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Synergistic effects of intermittent ethanol exposure and increased GIRK2 expression in NGN2 neurons

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE244985 | GEO | 2023/11/01

REPOSITORIES: GEO

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Project description:Purpose: Fetal alcohol spectrum disorders (FASD) result from ethanol exposure to the developing fetus. FASD occur in up to 1-5% of live births in the United States and there currently is no cure. Ethanol exposure to the developing central nervous system (CNS) has profound effects on learning and memory, impulse control, and motor function, resulting from neuropathology. The purpose of the current study was to perform transcriptome analysis to evaluate the effects of early postnatal ethanol exposure in the hippocampus and cerebellum. Methods: Postnatal C57BL/6 mice were treated with 4g/kg of ethanol from P4-P9, brains were harvested 24h after the final treatment at P10, hippocampi and cerebella were microdissected, RNA was isolated, and RNASeq analysis was performed. We compared differences in gene expression, sex-dependent expression, and global biological pathways associated with disruptions in hippocampal and cerebellar genes between the ethanol and vehicle treated neonates. Results: Ethanol caused both an up and down regulation of genes associated with the hippocampus and cerebellum, which may result in the disruption of normal circuitry and maturation and growth in these brain regions. Ethanol increased the expression of genes associated with the S phase of the cell cycle in both the hippocampus and cerebellum. In the cerebellum, ethanol increased effector gene expression, and in the hippocampus, genes associated with different interneuron lineages were altered. Postnatal ethanol exposure also resulted in altered expression of genes associated with oligodendrocyte lineages and myelination, along with alterations in microglia associated genes. Conclusion: Collectively, these data indicate that ethanol has profound effects on the hippocampus and cerebellum, resulting in alterations of gene expression and biological pathways regulating neurodevelopment. These studies may have important implications concerning alcohol-induced neuropathology and the neurological effects seen across the life span in FASD.

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Synergistic effects of intermittent ethanol exposure and increased GIRK2 expression in NGN2 neurons

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