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

神经肌肉接头突触后受体丰度调节机制的鉴定

• 批准号: 10091026
• 负责人: Jessica E Tanis
• 金额: $ 18.1万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10091026
• 关键词: Affect; Animal Model; Animals; Autoimmune; Biological Sciences; Biomechanics; Caenorhabditis elegans; Candidate Disease Gene; Cell membrane; Chemicals; Child; Cholinergic Receptors; Clathrin; Clinical; Communities; Congenital Myasthenic Syndromes; Contracts; Core Facility; Data; Defect; Delaware; Development; Disease; Endocytosis; Foundations; Future; Gene Targeting; Genes; Genetic; Genetic Models; Goals; Histopathology; Homeostasis; Homologous Gene; Hypersensitivity; Image; Individual; Lead; Levamisole; Locomotion; Maintenance; Mediating; Membrane; Mentors; Mentorship; Molecular; Motor Neurons; Movement; Muscle; Muscle Contraction; Muscle Weakness; Muscle function; Muscle relaxation phase; Muscular Dystrophies; Musculoskeletal; Myasthenia Gravis; Myopathy; Neuromuscular Junction; Neurosciences; Nicotinic Agonists; Nicotinic Receptors; Paralysed; Pathway interactions; Patients; Pharmacology; Physiology; Play; RNA interference screen; Research; Resource Development; Role; Scientist; Side; Signal Transduction; Skeletal Muscle; Synapses; Syndrome; System; Testing; Therapeutic; Time; Universities; Work; acetylcholine receptor agonist; base; career development; cholinergic; congenital myopathy; density; design; epsin; experimental study; genome-wide; innovation; kinematics; knock-down; levamisole resistance; muscular structure; mutant; neuromuscular; neuromuscular transmission; novel; optogenetics; postsynaptic; presynaptic neurons; programs; receptor; recruit; targeted treatment; trafficking

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.

项目概要 在神经肌肉接头 (NMJ)，突触后烟碱乙酰胆碱受体 (AChR) 转导 从胆碱能运动神经元释放的化学信号转化为电信号以诱导肌肉收缩。 胆碱能信号传导缺陷是患有以下疾病的个体严重肌无力的主要原因 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 一些先天性肌病和肌营养不良症的特征表明存在潜在的胆碱能信号传导 缺陷。总之，这凸显了确定如何通过 AChR 调节信号传导的重要性。 NMJ。虽然导致突触后 AChR 聚集的机制已得到充分研究，但人们知之甚少 关于如何控制受体插入和内吞作用以维持突触功效。 模式生物秀丽隐杆线虫的体壁肌肉在功能上与脊椎动物骨骼肌相当 肌肉。正弦运动是由于动物一侧突触后 AChR 激活而发生的， 引起肌肉收缩，同时刺激另一侧的 GABAA 受体 动物会引发肌肉放松。识别调节突触后胆碱能信号传导的新因素 我们对基因敲低进行了全基因组 RNAi 筛选，这些基因敲低改变了线虫对 AChR 的敏感性 激动剂左旋咪唑。一种引起左旋咪唑过敏的基因敲低是 epn-1，它是 哺乳动物 Epsin，其功能是招募特定货物并在过程中诱导膜弯曲 内吞作用。我们发现 epn-1 的缺失会导致 AChR 的增加，但令人惊讶的是，AChR 的减少 质膜上的 GABAA 受体。这使我们假设 EPN-1 以及其他一些 筛选分离物调节突触后受体的运输以维持适当的神经肌肉 传播。我们的首要目标是定义控制突触后受体丰度的机制 并通过表征我们筛选中识别的基因在 NMJ 进行定位。我们将采用综合方法， 进行创新的遗传、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贩运的机制有广泛的了解 NMJ，以及为未来的研究和治疗设计确定新的基因靶点。 我将利用我在遗传学、神经科学、生理学和线虫研究方面的坚实基础来开发 在 Velia Fowler 博士和 Robert 博士的指导下开展的一项全面且有意义的研究项目 Akins 在患有肌肉疾病的儿童骨骼肌收缩和 NMJ 发育方面拥有丰富的专业知识， 分别。本研究计划将在生物科学系及优秀核心开展 特拉华大学的设施。特拉华州肌肉骨骼研究中心将提供访问权限 强大的导师、职业发展资源和跨学科科学家协作社区。