AMPK/SIRT1/PGC-1α, a critical pathway in dry AMD

抽象的： ！ 年龄相关性黄斑变性 (AMD) 是导致 55 岁以上人群失明的主要原因 美国和发达国家。 AMD 的两种形式之一是“干”形式，目前尚无针对该形式的治疗方法。 有效的治疗。因此，开发新疗法的医疗需求尚未得到满足 AMD。包括 AMD 在内的许多视网膜疾病都与线粒体功能障碍有关5。 线粒体功能失调会导致活性氧 (ROS) 水平升高和代谢缺陷 活动。自噬丧失也会导致线粒体功能障碍，并被认为会增加对线粒体的易感性 氧化应激和AMD。我们最近发现自噬功能失调、ROS 增加和 来自 AMD 供体眼睛的 RPE 中线粒体功能障碍。然而，底层机制 诱导这些有缺陷的代谢稳态导致 AMD 的原因仍然未知。 过氧化物酶体增殖物激活受体-γ共激活剂 (PGC)-1α (PGC-1α) 发挥着 在线粒体生物发生和氧化代谢中起主要作用。它还调节自噬和线粒体自噬。 PGC-1α 活性受两个主要因素刺激：AMP 激活蛋白激酶 (AMPK) 和 NAD - 依赖性脱乙酰酶，SIRT1。我们实验室的初步证据表明 AMPK/SIRT- 1/PGC-1α 在 AMD RPE 中下调。根据我们的初步数据，我们假设 RPE 中的 AMPK/SIRT1/PGC-1α 通路受抑制会诱导线粒体、自噬功能障碍， ROS 产生增加，导致代谢活动异常、脂质和糖原积累，以及 玻璃膜疣的形成，导致AMD的病理生理学。为了检验我们的假设，我们开发了一个 通过从 AMD 捐赠者的眼睛中分离天然 RPE，然后 iPSC 的产生，以及随后分化为 RPE (AMD RPE-iPSC-RPE)。我们还有 从年龄匹配的正常供体 (正常 RPE-iPSC-RPE) 的 RPE 中生成 iPSC，作为对照。我们 证实 AMD RPE-iPSC-RPE 模仿其亲代供体的疾病表型， AMD RPE，验证了我们的模型。此外，我们建立了动物模型来测试PGC-1α的作用 对 RPE 和视网膜健康的抑制，并观察到 RPE 和光感受器变性。我们建议两个 目标：Aim1 将使用我们建立的方法来测试 AMPK/SIRT-1/PGC-1α 通路抑制在干性 AMD 中的作用。 AMD 捐赠者和 AMD 患者的体外模型。 Aim2将研究细胞和分子机制 PGC-1α 对小鼠模型中 RPE 和视网膜健康的作用。最终，这些研究将提供见解 深入了解干性 AMD 的分子机制，并可能促进新的治疗干预措施的开发。

ABSTRACT: ! Age-related macular degeneration (AMD) is the major cause of blindness in people over age 55 in the U.S. and the developed world. One of the two forms of AMD is the “dry” form for which currently there are no effective treatments. Consequently, there is an unmet medical need for development of new therapies for AMD. A number of retinal diseases including AMD are associated with mitochondrial dysfunction5. Dysfunctional mitochondria induce increased levels of reactive oxygen species (ROS), and defective metabolic activity. Autophagy loss also results in mitochondrial dysfunction and is suggested to increase susceptibility to oxidative stress and AMD. We have recently shown dysfunctional autophagy, increased ROS, and dysfunctional mitochondria in RPE derived from AMD donor eyes. However, the underlying mechanisms inducing these defective metabolic homeostases leading to AMD remain unknown. The Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha (PGC-1α) plays a major role in mitochondrial biogenesis and oxidative metabolism. It also regulates autophagy and mitophagy. PGC-1α activity is stimulated by two main factors: AMP-activated protein kinase (AMPK) and NAD+- dependent deacetylase, SIRT1. Preliminary evidence from our laboratory suggests that the AMPK/SIRT- 1/PGC-1α is down regulated in AMD RPE. Based on our preliminary data, we hypothesize that the repressed AMPK/SIRT1/PGC-1α pathway in RPE induces mitochondrial, autophagic dysfunction, and increased ROS production, which result in abnormal metabolic activity, lipid and glycogen accumulation, and drusen formation, leading to the AMD pathophysiology. To test our hypothesis, we have developed an inexhaustible AMD in vitro disease model by isolating native RPE from AMD donors' eyes followed by generation of iPSC, and their subsequent differentiation into RPE (AMD RPE-iPSC-RPE). We have also generated iPSC from RPE of age-matched normal donors (Normal RPE-iPSC-RPE) that serve as control. We confirmed that the AMD RPE-iPSC-RPE mimic the disease phenotypes of their parental donors, the primary AMD RPE, which validates our model. Additionally, we established an animal model to test the role of PGC-1α repression on RPE and retinal health and observed RPE and photoreceptor degeneration. We propose two aims: Aim1 will test the role of AMPK/SIRT-1/PGC-1α pathway inhibition in dry AMD using our established in vitro model from AMD donors and AMD patients. Aim2 will investigate the cellular and molecular mechanisms of PGC-1α actions on RPE and retinal health in a mouse model. Ultimately, these studies will provide insight into the molecular mechanisms of dry AMD and may facilitate development of new therapeutic interventions.