Elucidating the Role of KCNJ6 in a Human Neuronal Model of Alcohol Use Disorder

阐明 KCNJ6 在人类酒精使用障碍神经元模型中的作用

• 批准号: 10019308
• 负责人: Iya Prytkova
• 金额: $ 4.39万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-14 至 2022-09-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019308
• 关键词: Acute; Adolescent; Adult; Affect; Alcohols; Alleles; Attention; Autopsy; Brain; Brain region; Calcium; Cell Line; Cells; Chronic; Cognitive; Computer Analysis; Coupled; Data; Development; Disease; Doxycycline; Electroencephalogram; Electrophysiology (science); Equilibrium; Ethanol; Etiology; Event; Exhibits; GIRK4 subunit, G protein-coupled inwardly-rectifying potassium channel; GTP-Binding Proteins; Gene Expression; Genes; Genetic; Genetic Polymorphism; Genetic study; Glutamates; Heritability; Human; Image; Individual; Investigation; Lentivirus Vector; Maintenance; Measures; Messenger RNA; Modeling; Molecular; Molecular Biology; Monitor; Mus; Neurons; Neurophysiology - biologic function; Potassium Channel; Proteins; Public Health; Quantitative Trait Loci; RNA; Rewards; Risk; Role; Single Nucleotide Polymorphism; Techniques; Testing; Tissue-Specific Gene Expression; Training; Up-Regulation; Western Blotting; alcohol abuse therapy; alcohol effect; alcohol exposure; alcohol response; alcohol risk; alcohol use disorder; brain tissue; differential expression; disorder risk; dosage; endophenotype; excitatory neuron; frontal lobe; genome wide association study; genome-wide analysis; human subject; induced pluripotent stem cell; inter-individual variation; inward rectifier potassium channel; nerve stem cell; overexpression; patch clamp; relating to nervous system; therapeutic target; transcriptome; transcriptome sequencing; transcriptomics

Project Summary Alcohol Use Disorders (AUDs) are highly prevalent among adults and adolescents worldwide, and pose a significant public health problem. Furthermore, they are highly heritable, and remain largely untreated. This project aims to identify cellular and molecular mechanisms underlying AUD development through the investigation of KCNJ6 upregulation in human neurons. Multiple single nucleotide polymorphisms (SNPs) in KCNJ6 have a genome wide association with an AUD risk electroencephalogram endophenotype. Specifically, SNPs associated with a reduction in frontal theta event-related oscillations in measures of attention and inhibitory control are also associated with an upregulation of KCNJ6 mRNA in the frontal cortex. KCNJ6 encodes the G protein-activated inwardly rectifying potassium channel 2 (GIRK2), a protein contributing to the maintenance of inhibitory tone in the brain. Therefore, the first aim is to elucidate functional effects of upregulated KCNJ6 expression in neurons derived from control human induced pluripotent stem cells. This approach capitalizes on the translational potential of hiPSC-derived neurons and minimizes the confounds of inter-individual variability by comparing endogenous and elevated KCNJ6 expression in neurons with the same genetic background. Calcium imaging and patch clamp electrophysiology will be used to measure changes in neuronal activity associated with elevated GIRK2, such as decreased spontaneous activity and excitability. The second aim is to test the effects of acute and chronic ethanol exposure on these neural cultures using calcium imaging and RNA sequencing analyses of differential gene expression. Alcohol directly activates GIRK2 channels, and elevated levels of the protein could result in increased neuronal inhibition in response to alcohol exposure. Furthermore, exposure to alcohol and changes in neuronal activity may impact global gene expression as measured by RNAseq, supporting the hypothesis that elevated KCNJ6 impacts transcriptomic adaptations to alcohol treatment. Overall, this project provides a multifaceted characterization of human neurons in an etiologically-relevant model of alcohol use disorder, potentially identifying mechanisms suitable for therapeutic targeting.

项目摘要 酒精使用障碍(AUD)在世界各地的成年人和青少年中非常普遍， 这是一个重大的公共卫生问题。此外，它们具有高度的遗传性，而且在很大程度上仍未得到治疗。这 该项目旨在通过以下途径确定AUD发生的细胞和分子机制 KCNJ6在人神经细胞中上调的研究。中国汉族人多个单核苷酸多态(SNPs) KCNJ6与AUD危险脑电内表型有全基因组关联。具体来说， SNPs与注意和注意指标中额叶Theta事件相关振荡的减少相关 抑制性控制也与额叶皮质中KCNJ6基因的上调有关。KCNJ6 编码G蛋白激活的内向整流钾通道2(GIRK2)，这是一种有助于 维持大脑中的抑制性音调。因此，第一个目的是阐明其功能效应。 上调KCNJ6在对照人诱导的多能干细胞来源的神经元中的表达。这 该方法利用了HiPSC衍生神经元的翻译潜力，并最大限度地减少了 通过比较内源性和升高的KCNJ6在神经元中的表达 遗传背景。钙成像和膜片钳电生理学将被用来测量 与GIRK2升高相关的神经元活动，如自发活动和兴奋性降低。这个 第二个目的是测试急性和慢性酒精暴露对使用钙的神经培养的影响。 差异基因表达的成像和RNA测序分析。酒精直接激活GIRK2 通道和蛋白水平升高可导致神经元对酒精反应的抑制增加 曝光。此外，酒精暴露和神经元活动的变化可能会影响全球基因 RNAseq测量的表达，支持KCNJ6升高影响转录的假说 对酒精治疗的适应。总体而言，这个项目提供了人类的多方面特征 酒精使用障碍的病因学相关模型中的神经元，潜在地识别适合的机制 用于治疗靶向。