NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators

Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues in vivo. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms in vitro. In CLOCK-deficient fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.

CLOCK与BMAL1的异二聚体是哺乳动物昼夜节律时钟的主要转录激活因子。由于旁系同源基因NPAS2可在主昼夜节律起搏点视交叉上核（suprachiasmatic nucleus, SCN）中替代CLOCK的功能，因此CLOCK缺陷型小鼠在体内仍能维持行为与组织的昼夜节律。然而，据报道，从SCN中分离出的CLOCK缺陷型外周组织会呈现无节律状态，这表明视交叉上核与外周组织的昼夜节律时钟功能存在本质差异。令人意外的是，本研究通过发光检测法与针对PER2（Period 2）表达的单细胞生物发光成像技术，发现CLOCK缺陷型的分散SCN神经元与外周细胞在体外均能呈现出相似的稳定自主昼夜节律。在CLOCK缺陷型成纤维细胞中，敲低Npas2会导致节律缺失，这表明NPAS2同样可在外周细胞与SCN中代偿CLOCK的功能缺失。本研究数据推翻了"NPAS2在分子昼夜节律时钟中仅发挥视交叉上核特异性功能"的观点，转而表明在细胞层面，SCN神经元与外周细胞的昼夜节律时钟核心反馈环路本质上是相似的。