Regulation of Lysosomal pH in RPE cells

描述(申请人提供)：溶酶体酶需要酸性的pH来降解被吞噬的物质。我们在这里显示，与年龄匹配的对照组相比，ABCA4-/-小鼠RPE细胞的溶酶体pH升高。由于在Stargardt病中ABCA4基因发生了突变，由于RPE细胞内和周围部分降解物质的积累是该病的一个关键特征，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，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，显示出功能的改善。本研究探讨了cAMP、氯离子通道cftr和囊泡型质子泵VH+ATPase在恢复溶酶体酸性中的作用。它将确定年龄、A2E和ABCA4-/-小鼠RPE细胞溶酶体pH之间的关系，并确认cAMP在体外和体内治疗策略下恢复这些RPE细胞的溶酶体酸性。与公共卫生相关：Stargardt病是一种早发性黄斑变性，其特征是部分降解的物质在RPE细胞内和周围积聚。这项提案将探索从Stargardt病的ABCA4/-小鼠模型中新发现的RPE细胞溶酶体pH缺陷，该缺陷可能会减缓光感受器外节的处理，开发修复这一缺陷的治疗方法，并确定这些治疗方法是如何发挥作用的。

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.