CRCNS: Quantitative description of initial biochemical steps in LTP

描述(申请人提供)：LTP由通过NMDAR的钙输入触发；随后，钙激活钙调蛋白(CaM)，然后激活CaMKII。尽管广泛的研究表明CaMKII在LTP和记忆中起着关键作用，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了解这些机制。这需要在接近光学分辨率极限的单个脊柱中测量生化事件的方法，并需要一个复杂的建模框架来模拟这些反应。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性质进行测量。游离凸轮的水平将使用光学记录器进行测量。当使用计算模型对所涉及的多种平衡进行分析时，这些数据将产生对神经元中自由和结合的CaM池的第一次估计。Ng和CaMKII的总浓度也将被测量。目的2.要模拟钙调素的激活，需要了解脊髓中钙离子的时空梯度。谷氨酸的双光子去功能化将被用来以受控的方式激活NMDAR；由此产生的大量脊柱细胞质中的钙升高将在简化的条件下测量(见目标3)。NMDAR微域中CA2的升高对CaMKII的激活非常重要(见下文)。为了确定NR2A和NR2B微区的升高，将开发一个NMDAR激活的随机模型以及钙扩散和缓冲的模型来解释测量到的整体钙激活。然后，这个计算框架可以用来估计微域中的钙升高。目的3.利用最新发展的方法(荧光寿命法)，测量单棘CaMKII激活的时间过程。我们将研究微域在激活该激酶中的作用。我们的初步结果表明，意想不到的复杂性：1)需要微域和大宗钙进入；2)磷酸酶可能影响激酶激活；3)神经颗粒素的磷酸化，一种与CaM结合的蛋白质，可能会随着时间的推移而变化(见目标4)。为了简化系统，将禁止磷酸酶，并将禁用Ng调制。在这些简化的条件下，将测量CaMKII激活(和钙升高)。然后将使用计算机模拟来预测目标2中确定的钙离子升高如何导致CaMKII激活。这一预测将与测量的活化度进行比较。然后，这个高度受限的框架可用于研究所涉及的过程的不同模型(反应的局部化以及使CaM可用的扩散和非结合过程)。一旦了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。目的4.神经颗粒素(Ng)是一种丰富的突触后蛋白，与CaM结合，可能在控制CaM激活CaMKII中起重要作用。此外，还有一些调节过程可以使Ng磷酸化，改变其结合CaM的能力。为了确定这种调节过程是否真的影响可塑性，我们将研究激活代谢性谷氨酸受体的效果。以往的工作表明，这会导致Ng的磷酸化，并阻断LTP。为了确定Ng介导的CaM对LTP的控制是否介导了这一效应，我们将利用Ng基因敲除小鼠。细胞将被导入一种不能被PKC磷酸化的Ng形式。如果这阻断了mGluR对LTP的影响，将证明Ng磷酸化对CaM的调控可以调节可塑性。更广泛的影响：为了向公众传达在理解记忆方面的进展，将在布兰迪斯大学一个类似博物馆的空间和网站上安装一幅大型壁画，说明长期记忆的早期步骤。作为这笔赠款的一部分，我们将开发最先进的软件，用于对生化反应进行蒙特卡罗建模。该软件将可供使用。该项目将包括培训，部分是通过Posse基金会的少数族裔计划。这也有医学意义：了解突触可塑性的早期步骤可能会让我们对学习障碍有更深入的了解。Ng是待研究的蛋白质之一，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.