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: 10227901
• 负责人: Prosper N'Gouemo
• 金额: $ 34.76万
• 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/10227901
• 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.

全身性强直阵挛发作（GTCS）是酒精戒断引起的癫痫发作中最常见的类型 （AWSs），通常对当前的抗惊厥药具有耐药性，然而，其潜在机制是 不太了解。我们的长期目标是了解改变的Ca 2+信号传导如何有助于神经元的 过度兴奋，导致AWS易感性增加。我们以前曾报道过， 与下丘脑L型（CaV1.3）和P型（CaV2.1）电压门控Ca 2+（CaV）通道相关的蛋白质 当AWSs患病率达到峰值时，但在AWSs易感性开始之前，下丘（IC）神经元的变化不明显。的 CaV通道的上调导致细胞内Ca 2+（[Ca 2 +]i）增加和Ca 2+稳态改变 表明Na+/Ca 2+交换器，一种调节[Ca 2 +]i的双向膜离子转运蛋白， 优先以正向模式（NCXfwd）操作以挤出Ca 2+并恢复Ca 2+稳态。令人惊奇的是， NCX以反向模式（NCXrev）工作，导致AWSs发作前Ca 2+流入IC神经元 易感性和AWSs患病率达到峰值的时间。因此，NCX活性的变化可能在以下方面发挥关键作用： AWS启动。本文提出的实验将确定NCX 1型（NCX 1）对 神经元过度兴奋和AWS易感性，为开发新的治疗方法提供了特异性靶点。 我们的工作假设是，通过NCXrev的IC神经元中的Ca 2+内流对于产生癫痫样癫痫是必不可少的。 引发AWS的突发事件为了验证这一假设，并建立在我们的初步数据和先前的出版物上， 我们将联合收割机与分子遗传学，电生理学，分子生物学， 三个具体目标的行为分析。首先，我们将确定NCXrev在多大程度上有助于 在酒精戒断后IC神经元中产生癫痫样爆发;此外，我们将研究如何 细胞内Na+浓度以及L-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NCX 1基因的变化程度， IC神经元中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，我们将评估大鼠中产生的AWS，其中NCX 1 在IC神经元中选择性地缺失表达。此外，我们将在大鼠中测量AWS，其中 使用光遗传学抑制由多巴胺能IC神经元介导的过度兴奋。我们的发现将 提供了对NCX 1在癫痫发作活动中的作用、启动AWSs的机制以及 构成AWS和其他类型GTCS的基础。