CRCNS: Quantitative description of initial biochemical steps in LTP

描述（由申请人提供）：LTP由Ca 2+通过NMDAR进入触发;随后Ca 2+激活钙调蛋白（CaM），然后激活CaMKII。尽管广泛的研究证明了CaMK II在LTP和记忆中的关键作用，但其在活细胞中的激活机制尚不清楚。智力优势：拟议工作的目标是了解这些机制的定量细节。这就需要在光学分辨率的极限附近测量单个脊椎中的生化事件的方法，以及用于模拟这些反应的复杂建模框架。由于实验和计算方法以前不可用，这将是第一次尝试解释活细胞中酶的测量激活。目标1。将对系统的关键定量特性进行测量。将使用光学报告物测量游离CaM的水平。当使用所涉及的多重平衡的计算建模进行分析时，这些数据将产生神经元中游离和结合钙调素池的第一个估计值。还将测量Ng和CaMKII的总浓度。目标二。为了模拟钙调素激活需要的信息的空间/时间梯度的钙离子在脊柱。2-谷氨酸的光子释放将用于以受控的方式激活NMDAR;在简化的条件下测量大量棘细胞质中产生的Ca 2+升高（参见目的3）。NMDAR微结构域中的Ca 2+升高对于CaMKII活化是重要的（见下文）。为了确定NR 2A和NR 2B微域的升高，将开发NMDAR激活的随机模型以及Ca 2+扩散和缓冲的建模，以解释测量的整体Ca 2+激活。然后，可以使用该计算框架来估计微区中的Ca 2+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。将检查微区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）需要微结构域和大量Ca 2+进入; 2）磷酸酶可能影响激酶活化; 3）神经颗粒蛋白（一种结合CaM的蛋白质）的磷酸化可能随时间而变化（见目标4）。为了简化系统，磷酸酶将被抑制并且Ng调节将被禁用。将在这些简化条件下测量CaMKII激活（和Ca 2+升高）。然后，计算机模拟将用于预测目标2中确定的Ca 2+升高如何导致CaMKII激活。该预测将与测量的激活进行比较。这种高度约束的框架，然后可以用来研究不同的模型所涉及的过程（本地化的反应和扩散和解约束的过程，使钙调素可用）。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。神经颗粒蛋白（Ng）是一种丰富的突触后蛋白，其结合CaM并且可能在控制可用于激活CaMKII的CaM中是重要的。此外，存在磷酸化Ng并改变其结合CaM的能力的调节过程。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。先前的工作表明，这导致Ng磷酸化并阻断LTP。为了确定Ng介导的CaM控制是否介导这种对LTP的影响，将使用Ng敲除小鼠。将用不能被PKC磷酸化的Ng形式转染细胞。如果这阻断了mGluR对LTP的作用，则表明通过Ng磷酸化控制CaM可以调节可塑性。更广泛的影响：为了向公众传达理解记忆的进展，一幅大型壁画将展示LTP的早期步骤，该壁画将安装在布兰迪斯大学的博物馆式空间和网站上。作为这笔赠款的一部分，我们将开发最先进的软件，用于生物化学反应的蒙特卡罗建模。将提供该软件。该项目将包括培训，部分通过波塞基金会少数民族方案。这也有医学意义：了解突触可塑性的早期步骤可能有助于了解学习障碍。Ng是待研究的蛋白质之一，是精神分裂症的风险基因。

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.