PHAGOCYTOSIS AND MERTK FAMILY OF THE RECEPTOR TYROSINE KINASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 虽然脊椎动物的眼睛发育和功能分化是在怀孕中期开始的，但值得注意的是，视网膜可能是完成功能发育的最后一个器官。大多数视网膜分化发生在出生后，小鼠的视网膜发育直到出生后两周才完成。因此，视网膜的发育缺陷并不明显，直到出生后才明显。视网膜色素上皮(RPE)细胞是高度极化的，它们与发育中的视网膜感光细胞密切相关。一旦视网膜具有功能，RPE对于吞噬耗尽的光感受器远端外节(OS)尖端是必不可少的；这一功能对于维持光感受器内环境平衡是必不可少的。RPE早于视网膜发育，但RPE的发育缺陷直到出生后视网膜开始功能时才变得明显。RPE吞噬OS的发育缺陷导致光感受器死亡和出生后疾病视网膜色素变性(RP)。由于皇家外科医师学会(RCS)大鼠模型的出现，RPE在视杆OS转化中的作用已被广泛研究，该模型表现为RP表型。这些大鼠的RPE细胞携带着Rod OS吞噬功能的遗传缺陷，突变基因是MerTK受体。MerTK受体酪氨酸激酶是RPE吞噬功能所必需的。MerTK缺失突变不仅在RCS大鼠中引起光感受器变性，而且在MerTK基因敲除小鼠和人类RP患者中也是如此。此外，MerTK突变导致NK细胞分化、精子发生和巨噬细胞吞噬功能的普遍发育缺陷。为了了解MerTK在这些发育过程中的吞噬作用中的分子作用，我们进行了基因表达谱分析以确定MerTK的下游靶点。一种名为PTTG的基因在RPE和巨噬细胞中以MerTK突变的方式异位表达。有趣的是，PTTG调节分裂细胞的细胞骨架变化，PTTG稳定细胞骨架结构，如纺锤体，在有丝分裂期间分离姐妹染色单体。然而，PTTG必须被降解，这种细胞骨架结构才能在有丝分裂中期结束时解体。因此，PTTG是细胞骨架发生变化所必需的。我们现在已经将MerTK突变小鼠置于PTTG杂合背景中，以减少PTTG的异位表达，我们发现MerTK突变小鼠的光感受器丢失在很大程度上得到了防止。这些结果表明，PTTG的异位表达是MerTK突变体RPE缺陷的关键因素。我们假设，PTTG在MerTK突变体RPE中的异位表达阻止了启动吞噬作用所需的细胞骨架重排(方式类似于PTTG过表达时在有丝分裂中出现的细胞骨架重排缺陷)。我们现在重点研究PTTG在MerTK突变表型中异位表达的潜在作用，以及PTTG如何影响MerTK介导的吞噬作用。 具体目的1.体内检测PTTG表达的发育和昼夜节律调节及其在MerTK-/-RPE细胞吞噬光受体OS中的作用。 为此，我们将从mRNA和蛋白质水平研究PTTG在RPE细胞中的发育表达模式和昼夜节律调控。我们将研究光感受器退化的时间过程，并确定MerTK-/-PTTG+/-小鼠的吞噬功能是否恢复。 特定目的2.研究体外异位表达PTTG是否导致RPE吞噬功能缺陷。 我们将异位PTTG应用于正常RPE细胞，检测其是否会引起RPE细胞吞噬功能缺陷。相反，我们将通过RNA干扰在带有MerTK突变的RPE细胞中敲除PTTG的表达，并研究降低的PTTG表达是否足以恢复吞噬功能。 具体目的3.MerTK-/-和MerTK-/-PTTG+/-视网膜功能的视觉测试和ERG分析。 我们将对MerTK-/-PTTG+/-小鼠进行电生理和功能视力测量。这些生理测量将与视网膜标志物的详细形态分析和超微结构形态相关联。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While vertebrate eye development and functional differentiation is initiated in mid gestation, it is of note that the retina is perhaps the last organ to complete functional development. Most retinal differentiation occurs postnatally, and in the mouse retinal development is not completed until two weeks after birth. Thus, developmental defects in the retina are not evident functionally until significantly after birth. The retinal pigmenl epithelium (RPE) cells are highly polarized and they become tightly associated with the developing retinal photoreceptor cells. Once the retina becomes functional, the RPE is essential for phagocytic clearance of the spent photoreceptor distal outer segment (OS) tips; this function is essential to maintain photoreceptor homeostasis. The RPE develops well before the retina, but developmental defects in the RPE do not become evident until the postnatal period when the retina becomes functional. Developmental defects in RPE phagocytosis of the OS leads to photoreceptor death and the postnatal disease retinitis pigmentosa (RP). The role of the RPE in rod OS turnover has been extensively studied, due to the availability of the Royal College of Surgeons (RCS) rat model, which displays an RP phenotype. The RPE cells in these rats carry an inherited defect in rod OS phagocytosis and the mutant gene is the MerTK receptor. The MerTK receptor tyrosine kinase