Determining the mechanisms that regulate neurofascin155 distribution in myelin

描述(申请人提供)：神经吸引155(NF155)聚集在髓鞘外侧边缘，通过结合神经元异源二聚体Conactin/CASPR将鞘与轴突捆绑在一起。在多发性硬化症(MS)的早期阶段，NF155簇丢失，导致髓鞘-轴突黏附受损，导致脱髓鞘和毁灭性的运动和认知障碍的发展。因此，保持NF155簇的稳定性对于保护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。基于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簇和髓鞘稳定方面发挥重要的、但在时间上不同的作用。我的初步发现表明，在髓鞘形成过程中，NF155和脂类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结构域的稳定中发挥作用。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，我将使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。在确认特定的破坏后，我将使用各种洗涤剂来测试NF155集群的稳定性。根据我的实验室发表的工作，维持NF155簇需要脂类，特别是髓鞘糖脂硫脂。为了阐明脂类如何调节NF155的分布，我将产生几个带有和不带有棕榈酰化所需氨基酸突变的荧光标记NF155结构，棕榈酰化是一种翻译后修饰，已知介导蛋白质-脂质相互作用。在转导这些构建体之后，我将使用光漂白后的荧光恢复来量化Palmitylaiton在NF155簇维持的调节中所起的作用。与公共健康相关：Neurozoch 155聚集在髓鞘的副阳极上，并将鞘系在轴突上。在多发性硬化症的早期阶段，这些结旁核团会丢失，从而导致脱髓鞘以及随后的运动和认知能力下降。本提案中概述的研究旨在确定调节NF155分布的机制，因为这些机制似乎是疾病过程的目标。确定这些疾病的目标应有助于开发新的治疗策略，旨在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.