Jadali, A. | Search bioRxiv

bioRxiv2022

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.

neuroscience↗

bioRxiv2021

Analyses of the Autism-associated Neuroligin-3 R451C Mutation in Human Neurons Reveals a Gain-of-Function Synaptic Mechanism

Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C-mutation on human neurons has not been investigated. Here, we generated human knock-in neurons with the NLGN3 R451C-mutation. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C-mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses. No significant cell death and endoplasmic reticulum stress were detected. Importantly, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons that corresponded to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C-mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASDs.

neuroscience↗