Na+/Ca2+ exchanger remodeling in alcohol withdrawal seizures

酒精戒断发作中的 Na /Ca2 交换器重塑

基本信息

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
项目类别：
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

项目摘要

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.

普遍的强调持续性癫痫发作（GTCSS），最普遍的酒精戒断类型 - （AWSS）通常对当前的抗惊厥药具有抵抗力，但是它们的潜在机制是长期目标是了解CA2+信号变化如何有助于神经元过度兴奋的性能增加AWSSSSCEPTIBITY。与L型（CAV1.3）和P型（CAV2.1）电压门控Ca2+（CAV）通道相关的蛋白质 colliculus（IC）神经元时，Awen Awen Aws aws峰值，但在Susssspectity的发作之前就不会达到顶峰骑士的上调导致细胞内Ca2+（[Ca2+] i）增加并改变Ca2+稳态建议Na+/Ca2+交换器，一种调节[Ca2+] i的双向膜离子转运蛋白优先以向前模式（NCXFWD）运行以挤出Ca2+并恢复Ca2+稳态。 NCX以反向模式（NCXREV）运行，导致Ca2+ infux IC神经元优先于SSSS的发作敏感性Awen Awen Awen Awen Awen Aws AWSS的流行率。 AWS启动。神经元过度兴奋性和AWS的敏感性为开发新型新型治疗剂提供了特定的靶标。我们的工作假设是，通过NCXREV在IC神经元中的Ca2+涌入对于产生癫痫状态至关重要爆发了AWS，以测试这一假设我们将在体内药理学与分子遗传学，电生理学，分子生物学和D. 首先，我们将在三个特定目标中进行行为分析。戒酒后，在IC神经元中产生癫痫样爆发；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，我们将确定NCX1 Genee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发作易感性增加有关在戒酒过程中，我们将评估NCX1的大鼠产生的AWS 表达在IC神经元中被删除。使用光遗传学支持由谷氨酸能IC神经元介导的过度兴奋性。提供有关NCX1在癫痫活动，Initiale AWSS的机制和机制中的作用的关键见解这是AWSS和其他类型的GTCS的基础。