Upregulated GIRK2 Counteracts Ethanol-Induced Changes in Excitability and Respiration in Human Neurons.

Genome-wide association studies (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified noncoding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G-protein-coupled inwardly rectifying potassium channel that regulates neuronal excitability. We studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multielectrode arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 d of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-induced changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons.

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 channel subunit 2 (GIRK2), 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.

Comparison of K+ Channel Families.

K+ channels enable potassium to flow across the membrane with great selectivity. There are four K+ channel families: voltage-gated K (Kv), calcium-activated (KCa), inwardly rectifying K (Kir), and two-pore domain potassium (K2P) channels. All four K+ channels are formed by subunits assembling into a classic tetrameric (4x1P = 4P for the Kv, KCa, and Kir channels) or tetramer-like (2x2P = 4P for the K2P channels) architecture. These subunits can either be the same (homomers) or different (heteromers), conferring great diversity to these channels. They share a highly conserved selectivity filter within the pore but show different gating mechanisms adapted for their function. K+ channels play essential roles in controlling neuronal excitability by shaping action potentials, influencing the resting membrane potential, and responding to diverse physicochemical stimuli, such as a voltage change (Kv), intracellular calcium oscillations (KCa), cellular mediators (Kir), or temperature (K2P).

Dual Antidepressant Duloxetine Blocks Nicotinic Receptor Currents, Calcium Signals and Exocytosis in Chromaffin Cells Stimulated with Acetylcholine.

The inhibition of nicotinic acetylcholine receptors (nAChRs) has been proposed as a potential strategy to develop new antidepressant drugs. This is based on the observation that antidepressants that selectively block noradrenaline (NA) or serotonin (5-HT) reuptake also inhibit nAChRs. Dual antidepressants blocking both NA and 5-HT reuptake were proposed to shorten the delay in exerting their clinical effects; whether duloxetine, a prototype of dual antidepressants, also blocks nAChRs is unknown. Here we explored this question in bovine chromaffin cells (BCCs) that express native α3, α5, and α7 nAChRs and in cell lines expressing human α7, α3ß4, or α4ß2 nAChRs. We have found that duloxetine fully blocked the acetylcholine (ACh)-elicited nicotinic currents in BCCs with an IC50 of 0.86 µM. Such blockade seemed to be noncompetitive, voltage dependent, and partially use dependent. The ACh-elicited membrane depolarization, the elevation of cytosolic calcium ([Ca2+]c), and catecholamine release in BCCs were also blocked by duloxetine. This blockade developed slowly, and the recovery of secretion was also slow and gradual. Duloxetine did not affect Na+ or Ca2+ channel currents neither the high-K+-elicited [Ca2+]c transients and secretion. Of interest was that in cell lines expressing human α7, α3ß4, and α4ß2 nAChRs, duloxetine blocked nicotinic currents with IC50 values of 0.1, 0.56, and 0.85 µM, respectively. Thus, in blocking α7 receptors, which are abundantly expressed in the brain, duloxetine exhibited approximately 10-fold to 100- fold higher potency with respect to reported IC50 values for various antidepressant drugs. This may contribute to the antidepressant effect of duloxetine.

Upregulated GIRK2 counteracts ethanol-induced changes in excitability & respiration in human neurons.

Genome-wide association analysis (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified non-coding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G protein-coupled inwardly-rectifying potassium channel that regulates neuronal excitability. How changes in GIRK2 affect human neuronal excitability and the response to repeated ethanol exposure is poorly understood. Here, we studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multi-electrode-arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 days of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-dependent changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons. SIGNIFICANCE STATEMENT: Alcohol use disorder (AUD) is a major health problem that has worsened since COVID, affecting over 100 million people worldwide. While it is known that heritability contributes to AUD, specific genes and their role in neuronal function remain poorly understood, especially in humans. In the current manuscript, we focused on the inwardly-rectifying potassium channel GIRK2, which has been identified in an AUD-endophenotype genome-wide association study. We used human excitatory neurons derived from healthy donors to study the impact of GIRK2 expression. Our results reveal that elevated GIRK2 counteracts ethanol-induced increases in glutamate response and intracellular calcium, as well as deficits in activity-dependent mitochondrial respiration. The role of GIRK2 in mitigating ethanol-induced hyper-glutamatergic and mitochondrial offers therapeutic promise for treating AUD.

Tissue Microarray Lipidomic Imaging Mass Spectrometry Method: Application to the Study of Alcohol-Related White Matter Neurodegeneration.

Central nervous system (CNS) white matter pathologies accompany many diseases across the lifespan, yet their biochemical bases, mechanisms, and consequences have remained poorly understood due to the complexity of myelin lipid-based research. However, recent advances in matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) have minimized or eliminated many technical challenges that previously limited progress in CNS disease-based lipidomic research. MALDI-IMS can be used for lipid identification, semi-quantification, and the refined interpretation of histopathology. The present work illustrates the use of tissue micro-arrays (TMAs) for MALDI-IMS analysis of frontal lobe white matter biochemical lipidomic pathology in an experimental rat model of chronic ethanol feeding. The use of TMAs combines workload efficiency with the robustness and uniformity of data acquisition. The methods described for generating TMAs enable simultaneous comparisons of lipid profiles across multiple samples under identical conditions. With the methods described, we demonstrate significant reductions in phosphatidylinositol and increases in phosphatidylcholine in the frontal white matter of chronic ethanol-fed rats. Together with the use of a novel rapid peak alignment protocol, this approach facilitates reliable inter- and intra-group comparisons of MALDI-IMS data from experimental models and could be extended to human disease states, including using archival specimens.

5. Collaborative Study on the Genetics of Alcoholism: Functional genomics.

Alcohol Use Disorder is a complex genetic disorder, involving genetic, neural, and environmental factors, and their interactions. The Collaborative Study on the Genetics of Alcoholism (COGA) has been investigating these factors and identified putative alcohol use disorder risk genes through genome-wide association studies. In this review, we describe advances made by COGA in elucidating the functional changes induced by alcohol use disorder risk genes using multimodal approaches with human cell lines and brain tissue. These studies involve investigating gene regulation in lymphoblastoid cells from COGA participants and in post-mortem brain tissues. High throughput reporter assays are being used to identify single nucleotide polymorphisms in which alternate alleles differ in driving gene expression. Specific single nucleotide polymorphisms (both coding or noncoding) have been modeled using induced pluripotent stem cells derived from COGA participants to evaluate the effects of genetic variants on transcriptomics, neuronal excitability, synaptic physiology, and the response to ethanol in human neurons from individuals with and without alcohol use disorder. We provide a perspective on future studies, such as using polygenic risk scores and populations of induced pluripotent stem cell-derived neurons to identify signaling pathways related with responses to alcohol. Starting with genes or loci associated with alcohol use disorder, COGA has demonstrated that integration of multimodal data within COGA participants and functional studies can reveal mechanisms linking genomic variants with alcohol use disorder, and potential targets for future treatments.

ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex.

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

Advances in Targeting GIRK Channels in Disease.

G protein-gated inwardly rectifying potassium (GIRK) channels are essential regulators of cell excitability in the brain. While they are implicated in a variety of neurological diseases in both human and animal model studies, their therapeutic potential has been largely untapped. Here, we review recent advances in the development of small molecule compounds that specifically modulate GIRK channels and compare them with first-generation compounds that exhibit off-target activity. We describe the method of discovery of these small molecule modulators, their chemical features, and their effects in vivo. These studies provide a promising outlook on the future development of subunit-specific GIRK modulators to regulate neuronal excitability in a brain region-specific manner.