Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium (GIRK2) channel subunit 2, have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ('Affected: AF') and four control subjects without variants ('Unaffected: UN'). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol. Overall design: Pooled cultures of excitatory human neurons cultured on primary mouse glia were harvested and run through the 10X Genomics Chromium device for scRNAseq. Each pool mixed cells from four subjects, with one group (first character of sample name=C, control) having major-allele KCNJ6 haplotype and the other group (first character of sample name=A, affected) with minor-allele haplotype. One set of cultures from each group was exposed to intermitent ethanol (20 mM, 7 days; second character=7) and the other was untreated ( second character=C, control). Libraries were prepared using the 10X Genomics 3' GEM Reagent Kit v3.1. After aligning reads with a mixed human and mouse reference index in cellranger, and merging samples in Seurat, mouse-specific clusters were removed, leaving only human cell data for analysis. Cells from individual subjects were identified using SNP profiles and demuxlet.

编码G蛋白门控内向整流钾离子（G protein-gated inwardly rectifying potassium, GIRK2）通道亚基2的KCNJ6基因中的同义及非编码单核苷酸多态性（single nucleotide polymorphisms, SNPs），已被证实与酒精使用障碍（alcohol use disorder, AUD）受试者脑电图检测到的额叶θ事件相关振荡（electroencephalographic frontal theta event-related oscillations, ERO）增强存在关联。为在保留恰当遗传背景的前提下解析其分子与细胞机制，本研究从4名携带KCNJ6变异的AUD确诊受试者（命名为"受累组：AF"）及4名无变异的对照受试者（命名为"未受累组：UN"）的诱导多能干细胞（induced pluripotent stem cells, iPSCs）中，诱导生成了兴奋性谷氨酸能神经元（induced excitatory glutamatergic neurons, iN）。研究人员对神经元的基因表达、形态、兴奋性及生理特性开展了分析。单细胞RNA测序（single cell RNA sequencing, scRNAseq）结果显示，携带KCNJ6 AF变异的神经元存在突触传递与细胞投射形态发生模式的异常改变。实验结果证实，受累组神经元的GIRK2表达水平更低，神经突面积更大，且兴奋性显著升高。值得注意的是，暴露于中毒浓度乙醇可诱导GIRK2表达，并逆转受累组神经元的功能异常。仅通过异位过表达GIRK2即可模拟乙醇的作用，使诱导产生的兴奋性恢复正常。本研究得出结论：KCNJ6变异可降低GIRK2表达并提高神经元兴奋性，而乙醇可减弱乃至消除该效应。

实验总体设计：将培养于原代小鼠胶质细胞上的兴奋性人神经元混合培养物收集后，通过10X Genomics Chromium平台进行scRNAseq。每个混合样本包含4名受试者的细胞，其中一组（样本名称首字符为C，即对照组）携带KCNJ6主要等位基因单倍型，另一组（样本名称首字符为A，即受累组）携带次要等位基因单倍型。每组各有一批培养物暴露于间歇性乙醇（20 mM，处理7天；样本名称第二个字符为7），另一批未做处理（样本名称第二个字符为C，即对照组）。文库构建采用10X Genomics 3' GEM Reagent Kit v3.1试剂盒。使用cellranger将测序reads比对至人-鼠混合参考基因组索引，并通过Seurat合并样本后，移除小鼠特异性细胞簇，仅保留人类细胞数据用于后续分析。通过单核苷酸多态性（SNP）谱及demuxlet软件对来自不同受试者的细胞进行鉴定。