CRCNS: Quantitative description of initial biochemical steps in LTP

描述（由申请人提供）：LTP是由Ca2+通过NMDAR进入触发的；随后Ca2+激活钙调素（CaM）， CaM随后激活CaMKII。尽管大量研究表明CaMKII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。智力价值：建议工作的目标是定量详细地理解这些机制。这需要在接近光学分辨率极限的单个脊椎中测量生化事件的方法，以及模拟这些反应的复杂建模框架。由于以前没有实验和计算方法，这将是第一次尝试解释活细胞中酶的测量激活。目的1。将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进行测量。当使用所涉及的多个平衡的计算模型分析这些数据时，将产生神经元中自由和束缚的CaM池的第一个估计。同时测量Ng和CaMKII的总浓度。目标2。为了模拟CaM激活，需要关于Ca2+在脊柱中的空间/时间梯度的信息。将利用谷氨酸的双光子释放来可控地激活NMDARs；将在简化的条件下测量大量脊柱细胞质中产生的Ca2+升高（见目的3）。NMDAR微域Ca2+升高对CaMKII激活很重要（见下文）。为了确定NR2A和NR2B微域的升高，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。这个计算框架可以用来估计Ca2+在微域的升高。目标3。使用最近发展的方法（荧光寿命法（FLIM）），将测量单个棘中CaMKII激活的时间过程。微结构域在激酶活化中的作用将被检查。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的激活；神经粒蛋白（一种结合CaM的蛋白质）的磷酸化可能随时间而变化（见目的4）。为了简化系统，磷酸酶将被抑制，Ng调制将被禁用。CaMKII激活（和Ca2+升高）将在这些简化的条件下测量。然后，计算机模拟将用于预测在Aim 2中确定的Ca2+升高如何导致CaMKII激活。这个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以用来研究所涉及的过程的不同模型（反应的定位以及使CaM可用的扩散和解结合过程）。一旦这个简化的系统被理解，额外的实验和计算机模拟将用于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，可结合CaM，可能在控制可激活CaMKII的CaM中起重要作用。此外，还存在磷酸化Ng并改变其结合CaM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，将研究激活代谢性谷氨酸受体的影响。先前的研究表明，这导致Ng磷酸化并阻断LTP。为了确定Ng介导的CaM控制是否介导了这种对LTP的影响，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，这将证明通过Ng磷酸化控制CaM可以调节可塑性。更广泛的影响：为了向公众传达在理解记忆方面的进展，一幅描绘LTP早期步骤的大型壁画将被安装在布兰迪斯大学的一个类似博物馆的空间和一个网站上。作为拨款的一部分，我们将开发最先进的生化反应蒙特卡罗模型软件。该软件将提供。该计划将包括培训，部分通过波塞基金会少数族裔计划进行。这也有医学意义：理解突触可塑性的早期步骤可能会让我们深入了解学习障碍。Ng是一种被研究的蛋白质，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.