Targeting lysosome/RPE heterogeneity in AMD pathobiology as a novel therapy

摘要：老年性黄斑变性(AMD)导致的视力丧失是一个主要的、不断扩大的问题，原因是 人口老龄化。虽然可以对中度AMD和晚期湿性AMD进行治疗，但没有预防措施 或者，对于早期的AMD，可以进行治疗。虽然细胞的异质性可以帮助组织适应压力，但退化 衰老带来的异质性与疾病有关。目前的观点是RPE(视网膜色素沉着 上皮)退化是一个中心的、慢性的过程，不可避免地会导致细胞死亡。因此，先前的研究已经 重点探讨RPE细胞死亡对AMD病理生物学的影响。然而，RPE细胞的子集表现出一些特征 上皮-间充质转化(EMT)是一种使细胞在恶劣环境中生存的适应性过程。 RPE异质性已被认识到一段时间，但疾病驱动细胞亚群对AMD的影响 发病机制尚未被考虑。溶酶体功能障碍明显与几个年龄相关 包括AMD在内的疾病，但溶酶体损伤的异质性对 RPE细胞功能或异质性尚未被考虑。这项提议有别于常规。 因为它提出RPE的异质性包括早期AMD中伴有EMT的细胞的子集是由 溶酶体清除缺陷的异质性。EMT中的RPE对死亡具有抵抗力，因此可能导致AMD 很长一段时间内的病理生物学。另一方面，由于EMT可以逆转，EMT中的RPE是一种 令人信服的治疗目标，因为它的视力保护功能可以恢复。溶酶体功能和 自噬是由转录因子EB(TFEB)调控的。我们的实验室有 与年龄匹配的AMD患者相比，早期AMD患者RPE中的核TFEB染色减少 对照，从而将TFEB确定为AMD的潜在治疗靶点。考虑到异质性 溶酶体功能障碍导致RPE异质性，从EMT到变性，这项提议的目标是 确定恢复溶酶体功能和自噬对AMD相关RPE的影响 我们验证的溶酶体损伤的Cryba1 CKO啮齿动物模型中的异质性，该模型发展为干性AMD样 表型，以及人IPSC来源的RPE细胞。“使溶酶体恢复活力”的中心假说 激活TFEB的自噬功能将维持RPE细胞的功能，防止病理性RPE异质性 包括早期AMD的EMT“将以下列目标加以解决：目标1：测试TFEB的程度 激活使溶酶体/自噬功能恢复活力，并调节RPE异质性和RPE细胞健康。 目的：检测Cryba1 CKO小鼠视网膜色素上皮中TFEB活化对其AMD样表型的挽救程度。 目的3：检测溶酶体异质性在AMD中引起RPE异质性的程度。成功完成 这一提议的提出将确立溶酶体/自噬功能障碍作为RPE的关键驱动因素的新概念 早期AMD的异质性，并确定TFEB为一种新的治疗药物。

SUMMARY: Vision loss from age-related macular degeneration (AMD) is a major, expanding problem due to the aging population. While treatment is available for intermediate AMD and the advanced wet form, no prevention or treatment is available for early AMD. While cellular heterogeneity can help tissue adapt to stress, degenerative heterogeneity from aging is associated with disease. The current perspective is that RPE (retinal pigmented epithelium) degeneration is a central, chronic process that inevitably leads to cell death. Thus, prior studies have focused on the impact of RPE cell death on AMD pathobiology. However, a subset of RPE cells exhibit features of epithelial-mesenchymal transition (EMT), an adaptive process that allows cells to survive a harsh environment. RPE heterogeneity has been recognized for some time, but the impact of disease driving cell subsets on AMD pathogenesis has not been considered. Lysosomal dysfunction is clearly involved in several age-related diseases including AMD, but the impact of heterogeneity of lysosomal impairment, which can cause disease, on RPE cellular function or heterogeneity has not been considered. This proposal is a departure from the norm because it proposes that RPE heterogeneity including a subset of cells with EMT in early AMD is initiated by heterogeneity of lysosomal clearance defects. RPE in EMT are resistant to death, and thus, could drive AMD pathobiology for an extended period. On the other hand, since EMT can be reversed, RPE in EMT are a compelling treatment target because its vision preserving functions can be restored. Lysosomal function and autophagy are modulated by the master regulator transcription factor EB (TFEB). Our laboratory has demonstrated reduced nuclear TFEB staining in the RPE of early AMD globes compared to age-matched controls, thus identifying TFEB as a potential therapeutic target for AMD. Given the possibility that heterogeneity of lysosomal dysfunction drives RPE heterogeneity, from EMT to degeneration, the objective of this proposal is to determine the impact of restoring lysosomal function and autophagy on the AMD-associated RPE heterogeneity in our validated Cryba1 cKO rodent model of lysosomal impairment that develops a dry AMD-like phenotype, and in human-iPSC derived RPE cells. The central hypothesis of “rejuvenating lysosomal and autophagic function by activating TFEB will maintain RPE cell function and prevent pathologic RPE heterogeneity including EMT, in early AMD” will be addressed with the following aims: Aim 1: Test the extent that TFEB activation rejuvenates lysosomal/autophagy function, and modulates RPE heterogeneity and RPE cell health. Aim 2: Test the degree that TFEB activation in the RPE of Cryba1 cKO mice rescues its AMD-like phenotype. Aim 3: Test the degree that lysosomal heterogeneity leads to RPE heterogeneity in AMD. Successful completion of this proposal will establish the novel concept of lysosome/autophagy dysfunction as a key driver of RPE heterogeneity in early AMD and identify TFEB as a novel therapeutic agent.