Local calcium signalling in the postsynaptic density

在大脑中，数百亿个电活性细胞在称为突触的连接处相互通信。突触强度的长期变化被认为是学习和记忆的基础。研究的重点是了解突触可塑性是如何在包括海蛞蝓和老鼠在内的模式生物的分子水平上产生的。突触可塑性的“核心程序”的一些组成部分已经出现。值得注意的是，突触的持久增强和抑制都来自钙的内流。两种钙敏感酶对这一过程至关重要：一种在大量钙流入后修饰突触蛋白以加强突触，而另一种在少量钙流入后具有相反的作用。然而，这两种酶在试管中对钙表现出相同的敏感性，因此它们对突触内钙的反应不同的原因并不明显。一个重要的考虑因素是钙局部集中在靠近其进入点的细胞中。我们的研究将追求两个主要目标。首先，我们建议确定是否从钙进入点的两种酶的相对位置，在一个巨大的蛋白质组装称为突触后密度（PSD），解释为什么其中一种酶需要更大的钙流入激活。第二，我们的目标是确定为什么抑制突触强度的酶在大量钙离子流入后不活动，我们将采用互补的方法来检查这两种酶相对于PSD中钙离子进入点的位置。我们将化学交联PSD样品，并绘制蛋白质组装中不同蛋白质之间交联的确切位点。这将使我们能够建立准确的结构模型，这样我们就可以比较关键酶与钙进入点的相对位置。我们还将测量当与PSD内的不同蛋白质共定位时，钙传感器如何响应实验控制的钙流入。在追求我们的第二个主要目标，我们将集中在理解锚定蛋白如何调节钙敏感酶的活性，抑制突触强度。我们将确定锚定蛋白片段的高分辨率三维结构，以了解PSD内钙如何调节锚定蛋白。我们还将使用一种新的酶测定法来检测锚定蛋白是否能够调节引起长期突触抑制的酶，使其在低浓度而不是高浓度的钙下具有活性。这项研究将受益于与德国蛋白质组学专家的合作，从而巩固国际伙伴关系。除了解决大脑如何储存记忆的基本问题外，我们开发的新技术方法将广泛适用于生物学的其他领域。这项研究与人类大脑疾病高度相关，因为突触是导致神经系统疾病和精神疾病的突变位点，而异常的突触可塑性可能是许多大脑疾病的基础，包括阿尔茨海默氏症和自闭症。钙敏感酶和锚定蛋白是拟议研究的重点，都是药物靶点。总的来说，这项研究可以在分子水平上提供见解，为药物设计提供信息。

Within the brain, tens of billions of electrically active cells communicate with each other at junctions called synapses. Long-lasting changes in the strength of synapses are thought to underlie learning and memory. Research has focused on understanding how synaptic plasticity is brought about at the level of molecules in model organisms including sea slugs and rats. Some components of a 'core program' for synaptic plasticity have emerged. Notably, long-lasting potentiation and depression of synapses both follow from influx of calcium. Two calcium-sensitive enzymes are critical for this process: one modifies synaptic proteins to strengthen the synapse following large influxes of calcium whereas the other has the opposite effect following small influxes of calcium. However, both enzymes exhibit equal sensitivity to calcium in the test tube so it is not obvious why they respond differently to calcium within the synapse. An important consideration is that calcium is locally concentrated in cells near to its points of entry. Our research will pursue two major goals. First, we propose to determine whether the relative positions of the two enzymes from calcium entry points, within a massive protein assembly called the postsynaptic density (PSD), explain why one of the enzymes requires larger influxes of calcium for activation. Second, we aim to determine why the enzyme that depresses synaptic strength is inactive following large influxes of calcium.We will apply complementary approaches to examine the positions of the two enzymes relative to calcium entry points in the PSD. We will chemically crosslink PSD samples and map the exact sites of crosslinking between different proteins within the protein assembly. This will enable us to build accurate structural models so we can compare the relative positions of the key enzymes with calcium entry points. We will also measure how a calcium sensor responds to experimentally controlled influxes of calcium when co-localised with different proteins within the PSD. In pursuit of our second major aim, we will focus on understanding how an anchoring protein regulates the activity of the calcium-sensitive enzyme that depresses synaptic strength. We will determine the high-resolution three-dimensional structure of a fragment of the anchoring protein to understand how it is regulated by calcium within the PSD. We will also use a novel enzymatic assay to examine whether the anchoring protein is able to tune the enzyme that brings about long-term synaptic depression such that it is active at low but not high concentrations of calcium.The research will benefit from collaboration with proteomics specialists in Germany thereby cementing an international partnership. In addition to addressing a fundamental aspect of how the brain lays down memories, the novel technological approaches that we develop will be widely applicable to other areas of biology. The research is highly relevant to human brain disease as the synapse is a locus for mutations that cause neurological disease and psychiatric disorders, and aberrant synaptic plasticity may be fundamental to many brain disorders including Alzheimer's and autism. Both the calcium-sensitive enzymes and the anchoring protein that are the focus of the proposed research are drug targets. Overall, the research can provide insights at a molecular level that is informative for drug design.