Abstract
Alcohol is a potent pharmacological agent when consumed acutely at sufficient quantities and repeated overuse can lead to addiction and deleterious effects on health. Alcohol is thought to modulate neuronal function through low-affinity interactions with proteins, in particular with membrane channels and receptors. Paradoxically, alcohol acts as both a stimulant and a sedative. The exact molecular mechanisms for the acute effects of ethanol on neurons, as either a stimulant or a sedative, however remain unclear. We investigated the role that the heat shock transcription factor HSF-1 played in determining a stimulatory phenotype of Caenorhabditis elegans in response to physiologically relevant concentrations of ethanol (17 mM; 0.1% v/v). Using genetic techniques, we demonstrate that either RNA interference of hsf-1 or use of an hsf-1(sy441) mutant lacked the enhancement of locomotion in response to acute ethanol exposure evident in wild-type animals. We identify that the requirement for HSF-1 in this phenotype was IL2 neuron-specific and required the downstream expression of the α-crystallin ortholog HSP-16.48 Using a combination of pharmacology, optogenetics, and phenotypic analyses we determine that ethanol activates a Gαs-cAMP-protein kinase A signaling pathway in IL2 neurons to stimulate nematode locomotion. We further implicate the phosphorylation of a specific serine residue (Ser322) on the synaptic protein UNC-18 as an end point for the Gαs-dependent signaling pathway. These findings establish and characterize a distinct neurosensory cell signaling pathway that determines the stimulatory action of ethanol and identifies HSP-16.48 and HSF-1 as novel regulators of this pathway.
Highlights
Alcohol is one of the most prevalent addictive substances and its overuse produces a severe burden on society (WHO 2011)
In C. elegans, the hsf-1(sy441) allele is a viable loss-of-function point mutation in the hsf-1 gene that acts as an inhibitor of HSF-1 transcriptional activity (Hajdu-Cronin et al 2004), increasing temperature sensitivity and decreasing lifespan (Baird et al 2014)
Nematodes respond to high external ethanol concentration (400 mM) by a dose-dependent decrease in coordinated locomotion (Davies et al 2003; Graham et al 2009) and we have recently characterized a novel role for HSF-1 in determining this phenotype (Johnson et al 2016)
Summary
Alcohol is one of the most prevalent addictive substances and its overuse produces a severe burden on society (WHO 2011). We have recently shown that hsf-1 loss-of-function increases sensitivity to 400 mM ethanol (Johnson et al 2016). This hypersensitivity was partially the result of the downstream basal expression of HSP-16.48, an ortholog of the human small heat shock protein a-crystallin, in a process unrelated to an HSF-1-dependent heat shock stress response. Using a combination of pharmacology, genetics, and optogenetics, we determined further that this ethanol-dependent stimulation of motility acts via a Gas-cAMP-protein kinase A (PKA) signaling pathway within the IL2 sensory neurons and identifies the exocytotic protein UNC-18 as a downstream effector for PKA. Individual components of the Gas pathway have been linked previously to the neuronal effects of ethanol, this study uniquely characterizes the entire signaling pathway in ethanol-dependent stimulation
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