Mechanisms underlying the roles of cell adhesion molecules in the circadian timing system

昼夜节律系统调节内部生理以适应循环环境。光暗循环是生物体适应的最有效的同步器之一。在哺乳动物中，昼夜节律系统由许多时钟组成，它们协调着整体生理和行为（如活动、进食、睡眠）。下丘脑的视交叉上核（SCN）被认为是主要的昼夜节律振荡器，特别是因为它直接对光作出反应。SCN的活动/输出由光-暗循环和细胞-细胞相互作用的状态协调。事实上，SCN神经元和神经胶质在相互作用和交流特性上表现出巨大的差异。该计划提出，参与神经元可塑性的特定细胞粘附分子（CAMs）通过塑造SCN功能来调节昼夜节律系统。我们发现CAMs EphA4和NLGN1调节昼夜行为。更准确地说，我们发现EphA4和Nlgn1基因敲除小鼠在持续黑暗环境下具有更长的内源性轮跑活动周期，EphA4基因敲除小鼠对光的昼夜节律反应减弱。我们现在的目标是确定它们的作用是否起源于SCN，以及NLGN2是否具有类似的作用。该项目将通过3个目标测试不同cam对SCN功能的影响：第1个目标将确定小鼠SCN中EphA4、NLGN1和NLGN2的基因失活会破坏内源性昼夜节律、对光的昼夜节律反应和昼夜节律灵活性。这将通过病毒传递CRISPR/Cas9系统来独立地KO正常发育小鼠的CAMs，然后通过连续监测轮跑活动将其提交给昼夜节律表型。目的2将确定在相应KO小鼠的SCN中靶CAMs的遗传拯救将恢复内源性昼夜节律，对光的昼夜节律反应和昼夜节律灵活性。这将通过将CAMs病毒递送到成年小鼠的SCN来实现，同时使用轮式跑步活动监测进行昼夜节律表型分析。目的3将利用生化/分子生物学技术，在接受SCN CRISPR/ cas9介导的基因失活的小鼠或接受或未接受基因拯救的KO小鼠中，确定CAMs在SCN功能中作用的分子/细胞机制。这将包括对不同细胞类型的研究，特别是时钟和突触功能的标记。该计划将解开cam对昼夜节律系统功能的贡献。它代表了一个独特的研究领域，并在方法论上进行了创新，研究了性别的调节作用，研究了神经元可塑性和昼夜生理学元素之间复杂的分子联系。重要的是，这项研究汇集了一个杰出的合作者网络，并将以公平和包容的方式培养HQP的多样性。

The circadian system adjusts internal physiology to the cyclic environment. The light-dark cycle is among the most potent synchronizers to which organisms adjust. In mammals, the circadian system is composed of many clocks coordinating overall physiology and behaviors (eg, activity, feeding, sleep). The suprachiasmatic nucleus (SCN) of the hypothalamus is considered the chief circadian oscillator notably because it directly responds to light. SCN activity/outputs are coordinated by the light-dark cycle and according to the state of cell-cell interactions. Indeed, SCN neurons and glia demonstrate huge variations in their interaction and communication properties. This program proposes that specific cell adhesion molecules (CAMs) involved in neuronal plasticity regulate the circadian system by shaping SCN functioning. We discovered that the CAMs EphA4 and Neuroligin-1 (NLGN1) modulate circadian behaviors. More precisely, we found that mice knockout (KO) for EphA4 and Nlgn1 have a longer endogenous period of wheel-running activity in constant darkness, and that EphA4 KO mice have attenuated circadian responses to light. We now aim at defining whether their roles originate from the SCN, and whether NLGN2 has similar roles. The program will test the hypothesis that different CAMs contribute to SCN functioning via 3 aims: Aim 1 will establish that genetic inactivation of EphA4, NLGN1 and NLGN2 in the mouse SCN disrupt endogenous circadian rhythm, circadian responses to light and circadian flexibility. This will be done by viral delivery of a CRISPR/Cas9 system to independently KO the CAMs in normally developed mice, which will then be submitted to circadian phenotyping using continuous monitoring of wheel-running activity. Aim 2 will determine that genetic rescue of the target CAMs in the SCN of respective KO mice will restore endogenous circadian rhythm, circadian responses to light and circadian flexibility. This will be accomplished by viral delivery of CAMs to the SCN in adult mice KO for the respective CAMs together with circadian phenotyping using wheel-running activity monitoring. Aim 3 will identify the molecular/cellular mechanisms underlying the role of CAMs in SCN functioning using biochemical/molecular biology techniques in mice submitted to SCN CRISPR/Cas9-mediated genetic inactivation or in KO mice submitted or not to genetic rescue. This will include the investigation of different cell types and, in particular, of markers of clock and synaptic function. The program will disentangle the contributions of CAMs to the functioning of the circadian system. It represents a unique area of research and innovates with the methodological approach, the study of the modulatory role of sex, and the investigation of the complex molecular connections between elements of neuronal plasticity and circadian physiology. Importantly, this research assembles an outstanding network of collaborators and will train a diversity of HQP with equity and inclusion.