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

Abstract

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 ~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: ~90% were classified as melatonin-producing α- and β-pinealocytes (1:19 ratio). Non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular and leptomeningeal cells, and endothelial cells. α-Pinealocytes were distinguished from β-pinealocytes by ~3-fold higher levels of Asmt transcripts. In addition, α-pinealocytes have transcriptomic differences that likely enhance melatonin formation by increasing the availability of the Asmt cofactor S-adenosylmethionine, resulting from increased production of a precursor of S-adenosylmethionine, ATP. These transcriptomic differences include ~2-fold higher levels of the ATP-generating oxidative phosphorylation transcriptome and ~8-fold lower levels of the ribosome transcriptome, which is expected to reduce the consumption of ATP by protein synthesis. These findings suggest that α-pinealocytes have a specialized role in the pineal gland: efficiently O-methylating the N-acetylserotonin produced and released by β-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 β-pinealocytes and to a lesser degree in α-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.

Homeobox genes in melatonin‐producing pinealocytes: Otx2 and Crx act to promote hormone synthesis in the mature rat pineal gland

“…The cellular distribution of OTX2, CRX, and melatonin synthesis, e.g. ASMT, in the pineal gland, as reported here, supports the concept of two subpopulations of pinealocytes in the rat 27 ; in this context, OTX2 and CRX seem to exert their function in the large subpopulation β-pinealocytes devoted to melatonin synthesis.…”

“…Recent evidence suggests that the pineal gland contains two distinct populations of pinealocytes differentiated by the presence of ASMT. 27 To investigate whether OTX2 and CRX are present in the same pinealocytes and whether these produce melatonin, immunohistochemistry was performed on tissue sections of the pineal gland ( Figure 1). Exclusive colocalization between OTX2 and CRX in pineal cells was detected ( Figure 1A), and OTX2 10 also colocalized with ASMT 2 ( Figure 1B).…”

STAT1‐NFκB crosstalk triggered by interferon gamma regulates noradrenaline‐induced pineal hormonal production

“…Microglia and astrocytes play pivotal roles in regulating pineal response to immune signals. Recently, a study using single‐cell RNA sequencing of rat pineal gland reinforced the concept that pinealocytes are the cells expressing nocturnal Aanat . The formation of protein complexes composed by NA‐induced activation of CREB and STAT1 probably determines the normal levels of nocturnal melatonin production in healthy conditions.…”

“…Recently, a study using single-cell RNA sequencing of rat pineal gland reinforced the concept that pinealocytes are the cells expressing nocturnal Aanat. 49 The formation of protein complexes composed by NA-induced activation of CREB and STAT1 probably determines the normal levels of nocturnal melatonin production in healthy conditions. Further, pro-inflammatory signals such as TNF and LPS activate inhibitory NFκB p50 homodimers and block Aanat expression as well as pineal melatonin synthesis.…”

Neuromodulation of the Pineal Gland via Electrical Stimulation of Its Sympathetic Innervation Pathway

“…For example, astrocytes do not exhibit a biphasic positive-negative waveform composed of an initial segment-soma dendritic inflexion, whereas pinealocytes do. Pinealocytes are distributed uniformly throughout the gland and are predominantly responsible for the synthesis and secretion of melatonin in response to environmental lighting changes (Mays et al, 2018). Melatonin is considered the chemical expression of darkness and in the absence of light is secreted in response to signals from the suprachiasmatic nucleus (SCN).…”