No abstract

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.

SummaryLoss of spiral ganglion neurons (SGNs) significantly contributes to hearing loss. Otic progenitor cell transplantation is a potential strategy to replace lost SGNs. Understanding how key transcription factors promote SGN differentiation in otic progenitors accelerates efforts for replacement therapies. A pro-neural transcription factor, Neurogenin1 (Neurog1), is essential for SGN development. Using an immortalized multipotent otic progenitor (iMOP) cell line that can self-renew and differentiate into otic neurons, NEUROG1 was enriched at the promoter of cyclin-dependent kinase 2 (Cdk2) and neurogenic differentiation 1 (NeuroD1) genes. Changes in H3K9ac and H3K9me3 deposition at the Cdk2 and NeuroD1 promoters suggested epigenetic regulation during iMOP proliferation and differentiation. In self-renewing iMOP cells, overexpression of NEUROG1 increased CDK2 to drive proliferation, while knockdown of NEUROG1 decreased CDK2 and reduced proliferation. In iMOP-derived neurons, overexpression of NEUROG1 accelerated acquisition of neuronal morphology, while knockdown of NEUROG1 prevented differentiation. Our findings suggest that NEUROG1 can promote proliferation or neuronal differentiation.

Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP) cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.

The sensory hair cells of the inner ear are exquisitely sensitive to ototoxic insults. Loss of hair cells after exposure to ototoxic agents causes hearing loss. Chemotherapeutic agents such as cisplatin causes hair cell loss. Cisplatin forms DNA mono-adducts as well as intra- and inter-strand DNA crosslinks. DNA cisplatin adducts are repaired through the DNA damage response. The decision between cell survival and cell death following DNA damage rests on factors that are involved in determining damage tolerance, cell survival and apoptosis. Cisplatin damage on hair cells has been the main focus of many ototoxic studies, yet the effect of cisplatin on supporting cells has been largely ignored. In this study, the effects of DNA damage response in cochlear supporting cells were interrogated. Supporting cells play a major role in the development, maintenance and oto-protection of hair cells. Loss of supporting cells may indirectly affect hair cell survival or maintenance. Activation of the Phosphoinositide 3-Kinase (PI3K) signaling was previously shown to promote hair cell survival. To test whether activating PI3K signaling promotes supporting cell survival after cisplatin damage, cochlear explants from the neural subset (NS) Cre Pten conditional knockout mice were employed. Deletion of Phosphatase and Tensin Homolog (PTEN) activates PI3K signaling in multiple cell types within the cochlea. Supporting cells lacking PTEN showed increased cell survival after cisplatin damage. Supporting cells lacking PTEN also showed increased phosphorylation of Checkpoint Kinase 1 (CHK1) levels after cisplatin damage. Nearest neighbor analysis showed increased numbers of supporting cells with activated PI3K signaling in close proximity to surviving hair cells in cisplatin damaged cochleae. We propose that increased PI3K signaling promotes supporting cell survival through phosphorylation of CHK1 and increased survival of supporting cells indirectly increases hair cell survival after cisplatin damage.