mTORC1-TFEB pathway in degeneration of the RPE

本项目的长期目标是阐明细胞膜运输的调节机制， 视网膜色素上皮（RPE）细胞，并了解缺陷在视网膜色素上皮细胞中的作用， 包括年龄相关性黄斑变性（AMD）的疾病。RPE负责清除日常 通过吞噬作用脱落感光细胞外节（POS），这激活了严格控制的 膜运输和细胞器运输。在我们发表的和初步的研究中，我们发现， 吞噬激活由雷帕霉素（mTOR）的机制靶点介导的信号传导途径。下 在生理条件下，mTOR激活是短暂的。然而，衰老和变性使mTOR 早晨爆发后激活时间延长。在我们新开发的RPE特异性的小鼠模型中， mTORC 1上游抑制因子TSC 1的缺失，组成性高mTOR活性导致RPE， 光感受器变性，可能由失调的膜运输和延迟的POS降解引起。 我们进一步确定了VPS11，一个膜系连复合物的关键成分，在细胞中下调。 TSC 1缺陷型RPE细胞可能是由于对转录因子EB（TFEB）的抑制。基于那些 新的发现，我们假设mTORC1的过度激活可以导致RPE和感光细胞 由于膜运输缺陷而导致的变性。增强TFEB介导的RPE表达 运输蛋白可以恢复细胞功能，防止视网膜的退行性表型 TSC1 + RPE小鼠的生长曲线。这一假设将通过三个具体目标进行检验。目的1是进一步表征 RPE中TSC 1条件性敲除小鼠的视网膜表型。目标2是确定是否 mTOR的过度激活通过抑制TFEB介导的细胞内转运和膜融合来抑制RPE细胞内转运和膜融合。 VPS11表达。目的3：探讨TFEB基因治疗是否能预防或挽救RPE 退化预计拟议研究的结果将进一步确定mTOR在以下方面的关键作用： 控制RPE和感光细胞的相互作用。尽管抗VEGF治疗已经取得了前所未有的进展， 虽然成功，但大多数AMD患者具有疾病的萎缩形式，其病因仍然主要是 未知和治疗选择非常有限。鉴定mTOR下游的新靶点， 退化的RPE细胞可以促进治疗策略的设计，以防止或至少延迟视网膜色素变性。 在AMD的早期阶段疾病进展。

The long-term goal of this project is to elucidate the mechanisms by which membrane trafficking is regulated in retinal pigment epithelium (RPE) cells, and to understand the role of defects in this regulation in retinal diseases including age-related macular degeneration (AMD). The RPE is responsible for the removal of daily shed photoreceptor outer segments (POS) by phagocytosis, which activates tightly controlled processes of membrane trafficking and organelle transport. In our published and preliminary studies, we found that phagocytosis activated signaling pathway mediated by the mechanistic target of rapamycin (mTOR). Under physiological conditions, mTOR activation was transient. Aging and degeneration, however, rendered mTOR activation to become prolonged after morning burst. In our newly developed murine model of RPE-specific deletion of mTORC1 upstream suppressor TSC1, constitutively high mTOR activity led to RPE and photoreceptor degeneration, likely caused by deregulated membrane trafficking and delayed POS degradation. We further identified VPS11, a key component of the membrane tethering complexes, was downregulated in TSC1-deficient RPE cells possibly due to inhibition on the transcription factor EB (TFEB). Based on those novel findings, we hypothesize that hyperactivation of mTORC1 can cause RPE and photoreceptor degeneration due to defective membrane trafficking. Enhancing TFEB-mediated expression of RPE trafficking proteins can restore the cellular functions and prevent the degenerative phenotype in retina of TSC1RPE mice. The hypothesis will be tested by three specific aims. Aim 1 is to further characterize the retinal phenotype of mice with conditional knockout of TSC1 in the RPE. Aim 2 is to determine whether overactivation of mTOR inhibits RPE intracellular trafficking and membrane fusion by inhibiting TFEB-mediated VPS11 expression. Aim 3 is to determine whether TFEB gene therapy can prevent or rescue RPE degeneration. Results from the proposed studies are expected to further establish the critical roles of mTOR in controlling the RPE and photoreceptor interaction. Although anti-VEGF therapy has achieved unprecedented success, the majority of AMD patients have the atrophic form of the disease whose etiology is still largely unknown and treatment options are very limited. Identifying novel targets downstream of mTOR in degenerating RPE cells can facilitate the design of therapeutic strategies to prevent or at least delay the disease progression at early stage of AMD.