ALTERATIONS IN ADF/COFILIN ACTIVITY RELATED TO SEIZURES

与癫痫发作相关的 ADF/COFILIN 活性变化

• 批准号: 6227579
• 负责人: PETER J MEBERG
• 金额: $ 14.13万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6227579
• 关键词: actins; enzyme activity; epilepsy; gene expression; growth cones; immunocytochemistry; laboratory rat; phosphorylation; protein localization; synapses; tissue /cell culture; western blottings

Actin dynamics play an important role in the developmental outgrowth of neurites, the formation of synapses, and the modulation of synaptic activity at mature synapses. In addition, actin may play a significant role in neurodegenerative diseases, since actin depolymerization can protect against excitotoxicity and the sprouting of mossy fibers induced by epilepsy may be due to the re-engagement of developmental growth mechanisms. Actin-depolymerizing factor (ADF) is an important regulator of actin dynamics whose activity can be regulated by phosphorylation. ADF is localized to synapses and growth cones in neurons. ADF over-expression increases actin turnover and neurite outgrowth. The proposed experiments will test the hypothesis that the activity of ADF is modified by seizure activity, and changes in ADF activity in turn influence synaptic properties. The distribution of ADF and phosphorylated forms of ADF and cofilin in rat brain will first be determined by immunohistochemistry. Then changes in phosphorylation state and localization after kainate-induce seizures will be investigated using western blot and immunohistochemical analyses. To determine how changes in ADF activity affect synaptic properties a cell culture model of spontaneous seizure activity will be used. It will first be determined if seizure-induced changes in ADF phosphorylation and translocation in the cell culture model are similar to those observed in vivo. ADF activity will be altered in the cell culture system by recombinant adenovirus-mediated expression of mutant forms of ADF and LIM kinase, the ADF kinase. Effects of altered ADF activity on synaptic function will then be assessed by effects on F-actin distribution and turnover, calcium signaling and excitotoxicity.

肌动蛋白动力学在神经突起的发育、突触的形成以及成熟突触的突触活动调节中起着重要的作用。此外，肌动蛋白在神经退行性疾病中可能发挥重要作用，因为肌动蛋白解聚可以保护神经元免受兴奋性毒性，癫痫引起的苔藓纤维的萌发可能是由于发育生长机制的重新启动。肌动蛋白解聚因子(ADF)是肌动蛋白动力学的重要调节因子，其活性可通过磷酸化来调节。ADF定位于神经元中的突触和生长锥体。ADF的过表达增加了肌动蛋白的周转和轴突的生长。拟议的实验将验证ADF的活性被癫痫发作的活性改变，而ADF活性的变化反过来影响突触特性的假设。ADF及其磷酸化形式的ADF和cofilin在大鼠脑内的分布将首先通过免疫组织化学方法确定。然后用蛋白质印迹和免疫组织化学分析方法研究红藻氨酸诱导癫痫发作后磷酸化状态和定位的变化。为了确定ADF活性的变化如何影响突触特性，将使用自发癫痫活动的细胞培养模型。首先将确定癫痫发作诱导的ADF磷酸化和移位在细胞培养模型中的变化是否与体内观察到的相似。在细胞培养系统中，ADF的活性将通过重组腺病毒介导的突变形式的ADF和ADF的LIM激酶的表达而改变。ADF活性改变对突触功能的影响将通过对F-肌动蛋白分布和周转、钙信号和兴奋性毒性的影响来评估。