髓鞘的副极，并将鞘系在轴突上。在多发性硬化症的早期阶段，这些旁神经簇丢失，促进脱髓鞘和随后的运动和认知退化。本提案中概述的研究旨在确定调节NF155分布的机制，因为这些似乎是疾病过程的目标。确定这些疾病靶点应有助于开发新的治疗策略，以改善这种毁灭性疾病的影响。

DESCRIPTION (provided by applicant): Neurofascin155 (NF155) accumulates in that lateral edges of the myelin sheath and tethers the sheath to the axon by binding the neuronal heterodimer of contactin/caspr. In early stages of multiple sclerosis (MS), NF155 clusters are lost resulting in compromised myelin-axon adhesion leading to demyelination and the development of devastating motor and cognitive deficits. Thus, maintaining NF155 cluster stability is critical for preserving neuronal function. Unfortunately, the mechanisms that regulate NF155 cluster stability are unknown. Based on preliminary data from my laboratory, I have identified 2 potential methods of NF155 anchoring that may play essential, yet temporally distinct, roles in maintaining NF155 clusters and myelin stability. My initial findings suggest that intramembrane interactions between NF155 and lipids regulate the position of NF155 in the myelin sheath during myelin formation. Perhaps surprisingly, the actin cytoskeletal network does not appear to play a role in NF155 domain stabilization during this stage of development. With age, however, NF155 domain stability appears to be dependent on the actin network. It is yet to be determined what role, if any, the NF155-lipid interactions play in the mature sheath. To confirm these findings, I will employ a modified version of an in situ extraction method to disrupt either lipid domains or the actin network. Upon confirming specific disruption, I will use a variety of detergents to test NF155 cluster stability. Based on published work from my laboratory, lipids, particularly the myelin glycosphingolipid sulfatide, are required for maintaining NF155 clusters. To elucidate how lipids regulate NF155 distribution, I will generate several fluorescently labeled NF155 constructs with and without mutations to amino acids required for palmitoylation, a post translation modification known to mediate protein-lipid interactions. Following transfection of these constructs, I will employ Fluorescent Recovery After Photobleaching to quantify the role that palmitoylaiton plays in the regulation of NF155 cluster maintenance. PUBLIC HEALTH RELEVANCE: Neurofascin155 clusters in the paranode of the myelin sheath and tethers the sheath to the axon. In early stages of multiple sclerosis, these paranodal clusters are lost facilitating demyelination and subsequent motor and cognitive deterioration. The studies outlined in this proposal are designed to identify mechanisms that regulate NF155 distribution as these appear to be targets of the disease process. Identification of these disease targets should facilitate the development of novel therapeutic strategies designed to ameliorate the effects of this devastating disease.