Regulation of Lysosomal pH in RPE cells

描述（由申请人提供）：溶酶体酶需要酸性pH来降解吞没的物质。我们在这里表明，与年龄匹配的对照组相比，ABCA4-/-小鼠RPE细胞的溶酶体pH值升高。由于ABCA4基因在Stargardt病中发生突变，并且RPE细胞内和周围部分降解物质的积累是该疾病的一个关键特征，我们假设恢复溶酶体的酸性pH值将是有益的。高通量筛选表明，培养中受干扰的RPE细胞的溶酶体被细胞质cAMP和刺激与Gas蛋白偶联的受体的激动剂酸化。cAMP的升高增加了RPE细胞对外段的清除，显示了功能的改善。本研究探讨了cAMP、Cl-通道CFTR和囊泡质子泵vH+ atp酶对溶酶体酸度恢复的作用。本研究将确定ABCA4-/-小鼠RPE细胞中年龄、A2E和溶酶体pH之间的关系，并证实cAMP在体外和体内均能恢复这些RPE细胞的溶酶体酸度。公共卫生相关性：Stargardt病是一种早发性黄斑变性，其特征是RPE细胞内和周围部分降解物质的积累。本研究将探索来自Stargardt病ABCA4-/-小鼠模型的RPE细胞溶酶体pH中新发现的可能减慢光感受器外段加工的缺陷，开发恢复该缺陷的治疗方法，并确定这些治疗方法如何起作用。

DESCRIPTION (provided by applicant): Lysosomal enzymes require an acidic pH to degrade engulfed material. We show here that the lysosomal pH of RPE cells from ABCA4-/- mice is elevated as compared to age matched controls. As the ABCA4 gene is mutated in Stargardt's disease, and as accumulation of partially degraded material in and around RPE cells is a key characteristic of the disease, we hypothesize that restoring an acid pH to the lysosomes will be beneficial. High-throughput screening indicates that lysosomes of perturbed RPE cells in culture are acidified by cytoplasmic cAMP, and by agonists which stimulate receptors coupled to the Gas protein. Elevation of cAMP increases the clearance of outer segments by RPE cells, demonstrating an improvement in function. This proposal investigates the contribution of cAMP, the Cl- channel CFTR, and the vesicular proton pump vH+ATPase to the restoration of lysosomal acidity. It will determine the relationship between age, A2E and lysosomal pH in RPE cells from ABCA4-/- mice and confirm that cAMP restores lysosomal acidity the these RPE cells using both in vitro and in vivo treatment strategies. PUBLIC HEALTH RELEVANCE: Stargardt's disease is an early-onset form of macular degeneration characterized by the accumulation of partially degraded material in and around RPE cells. This proposal will explore a newly identified defect in the lysosomal pH of RPE cells from the ABCA4-/- mouse model of Stargardt's disease that may slow processing of photoreceptor outer segments, develop treatments to restore this defect, and determine how these treatments work.