A chemical screen to identify molecular mechanisms of rod outer segment renewal

描述(申请人提供)：这项研究的长期目标是识别和定义调节视网膜光感受器维持的分子途径和细胞机制。视网膜光感受器是一种独特的细胞，因为它们不断地更新自己的一部分--它们的外层部分，即接受光线的细胞器。虽然这种更新过程是光感受器功能的核心，但我们仍然对这些细胞如何更新其外部节段知之甚少。据观察，在感光细胞变性疾病期间，感光细胞的外节在感光细胞死亡之前逐渐缩短，问题是这些细胞是否可能因为失去外节而死亡。这项建议主要集中在确定控制杆状感光细胞外节段更新的分子机制。这一建议有一个特定的目的：进行高含量的筛选，以确定影响视杆外节更新的化合物和分子途径，包括邻近视网膜色素上皮对脱落视杆外段物质的吞噬和消化，视紫红质的运输和定位，视紫红质的运输和定位，以及视杆分化和形态发生。这个项目将使用化学库，其中包含许多具有已知分子目标的化合物。虽然该提案的主要目标是确定刺激外节生长的化合物，但屏幕的设计提供了独特和丰富的机会来确定影响杆状感光细胞生物学和维护的其他方面的化合物。该项目将以斑马鱼幼体作为模式生物。斑马鱼杆状感光器具有与哺乳动物相同的形态，并类似地更新其外节。斑马鱼为这项研究提供了几个优势。例如，这个拟议的筛查需要大量的双转基因幼虫，这些幼虫也是纯合突变(白化)，并且可以从少量的交配中产生大量的斑马鱼后代。小鼠在受精后6天就可以通过更新来维持其外节，相比之下，受精后数周才能达到相同的发育阶段。此外，与哺乳动物不同，斑马鱼的视网膜继续生长，这意味着我们还可以检查视网膜边缘的新视杆，以确定光感受器分化和初始光感受器形态发生的调控因素，例如内节和外节的形成，同时我们筛选外节更新的调控因子，吞噬和消化脱落的视杆外节物质的邻近视网膜色素上皮，视紫红质的运输和定位。如果如预期的那样，发现了通过刺激生长或抑制脱落来延长外节的化合物，然后可以在斑马鱼和人类视网膜退行性疾病的小鼠模型上测试它们维持外节、促进光感受器存活和延长视力的能力。

DESCRIPTION (provided by applicant): The long-term goal of this research is to identify and define the molecular pathways and cellular mechanisms that regulate retinal photoreceptor maintenance. Retinal photoreceptors are unique cells in that they are continually renewing a part of themselves - their outer segments, the light receptive organelle. Although this renewal process is central to the function of photoreceptors we still know very little about how these cell renew their outer segments. It is observed that the outer segments of photoreceptors progressively shorten before photoreceptors die during photoreceptor degeneration diseases and the question remains as to whether these cells might be dying because they lose their outer segments. This proposal focuses primarily on identifying the molecular mechanisms that control rod photoreceptor outer segment renewal. This proposal has one specific aim: perform a high content screen to identify compounds and molecular pathways affecting rod outer segment renewal, phagocytosis and digestion of shed rod outer segment material by the neighboring retinal pigmented epithelium, rod outer segment disc integrity, Rhodopsin trafficking and localization, and rod differentiation and morphogenesis. This project will use chemical libraries, containing many chemical compounds with known molecular targets. Although the primary objective of the proposal is to identify chemical compounds that stimulate outer segment growth, the design of the screen offers the unique and rich opportunity to identify compounds that affect additional aspects of rod photoreceptor cell biology and maintenance. This project will use young zebrafish larvae as a model organism. Zebrafish rod photoreceptors have the same morphology as mammalian species and similarly renew their outer segments. Zebrafish offers several advantages for this research. For example, this proposed screen requires large numbers of double transgenic larvae that also are homozygous mutants (albino) and large numbers of zebrafish offspring can be produced from a small number of matings. Rods are already maintaining their outer segments through renewal as soon as 6 days after fertilization, in comparison, it takes several weeks after fertilization for mouse rods to reach the same stage. In addition, unlike mammals, the retina continues to grow in zebrafish, which means that we also can examine new rods in the retinal margin to identify regulators of photoreceptor differentiation and initial photoreceptor morphogenesis, such as inner and outer segment formation at the same time that we screen for regulators of outer segment renewal, phagocytosis and digestion of shed rod outer segment material by the neighboring retinal pigmented epithelium, rod outer segment disc integrity, Rhodopsin trafficking and localization. If, as expected, chemical compounds are discovered that lengthen outer segments by either stimulating growth or suppressing shedding, these can then be tested in zebrafish and mouse models of human retinal degeneration disease for their ability to maintain outer segments, promote photoreceptor survival and prolong vision.