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Series GSE244985 Query DataSets for GSE244985
Status Public on Nov 01, 2023
Title Synergistic effects of intermittent ethanol exposure and increased GIRK2 expression in NGN2 neurons
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary Increased expression of G-coupled Inwardly Rectifying Potassium Channel Subunit 2 (GIRK2) has been linked to an electrophysiological endophenotype for alcohol use disorder. We have increased GIRK2 expression in human glutamatergic (NGN2-induced) neurons using two distinct strategies (CRISPRa targeting of the endogenous gene and ectopic expression with a lentiviral vector) to investigate the interplay between GIRK2 expression levels and neuronal response to alcohol (ethanol). We used six healthy donor cell lines (2 CRISPRa and 4 lenti) to draw comparisons within the same genetic background. Findings from functional data (calcium imaging, electrophysiology, and mitochondrial stress tests) indicate that increased GIRK2 expression, combined with prolonged (21-day) intermittent ethanol exposure (IEE), counteracts IEE-induced changes in glutamatergic activity and cellular respiration. To understand the transcriptomic underpinnings of these functional data, we conducted bulk RNAseq in the same donor cohort. Differential gene expression analysis indicates that both CRISPRa and lenti-increased GIRK2 interacted with IEE to produce unique differential regulation of synaptic genes, including glutamate receptors and voltage-gated ion channels, distinct from the effects of IEE or increased GIRK2 alone. Increased expression of G-coupled Inwardly Rectifying Potassium Channel Subunit 2 (GIRK2) has been linked to an electrophysiological endophenotype for alcohol use disorder. We have increased GIRK2 expression in human glutamatergic (NGN2-induced) neurons using two distinct strategies (CRISPRa targeting of the endogenous gene and ectopic expression with a lentiviral vector) to investigate the interplay between GIRK2 expression levels and neuronal response to alcohol (ethanol). We used six healthy donor cell lines (2 CRISPRa and 4 lenti) to draw comparisons within the same genetic background. Findings from functional data (calcium imaging, electrophysiology, and mitochondrial stress tests) indicate that increased GIRK2 expression, combined with prolonged (21-day) intermittent ethanol exposure (IEE), counteracts IEE-induced changes in glutamatergic activity and cellular respiration. To understand the transcriptomic underpinnings of these functional data, we conducted bulk RNAseq in the same donor cohort. Differential gene expression analysis indicates that both CRISPRa and lenti-increased GIRK2 interacted with IEE to produce unique differential regulation of synaptic genes, including glutamate receptors and voltage-gated ion channels, distinct from the effects of IEE or increased GIRK2 alone.
 
Overall design Two donor cohorts: CRISPRa (2 donors) and Lenti (4) donors were used to generate NGN2 neurons with endogenous and increased levels of GIRK2 expression. These neurons were then subject to 21 days of intermittent exposure to 17 mM ethanol. Two batches of neurons and three technical replicates/donor/batch were generated for RNA harvest and bulk RNAseq. The two cohorts were then analysed separately from one another, to separate donor effects from GIRK2 expression manipulation method. Additional analyses were performed on the pooled cohort, correcting for systematic noise and donor effects.

 
Contributor(s) Prytkova I, Liu Y, Goate A, Slesinger P
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Submission date Oct 10, 2023
Last update date Jan 04, 2024
Contact name Paul Slesinger
E-mail(s) paul.slesinger@mssm.edu
Organization name Icahn School of Medicine at Mount Sinai
Department Neuroscience
Street address 1425 Madison Ave
City New York
State/province NY
ZIP/Postal code 10029
Country USA
 
Platforms (1)
GPL20301 Illumina HiSeq 4000 (Homo sapiens)
Samples (138)
GSM7833121 Donor BJ endogenous GIRK2, alcohol-chronic, batch a, bio rep 1
GSM7833122 Donor BJ endogenous GIRK2, alcohol-chronic, batch a, bio rep 2
GSM7833123 Donor BJ endogenous GIRK2, alcohol-chronic, batch b, bio rep 1
Relations
BioProject PRJNA1026571

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Supplementary file Size Download File type/resource
GSE244985_salmon_cutadapt.tsv.gz 18.9 Mb (ftp)(http) TSV
GSE244985_star_gene.tsv.gz 7.2 Mb (ftp)(http) TSV
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