Calcium and Calmodulin Dependent Protein Kinase

描述(由申请人提供)： 突触传递的紊乱是几种神经和精神疾病的病理的一部分，包括癫痫、精神分裂症、抑郁症和阿尔茨海默病。我们正在研究调节突触传递的分子机制。在这里，我们建议研究钙/钙调蛋白依赖的蛋白激酶II(CaMKII)调控的谷氨酸能突触的调节通路。CaMKII主要集中在突触后密度，可通过NMDA受体被钙离子内流激活。该提案集中在CaMKII的两个主要突触后底物，SynGAP，一种集中在突触后密度中的Ras GTP酶激活蛋白，以及Densin，建议的CaMKII的对接位置。在第一个目标中，我们将检验SynGAP参与调节脑片突触细胞骨架的假设。我们之前已经证明SynGAP有助于调节培养神经元突触形成过程中的脊髓细胞骨架，并且在SynGAP杂合子的海马片中Kalirin/PAK激酶通路的位置和激活发生了改变。我们将使用野生型和SynGAP缺陷突变体的切片来定位SynGAP在突触刺激后的这一途径和相关途径中的作用。在第二个目标中，我们将使用来自野生型和条件性SynGAP缺陷小鼠的海马片的电生理学研究来研究SynGAP缺乏对LTP的影响是由于发育异常还是SynGAP的急性丢失。我们还将检验SynGAP参与MAP激酶调节树突状细胞兴奋性的假设。在第三个目标中，我们将研究CaMKII在PSD神经元培养中的募集，这些神经元培养来自缺失NMDA受体NR2A和NR2B亚基的羧基末端尾巴的敲入小鼠。我们还将研究在这些培养物中引入密度蛋白胞内尾巴的重组形式，或通过引入siRNA来减少密度蛋白表达后，CaMKII的招募情况。在第四个目标中，我们建议通过构建致密素的敲除突变体和条件性敲除突变体来确定致密素的生理重要性，并验证致密素是PSD中CaMKII的对接位点的假设。

DESCRIPTION (provided by applicant): Derangements in synaptic transmission are part of the pathology of several neurological and mental diseases including epilepsy, schizophrenia, depression, and Alzheimer's disease. We are studying the molecular mechanisms underlying regulation of synaptic transmission. Here we propose to study regulatory pathways at glutamatergic synapses governed by Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII is concentrated in the postsynaptic density where it can be activated by Ca 2+ influx through NMDA receptors. The proposal focuses on two principal postsynaptic substrates of CaMKII, SynGAP, a Ras GTPase-activating protein that is concentrated in the postsynaptic density, and densin, a proposed docking site for CaMKII. In the First Aim, we will test the hypothesis that synGAP participates in regulation of the cytoskeleton at synapses in brain slices. We have previously shown that synGAP helps to regulate the spine cytoskeleton during synapse formation in cultured neurons, and that the location and activation of the kalirin/PAK kinase pathway is altered in hippocampal slices heterozygous for synGAP. We will use slices from wild type and synGAP deficient mutants to map the role of synGAP in this and related pathways following synaptic stimulation. In the Second Aim, we will use electrophysiological studies in hippocampal slices from wild type and conditional synGAP deficient mice to investigate whether the effects of synGAP deficiency on LTP are a result of developmental abnormalities or of acute loss of synGAP. We will also test the hypothesis that synGAP participates in regulation of the modulation of dendritic excitability by MAP kinase. In the Third Aim, we will examine recruitment of CaMKII to the PSD in neuronal cultures prepared from knock-in mice that are missing the carboxyl terminal tails of the NR2A and NR2B subunits of the NMDA receptor. We will also examine recruitment of CaMKII in these cultures after introducing recombinant forms of the intracellular tails of densin, or reducing the expression of densin by introduction of siRNA. In the Fourth Aim, we propose to determine the physiological importance of densin, and test the hypothesis that densin is a docking site for CaMKII in the PSD by constructing knockout and conditional knockout mutants of densin.