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 ~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.脊椎动物的松果体（pineal gland）专门负责合成褪黑素（melatonin），该激素在夜间水平升高，进而调控昼夜节律与季节节律。这种激素水平的动态变化与松果体转录组（pineal transcriptome）的显著改变密切相关。本研究针对大鼠松果体转录组开展单细胞分析，通过对约17000个单个松果体细胞的信使RNA（mRNA）进行测序，旨在实现两大核心研究目标：其一，对构成松果体的各类细胞进行分型鉴定；其二，验证相关假说——即存在两种截然不同的松果体细胞群（pinealocytes），二者的表达差异以乙酰血清素O-甲基转移酶（Asmt）为标志，该基因编码的酶可将N-乙酰血清素转化为褪黑素。此外，本分析还为基因表达的细胞特异性昼夜动态变化提供了实证依据。研究共鉴定出9种转录组特征迥异的细胞类型：其中约90%为产褪黑素的α型与β型松果体细胞，二者比例为1:19。非松果体细胞（non-pinealocytes）则包含3种星形胶质细胞亚型（astrocyte subtypes）、2种小胶质细胞亚型（microglia subtypes）、血管软脑膜细胞以及内皮细胞（endothelial cells）。α型松果体细胞与β型的核心差异在于，其Asmt转录本水平约为β型的3倍。除此之外，α型松果体细胞还存在转录组层面的特异性差异：这类差异可通过提升S-腺苷甲硫氨酸（S-adenosylmethionine）的可获得性，增强褪黑素的合成效率——而S-腺苷甲硫氨酸的可获得性提升，源于其前体三磷酸腺苷（ATP）生成量的增加。此类转录组差异具体表现为：参与ATP生成的氧化磷酸化转录本（oxidative phosphorylation transcriptome）水平升高约2倍，而核糖体转录本（ribosome transcriptome）水平降低约8倍，该变化预计可减少蛋白质合成过程对ATP的消耗。上述研究结果表明，α型松果体细胞在松果体中承担着特殊功能：高效催化β型松果体细胞产生并释放的N-乙酰血清素发生O-甲基化修饰，从而提升整体褪黑素合成的效率。本研究还鉴定出7种细胞类型在昼夜交替间发生的转录组变化，其中多数变化发生于β型松果体细胞，α型松果体细胞的变化程度相对较轻；这类变化中的绝大多数可通过异丙肾上腺素（isoproterenol）介导的肾上腺素能刺激模拟。本研究揭示的松果体细胞异质性（cellular heterogeneity），为在单细胞分辨率（single-cell resolution）下解析松果体细胞生物学特性提供了全新的研究框架。