Circadian Rhythm In Cone Photoreceptors: Cellular Mechanisms

描述（由申请人提供）：黄斑变性是美国视力障碍的主要原因，与视锥光感受器死亡有关。黄斑变性中中心视力畸变的发病机制和细胞事件尚不清楚。众所周知，视网膜光感受器中的昼夜节律振荡器在调节光感受器功能和生理方面发挥着重要作用，这些昼夜节律振荡器的破坏会导致光感受器死亡，并可能导致黄斑变性的发病机制。本研究的目的是阐明导致锥体光感受器中l型电压依赖性钙通道（VDCCs）调节的昼夜节律振荡器的信号通路，因为l型VDCCs在调节视网膜中的神经递质释放中至关重要。例如，l型VDCC a1f亚基基因突变导致人类不完全先天性静止性夜盲症，其光感受器与二级神经元之间的神经传递存在缺陷。综合多种技术，包括电生理膜片钳记录，Western免疫印迹，免疫细胞化学共聚焦成像，定量实时RT- PCR和基因转染，将在本应用中使用，以实现以下四个特定目标：表征鸡视锥光感受器中VDCCs的昼夜节律性。l型vdcs在整个昼夜节律日的多个时间点的昼夜节律性将被详细表征。具体目标2。确定vdcs昼夜节律调节的细胞信号通路。将确定PKA， MAP激酶，CaMKII和PI3激酶作为导致vdcs昼夜节律调节的输出途径的作用。具体目标3。阐明肌动蛋白细胞骨架重排和蛋白质转运在vdcs昼夜节律调节中的作用。具体目标明确生长抑素对vdcs的相位依赖性调节的细胞机制。虽然生长抑素在其他神经元中作为VDCC抑制剂，但它在锥体中表现出l型VDCC的相位依赖性增强和抑制。该项目将是最早确定l型VDCCs的昼夜节律调节及其在视网膜光感受器中的分子机制的研究之一。了解视网膜功能昼夜节律调节的分子信号可能最终为制定治疗视网膜疾病的新策略，特别是年龄相关性黄斑变性，以及预防未来失明提供知识，这是PI的长期目标。

DESCRIPTION (provided by applicant): Macular degeneration is a major cause of visual impairment in the United States and is associated with the death of cone photoreceptors. The pathogenesis and the cellular events responsible for the distortion of central vision in macular degeneration are not understood. Circadian oscillators in retina photoreceptors are known to play important roles in regulating photoreceptor function and physiology, and disruption of these circadian oscillators leads to the death of photoreceptors and could contribute to the pathogenesis of macular degeneration. The objective of the proposed research is to elucidate the signaling pathways from the circadian oscillators leading to the regulation of L-type voltage-dependent calcium channels (VDCCs) in cone photoreceptors, since the L-type VDCCs are essential in regulating neurotransmitter release in the retina. For example, mutation of the L-type VDCC a1f subunit gene causes incomplete congenital stationary night blindness in humans, with a defect in neurotransmission between photoreceptors and second-order neurons. An integrative approach with multiple techniques including electrophysiological patch-clamp recordings, Western immunoblotting, immunocytochemistry with confocal imaging, quantitative real-time RT- PCR and gene transfection, will be used in this application to achieve the following four specific aims: Specific Aim 1. Characterize the circadian rhythmicity of VDCCs in chick cone photoreceptors. The circadian rhythmicity of L-type VDCCs at multiple time points throughout the entire circadian day will be characterized in detail. Specific Aim 2. Identify the cellular signaling pathways underlying the circadian regulation of VDCCs. The roles of PKA, MAP kinase, CaMKII and PI3 kinase as output pathways leading to the circadian regulation of VDCCs will be determined. Specific Aim 3. Elucidate the roles of actin cytoskeleton rearrangement and protein trafficking on circadian regulation of VDCCs. Specific Aim 4. Define the cellular mechanisms of the phase-dependent modulation of VDCCs by somatostatin. While somatostatin serves as a VDCC inhibitor in other neurons, it shows both phase-dependent enhancement and inhibition of L-type VDCCs in cones. This project will be among the very first studies to define the circadian regulation of L-type VDCCs and their molecular mechanisms in retina photoreceptors. Understanding the molecular signaling of circadian regulation of retina function may ultimately provide knowledge for developing new strategies to treat retinal diseases, especially age-related macular degeneration, and preventing blindness in the future, which are the long-term goals of the PI.