Identifying novel proteins in injured nerves that promote functional regeneration

雪旺细胞（Schwann cells，SC）在周围神经系统（peripheral nervous system，PNS）中作为一个细胞单元发挥功能的作用越来越明显 神经元来调节感觉功能。当PNS受损时，SC被激活进行修复。这 涉及显著的SC表型转化。如果这一过程异常或受到抑制，周围神经- 损伤可能导致慢性衰弱性疼痛，这是在一般人群中观察到的问题，包括许多 老兵慢性神经性疼痛的治疗选择是有限的。不像许多其他调查人员在 SC领域，我们假设，在响应损伤，SC激活是可变的，从细胞到细胞，使一个con- SC激活状态的连续体共存。我们还假设SC修复程序可能会增加 以改善治疗效果。这个研究项目的主要目标是确定我们是否可以 靶向SC LDL受体相关蛋白-1（LRP 1），我们已经确定该受体在 SC激活，以改善周围神经损伤后的疼痛结局。为了实现我们的目标，三个人... 提出了明确的目标。在具体目标1中，我们将应用先进的转录组分析技术和 现在“在手”的SC特异性RiboTag小鼠模型，以测试SC激活发生在一系列 步骤，并可以增加治疗。这一目标的核心是我们发现特异性LRP 1配体 能够进一步激活对SC修复程序至关重要的关键细胞信号通路。我们 假设这些配体可能是改善PNS损伤后结果的候选物。在特定 目的2，我们的目标是发现天然存在于坐骨神经中的新型LRP 1配体，主要是在 损伤，因此可能有助于激活SC修复程序。这种基于发现的目标适用于 我们已经建立了捕获技术和LC-MS/MS。我们已经开始在受伤的组织中鉴定新的配体。 神经，包括Pacsin 1和Decorin，将在验证实验中进行分析。我们的持续工作 应该揭示额外的配体，也将使用体外生物测定法进行验证，以确定最佳的可 过渡到翻译研究。在具体目标3中，我们将测试LRP 1配体是否具有 最佳性质（如特定目标2中所确定的）改善了小鼠PNS损伤后的结果， 通过测量诱发性和自发性疼痛来挖掘。将研究两类LRP 1配体。第一、 将研究在受损神经中鉴定的LRP 1配体，其可作为内源性SC LRP 1激活剂。 其次，我们建议检查已知的LRP 1配体，这些配体已经被基因工程改造以捕获 有利的活动，同时消除不利的脱靶效应。Aim中提出的实验 已发表的研究表明，当小鼠SC中的LRP 1缺失时， 在野生型小鼠中研究表明SC LRP 1活性可以在体内增强。我们 我们认为这个项目是创新的，因为我们靶向干细胞，而不是神经元来治疗慢性疼痛。这项工作是 翻译是因为它有很好的机会来改善我们的VA患者的慢性疼痛管理。

It is increasingly apparent that Schwann cells (SCs) in the peripheral nervous system (PNS) function as a unit with neurons to regulate sensory function. When the PNS is injured, SCs become activated for repair. This involves dramatic SC phenotypic transformation. If this process is abnormal or inhibited, peripheral nerve in- jury may result in chronic debilitating pain, a problem observed in the general population, including numerous Veterans. Treatment options for chronic neuropathic pain are limited. Unlike many other investigators in the SC field, we hypothesize that in response to injury, SC activation is variable from cell to cell so that a con- tinuum of SC activation states co-exist. We also hypothesize that the SC Repair Program may be augmented therapeutically to improve outcomes. The major goal of this research project is to determine whether we can target SC LDL Receptor-related Protein-1 (LRP1), a receptor that we have identified as playing a central role in SC activation, to improve pain outcomes following peripheral nerve injury. To accomplish our goals, three spe- cific aims are proposed. In Specific Aim 1, we will apply advanced transcriptome profiling technology and a now “in hand” SC-specific RiboTag mouse model to test the hypothesis that SC activation occurs in a series of steps and may be augmented therapeutically. Central to this Aim is our discovery that specific LRP1 ligands are capable of further activating key cell-signaling pathways that are essential to the SC Repair Program. We hypothesize that these ligands may be candidates for improving outcomes following PNS injury. In Specific Aim 2, our goal is to discover novel LRP1 ligands that are naturally present in the sciatic nerve, mainly after injury, and thus may contribute to activation of the SC Repair Program. This discovery-based Aim applies established capture technology and LC-MS/MS. We have already begun to identify novel ligands in the injured nerve, including Pacsin1 and Decorin, which will be analyzed in validation experiments. Our continuing work should reveal additional ligands that also will be validated using in vitro bioassays to determine optimal can- didates to transition into translational studies. In Specific Aim 3, we will test whether LRP1 ligands with optimal properties (as determined in Specific Aim 2) improve outcomes following PNS injury in mice, as deter- mined by measuring evoked and spontaneous pain. Two categories of LRP1 ligands will be studied. First, LRP1 ligands identified in injured nerves, which may serve as endogenous SC LRP1 activators, will be studied. Second, we propose to examine known LRP1 ligands that have been genetically engineered to capture advantageous activities while eliminating disadvantageous off-target effects. The experiments proposed in Aim 3 are justified by published studies showing that when LRP1 is deleted in SCs in mice, pain-related behaviors increase and by studies in wild-type mice showing that SC LRP1 activity may be augmented in vivo. We consider this project innovative because we target SCs, instead of neurons to treat chronic pain. This work is translational in that it has excellent opportunity to improve management of chronic pain for our VA patients.