Circadian Rhythm in Cone Photoreceptors: Cellular Mechanisms

描述(申请人提供)：了解维持光感受器健康和存活的机制至关重要，因为目前还没有逆转光感受器的治疗方法。 与黄斑变性(MD)等眼部疾病相关的死亡，黄斑变性是视力受损和失明的主要原因。这种毁灭性的疾病通常与视锥感光细胞的死亡有关，视锥感光细胞高度集中在黄斑中。光感受器的昼夜节律机制被认为是调节其功能和生理的机制，而光感受器昼夜节律的破坏会导致光感受器死亡。然而，光感受器昼夜节律是如何调节的，以及为什么光感受器对夜间的光损伤更敏感，目前还不完全清楚。因此，迫切需要了解锥体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的关键因素。这一假设的核心假设是，蛋白激酶B(Akt)相关的信号通路是调节光感受器L-VGCCs昼夜节律的主要途径，夜间强光刺激引起Akt依赖的信号和L-VGCCs的剧烈变化，从而触发光感受器的凋亡。光感受器L-VGCCs在调节神经递质释放中起重要作用，通过L-VGCCs的钙离子内流触发后续的钙依赖事件，影响细胞内钙稳态。细胞内钙稳态失衡被认为是触发细胞凋亡的重要因素。因此，对L-VGCC的精确调控对光感受器的生存和健康至关重要。这项研究的长期目标是了解光感受器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。本课题的目的是阐明MicroRNAs(MiRNAs)如何整合到Akt信号中来调节光感受器L-VGCC的昼夜节律，并揭示L-VGCC的昼夜节律特别是在细胞内钙稳态中的功能意义。生物信息学分析、电生理学、分子/生化分析和先进的高分辨率显微镜将用于研究以下特定目标。目的1.阐明miRNAs对光感受器L-VGCCs昼夜节律的调控。目的2.研究夜间强光如何比白天更严重地引起光感受器细胞凋亡。目的3.研究L-VGCC、维甲酸(Rs1)和质膜Ca~(2+)-ATPase(PMCA_1)对L-VGCC膜保留率和钙动态平衡的影响。L-VGCCs、视黄鞘蛋白和PMCA1之间存在物理相互作用，但这些相互作用的功能尚不清楚。这些研究结果将揭示miRNAs和整合Akt相关信号在离子通道和昼夜节律调节中的新机制，以及光感受器L-VGCCs的昼夜调节如何有助于钙稳态、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由光感受器死亡引起的失明提供新的知识。 与公共健康相关：拟议的研究结果将揭示对维持视网膜光感受器健康和存活至关重要的新机制。这项拟议的研究与公众健康有关，因为了解视网膜光感受器昼夜节律调节的细胞机制最终将为开发治疗眼部疾病的新策略，特别是预防黄斑变性引起的失明提供知识，因为视网膜昼夜节律的破坏会导致光感受器死亡，从而可能导致黄斑变性。因此，申请中提出的研究与美国国立卫生研究院国家眼科研究所获取有助于促进眼部疾病预防和治疗的基础知识的使命的一部分有关。

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.