Identification of mechanisms that regulate postsynaptic receptor abundance at the neuromuscular junction

项目摘要 在神经肌肉接头(NMJ)，突触后烟碱型乙酰胆碱受体(AChRs)转导 从胆碱能运动神经元释放的化学信号转变为电信号以诱导肌肉收缩。 胆碱能信号缺陷是严重肌肉无力的主要原因 先天性肌无力综合征和自身免疫综合征重症肌无力。此外，临床上 一些先天性肌病和肌营养不良的特征提示潜在的胆碱能信号 缺陷。总而言之，这突显了确定如何通过AChRs在 NMJ。虽然导致突触后AChR聚集的机制已经被很好地研究，但对此知之甚少 关于如何控制受体插入和内吞以维持突触效能。 模式生物线虫的体壁肌肉在功能上与脊椎动物的骨骼相当 肌肉。正弦运动是动物一侧突触后AChR激活的结果， 导致肌肉收缩，同时刺激对侧的GABAA受体 这种动物会引发肌肉放松。识别调节突触后胆碱能信号的新因素 我们进行了全基因组RNAi筛选，寻找改变线虫对AChR敏感性的基因敲除 激动剂左旋咪唑。导致左旋咪唑过敏的一种基因敲除是epn-1，它是 哺乳动物的Epsin，它的功能是招募特定的货物，并在 内吞作用。我们发现，epn-1的丢失导致AChRs的增加，但令人惊讶的是， 质膜上的GABAA受体。这导致我们假设EPN-1和其他一些 筛选分离株调节突触后受体的运输以维持适当的神经肌肉 变速箱。我们的首要目标是确定控制突触后受体丰度的机制 并通过表征我们屏幕上识别的基因在NMJ进行定位。我们将使用一个综合的方法， 进行创新的遗传学、成像、生物力学分析和光遗传学实验。我们的研究将 使我们能够对突触后受体运输的机制有一个更广泛的理解 NMJ，以及为未来的研究和治疗设计确定新的基因靶点。 我将在我在遗传学、神经科学、生理学和线虫研究方面的坚实基础上发展 在Velia Fowler博士和Robert博士的指导下进行的全面而有意义的研究计划 在骨骼肌收缩和患有肌肉疾病的儿童的NMJ发育方面拥有专业知识的Akins， 分别进行了分析。这项研究计划将在生物科学系和卓越核心进行 特拉华大学的设施。特拉华州肌肉骨骼研究中心将提供 强大的导师、职业发展资源和协作的跨学科科学家社区。

Project Summary At the neuromuscular junction (NMJ), postsynaptic nicotinic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. Defects in cholinergic signaling are the primary cause of severe muscle weakness observed in individuals with congenital myasthenic syndromes and the autoimmune syndrome myasthenia gravis. In addition, clinical features of some congenital myopathies and muscular dystrophies suggest underlying cholinergic signaling defects. Together, this highlights the importance of determining how signaling through AChRs is regulated at the NMJ. While mechanisms that lead to the clustering of postsynaptic AChRs have been well studied, little is known about how receptor insertion and endocytosis is controlled to maintain synaptic efficacy. The body wall muscles in the model organism C. elegans are functionally comparable to vertebrate skeletal muscles. Sinusoidal locomotion occurs as a result of activation of postsynaptic AChRs on one side of the animal, which causes muscle contraction, while simultaneous stimulation of GABAA receptors on the opposite side of the animal triggers muscle relaxation. To identify novel factors that regulate postsynaptic cholinergic signaling we performed a genome wide RNAi screen for gene knockdowns that altered C. elegans sensitivity to the AChR agonist levamisole. One knockdown that caused levamisole hypersensitivity was epn-1, the homolog of mammalian Epsin, which functions to recruit specific cargoes and induce membrane curvature during endocytosis. We discovered that loss of epn-1 resulted in an increase in AChRs, but surprisingly, a decrease in GABAA receptors on the plasma membrane. This led us to hypothesize that EPN-1 as well as some of the other screen isolates regulate trafficking of postsynaptic receptors to maintain appropriate neuromuscular transmission. Our overarching goal is to define the mechanisms that control postsynaptic receptor abundance and localization at the NMJ by characterizing genes identified in our screen. We will use an integrated approach, performing innovative genetic, imaging, biomechanical profiling, and optogenetic experiments. Our study will enable us to develop a broad understanding of mechanisms underlying postsynaptic receptor trafficking at the NMJ, as well as identify novel gene targets for future studies and therapeutic design. I will build upon my strong foundation in genetics, neuroscience, physiology, and C. elegans research to develop a comprehensive and meaningful research program under the mentorship of Dr. Velia Fowler and Dr. Robert Akins who have expertise in skeletal muscle contraction and NMJ development in children with muscle diseases, respectively. This research plan will be carried out in the Department of Biological Sciences and excellent core facilities at the University of Delaware. The Delaware Center for Musculoskeletal Research will provide access to strong mentors, career development resources, and a collaborative interdisciplinary community of scientists.