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

昼夜节律系统调整内部生理以适应循环环境。明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，协调整体的生理和行为(例如，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做出反应。SCN的活动/输出由明暗周期和根据细胞-细胞相互作用的状态来协调。事实上，SCN神经元和神经胶质细胞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黏附分子(CAM)通过塑造SCN的功能来调节昼夜节律系统。我们发现，细胞黏附分子EphA4和神经连接蛋白-1(NLGN1)调节昼夜节律行为。更准确地说，我们发现EphA4和NLGN1基因敲除的小鼠在持续的黑暗中有更长的内源性车轮活动周期，并且EphA4 KO小鼠对光的昼夜反应减弱。我们现在的目标是定义他们的角色是否源于SCN，以及NLGN2是否具有类似的角色。该程序将检验不同的CaM通过三个目标促进SCN功能的假设：目标1将建立小鼠SCN中EphA4、NLGN1和NLGN2的遗传失活，扰乱内源性昼夜节律、昼夜节律对光的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正常发育的小鼠的凸轮，然后通过持续监测轮子运行活动来提交给昼夜节律表型。目的2将确定在相应的KO小鼠的SCN中对目标CaM的遗传挽救将恢复内源性的昼夜节律、昼夜节律对光的反应和昼夜节律的灵活性。这将通过病毒向成年小鼠的SCN传递相应的CAM的KO，以及使用轮子运行活动监测的昼夜表型来实现。目的3将利用生化/分子生物学技术，在接受SCN CRISPR/Cas9介导的遗传失活的小鼠或接受或不接受基因拯救的KO小鼠中，鉴定CaM在SCN功能中的分子/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。该程序将解开凸轮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，在方法上进行了创新，研究了性别的调节作用，并研究了神经元可塑性因素和昼夜生理之间的复杂分子联系。重要的是，这项研究汇集了一个优秀的合作者网络，并将培养出具有公平性和包容性的多样化的HQP。

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.