is essential for RPE phagocytosis. MerTK null mutation causes photoreceptor degeneration not only in the RCS rat, but also in MerTK gene knockout mice and human RP patients. In addition, the MerTK mutation leads to general developmental defects in NK cell differentiation, spermatogenesis, and macrophage phagocytosis. To understand the molecular role of MerTK in phagocytosis in these developmental process, we performed gene expression profiling to identify downstream targets of MerTK. One gene known as PTTG was ectopically expressed with MerTK mutation in RPE and macrophages. Interestingly, PTTG regulates cytoskeletal changes in dividing cells, PTTG stabilizes cytoskeletal structures such as the spindle apparatus, which separates sister chromatids during mitosis. However, PTTG must be degraded for this cytoskeletal structure to be disassembled at the end of mitotic metaphase. Thus, PTTG is required for cytoskeletal changes to occur in the cell. We have now crossed the MerTK mutant mice into a PTTG heterozygous background to reduce ectopic expression of PTTG, and we have found that photoreceptor loss in the MerTK mutant is largely prevented. These results point toward ectopic expression of PTTG as a critical factor in the RPE defects in the MerTK mutant. We hypothesize that ectopic expression of PTTG in the MerTK mutant RPE prevents the cytoskeletal rearrangements required to initiate phagocytosis (in a fashion analogous to that seen in cytoskeletal rearrangement defects seen in mitosis when PTTG becomes overexpressed). We now focus this proposal on investigation of the potential role for PTTG ectopic expression in the MerTK mutant phenotypes, and how the PTTG affect MerTK-mediated phagocytosis. Specific Aim 1. Examination of developmental and circadian regulation of PTTG expression and its role in phagocytosis of photoreceptor OS in the MerTK-/- RPE cells in vivo. In this Specific Aim, we will investigate the developmental expression pattern and circadian regulation of PTTG in RPE cells at both mRNA and protein level. We will study the photoreceptor degeneration time course and determine if phagocytosis is restored in MerTK-/-PTTG+/- mice. Specific Aim 2. Investigate whether in vitro ectopic expression of PTTG leads to defects in RPE phagocytosis. We will ectopically PTTG in normal RPE cells to examine if it will cause RPE cell phagocytic defects. Conversely, we will knockdown PTTG expression by RNA interference in the RPE cells with MerTK mutation and investigate whether decreased PTTG expression is sufficient to restore phagocytosis. Specific Aim 3. Functional studies of the MerTK-/- and MerTK-/-PTTG+/- retina by visual test and ERG analysis. We will perform electrophysiology and functional vision measurements on the MerTK-/-PTTG+/- mice. These physiological measurements will be correlated with a detailed morphological analysis of retinal markers and ultrastructual morphology.