Lipofuscin and the complement system in retinal pigment epithelial cell biology

视网膜色素上皮细胞生物学中的脂褐素和补体系统

• 批准号: MR/M02282X/1
• 负责人: Stephen Moss
• 金额: $ 57.7万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FM02282X%2F1
• 关键词: Lipofuscin; complement; system; retinal; pigment

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in developed countries. The disease is characterised by progressive degeneration of retinal pigment epithelial (RPE) cells and photoreceptors, leading to vision-threatening complications. Genetic studies a decade ago revealed that variants in a number of genes involved in the complement system are associated with increased susceptibility to AMD. The complement system is part of our immune system, and while it normally plays a vital role in defence against infection, it is now clear that defects in this system can lead to the development of AMD. In normal healthy individuals the complement system works by assembling a number of complement proteins into a complex known as C5b-9, on the surface of the target cells such as bacteria. The C5b-9 complex forms a pore that helps to destroy the target cell. However, over-activation of the system can result in C5b-9 forming on our own cells, leading to changes in cell function and ultimately cell death. RPE cells have evolved mechanisms to protect themselves from complement attack, such as the internalization and destruction of C5b-9, but with advancing age cells appear to be more susceptible to complement attack. The hypothesis to be tested in this proposal is that this increased susceptibility is due, at least in part, to lipofuscin accumulation. The slow accumulation of lipofuscin within RPE cells is a feature of normal physiological ageing but high levels are strongly correlated with AMD. Lipofuscin is a complex mixture of oxidised lipids, proteins and autofluorescent material mainly derived from breakdown products of the visual cycle and photoreceptor outer segments (POS). The tips of the POS detach from photoreceptors every day, to be engulfed and digested by the adjacent RPE cells. Waste products are disposed of, but useful POS constituents are recycled back to the photoreceptors. POS are rich in polyunsaturated fatty acids, which are highly susceptible to oxidation, for example by exposure to ultraviolet (UV) light, and changes in these lipids may lead to their incomplete digestion by the RPE cells. This would favour the accumulation of insoluble deposits such as lipofuscin within the cell. To test our hypothesis we will establish an AMD model that combines lipofuscin accumulation and complement attack, in order to gain insight into the cellular mechanisms of RPE dysfunction in AMD. In preliminary work we have shown that POS may be exposed to an ultraviolet (UV) light source to alter their structure. Here, cultured RPE cells will be fed with UV-irradiated POS and then treated with complement proteins to induce C5b-9 formation. Biochemical tests on these cells will reveal whether this combination of insults leads to changes similar to those associated with AMD. We will also establish an in vivo model that mimics the features of AMD by crossing a mouse that accumulates lipofuscin with another that has increased complement activation in the retina. We will analyse whether these mice exhibit the signs of inflammation and/or abnormal blood vessel growth that are seen in AMD. In addition we will use ocular imaging techniques to study subretinal deposits, and light and electron microscopy to investigate the integrity of subcellular structures. These investigations will provide insight into the underlying mechanism of how lipofuscin-loaded RPE cells react to increased complement activation, and whether lipofuscin-free cells are better able to defend themselves.The results of our studies will help to explain some of the underlying mechanisms involved in the pathogenesis of AMD, a key step toward the development of more effective and better targeted therapeutics.

视网膜相关性黄斑变性（AMD）是发达国家老年人失明的主要原因。这种疾病的特征是视网膜色素上皮（RPE）细胞和光感受器的进行性变性，导致威胁视力的并发症。十年前的遗传学研究表明，补体系统中涉及的许多基因的变异与AMD易感性增加有关。补体系统是我们免疫系统的一部分，虽然它通常在防御感染方面起着至关重要的作用，但现在很清楚，该系统的缺陷可能导致AMD的发展。在正常的健康个体中，补体系统通过将许多补体蛋白组装成称为C5 b-9的复合物，在靶细胞如细菌的表面上起作用。C5 b-9复合物形成有助于破坏靶细胞的孔。然而，该系统的过度激活可能导致C5 b-9在我们自己的细胞上形成，导致细胞功能的变化并最终导致细胞死亡。RPE细胞已经进化出保护自身免受补体攻击的机制，例如C5 b-9的内化和破坏，但随着年龄的增长，细胞似乎更容易受到补体攻击。在这个提议中要测试的假设是，这种增加的易感性是由于，至少部分是由于脂褐质积累。RPE细胞内脂褐素的缓慢积累是正常生理老化的特征，但高水平与AMD密切相关。脂褐素是氧化脂质、蛋白质和主要来自视觉周期和光感受器外节（POS）的分解产物的自发荧光物质的复杂混合物。POS的尖端每天从光感受器分离，被邻近的RPE细胞吞噬和消化。废物被处理掉，但有用的POS成分被回收回到感光体。POS富含多不饱和脂肪酸，这些脂肪酸对氧化非常敏感，例如通过暴露于紫外线（UV）光，并且这些脂质的变化可能导致它们被RPE细胞不完全消化。这将有利于不溶性沉积物如脂褐素在细胞内的积累。为了验证我们的假设，我们将建立一个结合脂褐素积累和补体攻击的AMD模型，以深入了解AMD中RPE功能障碍的细胞机制。在初步工作中，我们已经表明，POS可能会暴露于紫外线（UV）光源，以改变其结构。在这里，培养的RPE细胞将用UV照射的POS喂养，然后用补体蛋白处理以诱导C5 b-9形成。对这些细胞的生化测试将揭示这种损伤的组合是否会导致类似于AMD相关的变化。我们还将建立一个体内模型，通过交叉积累脂褐素的小鼠与另一个增加了视网膜中补体激活的小鼠来模拟AMD的特征。我们将分析这些小鼠是否表现出在AMD中观察到的炎症和/或异常血管生长的迹象。此外，我们将使用眼部成像技术来研究视网膜下沉积物，并使用光学和电子显微镜来研究亚细胞结构的完整性。这些研究将提供深入了解脂褐素负载的RPE细胞如何对增加的补体激活反应的潜在机制，以及无脂褐素的细胞是否能够更好地保护自己。我们的研究结果将有助于解释AMD发病机制中涉及的一些潜在机制，这是开发更有效和更有针对性的治疗方法的关键一步。

项目成果

Complement Stimulates Retinal Pigment Epithelial Cells to Undergo Pro-Inflammatory Changes.

补体刺激视网膜色素上皮细胞发生促炎症变化。

• DOI: 10.1159/000439596
• 发表时间: 2015
• 期刊: Ophthalmic research
• 影响因子: 2.1
• 作者: Lueck K

Retinal Pigment Epithelial Cells Mitigate the Effects of Complement Attack by Endocytosis of C5b-9.

• DOI: 10.4049/jimmunol.1500937
• 发表时间: 2015-10-01
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Georgiannakis A;Burgoyne T;Lueck K;Futter C;Greenwood J;Moss SE
• 通讯作者: Moss SE

Regulation of retinal pigment epithelial cell phenotype by Annexin A8.

• DOI: 10.1038/s41598-017-03493-3
• 发表时间: 2017-07-05
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Lueck K;Carr AF;Stampoulis D;Gerke V;Rescher U;Greenwood J;Moss SE
• 通讯作者: Moss SE