Determining a Role for Protein Kinase A in Dendrite Development using a FRET-based Sensor

使用基于 FRET 的传感器确定蛋白激酶 A 在树突发育中的作用

• 批准号: 10227346
• 负责人: Nada Boustany
• 金额: $ 43.14万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227346
• 关键词: Action Potentials; Affect; Amides; Bioenergetics; Biological Models; Brain-Derived Neurotrophic Factor; Cell Nucleus; Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Dendrites; Development; Diffusion; Dose; Drosophila genus; Embryo; Fibrinogen; Fluorescence; Fluorescence Resonance Energy Transfer; Glean; Goals; Growth; Hippocampus (Brain); Image; In Vitro; Individual; Injury; Investigation; Knowledge; Light; Measurement; Measures; Mediating; Methods; Microtubules; Mitochondria; Morphology; Mus; Neurodegenerative Disorders; Neuromodulator; Neurons; Neurophysiology - biologic function; Nuclear; Nuclear Protein; Pattern; Pharmacological Treatment; Phosphotransferases; Play; Positioning Attribute; Process; Protein Kinase; Proteins; Rattus; Regulation; Reporter; Reporting; Role; Shapes; Signal Transduction; Site; Solid; Stroke; Synapses; System; Techniques; Testing; Time; Transfection; Work; base; experience; experimental study; extracellular; imaging modality; imaging platform; information processing; inhibitor/antagonist; injured; neural circuit; neuronal cell body; neuronal growth; protein activation; protein distribution; protein kinase inhibitor; regenerative approach; relating to nervous system; repair strategy; response; sensor; sex; spatiotemporal; targeted treatment; valosin-containing protein

PROJECT SUMMARY Dendrite morphology determines many aspects of neuronal function, including action potential propagation and information processing. Although emerging evidence suggests that dendrite growth and branching is regulated locally, the lack of optimal measurements at local sites exists. Brain-derived neurotrophic factor (BDNF) is one of the most studied regulators of dendrite development, and we reported that BDNF exerts distinct local effects on the dendritic arbor depending on where on the arbor it is applied. BDNF triggers PKA activation to regulate dendrite branching, yet not much is known about how BDNF activates PKA to promote local dendrite branching. The proposed work aims to utilize cAMP-dependent protein kinase (PKA) activation sensors as part of a Fӧrster resonance energy transfer (FRET)-based imaging platform to study the spatiotemporal regulation of the dendritic arbor by PKA activation. First, we will locally apply PKA activator or inhibitor to the dendrite and show a functional relationship between PKA activity and dendrite branching. A microtubule targeted A-kinase activity reporter (tAKAR4α) that shows high sensitivity and dynamic range and is activated by neuromodulators will be expressed in cultured embryonic rat hippocampal neurons of both sexes. We will measure the time-dependent dynamic distribution of PKA in neurons as dendrites develop and branch over time. Second, it has been reported that nuclear signaling of activated PKA occurs and is highest after stimulation of secondary versus other order dendrites, regardless of distance from the soma. As such, we will construct a new PKA activation sensor, tAKAR4ν, that will be targeted to the nucleus and use this new FRET sensor to determine whether nuclear PKA activity increases when secondary, but not other order, dendrites are stimulated with PKA activator. Third, we will use the tAKAR4α FRET sensor and nuclear- targeted AKAR4 probe to determine the mechanism by which BDNF locally regulates the arbor. These studies will shed light on mechanisms that shape neuronal morphology that can then be targeted for therapeutics to restore neuronal connectivity and circuitry after injury due to stroke or TBI or as a result of neurodegenerative diseases.

项目概要 树突形态决定神经元功能的许多方面，包括动作电位传播 和信息处理。尽管新出现的证据表明树突生长和分支是 由于当地监管，当地缺乏最佳测量。脑源性神经营养因子 (BDNF) 是树突发育中研究最多的调节因子之一，我们报道 BDNF 发挥作用 对树突心轴的不同局部影响取决于其应用在心轴上的位置。 BDNF 触发 PKA BDNF 激活调节树突分支，但目前对于 BDNF 如何激活 PKA 促进树突分支知之甚少。 局部树突分支。拟议的工作旨在利用 cAMP 依赖性蛋白激酶 (PKA) 激活 传感器作为基于费斯特共振能量转移 (FRET) 的成像平台的一部分，用于研究 PKA 激活对树突乔木的时空调节。首先，我们会在本地应用PKA激活剂或 树突抑制剂，并显示 PKA 活性和树突分支之间的功能关系。一个 微管靶向 A 激酶活性报告基因 (tAKAR4α)，具有高灵敏度和动态范围， 被神经调节剂激活后，将在培养的胚胎大鼠海马神经元中表达 性别。我们将测量随着树突的发育和神经元中 PKA 的时间依赖性动态分布 随着时间的推移分支。其次，据报道，激活的PKA的核信号传导发生并且是最高的 在刺激二级树突与其他级树突之后，无论距胞体的距离如何。因此，我们 将构建一种新的 PKA 激活传感器 tAKAR4ν，该传感器将针对细胞核并使用这种新的 FRET 传感器确定核 PKA 活性是否在二级时增加，但不是其他顺序， 树突受到 PKA 激活剂的刺激。第三，我们将使用 tAKAR4α FRET 传感器和核- 靶向 AKAR4 探针以确定 BDNF 局部调节乔木的机制。这些研究 将揭示塑造神经元形态的机制，然后可以将其作为治疗的目标 恢复中风、创伤性脑损伤或神经退行性损伤后的神经元连接和电路 疾病。