Dynamic temporal regulation of astrocyte coupling to shape neuronal activity during acquired epilepsy development

项目摘要 星形胶质细胞连接成数百个细胞的网络。受损的星形胶质细胞偶联与 癫痫，但是否减少耦合促进或抵消异常的神经元 活动，在癫痫发作之前。缺乏星形胶质细胞偶联可以促进癫痫发作;1然而，相反的情况也 已经显示：在偶联被抑制后，异常神经元活动和癫痫发作减少1 -3。一个 综合考虑这两个发现，需要揭示星形胶质细胞耦合如何调节癫痫。 在获得性癫痫中，这是由神经损伤如创伤性脑损伤（TBI）引起的， 研究已经证明间隙连接（GJ）功能障碍和连接蛋白43（Cx43）调节异常。Cx43 负责星形胶质细胞偶联的GJ形式，从而得出结论，偶联减少可能有助于 癫痫发作在Cx43基因敲除小鼠中的研究表明，星形胶质细胞偶联减少的时间和持续时间可能 确定异常的神经元活动是否被促进或抵消。由于缺乏 动态调节耦合的工具。为了动态地减少或恢复星形胶质细胞偶联，我们生成了 病毒构建体可以在不同的温度下诱导表达功能性或突变的Cx43可变的持续时间， 获得性癫痫的阶段。这些Cx43突变体抑制星形胶质细胞偶联并诱导神经元凋亡。 体内兴奋过度。为了评估星形胶质细胞偶联和神经元活性之间的关系，我们将 使用获得性癫痫的模型，在没有许多癫痫的情况下， 混杂因素5.该模型概括了Cx43病理学的三个关键方面： 减少，Cx43蛋白增加，Cx43丝氨酸368处的磷酸化增加。这种翻译后 修饰改变了GJ电导率，并且与GJ的内化有关。然而，关键的上游 导致星形胶质细胞偶联减少的信号传导及其对不同阶段神经元活动的影响 必须揭示获得性癫痫，作为未来治疗靶向的基础。该提案将产生 获得性癫痫中星形胶质细胞耦合调制的综合模型，旨在统一以前的研究结果。 Cx43功能将被动态操纵以1）确定减少的星形胶质细胞偶联的时间是否为 调节异常的神经元活动2）鉴定控制减少的星形胶质细胞偶联的信号级联 以及3）确定恢复星形胶质细胞偶联是否以及何时防止获得性癫痫。获得性癫痫影响 全世界有6500万人患有这种疾病，并且是出了名的难以治疗。即使经过几十年的研究和 尽管开发了靶向神经元的新型抗癫痫药物，但三分之一的患者仍患有耐药性 癫痫针对星形胶质细胞Cx43可能是一种选择，但治疗的第一步是确定何时 减少耦合是自适应的，当它是不适应的。

Project Summary Astrocytes couple into networks of hundreds of cells. Impaired astrocyte coupling is associated with epilepsy, but there is no consensus on whether reduced coupling promotes or counteracts abnormal neuronal activity, which precedes seizures. A lack of astrocyte coupling can promote seizures;1 yet, the opposite has also been shown: abnormal neuronal activity and seizures were reduced after coupling was inhibited1-3. An integrated view that accounts for both findings is needed to reveal how astrocyte coupling modulates epilepsy. In acquired epilepsy, which is initiated by a neurological insult such as traumatic brain injury (TBI), many studies have demonstrated dysfunction of gap junctions (GJs) and dysregulation of Connexin43 (Cx43). Cx43 forms GJ responsible for astrocyte coupling leading to the conclusion that reduced coupling may contribute to seizures. Studies in Cx43 knockout mice suggest that the timing and duration of reduced astrocyte coupling may determine if abnormal neuronal activity is promoted or counteracted. This has not been tested due to a lack of tools that dynamically modulate coupling. To reduce or restore astrocyte coupling dynamically, we generated viral constructs that can be induced to express functional or mutated Cx43 for variable durations at different stages of acquired epilepsy. These Cx43 mutants inhibited astrocyte coupling and induced neuronal hyperexcitability in vivo. To assess the relationship between astrocyte coupling and neuronal activity, we will use a model of acquired epilepsy that progresses to spontaneous seizures after mild TBI in the absence of many confounding factors5. This model recapitulates three key aspects of Cx43 pathology: astrocyte coupling is reduced, Cx43 protein is increased and increased phosphorylation at Cx43 serine 368. This post-translational modification alters GJ conductivity, and is associated with internalization of GJ. Yet, the critical upstream signaling causing reduced astrocyte coupling and its effects on neuronal activity during different stages of acquired epilepsy must be revealed as a foundation for future therapeutic targeting. This proposal will generate an integrated model of astrocyte coupling modulation in acquired epilepsy that aims to unify previous findings. Cx43 function will be dynamically manipulated to 1) determine if the timing of reduced astrocyte coupling modulates abnormal neuronal activity 2) identify the signaling cascade controlling reduced astrocyte coupling and 3) determine if and when restoring astrocyte coupling prevents acquired epilepsy. Acquired epilepsy affects 65 million people worldwide and is notoriously difficult to treat. Even after decades of research and the development of new anti-epileptic drugs targeting neurons, one third of patients still suffer from drug-resistant epilepsy. Targeting astrocytic Cx43 might be an option, but the first step towards therapy is determining when reduced coupling is adaptive and when it is maladaptive.