CRCNS: Quantitative description of initial biochemical steps in LTP

描述（由申请人提供）：LTP 由 Ca2 通过 NMDAR 进入而触发；随后 Ca2 激活钙调蛋白 (CaM)，然后钙调蛋白激活 CaMKII。尽管大量研究表明 CaMKII 在 LTP 和记忆中发挥着关键作用，但其在活细胞中的激活机制尚不清楚。智力价值：拟议工作的目标是定量详细地理解这些机制。这需要测量接近光学分辨率极限的单个脊柱中的生化事件的方法，以及用于模拟这些反应的复杂的建模框架。由于之前无法使用实验和计算方法，因此这将是首次尝试解释活细胞中酶的测量激活。目标 1. 对系统的关键定量特性进行测量。游离 CaM 的水平将使用光学报告仪测量。当使用所涉及的多个平衡的计算模型进行分析时，该数据将产生神经元中游离和结合 CaM 池的第一个估计。还将测量 Ng 和 CaMKII 的总浓度。目标 2. 要模拟 CaM 激活，需要有关棘中 Ca2 的空间/时间梯度的信息。谷氨酸的 2 光子解禁将用于以受控方式激活 NMDAR；由此产生的大体棘细胞质中的 Ca2+ 升高将在简化的条件下进行测量（参见目标 3）。 NMDAR 微域中 Ca2 的升高对于 CaMKII 激活很重要（见下文）。为了确定 NR2A 和 NR2B 微区的升高，将开发 NMDAR 激活的随机模型以及 Ca2 扩散和缓冲模型，以解释测量的大量 Ca2 激活。然后可以使用该计算框架来估计微域中的 Ca2+ 升高。目标 3. 使用最近开发的方法（荧光寿命方法 (FLIM)），将测量单个棘中 CaMKII 激活的时间过程。将检查微结构域在激酶激活中的作用。我们的初步结果表明了意想不到的复杂性：1）微域和大量 Ca2 进入都是必需的； 2) 磷酸酶可能影响激酶激活； 3) 神经颗粒素（一种结合 CaM 的蛋白质）的磷酸化可能会随时间而变化（参见目标 4）。为了简化系统，磷酸酶将被抑制，并且 Ng 调制将被禁用。 CaMKII 激活（和 Ca2+ 升高）将在这些简化的条件下进行测量。然后，计算机模拟将用于预测 Ca2 的升高（如目标 2 中确定的那样）如何导致 CaMKII 激活。该预测将与测量的激活进行比较。然后，这个高度受限的框架可用于研究所涉及过程的不同模型（反应的局部化以及使 CaM 可用的扩散和释放过程）。一旦理解了这个简化的系统，将使用额外的实验和计算机模拟来理解通常调节和调节这些步骤的更复杂的过程。目标 4. Neurogranin (Ng) 是一种丰富的突触后蛋白，可结合 CaM，并且可能在控制可用于激活 CaMKII 的 CaM 方面发挥重要作用。此外，还有一些调节过程可以磷酸化 Ng 并改变其结合 CaM 的能力。为了确定这种调节过程是否确实影响可塑性，将研究激活代谢型谷氨酸受体的影响。之前的研究表明这会导致 Ng 磷酸化并阻断 LTP。为了确定 Ng 介导的 CaM 控制是否介导对 LTP 的这种影响，将使用 Ng 敲除小鼠。细胞将被转染一种不能被 PKC 磷酸化的 Ng。如果这阻断了 mGluR 对 LTP 的影响，则表明 Ng 磷酸化对 CaM 的控制可以调节可塑性。更广泛的影响：为了向公众传达在理解记忆方面取得的进展，布兰代斯大学博物馆般的空间和网站上将悬挂一幅大型壁画，展示 LTP 的早期步骤。作为这笔赠款的一部分，我们将开发最先进的生化反应蒙特卡罗建模软件。该软件将可供使用。该项目将涉及培训，部分通过 Posse 基金会少数族裔项目进行。它还具有医学意义：了解突触可塑性的早期步骤可能有助于深入了解学习障碍。 Ng 是待研究的蛋白质之一，它是精神分裂症的危险基因。

DESCRIPTION (provided by applicant): LTP is triggered by Ca2+ entry through the NMDAR; subsequently Ca2+ activates calmodulin (CaM), which then activates CaMKII. Despite extensive studies demonstrating the pivotal role of CaMKII in LTP and memory, the mechanisms of its activation in living cells is not known. Intellectual Merit: The goal of the proposed work is to understand these mechanisms in quantitative detail. This requires methods to measure biochemical events in single spines near the limit of optical resolution and a sophisticated modeling framework for simulating these reactions. Because the experimental and computational methods were not previously available, this will be the first attempt to account for the measured activation of an enzyme in a living cell. Aim 1. Measurements will be made of critical quantitative properties of the system. The levels of free CaM will be measured using an optical reporter. This data, when analyzed using a computational modeling of the multiple equilibria involved, will yield the first estimate of free and bound CaM pools in neurons. The total concentration of Ng and CaMKII will also be measured. Aim 2. To model CaM activation requires information about the spatial/temporal gradients of Ca2+ in spines. 