Neuromuscular Junction Formation

描述(申请人提供)：我们实验室的长期目标是了解突触形成和维持的分子机制。我们的模型是NMJ，运动神经元和肌肉细胞之间的胆碱能突触。NMJ表现出化学突触的高度亚细胞特化特征。尽管我们对NMJ的解剖结构的了解有了很大的进步，但对各种成分如何组合在一起的机制却知之甚少。在初步研究中，我们使用条件性基因敲除策略抑制其在骨骼肌中的表达，研究了β-连环蛋白在NMJ形成中的作用。结果表明，肌肉β-连环素缺乏可导致突触后和突触前缺陷，包括膈神经初级分支脱位、AChR簇增大和神经传递功能障碍。相反，运动神经元中β-连环素的失活似乎对NMJ的形态和传递几乎没有影响。这些观察证明了肌肉？-连环蛋白在NMJ发育中的作用，确定了一种可能的？-连环蛋白依赖的逆行机制。为了进一步验证这一假设，我们提出了以下三个实验目标。我们将利用电子显微镜技术鉴定HSA-？-CAT-/-小鼠的NMJ；研究HSA-2？-CAT-/-小鼠神经传递缺陷的机制；以及肌肉B-连环蛋白调节NMJ形成的机制。这些实验结果将有助于更好地了解肌肉β-连环蛋白调节NMJ形成的机制，并为识别β-连环蛋白的效应因子和可能的逆行信号铺平道路。这些研究将有助于更好地了解神经肌肉疾病的病理生理学，并开发针对这些疾病的基因治疗和诊断工具的策略。与公共卫生相关：我们实验室的长期目标是了解突触形成和维持的分子机制。本研究旨在研究肌肉连环蛋白在NMJ形成中的作用。这些实验结果将有助于更好地理解肌肉β-连环蛋白调节NMJ形成的机制，并为识别b-连环蛋白的效应因子和可能的逆行信号铺平道路。这些研究将有助于更好地了解神经肌肉疾病的病理生理学，并开发针对这些疾病的基因治疗和诊断工具的策略。

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to understand molecular mechanisms underlying the formation and maintenance of synapses. Our model is the NMJ, a cholinergic synapse between motoneurons and muscle cells. The NMJ exhibits the high degree of subcellular specialization characteristic of chemical synapses. Despite significant progress in our understanding of anatomic structures of the NMJ, much less is known about mechanisms of how various components are put together. In preliminary studies, we investigated the role of ? -catenin in NMJ formation using a conditional knockout strategy to suppress its expression in skeletal muscles. The results demonstrate that muscle ? -catenin deficiency caused post- and pre-synaptic defects including dislocation of phrenic nerve primary branches, enlarged AChR clusters, and impaired neurotransmission. In contrast, ? -catenin inactivation in motoneurons appeared to have little effect on NMJ morphology and transmission. These observations demonstrate a role of muscle ? -catenin in NMJ development, identifying a possible ? -catenin-dependent retrograde mechanism. To further test this hypothesis, we propose the following three aims of experiments. We will characterize NMJs in HSA-?? -cat-/- mice using electron microscopic techniques; investigate the mechanisms of defective neurotransmission in HSA-2 ? -cat-/- mice; and investigate mechanisms of how muscle B-catenin regulates NMJ formation. Results of these experiments will contribute to a better understanding of mechanisms of muscle ? -catenin in regulating NMJ formation and pave the way to the identification of effectors of ? -catenin and possible retrograde signals. These studies will contribute to a better understanding of pathophysiology of the neuromuscular disorders and to developing strategies of gene therapy and of diagnostic tools for these diseases. PUBLIC HEALTH RELEVANCE: The long-term goal of our laboratory is to understand molecular mechanisms underlying the formation and maintenance of synapses. This proposal is to study the role of muscle ?-catenin in NMJ formation. Results of these experiments will contribute to a better understanding of mechanisms of muscle ?-catenin in regulating NMJ formation and pave the way to the identification of effectors of b-catenin and possible retrograde signals. These studies will contribute to a better understanding of pathophysiology of the neuromuscular disorders and to developing strategies of gene therapy and of diagnostic tools for these diseases.