Na+/Ca2+ exchanger remodeling in alcohol withdrawal seizures

酒精戒断发作中的 Na /Ca2 交换器重塑

• 批准号: 9915824
• 负责人: Prosper N'Gouemo
• 金额: $ 33.05万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9915824
• 关键词: Acoustics; Acute; Adjuvant Therapy; Alcohol Withdrawal Seizures; Alcohol withdrawal syndrome; Anticonvulsants; Behavioral; Benzodiazepines; Brain; Buffers; Cell Surface Proteins; Cell surface; Couples; Data; Dependovirus; Development; Dose; Electrophysiology (science); Epilepsy; Epileptogenesis; Exhibits; Fractionation; Functional disorder; Gene Expression; Generations; Glutamates; Goals; Homeostasis; Inferior Colliculus; Infusion procedures; Intensive Care Units; Ions; Measures; Mechanical ventilation; Mediating; Membrane; Membrane Proteins; Messenger RNA; Microinjections; Modeling; Molecular; Molecular Biology; Molecular Genetics; Molecular Target; Monitor; Movement; Neurons; Outcome; Patients; Pattern; Pharmacology; Phenobarbital; Play; Predisposition; Prevalence; Propofol; Proteins; Publications; Rattus; Refractory; Reporting; Research; Resistance; Risk; Role; Seizures; Severities; Signal Transduction; Site; Slice; Small Interfering RNA; Testing; Tonic - clonic seizures; Up-Regulation; Western Blotting; aggressive therapy; alcohol abuse therapy; alcohol prevention; alcohol use disorder; alcohol use initiation; base; density; effective therapy; experience; experimental study; in vivo; insight; mRNA Expression; novel; novel therapeutics; optogenetics; patch clamp; prevent; protein expression; selective expression; voltage

Generalized tonic-clonic seizures (GTCSs), the most prevalent type of alcohol withdrawal‒induced seizures (AWSs), are commonly resistant to current anticonvulsants, nevertheless, their underlying mechanisms are poorly understood. Our long-term goal is to understand how altered Ca2+ signaling contributes to the neuronal hyperexcitability that causes increased AWSs susceptibility. We have previously reported upregulation of proteins associated to L-type (CaV1.3) and P-type (CaV2.1) voltage-gated Ca2+ (CaV) channels in inferior colliculus (IC) neurons when AWSs prevalence peaks but not before the onset of AWSs susceptibility. The upregulation of CaV channels leading to increased intracellular Ca2+ ([Ca2+]i) and altered Ca2+ homeostasis suggests that the Na+/Ca2+ exchanger, a bidirectional membrane ion transporter that regulates [Ca2+]i would preferentially operate in the forward mode (NCXfwd) to extrude Ca2+ and restore Ca2+ homeostasis. Surprisingly, NCX operates in the reverse mode (NCXrev) causing Ca2+ influx into IC neurons prior to the onset of AWSs susceptibility and when AWSs prevalence peaks. Thus, changes in NCX activity possibly play a critical role in AWS initiation. The experiments proposed here will determine the precise contribution of NCX type1 (NCX1) to neuronal hyperexcitability and AWSs susceptibility, providing specific target for developing novel therapeutics. Our working hypothesis is that Ca2+ influx in IC neurons via NCXrev is essential for generating the epileptiform bursts that initiate an AWS. To test this hypothesis—and building on our preliminary data and prior publications— we will combine in vivo pharmacology with molecular genetics, electrophysiology, molecular biology, and behavioral analysis in three specific aims. First, we will determine the extent to which NCXrev contributes to generating the epileptiform bursts in IC neurons following alcohol withdrawal; in addition, we will investigate how intracellular Na+ concentration as well as L- and P-type CaV channels contribute to increased NCXrev activity during the course of alcohol withdrawal. Second, we will determine the extent to which changes in NCX1 gene expression and cell surface protein levels in IC neurons are associated with increased seizure susceptibility during the course of alcohol withdrawal. Third, we will evaluate AWSs generated in rats in which NCX1 expression is deleted selectively in IC neurons. Furthermore, we will measure AWSs in rats in which hyperexcitability mediated by glutamatergic IC neurons is suppressed using optogenetics. Our findings will provide key insight into NCX1 role on seizure activity, the mechanisms that initiate AWSs, and the mechanisms that underlie AWSs and other types of GTCSs.

全身强直阵挛性癫痫发作 (GTCS)，最常见的酒精戒断诱发癫痫发作类型 （AWS），通常对当前的抗惊厥药物有抵抗力，然而，它们的潜在机制是 不太了解。我们的长期目标是了解改变的 Ca2+ 信号如何影响神经元 过度兴奋导致 AWS 敏感性增加。我们之前曾报道过上调 与下壁 L 型 (CaV1.3) 和 P 型 (CaV2.1) 电压门控 Ca2+ (CaV) 通道相关的蛋白质 当AWSs患病率达到峰值时，而不是在AWSs易感性出现之前，丘（IC）神经元。这 CaV 通道上调导致细胞内 Ca2+ ([Ca2+]i) 增加并改变 Ca2+ 稳态 表明 Na+/Ca2+ 交换器（一种调节 [Ca2+]i 的双向膜离子转运蛋白）会 优先以正向模式 (NCXfwd) 运行以挤出 Ca2+ 并恢复 Ca2+ 稳态。出奇， NCX 以反向模式 (NCXrev) 运行，导致在 AWS 出现之前 Ca2+ 流入 IC 神经元 易感性以及 AWS 患病率何时达到峰值。因此，NCX 活性的变化可能在 AWS启动。这里提出的实验将确定 NCX type1 (NCX1) 对 神经元过度兴奋和 AWS 敏感性，为开发新疗法提供了具体目标。 我们的工作假设是，Ca2+ 通过 NCXrev 流入 IC 神经元对于产生癫痫样症状至关重要 启动 AWS 的突发。为了检验这一假设——并以我们的初步数据和之前的出版物为基础—— 我们将体内药理学与分子遗传学、电生理学、分子生物学和 行为分析的三个具体目标。首先，我们将确定 NCXrev 的贡献程度 酒精戒断后，IC 神经元会产生癫痫样爆发；此外，我们将研究如何 细胞内 Na+ 浓度以及 L 型和 P 型 CaV 通道有助于增加 NCXrev 活性 在戒酒过程中。其次，我们将确定 NCX1 基因的变化程度 IC神经元的表达和细胞表面蛋白水平与癫痫易感性增加相关 在戒酒过程中。第三，我们将评估 NCX1 在大鼠中产生的 AWS IC 神经元中的表达被选择性删除。此外，我们将测量大鼠的 AWS，其中 使用光遗传学抑制谷氨酸能 IC 神经元介导的过度兴奋。我们的研究结果将 提供有关 NCX1 在癫痫发作活动中的作用、启动 AWS 的机制以及机制的重要见解 是 AWS 和其他类型 GTCS 的基础。