ALTERATIONS IN ADF/COFILIN ACTIVITY RELATED TO SEIZURES

肌动蛋白动力学在神经突的发育生长、突触的形成和成熟突触的突触活动调节中起重要作用。此外，肌动蛋白可能在神经退行性疾病中发挥重要作用，因为肌动蛋白解聚可以防止兴奋性毒性，并且癫痫诱导的苔藓纤维的发芽可能是由于发育生长机制的重新参与。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调节。ADF定位于神经元中的突触和生长锥。ADF过度表达增加肌动蛋白周转和神经突生长。拟议的实验将测试的假设，即ADF的活动是由癫痫活动修改，并在ADF活动的变化反过来影响突触特性。首先通过免疫组织化学确定ADF和磷酸化形式的ADF和cofilin在大鼠脑中的分布。然后用免疫印迹和免疫组化分析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性质，将使用自发性癫痫发作活性的细胞培养模型。首先将确定细胞培养模型中的尿素诱导的ADF磷酸化和易位的变化是否与体内观察到的变化相似。通过重组腺病毒介导的ADF和LIM激酶（ADF激酶）突变形式的表达，ADF活性将在细胞培养系统中改变。然后通过对F-肌动蛋白分布和周转、钙信号传导和兴奋性毒性的影响来评估改变的ADF活性对突触功能的影响。

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.