GSE203530
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.