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

项目摘要 树突形态决定了神经元功能的许多方面，包括动作电位的传播 和信息处理。虽然新出现的证据表明，枝晶生长和分支是 由于在地方上受到管制，在地方上缺乏最佳的测量方法。脑源性神经营养因子 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，我们报道了BDNF对树突发育的影响。 对树枝状乔木的不同局部效果取决于其施加在乔木上的位置。BDNF触发PKA 激活来调节树枝状突起分支，但关于脑源性神经营养因子如何激活PKA来促进树枝状突起分支的知之甚少 局部枝晶分支。这项工作的目的是利用cAMP依赖的蛋白激酶（PKA）激活 传感器作为一个F rster共振能量转移（FRET）为基础的成像平台的一部分，研究 PKA激活对树突状乔木的时空调控。首先，我们将在本地应用PKA激活剂或 抑制剂的树突，并显示PKA活性和树突分支之间的函数关系。一 微管靶向A激酶活性报告基因（tAKAR 4 α），显示出高灵敏度和动态范围， 被神经调质激活后，将在培养的胚胎大鼠海马神经元中表达， 性别我们将测量PKA在神经元中随着树突发育的时间依赖性动态分布， 分支。第二，据报道，激活PKA的核信号传导发生并最高 在刺激次级树突与其他级树突之后，无论与索马的距离如何。因此我们 将构建一种新的PKA激活传感器tAKAR 4 ν，它将靶向细胞核，并使用这种新的 FRET传感器，以确定是否核PKA活性增加时，二级，但不是其他顺序， 树突用PKA激活剂刺激。第三，我们将使用tAKAR 4 α FRET传感器和核- 靶向AKAR 4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，然后可以靶向治疗， 在由于中风或TBI或由于神经退行性疾病而受伤后恢复神经元连接和电路 疾病

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.