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

获得性癫痫发展过程中星形胶质细胞耦合塑造神经元活动的动态时间调节

• 批准号: 10366826
• 负责人: Stefanie Robel
• 金额: $ 34.63万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366826
• 关键词: Affect; Antiepileptic Agents; Astrocytes; Cells; Chemicals; Chronic; Connexin 43; Connexins; Consensus; Coupling; Data; Development; Disease; Drug Targeting; Electroencephalography; Electrophysiology (science); Epilepsy; Foundations; Functional disorder; Future; Gap Junctions; Impairment; In Situ; Intractable Epilepsy; Knock-in Mouse; Knockout Mice; Measurement; Measures; Modeling; Monitor; Mus; Mutate; Mutation; Neurologic; Neurons; Pathology; Patients; Persons; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Regulation; Seizures; Serine; Shapes; Signal Pathway; Signal Transduction; Silicon; Techniques; Technology; Testing; Time; Traumatic Brain Injury; Viral; Virus; Western Blotting; acquired epilepsy; in vivo; mild traumatic brain injury; mimetics; mouse model; mutant; prevent; research and development; therapeutic target; tool

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.

项目摘要 星形胶质细胞连接成数百个细胞的网络。星形胶质细胞偶联受损与 癫痫，但对于偶联减少是促进还是抵消异常神经元还没有达成共识 活动，这是癫痫发作之前的。缺乏星形胶质细胞偶联可以促进癫痫发作；然而，相反的情况也会 结果显示：抑制偶联作用后，异常神经元活动和癫痫发作减少1-3。一个 解释这两个发现的综合观点需要揭示星形胶质细胞耦合是如何调节癫痫的。 在获得性癫痫中，由诸如创伤性脑损伤(TBI)等神经侮辱引发的许多 研究表明，缝隙连接功能障碍(GJS)和连接蛋白43(Cx43)调节失调。Cx43 负责星形胶质细胞偶联的GJ形式导致了以下结论：偶联减少可能有助于 癫痫发作。对Cx43基因敲除小鼠的研究表明，星形胶质细胞偶联减弱的时间和持续时间可能 确定异常神经元活动是被促进还是被抵消。这还没有经过测试，因为缺乏 动态调整耦合的工具。为了动态减少或恢复星形胶质细胞的耦合，我们生成了 可诱导表达功能性或突变的Cx43的病毒构建物在不同的不同持续时间内 获得性癫痫的各个阶段。这些Cx43突变体抑制星形胶质细胞偶联并诱导神经元 体内的超兴奋性。为了评估星形胶质细胞偶联和神经元活动之间的关系，我们将 使用获得性癫痫模型，该模型在轻度脑外伤后进展为自发性癫痫，但缺乏许多 混杂因素5.这个模型概括了Cx43病理学的三个关键方面：星形胶质细胞偶联是 减少，Cx43蛋白增加，并增加Cx43丝氨酸368位的磷酸化。这个翻译后版本 修饰改变了GJ的导电性，并与GJ的内化有关。然而，关键的上游 星形胶质细胞偶联减弱的信号转导及其对神经元活动的影响 获得性癫痫必须被揭示为未来治疗靶向的基础。这项提议将产生 旨在统一先前发现的获得性癫痫的星形胶质细胞偶联调节的综合模型。 Cx43功能将被动态操纵以1)确定星形胶质细胞偶联减弱的时机 调节异常神经元活动2)识别控制星形胶质细胞偶联减弱的信号级联 以及3)确定是否以及何时恢复星形胶质细胞偶联可预防获得性癫痫。获得性癫痫影响 全世界有6500万人，而且出了名的难以治疗。即使经过几十年的研究和 针对神经元的新型抗癫痫药物的开发，三分之一的患者仍存在耐药性 癫痫。靶向星形细胞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，当它是不适应的时候。