Regulation of Lysosomal pH in RPE cells

性状（由申请方提供）：溶酶体酶需要酸性pH值才能降解吞噬物质。我们在此表明，与年龄匹配的对照相比，来自ABCA 4-/-小鼠的RPE细胞的溶酶体pH升高。由于ABCA 4基因在Stargardt病中发生突变，并且由于RPE细胞内和周围部分降解物质的积累是该疾病的关键特征，我们假设恢复溶酶体的酸性pH将是有益的。高通量筛选表明，扰动的RPE细胞在培养中的溶酶体被细胞质cAMP酸化，并通过激动剂刺激受体偶联到气体蛋白。cAMP的升高增加了RPE细胞对外节的清除，证明了功能的改善。该提议研究了cAMP、Cl-通道CFTR和囊泡质子泵vH+ ATP酶对溶酶体酸度恢复的贡献。它将确定来自ABCA 4-/-小鼠的RPE细胞中年龄、A2 E和溶酶体pH之间的关系，并使用体外和体内治疗策略证实cAMP恢复这些RPE细胞的溶酶体酸性。公共卫生相关性：Stargardt病是一种早发性黄斑变性，其特征在于部分降解的物质在RPE细胞中和周围的积累。该提案将探索新发现的来自斯塔加特病ABCA 4-/-小鼠模型的RPE细胞溶酶体pH值缺陷，该缺陷可能会减缓感光细胞外节的加工，开发修复该缺陷的治疗方法，并确定这些治疗方法的工作原理。

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.