Altered phosphorylation of connexin 43 contributes to increased gap-junction coup

描述（由申请方提供）：匹鲁卡品诱导的癫痫持续状态（SE）诱导反应性神经胶质增生，并以时间依赖性方式激活海马星形胶质细胞中的钙信号传导（1，2）。初步研究表明SE后48小时和SE后30天慢性癫痫大鼠连接蛋白磷酸化显著降低。该数据与先前的研究相结合，表明阵发性去极化转变可以由星形胶质细胞释放谷氨酸启动3，表明通过海马星形胶质细胞中连接蛋白43的去磷酸化增加间隙连接半通道的开放概率可能有助于癫痫发生的进展。该提议的主要目标是表征丝氨酸/苏氨酸激酶和磷酸酶在尿素诱导的连接蛋白43的去磷酸化中的作用，具体地，评估癫痫持续状态急性激活磷酸酶的假设（并可能在特定时间点减少激酶），其使连接蛋白43去磷酸化，随后增加含连接蛋白43的半通道开放概率和星形胶质细胞间隙-海马癫痫发生过程中的连接偶联。将使用分子方法来表征磷酸酶和激酶活化的时间过程。在SE后的多个时间点，将对海马切片中激酶和磷酸酶活性之间的平衡变化做出响应，定量测量间隙连接偶联的变化。将对激酶和磷酸酶活性进行基因调控，以确定自发性癫痫发作之前和之后，导致癫痫诱导的连接蛋白43去磷酸化的主要机制以及对间隙连接偶联、钾电流和谷氨酸转运蛋白电流的下游影响。慢性视频EEG监测和冷冻断裂复制免疫金标记（FRIL）将用于确定是否可以通过药理学或转基因方法预防癫痫诱导的磷酸化连接蛋白43膜表达的改变和与磷酸化介导的半通道开放相关的癫痫发作频率的潜在增加。该提案将提供有关磷酸酶抑制剂或激酶激动剂如何被利用来防止癫痫发生的进展并实现疾病修饰的有价值的翻译信息。

DESCRIPTION (provided by applicant): Pilocarpine-induced status epileptics (SE) induce reactive gliosis and activate calcium signaling in astrocytes of the hippocampus in a time-dependent manner (1, 2). Primary studies demonstrate significant decreases in connexin phosphorylation 48 h after SE and in chronically epileptic rats 30 d after SE. This data combined with previous studies showing that paroxysmal depolarization shifts can be initiated by release of glutamate from astrocytes3 suggests that increased open probability of gap- junction hemi channels via dephosphorylating of connexin 43 in hippocampal astrocytes may contribute to the progression of epileptogenesis. The major goal of this proposal is to characterize the role of serine/threonine kinases and phosphatases in seizure-induced dephosphorylating of connexin 43 and specifically, to evaluate the hypothesis that status epileptics acutely activates phosphatases (and may decrease kinases at specific time points) in glyptic astrocytes which dephosphorylates connexin 43 to subsequently increase connexin 43-containing hemi channel open probability and astrocytes gap- junction coupling during epileptogenesis in the hippocampus. Molecular methods will be used to characterize the time course of phosphatase and kinase activation. Alterations in gap-junction coupling will be quantitatively measured in response to equilibrium shifts between kinase and phosphatase activity in hippocampal slices at multiple time points following SE. Kinase and phosphatase activity will be pharmacologically and genetically modulated to determine the primary mechanism responsible for the seizure-induced dephosphorylating of connexin 43 and downstream effects on gap-junction coupling, potassium currents, and glutamate transporter currents prior to and after the onset of spontaneous seizures. Chronic video-EEG monitoring and freeze-fracture replica immunogold labeling (FRIL) will be used to determine whether seizure-induced alterations in phosphorylated connexin 43 membrane expression and potential increases in seizure frequency associated with phosphorylation-mediated hemi channel opening can be prevented with pharmacological or transgenic methods. The proposal will provide valuable translational information about how phosphatase inhibitors or kinase agonists may be exploited to prevent the progression of epileptogenesis and achieve and disease-modification.