Bmal1 Is an Essential Regulator for Circadian Cytosolic Ca2+ Rhythms in Suprachiasmatic Nucleus Neurons
Bmal1 Is an Essential Regulator for Circadian Cytosolic Ca2+ Rhythms in Suprachiasmatic Nucleus Neurons
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DOI：
10.1523/jneurosci.5158-13.2014
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发表时间：
2014-09-03
影响因子：
5.3
通讯作者：
Ikeda, Masaaki
中科院分区：
文献类型：
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作者：
Ikeda, Masayuki;Ikeda, Masaaki
下丘脑视交叉上核（SCN）在哺乳动物生物钟系统中起着关键作用。Bmal1是一个时钟基因，驱动自身和其他基因的转录-翻译反馈回路（ttfl），几乎在所有SCN神经元中表达。尽管有强有力的证据表明Bmal1缺失突变小鼠在持续黑暗下表现出不规律的行为，但Bmal1在神经元活动中的功能尚不清楚。最近，细胞内信号信使水平的周期性变化，如胞浆Ca2+和cAMP，被认为调节ttfl。然而，时钟基因ttfl如何调节细胞质信号传导的相反方面仍不清楚。为了研究Bmal1扰动下细胞内Ca2+动态，我们使用适用于小鼠器官型培养的基因枪将一些SCN神经元与野生型或显性阴性Bmal1一起共转染。与BMAL1相关的标记蛋白免疫荧光染色显示野生型BMAL1在SCN神经元中核表达，并在转染后1周内降解。然而，显性阴性的BMAL1并没有转移到细胞核中，细胞质信号持续超过1周。一致地，SCN神经元的昼夜Ca2+节律被显性阴性Bmal1过表达抑制更长时间。此外，转染Bmal1 shRNA的SCN神经元延长，而过度表达野生型Bmal1的SCN神经元缩短Ca2+节律周期，其振幅显著降低。在器官型培养中，BMAL1的表达在大多数邻近神经元中是完整的。因此，我们得出结论，适当的内在Bmal1表达，而不是通过细胞间相互作用的被动信号，是SCN神经元中昼夜节律Ca2+的决定因素。
The hypothalamic suprachiasmatic nucleus (SCN) plays a pivotal role in the mammalian circadian clock system. Bmal1 is a clock gene that drives transcriptional-translational feedback loops (TTFLs) for itself and other genes, and is expressed in nearly all SCN neurons. Despite strong evidence that Bmal1-null mutant mice display arrhythmic behavior under constant darkness, the function of Bmal1 in neuronal activity is unknown. Recently, periodic changes in the levels of intracellular signaling messengers, such as cytosolic Ca2+ and cAMP, were suggested to regulate TTFLs. However, the opposite aspect of how clock gene TTFLs regulate cytosolic signaling remains unclear. To investigate intracellular Ca2+ dynamics under Bmal1 perturbations, we cotransfected some SCN neurons with yellow cameleon together with wild-type or dominant-negative Bmal1 using a gene-gun applied for mouse organotypic cultures. Immunofluorescence staining for a tag protein linked to BMAL1 showed nuclear expression of wild-type BMAL1 and its degradation within 1 week after transfection in SCN neurons. However, dominant-negative BMAL1 did not translocate into the nucleus and the cytosolic signals persisted beyond 1 week. Consistently, circadian Ca2+ rhythms in SCN neurons were inhibited for longer periods by dominant-negative Bmal1 overexpression. Furthermore, SCN neurons transfected with a Bmal1 shRNA lengthened, whereas those overexpressing wild-type Bmal1 shortened, the periods of Ca2+ rhythms, with a significant reduction in their amplitude. BMAL1 expression was intact in the majority of neighboring neurons in organotypic cultures. Therefore, we conclude that proper intrinsic Bmal1 expression, but not passive signaling via cell-to-cell interactions, is the determinant of circadian Ca2+ rhythms in SCN neurons.