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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：
10599223
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 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10599223
关键词：
Acoustics Acute Adjuvant Therapy Alcohol Withdrawal Seizures Alcohol withdrawal syndrome Anticonvulsants Behavioral Benzodiazepines Binding Brain Buffers Cell Surface Proteins Cell surface Couples Data Dependovirus Development Dissociation 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 P-Q type voltage-dependent calcium channel 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 density effective therapy epileptiform 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.

全身性强直-阵挛发作(GTCSs)，最常见的酒精戒断诱发癫痫类型 (AWSS)通常对当前的抗惊厥药物具有抵抗力，然而，它们的基本机制是人们对此知之甚少。我们的长期目标是了解改变的钙信号如何影响神经元导致AWSS易感性增加的过度兴奋性。我们之前曾报道过上调下层L(CaV1.3)和P型(CaV2.1)电压门控钙通道相关蛋白的研究当AWSS患病率达到高峰时，但不是在AWSS易感性开始之前，丘(IC)神经元。这个 Cav通道上调导致细胞内钙离子浓度升高和钙离子稳态改变提示Na+/Ca~(2+)交换器，一种调节[Ca~(2+)]i的双向膜离子转运体优先工作在正向模式(NCXfwd)，以挤出Ca~(2+)，恢复钙稳态。令人惊讶的是， NCX以相反的模式工作(NCXrev)，导致在AWSS发作之前钙离子内流到IC神经元易感性和AWSS流行率达到顶峰的时间。因此，NCX活动的变化可能在 AWS启动。这里提出的实验将确定NCX类型1(NCX1)对神经元的超兴奋性和AWSS的易感性，为开发新的治疗药物提供了特异性靶点。我们的工作假设是，通过NCXrev的内流是癫痫样发作所必需的引发AWS的突发事件。为了验证这一假设，并基于我们的初步数据和之前的出版物，我们将把体内药理学与分子遗传学、电生理学、分子生物学以及三个具体目标的行为分析。首先，我们将确定NCXrev对酒精戒断后IC神经元产生癫痫样放电；此外，我们还将研究细胞内Na+浓度以及L和P型钙通道参与NCXrev活性的增加在戒酒过程中。第二，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 在IC神经元中，表达被选择性地删除。此外，我们将在大鼠身上测量AWS 利用光遗传学抑制谷氨酸能神经元介导的超兴奋性。我们的发现将提供对NCX1在癫痫发作活动中的作用、启动AWS的机制以及机制的关键见解这是AWS和其他类型的GTCS的基础。