Cellular and Molecular Analysis of Defects at Aging Neuromuscular Synapses

描述(申请人提供)：神经系统会随着年龄的增长发生令人不安的变化。为了阐明与年龄相关的神经功能下降的关键机制，我们将检查神经肌肉系统，它非常容易获得，相对简单，也是临床上与年龄相关的功能下降的显著部位。我们的初步分析导致了四组结果：1)随着年龄的增长，神经肌肉接头(NMJ)经历了许多结构和分子变化。2)运动轴突前终末部分出现异常变薄、膨大和发芽的区域。3)衰老肌肉的萎缩和突触变化在逐个纤维的基础上是相关的。4)尽管大多数肌肉中的NMJ随着年龄的增长而受到破坏，但少数肌肉中的NMJ却幸免于难。在这里，我们提出了一些研究，旨在探索这些变化之间的关系，确定它们背后的分子缺陷，并测试一种逆转它们的方法。首先，我们将跟踪初步观察到，在衰老的NMJ中，三种已知的突触组织分子--层粘连蛋白4和层粘连蛋白2以及集聚蛋白--的水平显著而具体地下降。我们将把这些蛋白质水平和分布的变化与结构变化联系起来，并询问这些蛋白质水平降低的靶向零突变或条件突变是否表明突触过早老化。其次，我们将通过相关的光学和电子显微镜来寻找轴突营养不良背后的运输缺陷，以及使用标记线粒体和突触小泡的新型转基因小鼠。第三，我们将确定突触异常和骨质疏松症之间的关系，骨质疏松症是临床上与年龄相关的肌肉质量和力量的显著下降。在转基因小鼠中，选择性地标记特定类型的单个运动轴突和肌肉纤维，我们将评估肌源性和神经源性石棺减少的决定因素。第四，我们将跟踪我们的观察结果，即眼外肌不会受到年龄相关性神经肌肉衰退的影响。这一结果很耐人寻味，因为在总是致命的肌萎缩侧索硬化症(ALS)中，眼外也是幸免于难的，这表明年龄相关和神经退行性缺陷的机制是平行的。最后，我们将使用转基因挽救技术来询问重新引入层粘连蛋白或集聚蛋白是否可以减轻与年龄相关的突触紊乱。总之，这些研究将为与年龄相关的神经缺陷提供见解，这些缺陷不仅可能提供改善骨质疏松症的方法，而且普遍适用于神经系统公共卫生相关性：神经系统随着年龄的增长经历令人不安的变化。研究人员建议使用神经肌肉系统来阐明与年龄相关的神经功能下降的关键机制。该系统非常方便，相对简单，临床上存在明显的与年龄相关的功能下降。首先，他们将对衰老神经肌肉接头(NMJ)的已知发育重要分子(层粘连蛋白和集聚蛋白)水平的戏剧性和特异性下降进行初步观察。NMJ是由运动神经元在肌肉纤维上形成的突触。他们将把这些蛋白质水平和分布的变化与结构变化联系起来，并询问这些蛋白质水平降低的突变小鼠是否表现出突触过早老化。其次，他们将寻找沿着神经纤维将材料输送到NMJ的缺陷。第三，他们将确定突触异常和骨质疏松症之间的关系，骨质疏松症是临床上与年龄相关的肌肉质量和力量显著下降。第四，他们将探索老年小鼠的神经肌肉变化与总是致命的肌萎缩侧索硬化症(ALS)之间在症状和肌肉特异性易感性方面有趣的相似之处。最后，他们会问，层粘连蛋白或集聚蛋白的重新引入是否可以减轻与年龄相关的突触紊乱。总之，这些研究将为与年龄相关的神经缺陷提供见解，这些缺陷不仅可能提供改善骨质疏松症的方法，而且普遍适用于神经系统。

DESCRIPTION (provided by applicant): The nervous system undergoes disturbing changes with age. To elucidate key mechanisms underlying age-related decline in neural function, we will examine the neuromuscular system, which is very accessible, relatively simple, and the site of clinically significant age-related functional decline. Our initial analysis has led to four sets of results: 1) Neuromuscular junctions (NMJs) undergo many structural and molecular alterations as they age. 2) Preterminal portions of motor axons exhibit regions of abnormal thinning, distension and sprouting. 3) Atrophy and synaptic changes in aged muscles are correlated on a fiber-by-fiber basis. 4) Although NMJs in most muscles are ravaged by age, those in a few are spared. Here, we propose studies designed to explore the relationships among these changes, identify molecular defects that underlie them, and test one way to reverse them. First, we will follow up preliminary observation of a dramatic and specific decline in levels of three known synaptic organizing molecules at aging NMJs -laminins 4 and 2 and agrin. We will correlate changes in the levels and distribution of these proteins with structural alterations, and ask whether targeted null or conditional mutants with decreased levels of these proteins show premature synaptic aging. Second, we will seek transport defects that underlie axonal dystrophy by correlative light and electron microscopy, along with use of new transgenic mice in which mitochondria and synaptic vesicles are labeled. Third, we will determine the relationship between the synaptic abnormalities and sarcopenia, the clinically significant age-related decline in muscle mass and strength. Using transgenic mice in which single motor axons and muscle fibers of specific types are selectively labeled we will assess myogenic and neurogenic determinants of sarcopenia. Fourth, we will follow up our observation that extraocular muscles are spared from age-related neuromuscular decline. This result is intriguing because extraoculars are also spared in the invariably fatal disease, amyotrophic lateral sclerosis (ALS), suggesting parallel mechanisms underlying age-related and neurodegenerative defects. Finally, we will use transgenic rescue techniques to ask whether reintroduction of laminins or agrin attenuates age- related synaptic disorganization. Together, these studies will provide insights into age- related neural defects that may not only provide ways to ameliorate sarcopenia but also be generally applicable to the nervous system PUBLIC HEALTH RELEVANCE: The nervous system undergoes disturbing changes with age. The investigators propose to use the neuromuscular system to elucidate key mechanisms underlying age-related decline in neural function. This system is very accessible, relatively simple, and the site of clinically significant age-related functional decline. First, they will follow up preliminary observation of a dramatic and specific decline in levels of known developmentally important molecules (laminins and agrin) at aging neuromuscular junction (NMJs), the synapses made by motoneurons on muscle fibers. They will correlate changes in the levels and distribution of these proteins with structural alterations, and ask whether mutant mice with decreased levels of these proteins show premature synaptic aging. Second, they will seek defects in the transport of materials along nerve fibers to the NMJ. Third, they will determine the relationship between the synaptic abnormalities and sarcopenia, the clinically significant age-related decline in muscle mass and strength. Fourth, they will explore intriguing similarities in symptoms and muscle-specific susceptibility between neuromuscular changes in aged mice and those in the invariably fatal disease, amyotrophic lateral sclerosis (ALS). Finally, they will ask whether reintroduction of laminins or agrin attenuates age-related synaptic disorganization. Together, these studies will provide insights into age-related neural defects that may not only provide ways to ameliorate sarcopenia but also be generally applicable to the nervous system.