Cholesterol Crystalline Domain Function in Eye Lens: EPR Spin-Labeling Studies

描述（由申请人提供）：人眼透镜的纤维细胞膜被胆固醇饱和（显示皮质中的胆固醇与磷脂摩尔比为1，核中高达4），这导致在这些膜内形成不混溶的胆固醇结晶结构域（CCD）。在胆固醇含量最高时，多达50%的细胞膜表面可以被CCD占据。这些条件存在于透镜核中，其中胆固醇含量可显著超过膜的溶解度阈值。CCD的出现通常是一种病理的迹象;然而，只有在眼睛的透镜中，CCD才能发挥积极的生理功能，保持透镜的透明度，并可能防止白内障的形成。 这项提案的长期目标是更好地了解胆固醇在纤维细胞膜中的功能。在短期内，我们将（i）检查高胆固醇含量如何影响磷脂透镜膜的横向组织，特别注意CCD的形成，和（ii）测试假设CCD的存在决定了周围散装磷脂-胆固醇膜的性质。(iii)此外，实验将揭示透镜膜的磷脂成分是否影响CCD的形成、尺寸和稳定性。(iv)最后，在模型膜系统中开发和测试的方法将被应用于检测和表征在成熟、老化和白内障形成过程中透镜纤维细胞膜中共存的胆固醇结晶和大量磷脂胆固醇结构域。对于从眼透镜分离的膜以及纤维细胞膜模型中的共存域的研究，将使用氧转运（DOT）方法进行区分。的DOT方法，这是基于电子顺磁共振（EPR）自旋标记技术，允许歧视不同的膜域，并提供信息的结构和分子动力学作为膜深度的函数在共存的域，而不需要他们的分离。它还允许获得关于膜域内和跨膜域的氧转运的信息。 公共卫生相关性：在发展中国家，与糖尿病相关的白内障是致盲的主要原因。白内障发病的原因尚不清楚，但大量证据表明，眼睛透镜中胆固醇晶体结构域的存在有助于保持透明度和防止白内障形成。拟议的研究将产生重要的基本信息，地形和年龄相关的差异，胆固醇-膜相互作用的眼睛透镜，这将增加我们的理解的作用，胆固醇发挥，反过来，有助于预防年龄相关的核性白内障。

DESCRIPTION (provided by applicant): Fiber-cell membranes of the human eye lens are saturated with cholesterol (showing cholesterol-to- phospholipid mole ratios from 1 in the cortex to as high as 4 in the nucleus), which leads to the formation of immiscible cholesterol crystalline domains (CCDs) within these membranes. At the highest cholesterol content, up to 50% of the cell-membrane surface can be occupied by CCDs. These conditions exist in the lens nucleus, where the cholesterol content can significantly exceed the solubility threshold of the membrane. The appearance of CCDs is usually a sign of pathology; however, only in the eye lens can CCDs play a positive physiological function, maintaining lens transparency and possibly protecting against cataract formation. The long-term objective of this proposal is to achieve a greater understanding of cholesterol's function in fiber-cell membranes. In the short-term, we will (i) examine how high cholesterol content affects the lateral organization of phospholipid lens membranes, with special attention paid to the formation of CCDs, and (ii) test the hypothesis that the presence of CCDs determines the properties of the surrounding bulk phospholipid- cholesterol membranes. (iii) Additionally, experiments will reveal if the phospholipid composition of the lens membrane affects the formation, size, and stability of CCDs. (iv) Finally, the methodology developed and tested in model-membrane systems will be applied to detect and characterize coexisting cholesterol crystalline and bulk phospholipid-cholesterol domains in lens fiber-cell membranes during maturation, aging, and cataract formation. For studies of coexisting domains in membranes isolated from the eye lens, as well as in models of fiber-cell membranes, the discrimination by oxygen transport (DOT) method will be used. The DOT method, which is based on electron paramagnetic resonance (EPR) spin-labeling techniques, permits discrimination of different membrane domains and gives information about structure and molecular dynamics as a function of the membrane depth in coexisting domains without the need for their separation. It also allows information about oxygen transport within and across membrane domains to be obtained. PUBLIC HEALTH RELEVANCE: Age-related cataracts are a major cause of blindness in developing countries. The reason for the onset of cataracts is unknown, but a great deal of evidence suggests that the presence of the cholesterol crystalline domain in the eye lens helps to maintain transparency and prevent cataract formation. The proposed studies will generate important fundamental information about topographical and age-related differences in cholesterol- membrane interactions in the eye lens that will increase our understanding of the role cholesterol plays and, in turn, help contribute to the prevention of age-related nuclear cataracts.