The nitric oxide signaling pathway inhibits intracellular calcium release to prevent neurodevelopmental alcohol toxicity

In the context of fetal alcohol spectrum disorders, we investigated how the nitric oxide (NO) signaling pathway influences intracellular calcium (Ca²⁺) to mediate alcohol resistance, using a primary cell culture model of cerebellar granule neurons (CGN). Alcohol during fetal brain development triggers abnormally high apoptotic cell death in vulnerable neuronal populations, culminating in serious behavioral and cognitive deficits that persist into adulthood. Prior studies demonstrated that the NO signaling pathway [neuronal nitric oxide synthase → NO → soluble guanylyl cyclase → cyclic guanosine monophosphate → protein kinase G (PKG)] mitigates alcohol toxicity, consequently diminishing neuronal loss both in vivo and in vitro. Endoplasmic reticulum (ER) Ca²⁺ release, a key apoptotic mechanism, requires the inositol 1,4,5-trisphosphate receptor (IP₃R), a known PKG substrate. Our studies focused on this crucial intersection point where the NO signaling cascade can influence Ca²⁺-mediated apoptotic mechanisms, and exposed a downstream mechanism where NO can moderate alcohol neurotoxicity.

We hypothesized that as alcohol disturbs neuronal Ca²⁺ homeostasis to trigger cell death, the NO signaling pathway counters it by limiting Ca²⁺ release from the ER. We examined first the role of the phospholipase C (PLC) pathway [PLC → inositol 1,4,5-trisphosphate → IP₃R → Ca²⁺] in developmental neurotoxicity through our in vitro CGN model, extending previous in vivo studies. We found that alcohol terminates developing neurons by eliciting abnormal Ca²⁺ release from the ER rather than from an extracellular source, via a PLC - IP₃R-dependent signaling mechanism. Inhibiting either calcineurin or Ca²⁺ / calmodulin-dependent protein kinase ii (CaMKii), which participate in parallel Ca²⁺-activated apoptotic cascades, shielded CGN cultures from alcohol. Blocking the mitochondrial Ca²⁺ uniporter or the mitochondrial permeability transition pore also provided neuroprotection. That the activated pathways must interact to generate cell death likely explains why inhibiting one of multiple parallel signaling cascades limits alcohol toxicity.

We next demonstrated that activating the NO pathway downstream at PKG eliminated both alcohol-related neuronal death and the accompanying rapid rise in intracellular Ca²⁺, an effect that markedly resembled IP₃R inhibition. Experiments that temporally manipulated the addition of PKG activators in relation to alcohol exposure linked PKG's obstruction of alcohol-induced Ca²⁺ elevations to alcohol resistance. In contrast, brain-derived neurotrophic factor (BDNF), which does not rely on PKG to provide neuroprotection, failed to block alcohol-induced Ca²⁺ elevations while preventing alcohol toxicity. This indicates that although PKG blocks alcohol-induced Ca²⁺ elevations, averting these Ca²⁺ elevations is not necessary for neuroprotection. BDNF may confer alcohol resistance through an as yet unidentified process downstream from the disruption of intracellular Ca²⁺.

In summary, we established that 1) alcohol induces toxic Ca²⁺ elevations originating from the ER through a PLC - IP₃R-dependent pathway, and that 2) PKG-mediated alcohol resistance is linked to preventing the intracellular Ca²⁺ surges. These findings support the hypothesis that the NO signaling pathway shields developing neurons from alcohol by limiting Ca²⁺ release from the ER.