2-photon uncaging of glutamate will be used to activate NMDARs in a controlled way; the resulting Ca2+ elevation in the bulk spine cytoplasm will be measured under simplified conditions (see Aim 3). Ca2+ elevation in microdomains of the NMDAR is important for CaMKII activation (see below). To determine the elevation in microdomains of NR2A and NR2B, a stochastic model of NMDAR activation together with a modeling of Ca2+ diffusion and buffering will be developed to account for the measured bulk Ca2+ activation. This computational framework can then be used to estimate Ca2+ elevation in the microdomains. Aim 3. Using recently developed methods (fluorescence lifetime methodology (FLIM)), the time course of CaMKII activation in single spines will be measured. The role of microdomains in activation of the kinase will be examined. Our preliminary results suggest unexpected complexity: 1) both microdomain and bulk Ca2+ entry are required; 2) phosphatases may influence kinase activation; 3) phosphorylation of neurogranin, a protein that binds CaM, may vary over time (see Aim 4). To simplify the system, phosphatases will be inhibited and Ng modulation will be disabled. CaMKII activation (and Ca2+ elevation) will be measured under these simplified conditions. Computer simulations will then be used to predict how the elevation of Ca2+, as determined in Aim 2, leads to CaMKII activation. This prediction will be compared to the measured activation. This highly constrained framework can then be used to investigate different models of the processes involved (localization of reactions and the diffusional and unbinding processes that make CaM available). Once this simplified system is understood, additional experiments and computer simulations will be used to understand the more complex processes that normally regulate and modulate these steps. Aim 4. Neurogranin (Ng) is an abundant postsynaptic protein that binds CaM and may be important in controlling the CaM that is available to activate CaMKII. Moreover, there are modulatory processes that phosphorylate Ng and alter its ability to bind CaM. To determine whether such modulatory processes indeed affect plasticity, the effects of activating the metabotropic glutamate receptor will be studied. Previous work indicates that this leads to Ng phosphorylation and blocks LTP. To determine whether the Ng-mediated control of CaM mediates this effect on LTP, a Ng knockout mouse will be utilized. Cells will be transfected with a form of Ng that cannot be phosphorylated by PKC. If this blocks the effect of mGluR on LTP, it would demonstrate that the control of CaM by Ng phosphorylation can modulate plasticity. Broader Impact: To convey to the public progress in understanding memory, a large-scale mural illustrating the early steps in LTP will be mounted in a museum-like space at Brandeis University and on a website. As part of this grant, we will develop state-of-the-art software for Monte Carlo modeling of biochemical reactions. This software will be made available. The project will involve training, in part through the Posse Foundation minority program. There are also medical implications: understanding the early steps of synaptic plasticity may give insight into learning disorders. Ng, one of the proteins to be studied, is a risk gene for schizophrenia.