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.

The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase inAppgene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. GCaMP8f Ca2+reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown ofAppblock the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ – either fibrillar or oligomeric – has no effect. In culture, APPSwe(a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model – both females and males – neurons have elevated levels of APP protein that invade the AIS. In vivo as in vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD.SIGNIFICANCE:While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. In vivo and in vitro, modest amounts of excess APP alter the properties of the axon initial segment (AIS). The Aβ peptide derived from APP is without effect. Consistent with the observed changes in the AIS which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the AIS suggesting a relationship with AD dementia.

The small Aβ peptide has been hypothesized to be the main driver of Alzheimer's disease (AD). Aβ is a proteolytic cleavage product of a larger protein, the amyloid precursor protein (APP), whose normal functions remain largely unexplored. We report here activities of the full-length APP protein that relate directly to the etiology of AD. Increasing neuronal activity leads to a rapid increase in App gene expression. In both cultures of mouse cortical neurons and human iPSC-derived neurons, elevated APP protein changes the structure of the axon initial segment (AIS), the site of action potential initiation. In neurons with elevated APP, the AIS shortens in length and shifts in position away from the cell body. Both changes would be expected to reduce neuronal excitability. The AIS effects are due to the cell-autonomous actions of APP; exogenous Aβ - either fibrillar or oligomeric - has no effect. The findings relate directly to AD in several ways. In culture, APP carrying the Swedish familial AD mutation (APPSwe) induces stronger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, and APPSwe binds more avidly than wild type APP. Finally, neurons in the frontal cortex of humans with sporadic AD reveal histologically elevated levels of APP protein that invade the domain of the AIS, whose length is significantly shorter than that found in healthy control neurons. The findings offer an alternative explanation for the effects of at least some familial AD mutations.

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.