Circadian Rhythm in Cone Photoreceptors: Cellular Mechanisms

描述（由申请人提供）：了解维持感光细胞健康和存活的机制至关重要，因为没有逆转感光细胞的治疗方法 黄斑变性（MD）是导致视力受损和失明的主要原因。这种毁灭性的疾病通常与高度集中在黄斑中的视锥光感受器的死亡特别相关。已知光感受器中的昼夜时间保持机制调节其功能和生理，并且光感受器昼夜节律的破坏导致光感受器死亡。然而，人们仍然没有完全理解光感受器的昼夜节律是如何调节的，以及为什么光感受器在夜间对光损伤更敏感。因此，有一个关键的需要，以了解负责锥光感受器昼夜节律的细胞机制，这将揭示导致光感受器死亡和MD的预防的关键因素。该提议的中心假设是蛋白激酶B（Akt）相关的信号传导是调节光感受器L-VGCC的昼夜节律的主要途径，并且夜间的强光刺激引起Akt依赖性信号传导和L-VGCC的剧烈变化，其随后触发光感受器凋亡。光感受器L-VGCC在门控神经递质释放中是必不可少的，并且通过L-VGCC的钙（Ca 2+）内流触发随后的Ca 2+依赖性事件并影响细胞内Ca 2+稳态。细胞内Ca 2+稳态失衡会触发细胞凋亡。因此，精确调节L-VGCC对于感光细胞的存活和健康至关重要。这项研究的长期目标是了解负责感光细胞昼夜节律的分子机制，以及这种节律如何影响其生存和健康。本研究的目的是阐明microRNA（miRNAs）是如何整合到Akt信号中来调节光感受器L-VGCC的昼夜节律的，并揭示L-VGCC昼夜节律的功能意义，特别是在细胞内Ca 2+稳态中的作用。生物信息学分析、电生理学、分子/生物化学测定和高级高分辨率显微镜的组合将用于研究以下特定目标。目标1.阐明miRNAs如何调节光感受器L-VGCC的昼夜节律。目标2.研究夜间明亮的光线如何比白天更严重地导致感光细胞凋亡。目标3。确定动态三重- L-VGCC，视网膜分裂素（rs 1）和质膜Ca 2 +-ATP酶（PMCA 1）-如何促进L-VGCC的膜保留和Ca 2+稳态。L-VGCC、retinoschisin和PMCA 1之间存在物理相互作用，但这些相互作用的功能尚不清楚。这些研究的结果将揭示miRNA和整合的Akt相关信号在离子通道和昼夜节律调节中的新机制，以及感光细胞L-VGCC的昼夜调节如何有助于Ca 2+稳态，细胞存活和光敏性。了解miRNAs的新功能和整合的信号通路将最终为预防感光细胞死亡导致的失明提供新的知识。 公共卫生相关性：这项研究的结果将揭示对维持视网膜感光细胞健康和存活至关重要的新机制。拟议的研究与公共卫生有关，因为了解视网膜光感受器昼夜节律调节的细胞机制将最终为开发治疗眼部疾病的新策略提供知识，特别是预防黄斑变性引起的失明，因为视网膜昼夜节律的破坏会导致感光细胞死亡，可能导致黄斑变性。因此，本申请中提出的研究与NIH国家眼科研究所的使命的一部分有关，即获得有助于促进眼部疾病预防和治疗的基础知识。

DESCRIPTION (provided by applicant): Understanding the mechanisms that maintain photoreceptor health and survival is critical, since there is no treatment to reverse photoreceptor death associated with ocular diseases such as Macular Degeneration (MD), the leading cause of vision impairment and blindness. This devastating disease is often specifically associated with the death of cone photoreceptors, which are highly concentrated in the macula. The circadian time-keeping mechanism in photoreceptors is known to regulate its function and physiology, and disruption of photoreceptor circadian rhythms leads to photoreceptor death. However, it is still not completely understood how photoreceptor circadian rhythms are regulated, and why photoreceptors are more sensitive to light damage at night. Hence, there is a critical need to understand the cellular mechanisms responsible for cone photoreceptor circadian rhythms, which will uncover key elements leading to the prevention of photoreceptor death and MD. The central hypothesis of this proposal is that protein kinase B (Akt)-related signaling is the major pathway that regulates the circadian rhythm of photoreceptor L-VGCCs, and intense light stimulation at night causes drastic changes of Akt- dependent signaling and L-VGCCs that subsequently triggers photoreceptor apoptosis. Photoreceptor L-VGCCs are essential in gating neurotransmitter release, and the calcium (Ca2+) influx through L-VGCCs triggers subsequent Ca2+- dependent events and affects intracellular Ca2+ homeostasis. Imbalanced intracellular Ca2+ homeostasis is known to trigger cell apoptosis. Hence, precise regulation of L-VGCCs is critical for photoreceptor survival and health. The long-term goals of this research are to understand the molecular mechanisms responsible for the photoreceptor circadian rhythm, and how this rhythm impacts its survival and health. The objectives of this project are to elucidate how microRNAs (miRNAs) integrate into Akt signaling to regulate the circadian rhythm of photoreceptor L-VGCCs and to reveal the functional significance of L-VGCC circadian rhythms especially in intracellular Ca2+ homeostasis. Combinations of bioinformatics analysis, electrophysiology, molecular / biochemical assays, and advanced high resolution microscopy will be used to investigate the following specific aims. Aim 1. Elucidate how miRNAs regulate the circadian rhythms of photoreceptor L-VGCCs. Aim 2. Investigate how bright light at night causes photoreceptor apoptosis more severely than during the day. Aim 3. Determine how the dynamic trio - L-VGCC, retinoschisin (rs1), and plasma membrane Ca2+-ATPase (PMCA1) - promote membrane retention of L-VGCCs and Ca2+ homeostasis. There are physical interactions between L-VGCCs, retinoschisin, and PMCA1, but the functions of these interactions are not clear. The outcomes of these studies will reveal new mechanisms on miRNAs and integrated Akt-related signaling in the regulation of ion channels and circadian rhythms, and how circadian regulation of photoreceptor L- VGCCs contribute to Ca2+ homeostasis, cell survival, and photosensitivity. Understanding the novel functions of miRNAs and integrated signaling pathways will ultimately provide new knowledge for preventing blindness caused by photoreceptor death. PUBLIC HEALTH RELEVANCE: The outcome of the proposed research will reveal new mechanisms that are critical in maintaining retinal photoreceptor health and survival. The proposed research is relevant to public health because understanding the cellular mechanisms of circadian regulation in retinal photoreceptors will ultimately provide knowledge for developing new strategies to treat ocular diseases and especially for preventing blindness caused by macular degeneration, since disruption of circadian rhythms in the retina causes photoreceptor death that could lead to macular degeneration. Therefore, the research proposed in this application is pertinent to part of the mission of the National Eye Institute at NIH in garnering fundamental knowledge that will help promote the prevention and treatment of ocular diseases.