Serotonergic neuron regulation informed by in vivo single-cell transcriptomics.

Despite the recognized importance of the dorsal raphe (DR) serotonergic (5-HT) nuclei in the pathophysiology of depression and anxiety, the molecular components/putative drug targets expressed by these neurons are poorly characterized. Utilizing the promoter of an ETS domain transcription factor that is a stable marker of 5-HT neurons (Pet-1) to drive 5-HT neuronal expression of YFP, we identified 5-HT neurons in live acute slices. We isolated RNA from single 5-HT neurons in the ventromedial and lateral wings of the DR and performed single-cell RNA-Seq analysis identifying >500 G-protein coupled receptors (GPCRs) including receptors for classical transmitters, lipid signals, and peptides as well as dozens of orphan-GPCRs. Using these data to inform our selection of receptors to assess, we found that oxytocin and lysophosphatidic acid 1 receptors are translated and active in costimulating, with the α1-adrenergic receptor, the firing of DR 5-HT neurons, while the effects of histamine are inhibitory and exerted at H3 histamine receptors. The inhibitory histamine response provides evidence for tonic in vivo histamine inhibition of 5-HT neurons. This study illustrates that unbiased single-cell transcriptomics coupled with functional analyses provides novel insights into how neurons and neuronal systems are regulated.

Elk-1 phosphorylated at threonine-417 is present in diverse cancers and correlates with differentiation grade of colonic adenocarcinoma.

Elk-1 is a member of the Ets family of transcription factors, which are identified by a conserved Ets DNA-binding domain that mediates transcriptional regulation at Ets sequence--containing promoters. The activation domain of Elk-1 is important for executing its physiologic functions and contains many phosphorylation sites targeted by various MAP kinases following exposure to cell stressors or mitogenic stimuli. The different combinations of phosphorylated sites allow specificity of cellular responses mediated through redundant signaling pathways activated by distinct stimuli. Through phosphorylation of S383, mitogen-activated protein kinase (MAPK)-activating stimuli have been shown to regulate various processes important in carcinogenesis through transcriptional regulation in various cell lines, including proliferation. Phosphorylation at the T417 site (pT417), but not the S383 site, is involved in neuronal apoptosis induced through dendritic signaling mechanisms and associates with neuronal lesions in many Lewy body diseases. This points to distinct roles for these different phosphorylation sites in pathophysiologic pathways. However, the S383 site remains the best characterized in the context of normal function and carcinogenesis in cell lines, and less is known about the biochemistry of other phosphorylation sites, particularly in more biochemically relevant models. Here, we show that Elk-1 pT417 is present in epithelial cell nuclei of various normal and cancer tissues and that the number of pT417-positive cells correlates with differentiation grade of colonic adenocarcinomas. This nuclear localization and correlation with tumor differentiation in adenocarcinoma suggests a potentially important transcriptional and biochemical role of this phosphorylation site in carcinogenesis of this tumor type.