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

细胞粘附分子在昼夜节律系统中的作用机制

• 批准号: RGPIN-2020-05262
• 负责人: Mongrain, Valérie
• 金额: $ 4.23万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739772
• 关键词: Mechanisms; underlying; roles; cell; adhesion

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.

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协调整体生理和行为（例如，活动，进食，睡眠）的时钟组成。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线做出反应。SCN活性/输出由亮-暗循环和根据细胞-细胞相互作用的状态来协调。事实上，SCN神经元和神经胶质在它们的相互作用和通信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（CAM）通过塑造SCN功能来调节昼夜节律系统。我们发现CAMs EphA 4和Neuroligin-1（NLGN 1）调节昼夜节律行为。更确切地说，我们发现EphA 4和Nlgn 1敲除（KO）小鼠在恒定黑暗中具有较长的内源性轮跑活动期，并且EphA 4 KO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角色是否源于SCN，以及NLGN 2是否具有类似的角色。该计划将通过3个目标来测试不同CAM有助于SCN功能的假设：目标1将确定小鼠SCN中EphA 4、NLGN 1和NLGN 2的遗传失活破坏内源性昼夜节律、对光的昼夜节律响应和昼夜节律灵活性。这将通过CRISPR/Cas9系统的病毒递送来完成，以独立地KO正常发育的小鼠中的CAM，然后使用连续监测轮运行活动来进行昼夜节律表型分析。目的2将确定各KO小鼠SCN中靶CAM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CAM病毒递送至成年小鼠中的SCN来实现，对于相应的CAM KO以及使用轮运行活动监测的昼夜节律表型分析。目的3将在接受SCN CRISPR/Cas9介导的遗传失活的小鼠或接受或不接受遗传拯救的KO小鼠中，使用生物化学/分子生物学技术鉴定CAM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，特别是对生物钟和突触功能的标记的研究。该计划将解开CAMs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、性别调节作用的研究以及神经元可塑性和昼夜生理学元素之间复杂分子联系的研究方面进行了创新。重要的是，这项研究汇集了一个优秀的合作者网络，并将培养公平和包容的HQP的多样性。