Determining the role of dysregulated GABA uptake by reactive astrocytes in thalamic circuit hyperexcitability and seizures

确定反应性星形胶质细胞摄取失调的 GABA 在丘脑回路过度兴奋和癫痫发作中的作用

• 批准号: 10007592
• 负责人: Frances Cho
• 金额: $ 4.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10007592
• 关键词: Adult; Affect; Astrocytes; Brain; Brain Injuries; Bypass; CRISPR/Cas technology; Cells; Child; Chronic; Complex; Dependovirus; Development; Disease; Elderly; Electrophysiology (science); Epilepsy; Epileptogenesis; Functional disorder; GABA Receptor; GABA transporter; Generalized seizures; Genetic; In Vitro; Inflammation; Inflammatory; Injury; Intractable Epilepsy; Knowledge; Lead; Lesion; Link; Literature; Mediating; Modeling; Mus; Neurologic; Neurons; Pathologic; Pathway interactions; Phenocopy; Post-Traumatic Epilepsy; Predisposition; Prevention strategy; Property; Rodent Model; Role; Seizures; Signal Transduction; Slice; Source; Stroke; Synaptic Transmission; Testing; Thalamic structure; Transgenic Organisms; Traumatic Brain Injury; Viral; Whole-Cell Recordings; Wild Type Mouse; Work; astrogliosis; awake; base; comorbidity; effective therapy; electrical property; gamma-Aminobutyric Acid; in vivo; knock-down; nervous system disorder; neural circuit; neuron loss; neuronal circuitry; overexpression; prevent; response; stroke model; therapeutic target; tool; transcriptomics; treatment strategy; uptake; voltage clamp

PROJECT SUMMARY & ABSTRACT Acquired epilepsies can occur following brain lesions such as stroke or traumatic brain injury, and particularly affects elderly people children. However, there is no effective treatment or prevention strategy for post-traumatic epilepsy (PTE). Key to finding therapeutic targets is understanding epileptogenesis, the latent period between the initial injury and the development of epilepsy. Reactive astrocytes, or astrogliosis, form in response to neurological insults, and are strongly associated with epileptogenesis and with intractable, drug-resistant, epilepsies. In order to understand how reactive astrocytes contribute to diseases characterized by circuit abnormalities such as seizures, there is an urgent need to understand how they affect complex neuronal activity. The thalamus has been implicated in PTE—following cortical injuries and stroke, the thalamus becomes hyperexcitable, and develops chronic astrogliosis preceding the onset of seizures. Using a viral model of selectively induced astrogliosis that I previously characterized, I will investigate the cellular and circuit mechanisms by which reactive astrocytes drive circuit hyperexcitability in the thalamus and enable seizures. My preliminary electrophysiological and transcriptomic studies have suggested a direct, mechanistic link between astrocyte dysfunction and neural circuit hyperexcitability, in the context of inflammation. In this proposal, I will test the working hypothesis that thalamic reactive astrocytes downregulate GABA uptake via reduction of GABA transporters, which leads to enhanced tonic GABA currents in thalamocortical neurons, ultimately resulting in thalamic circuit hyperexcitability and seizures. To test this hypothesis, I will characterize the effects of bidirectional manipulation of astrocytic GABA uptake on GABAergic signaling in thalamocortical neurons, rhythmogenesis of the intrathalamic circuit in vitro, and seizure susceptibility in awake, behaving mice. I will use a dual adeno-associated virus (AAV) CRISPR- Cas9 approach to decrease astrocytic GABA transporter expression in wild-type mice (Aim 1), and an AAV- mediated overexpression approach to selectively enhance astrocytic GABA transporter expression in wild-type mice that have thalamic astrogliosis and abnormal thalamocortical hyperexcitability (Aim 2). The proposed work will harness the selectivity of the viral astrogliosis approach in combination with slice and in vivo electrophysiological assessments of neuronal circuits. These results will elucidate our basic understanding of astrocytic GABA uptake and neural circuit plasticity. Furthermore, by investigating the functional changes in reactive astrocytes that enable pathological circuit activity and mediate epileptogenesis, the proposed work will identify potential therapeutic targets to intervene and prevent PTE.

项目概要和摘要 获得性癫痫可发生在脑损伤后，如中风或创伤性脑损伤， 特别是对老年人和儿童的影响。然而，没有有效的治疗或预防策略 创伤后癫痫（PTE）寻找治疗靶点的关键是了解癫痫的发生， 初始损伤和癫痫发展之间的潜伏期。反应性星形胶质细胞，或星形胶质细胞增生， 形成于对神经损伤的反应，并且与癫痫发生和顽固性， 抗药性癫痫为了了解反应性星形胶质细胞如何导致疾病， 由电路异常，如癫痫发作，有迫切需要了解他们如何影响复杂的 神经元活动 丘脑与PTE有关，在皮质损伤和中风后，丘脑变得 过度兴奋，并在癫痫发作前发展为慢性星形胶质细胞增生。使用病毒模型， 选择性诱导的星形胶质细胞增生，我以前的特点，我将研究细胞和电路 反应性星形胶质细胞驱动丘脑回路过度兴奋并使癫痫发作的机制。我 初步的电生理学和转录组学研究表明， 星形胶质细胞功能障碍和神经回路过度兴奋，在炎症的背景下。在这份提案中，我将 测试工作假设，丘脑反应性星形胶质细胞下调GABA的摄取，通过减少 GABA转运蛋白，导致增强的紧张性GABA电流在丘脑皮质神经元，最终 导致丘脑回路过度兴奋和癫痫发作。 为了验证这一假设，我将描述双向操纵星形胶质细胞GABA的影响， 对丘脑皮质神经元GABA能信号传导的摄取，体外丘脑内回路的节律发生， 和癫痫易感性之间的关系。我将使用双重腺相关病毒（AAV）CRISPR- Cas9方法降低野生型小鼠中星形胶质细胞GABA转运蛋白表达（Aim 1），以及AAV- 一种选择性增强野生型星形胶质细胞GABA转运蛋白表达的介导过表达方法 具有丘脑星形胶质细胞增生和异常丘脑皮质过度兴奋的小鼠（Aim 2）。 拟议的工作将利用选择性的病毒星形胶质细胞增生的方法结合切片 以及神经元回路的体内电生理学评估。这些结果将阐明我们的基本 理解星形胶质细胞GABA摄取和神经回路可塑性。此外，通过调查 反应性星形胶质细胞的功能变化使病理性回路活动和介导癫痫发生， 建议的工作将确定潜在的治疗靶点，以干预和预防PTE。