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: 10224817
• 负责人: Iya Prytkova
• 金额: $ 4.44万
• 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 2022-08-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224817
• 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; Induced pluripotent stem cell derived neurons; 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（AUDs）在全世界的成年人和青少年中非常普遍， 一个重大的公共卫生问题。此外，它们是高度遗传的，并且在很大程度上仍然未经治疗。这 该项目旨在通过以下方式确定AUD发展的细胞和分子机制： KCNJ6在人类神经元中上调的研究。多个单核苷酸多态性（SNPs）， KCNJ6与AUD风险脑电图内表型具有全基因组关联。具体地说， 在注意力和行为的测量中，与额叶θ事件相关振荡减少相关的SNP 抑制性控制也与额叶皮质中KCNJ6 mRNA的上调有关。kcnj6 编码G蛋白激活的内向整流钾通道2（GIRK2），一种有助于 维持大脑中的抑制性张力。因此，第一个目的是阐明功能的影响， 在来源于对照人诱导多能干细胞的神经元中上调KCNJ6表达。这 这种方法利用了hiPSC衍生的神经元的翻译潜力，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用于测量 与GIRK2升高相关的神经元活动，如自发活动和兴奋性降低。的 第二个目的是测试急性和慢性乙醇暴露对这些神经培养物的影响， 差异基因表达的成像和RNA测序分析。酒精直接激活GIRK2 通道，蛋白质水平升高可能导致对酒精反应的神经元抑制增加 exposure.此外，暴露于酒精和神经元活动的变化可能会影响整体基因表达。 通过RNAseq测量的表达，支持了升高的KCNJ6影响转录组学的假设。 适应酒精治疗。总的来说，这个项目提供了一个多方面的特点，人类 神经元在酒精使用障碍的病因学相关模型，潜在地识别适合的机制 用于治疗靶向。