Identifying the Mechanisms and Localization of Activity-Dependent CaMKII Synthesis

确定活性依赖性 CaMKII 合成的机制和定位

• 批准号: 10750472
• 负责人: Kelsey Clements
• 金额: $ 3.86万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-16 至 2026-07-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750472
• 关键词: 3' Untranslated Regions; Adult; Affect; Affinity Chromatography; Alleles; Alzheimer' s Disease; Axon; Binding; Binding Proteins; Binding Sites; Biological Models; Brain Diseases; CRISPR/Cas technology; Ca(2+)-Calmodulin Dependent Protein Kinase; Catalytic Domain; Cells; Chemosensitization; Communication; Cycloheximide; Data; Dendrites; Drosophila genus; Elements; Excision; Frequencies; Functional disorder; Genes; Genetic; Genetic Recombination; Goals; Growth; Image Analysis; Imaging Device; Intellectual functioning disability; Invertebrates; Learning; Link; Mammals; Memory; MicroRNAs; Modeling; Modification; Molecular; Motor Neurons; Movement; Muscle; Neuromuscular Junction; Neurons; Physiologic pulse; Potassium; Process; Protein Biosynthesis; Protein translocation; Proteins; Protocols documentation; RNA; RNA-Binding Proteins; Reporter; Research; Research Design; Research Proposals; Role; Side; Spatial Distribution; Statistical Data Interpretation; Stimulus; Structure; Synapses; System; Training; Trans-Activators; Translations; Untranslated Regions; calmodulin-dependent protein kinase II; experimental study; fly; inhibitor; insight; nano; postsynaptic; presynaptic; protein transport; skills; superresolution imaging; superresolution microscopy; synaptic function; tool

Project Summary At the Drosophila neuromuscular junction (NMJ), repetitive stimulation has been shown to induce various forms of protein synthesis-dependent plasticity, including growth of immature “ghost” boutons and potentiation of miniature excitatory junction potential (mEJP) frequency. There is a concurrent increase in calcium/calmodulin-dependent protein kinase II (CaMKII) protein abundance, which is required for plasticity. The molecular mechanisms of this increase in CaMKII are not well understood, and previous studies examining this effect did not differentiate between the presynaptic and postsynaptic sides of the NMJ. The Drosophila NMJ has a wide variety of genetic tools to separate the motoneuron and muscle, making it an ideal model for separating presynaptic and postsynaptic mechanisms. Preliminary data indicate that after spaced depolarization with a high potassium solution, CaMKII levels increase in both the presynaptic and postsynaptic sides of the NMJ. Additionally, this increase was blocked by application of cycloheximide, a translation inhibitor, indicating that CaMKII accumulation is dependent on protein synthesis and is unlikely to be due to protein translocation or transport. The goal of this research proposal is to identify the molecular mechanisms and localization of CaMKII synthesis on both sides of the synapse. In the first part of Aim 1, GFP reporter constructs will be used to determine the sequences in the CaMKII 3’UTR that are required for activity- dependent translation. In the second part of Aim 1, the trans-acting proteins that bind to the UTR will be identified using RNA affinity purification. The results from these experiments will elucidate the specific cis- and trans-acting factors that are required for activity-dependent CaMKII protein synthesis, and determine whether similar mechanisms act pre- and post-synaptically. In Aim 2, Flp-Frt recombination will be used to delete either the CaMKII catalytic domain or the 3’UTR and the effect on potentiation of mEJP frequency will be determined. The excision of the domains will be done exclusively in the presynaptic or postsynaptic compartment, so that the contribution of either the motoneuron or muscle can be clearly separated. In Aim 3, the subcellular localization of newly-synthesized CaMKII will be compared to that of pre-existing CaMKII. The function of this newly-synthesized CaMKII pool is unknown, and this experiment will determine whether the newly-synthesized CaMKII is targeted to a distinct subcellular nanodomain. Ultimately, this research plan will clarify how compartment-specific CaMKII synthesis may be contributing to long-term functional changes in the synapse and may identify new avenues of research into CaMKII-dependent plasticity. While conducting this research, in-depth training will be provided in a variety of crucial skills, including research design, statistical analysis, communication skills, super-resolution microscopy, and image analysis.

项目概要 在果蝇神经肌肉接头（NMJ），重复刺激已被证明可以诱导各种 蛋白质合成依赖性可塑性的形式，包括未成熟“幽灵”布顿的生长和增强 微型兴奋性结电位 (mEJP) 频率同时增加。 钙/钙调蛋白依赖性蛋白激酶 II (CaMKII) 蛋白质丰度，这是可塑性所必需的。 CaMKII 增加的分子机制尚不清楚，之前的研究 检查这种效应并没有区分 NMJ 的突触前侧和突触后侧。 果蝇 NMJ 具有多种遗传工具来分离运动神经元和肌肉，使其成为理想的 分离突触前和突触后机制的模型初步数据表明，在间隔之后。 用高钾溶液去极化，突触前和突触后的 CaMKII 水平增加 此外，这种增加被放线菌酮（一种翻译）的应用所阻止。 抑制剂，表明 CaMKII 积累依赖于蛋白质合成，不太可能是由于 本研究计划的目标是确定蛋白质易位或运输的分子机制。 以及突触两侧 CaMKII 合成的定位 在目标 1 的第一部分中，GFP 报告基因。 构建体将用于确定 CaMKII 3'UTR 中活性所需的序列 在目标 1 的第二部分中，与 UTR 结合的反式作用蛋白将是 这些实验的结果将阐明特定的顺式和 活性依赖性 CaMKII 蛋白合成所需的反式作用因子，并确定是否 在目标 2 中，Flp-Frt 重组将用于删除任一突触前和突触后的类似机制。 将确定 CaMKII 催化结构域或 3'UTR 以及对 mEJP 频率增强的影响。 域的切除将仅在突触前或突触后区室中进行，以便 在目标 3 中，可以清楚地区分运动神经元或肌肉的贡献。 新合成的 CaMKII 的定位将与先前存在的 CaMKII 的功能进行比较。 新合成的CaMKII池是未知的，本实验将确定新合成的CaMKII池是否 最终，该研究计划将阐明 CaMKII 的目标是一个独特的亚细胞纳米域。 室特异性 CaMKII 合成可能有助于突触的长期功能变化 在进行这项研究时，可能会发现 CaMKII 依赖性可塑性的新研究途径。 将提供各种关键技能的深入培训，包括研究设计、统计分析、 沟通技巧、超分辨率显微镜和图像分析。