Investigating the compartment-specific functions of G protein-coupled receptor kinase 2 in the central circadian clock of mice

大多数生物体拥有一种内在的计时机制，称为生物钟，它将行为和生理过程临时组织到近24小时的周期。生物钟以一种自我维持的方式振荡，但能够根据光线或其他外部时间提示重新设置其相位。这种使行为和生理与环境明暗周期同步(或引导)的能力对于生物钟的主要功能至关重要：作为外部时间的内部预报器，并在这样做的过程中促进对环境变化的适应。在哺乳动物中，中央昼夜节律起搏器位于下丘脑的视交叉上核(SCN)，它是光作用于昼夜节律系统其余部分的门户。细胞间和细胞内的通信对于SCN时钟的正常运行是必不可少的。我的研究计划的首要目标是识别和理解支配SCN时钟携带和节律性的分子机制。利用PAN-SCN GRK2基因敲除小鼠，我们最近发现了G蛋白偶联受体(GPCR)激酶GRK2在微调SCN的夹带、幅度和周期属性中的作用。我们的数据表明，GRK2可能与不同的GPCR和核心时钟元件在不同的水平上相互作用，以调节SCN时钟。然而，其确切的分子机制仍有待阐明。此外，鉴于SCN在解剖学上被分为光响应(核心)和强烈节律(壳)区域，目前尚不清楚特定区域的GRK2活动如何影响SCN的携带和振荡特性，也不清楚GRK2的隔室特定效应背后的机制。因此，这项建议旨在通过选择性地消融SCN神经元亚群中的GRK2来剖析GRK2在SCN中的确切作用，这些SCN神经元包括两个功能不同的隔室，并通过结合行为、药理学、生化、组织学、细胞培养和生物发光成像技术的多管齐下的方法来解决机制问题。我们将研究GRK2和关键神经肽-GPCR系统之间的功能相互作用，以及GRK2和核心时钟蛋白之间的相互作用，在SCN外壳和核心区。通过这样做，我们将提供新的机械性见解，了解GRK2如何调节SCN对环境光提示的反应能力，并通过定义但可调的特征保持节奏性。总体而言，这项拟议的研究为培养研究生和本科生提供了难得的机会，将极大地扩展我们目前对正常时钟功能所必需的细胞机制的知识，并将导致重要的见解，对受GRK2调控的各种生物系统具有广泛的相关性。***********

Most living organisms possess an intrinsic timekeeping mechanism, known as the circadian clock, that temporally organizes behavioural and physiological processes to near-24h cycles. The circadian clock oscillates in a self-sustaining fashion, but is able to reset its phase in response to light or other external time cues. This ability to synchronize (or entrain) behaviour and physiology to environmental light-dark cycles is vital for the principal function of the circadian clock: to serve as an internal predictor of external time, and in doing so to facilitate adaptation to environmental changes. In mammals, the central circadian pacemaker resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, which serves as the gateway through which light acts on the rest of the circadian timing system. Inter- as well as intra-cellular communication are essential for the proper functioning of the SCN clock. The overarching goal of my research program is to identify and understand the molecular mechanisms that govern SCN clock entrainment and rhythmicity. Using pan-SCN grk2 knockout mice, we recently uncovered a role of the G protein-coupled receptor (GPCR) kinase, GRK2, in fine-tuning the entrainment, amplitude and period properties of the SCN. Our data suggest that GRK2 may be interacting at various levels with different GPCRs and core clock elements to regulate the SCN clock. However, the exact molecular mechanisms remain to be clearly elucidated. Moreover, given that the SCN is anatomically divided into light-responsive (core) and robustly rhythmic (shell) regions, it is unclear how region-specific GRK2 activity influences entrainment and oscillatory properties of the SCN, or what the mechanisms are that underlie compartment-specific effects of GRK2. Hence, this proposal aims to dissect the precise roles of GRK2 in the SCN by selectively ablating GRK2 in the subsets of SCN neurons that comprise the two functionally distinct compartments, and by using a multi-pronged approach that incorporates behavioural, pharmacological, biochemical, histological, cell culture-based and bioluminescent imaging techniques to tackle mechanistic questions. We will examine the functional interactions between GRK2 and key neuropeptide-GPCR systems, as well as interactions between GRK2 and core clock proteins, in the SCN shell and core regions. In doing so, we will provide fresh mechanistic insights into how GRK2 regulates the ability of the SCN to respond to environmental light cues and maintain rhythmicity with defined yet tunable characteristics. Overall, the proposed research, which offers exceptional opportunities for training graduate and undergraduate students, will greatly expand our current knowledge of the cellular mechanisms that are essential for proper clock function, and will lead to important insights of broad relevance to diverse biological systems that are regulated by GRK2. ***********