Synaptic and Nerve Terminal Changes and Associated Muscle Weakness of Burn Injury

 描述（由申请人提供）：烧伤（BI）导致神经肌肉功能障碍（NMD）和肌无力（MW）。BI后运动神经末梢和突触变化与MW的关系尚不清楚。神经肌肉接头（NMJ）的完整性取决于经由突触乙酰胆碱受体（AChR）介导的从神经到肌肉的向性信号，以及来自肌肉的逆行线索，包括经由Akt的关键信号传导（也称为，PKB）和糖原合成酶激酶-3 β（GSK-3 β）参与突触。末端雪旺细胞（TSC）的功能也是NMJ完整性所必需的。我们认为，BI的炎症反应和NMJ组织的线粒体功能障碍（MD）钝化了嗜性信号传导，TSC功能导致神经和突触紊乱，这些紊乱是NMD和MW的中心。主要的挑战是如何有效地逆转炎症反应介导的NMJ变化。TSC组成性表达α 7 AChRs亚型，BI后肌肉上调α 7 AChRs。发表，我们的新数据表明，α 7 AChRs刺激与特定的激动剂，GTS-21，减轻炎症反应，MD和增强信号通过Akt/GSK-3 β。检验的假设是，GTS-21刺激α 7 AChR，通过其多能作用，将保护BI小鼠的NMJ变化。具体目标1检验BI的突触和神经畸变导致NMD和MW的假设。使用Thy 1-YFP（黄色荧光蛋白）转基因小鼠（描绘运动神经）和荧光银环蛇毒素标记突触AChR，将表征BI后时间依赖性神经和突触形态学变化，以及它们与肌肉功能（张力和疲劳性）的关系。使用描绘神经MD功能的Thy 1-mito CFP（青色荧光蛋白）转基因小鼠和反映TSC状态的S100-GFP（绿色荧光蛋白）转基因小鼠将阐明BI诱导的神经MD和TSC变化对MW的作用。特定目的2检验了以下假设：α 7 AChR通过调节异常炎症反应、MD和BI的受损Akt/GSK-3 β信号传导，在维持NMJ完整性方面发挥保护作用。在基于特定目标1结果的固定时间点，将在BI早期损伤阶段和后期恢复阶段期间，在野生型和α 7 AChR敲除小鼠中研究NMJ的形态学和功能变化。具体目标3检验了以下假设：用GTS-21（α 7 AChR的特异性激动剂）治疗将减轻BI的突触和神经末梢变化。还将阐明GTS-21的功能和形态学变化、特异性和介导有益作用的信号通路。这些研究将表征BI的NMJ变化、其与MW的关系以及α 7 AChRs在NMJ完整性中的关键保护作用。通过α 7 AChR刺激的GTS-21治疗是一种未经测试的恢复BI的突触和神经末梢变化和MW的新方法。

 DESCRIPTION (provided by applicant): Burn injury (BI) leads to neuromuscular dysfunction (NMD), and muscle weakness (MW). The relationship of motor nerve terminal and synaptic changes to MW after BI is unknown. Integrity of neuromuscular junction (NMJ) depends on tropic signals from nerve to muscle mediated via synaptic acetylcholine receptors (AChRs), and on retrograde cues from muscle including pivotal signaling via Akt (a.k.a., PKB) and glycogen synthase kinase-3ß (GSK-3ß) to synapse. Terminal Schwann cells (TSC) function is also essential to NMJ integrity. We posit that inflammatory responses of BI, and mitochondrial dysfunction (MD) of NMJ tissues blunt tropic signaling, and TSC function leading to nerve and synaptic derangements, and these derangements are central to the NMD and MW. The major challenge is how to effectively reverse the inflammatory responses-mediated NMJ changes. TSCs constitutively express α7AChRs isoform, and muscles up-regulate α7AChRs after BI. Published, and our new data show that α7AChRs stimulation with specific agonist, GTS-21, mitigates inflammatory responses, MD and enhances signaling via Akt/GSK-3ß. The hypothesis tested is that stimulation with GTS-21 of α7AChRs, via its pluri-potent actions, will protect NMJ changes in BI mice. Specific Aim 1 tests the hypothesis that synaptic and nerve aberrations of BI lead to NMD and MW. Using Thy1-YFP (yellow fluorescent protein) transgenic mice (depicting motor nerve) and fluorescent-bungarotoxin to label synaptic AChRs, time-dependent nerve and synaptic morphological changes after BI, and their relationship to muscle function (tension and fatigability) will be characterized. Use of Thy1-mito CFP (cyan fluorescent protein) transgenic mice depicting nerve MD function, and S100-GFP (green fluorescent protein) transgenic mice reflecting TSC status will elucidate the role of BI-induced nerve MD and TSC changes to MW. Specific Aim 2 tests the hypothesis that α7AChRs play a protective role in maintenance of NMJ integrity by modulating aberrant inflammatory responses, MD, and impaired Akt/GSK-3ß signaling of BI. At fixed time points based on Specific Aim 1 results, morphological and functional changes at NMJ will be studied in wild type and α7AChR knockout mice during early injury-phase and later recovery-phase of BI. Specific Aim 3 tests the hypothesis that treatment with GTS-21, a specific agonist of α7AChRs, will mitigate synaptic and nerve terminal changes of BI. The functional and morphological changes, the specificity and signaling pathways mediating beneficial effects of GTS-21 will also be elucidated. These studies will characterize the NMJ changes of BI, their relationship to MW, and pivotal protective role of α7AChRs in NMJ integrity. Therapeutics with GTS-21 via a7AChRs stimulation is an untested novel approach to restore synaptic, and nerve terminal changes and MW of BI.