Cellular and Molecular Analysis of Defects at Aging Neuromuscular Synapses

描述（由申请人提供）：随着年龄的增长，神经系统会发生令人不安的变化。为了阐明与年龄相关的神经功能衰退的关键机制，我们将研究神经肌肉系统，这是非常容易接近的，相对简单的，也是临床显著的与年龄相关的功能衰退的部位。我们的初步分析得出了四组结果：1)神经肌肉连接（NMJs）随着年龄的增长经历了许多结构和分子改变。2)运动轴突前端部出现异常变薄、膨胀和萌发区。3)衰老肌肉的萎缩和突触变化在纤维的基础上相互关联。4)尽管大多数肌肉中的NMJs会随着年龄的增长而受损，但少数肌肉中的NMJs却能幸免于难。在这里，我们提出了旨在探索这些变化之间关系的研究，确定其背后的分子缺陷，并测试一种逆转它们的方法。首先，我们将跟踪初步观察到的三种已知的突触组织分子-层粘胶蛋白4和2和agrin在衰老NMJs中的水平急剧下降。我们将把这些蛋白水平和分布的变化与结构改变联系起来，并询问这些蛋白水平降低的靶向零突变或条件突变是否表现出突触过早老化。其次，我们将通过相关的光镜和电子显微镜，以及使用标记线粒体和突触囊泡的新型转基因小鼠，寻找导致轴突营养不良的运输缺陷。第三，我们将确定突触异常与肌肉减少症之间的关系，肌肉减少症是临床上与年龄相关的肌肉质量和力量下降。使用转基因小鼠，其中单个运动轴突和特定类型的肌纤维被选择性标记，我们将评估肌少症的肌源性和神经源性决定因素。第四，我们将继续观察眼外肌免受与年龄相关的神经肌肉衰退的影响。这一结果很有趣，因为眼外肌在致命性疾病肌萎缩性侧索硬化症（ALS）中也不受影响，这表明了与年龄相关的和神经退行性缺陷的平行机制。最后，我们将使用转基因拯救技术来询问重新引入层粘连蛋白或agrin是否会减弱与年龄相关的突触紊乱。总之，这些研究将提供与年龄相关的神经缺陷的见解，这不仅可能提供改善肌肉减少症的方法，而且也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而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衰退的关键机制。这个系统是非常容易接近的，相对简单，和临床显著的年龄相关的功能衰退。首先，他们将对衰老的神经肌肉连接处（NMJs）的已知发育重要分子（层粘连蛋白和agrin）水平急剧下降的初步观察进行追踪。NMJs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改变联系起来，并询问这些蛋白质水平下降的突变小鼠是否表现出过早的突触老化。其次，他们将寻找沿神经纤维向NMJ输送物质的缺陷。第三，他们将确定突触异常与肌肉减少症之间的关系，肌肉减少症是临床上显著的与年龄相关的肌肉质量和力量下降。第四，他们将探索老年小鼠的神经肌肉变化与不可避免的致命疾病肌萎缩侧索硬化症（ALS）的症状和肌肉特异性易感性之间有趣的相似性。最后，他们将询问重新引入层粘连蛋白或agrin是否会减弱与年龄相关的突触紊乱。总之，这些研究将提供与年龄相关的神经缺陷的见解，这不仅可能提供改善肌肉减少症的方法，而且也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.