Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression

The vertebrate pineal gland is dedicated to the production of the hormone melatonin, which increases at night to influence circadian and seasonal rhythms. This increase is associated with dramatic changes in the pineal transcriptome. Here, single-cell analysis of the rat pineal transcriptome was approached by sequencing mRNA from similar to 17,000 individual pineal cells, with the goals of profiling the cells that comprise the pineal gland and examining the proposal that there are two distinct populations of pinealocytes differentiated by the expression of Asmt, which encodes the enzyme that converts N-acetylserotonin to melatonin. In addition, this analysis provides evidence of cell-specific time-of-day dependent changes in gene expression. Nine transcriptomically distinct cell types were identified: similar to 90% were classified as melatonin-producing alpha- and beta-pinealocytes (1:19 ratio). Non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular and leptomeningeal cells, and endothelial cells. alpha-Pinealocytes were distinguished from beta-pinealocytes by similar to 3-fold higher levels of Asmt transcripts. In addition, alpha-pinealocytes have transcriptomic differences that likely enhance melatonin formation by increasing the availability of the Asmt cofactor S-adenosyl-methionine, resulting from increased production of a precursor of S-adenosylmethionine, ATP. These transcriptomic differences include similar to 2-fold higher levels of the ATP-generating oxidative phosphorylation transcriptome and similar to 8-fold lower levels of the ribosome transcriptome, which is expected to reduce the consumption of ATP by protein synthesis. These findings suggest that alpha-pinealocytes have a specialized role in the pineal gland: efficiently O-methylating the N-acetylserotonin produced and released by beta-pinealocytes, thereby improving the overall efficiency of melatonin synthesis. We have also identified transcriptomic changes that occur between night and day in seven cell types, the majority of which occur in beta-pinealocytes and to a lesser degree in alpha-pinealocytes; many of these changes were mimicked by adrenergic stimulation with isoproterenol. The cellular heterogeneity of the pineal gland as revealed by this study provides a new framework for understanding pineal cell biology at single-cell resolution.

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