Partial epigenetic reprogramming of retinal pigment cells in age-related degeneration, dysfunction, and injury

项目总结 视网膜色素上皮(RPE)细胞功能障碍和损害是老年性黄斑的特征 变性(AMD)是导致老年人失明的主要原因。虽然抗血管内皮生长因子已经彻底改变了 湿性或渗出性AMD的治疗，目前还没有治疗非渗出性AMD的方法。老龄化 是RPE变性和AMD发生发展的主要危险因素。在衰老过程中，表观遗传变化 破坏“年轻”的DNA甲基化模式，导致细胞功能障碍和衰老。部分表观遗传 异位诱导三种山中因子Oct4、Sox2、Klf4(OSK)在视网膜神经节重编程 最近有报道称，细胞可以重新编程并逆转表观遗传年龄，从而提高老年人的视力。 在小鼠和小鼠青光眼模型中也是如此。我建议测试部分表观遗传学的假设 重编程可在体内挽救衰老相关的RPE细胞退化和功能，防止RPE细胞损伤 在体外诱导的一系列侮辱，并抑制AMD样病理形成的小鼠模型 目标有以下几点。(I)确定OSK介导的表观遗传重编程是否可以恢复RPE 老年小鼠的形态和功能，我将检测老年小鼠的视觉功能和RPE的形态 在有和没有OSK诱导的情况下，进行视神经运动、ERG、RPE平铺和透射电子显微镜检查。在……里面 此外，我将比较有无OSK的老年小鼠RPE的转录本和表观基因组 诱导评估OSK的表达效果。(2)检测OSK诱导抢救小学生的能力 人视网膜色素上皮细胞的形态和功能变化，以及不同AMD后的细胞死亡- 与体外相关的侮辱，包括氧化应激，氧化低密度脂蛋白和香烟吸烟提取物。我还会检查 OSK对AMD高危和低危供者IPSC-RPE细胞的诱导作用。(Iii) 检测OSK在NaIO3处理的小鼠中的作用，这是一种诱导局部RPE的短期损伤模型 在高脂肪、高胆固醇饲料喂养的RPE特异性老龄CFH杂合子(CFH+/-)小鼠中， 有类似早期干性AMD的病变。这些调查的结果将揭示 表观遗传重编程作为体内RPE再生的治疗策略的有效性和可行性。我的 指导小组和拟议的培训计划将使我掌握学习年龄的新知识和技能- 与视网膜相关的疾病，并将催化我过渡到作为一名独立调查人员的职业生涯。

PROJECT SUMMARY Retinal pigment epithelial (RPE) cell dysfunction and damage are hallmarks of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. While anti-VEGF has revolutionized the management of wet or exudative AMD, there is currently no treatment available for non-exudative AMD. Aging is the major risk factor for RPE degeneration and the development of AMD. During aging, epigenetic changes disrupt "youthful" DNA methylation patterns, leading to cellular dysfunction and senescence. Partial epigenetic reprogramming via ectopic induction of three Yamanaka factors, Oct4, Sox2, Klf4 (OSK), in retinal ganglion cells was recently reported to reprogram and reverse epigenetic age, leading to vision improvement in aged mice as well as in a mouse glaucoma model. I propose to test the hypothesis that partial epigenetic reprogramming can rescue age-related RPE cell degeneration and function in vivo, prevent RPE cell injury induced by a range of insults in vitro, and inhibit the formation of AMD-like pathologies in a mouse model with the following aims. (i) To determine whether OSK-mediated epigenetic reprogramming can restore RPE morphology and function in aged mice, I will examine the visual function and RPE morphology in aged mice with and without OSK induction by optomotor, ERG, RPE flatmount, and transmission electron microscopy. In addition, I will compare the transcriptomes and epigenomes of RPE from aged mice with and without OSK induction to assess the effect of OSK expression. (ii) To test the ability of OSK-induction in rescuing primary human RPE cells from morphologic and functional changes, as well as cell death following various AMD- associated insults in vitro, including oxidative stress, ox-LDL, and cigarette smoking extract. I will also examine the effect of OSK-induction in iPSC-RPE cells derived from high-risk and low-risk donors with AMD. (iii) Examine the role of OSK induction in NaIO3-treated mice, a short-term injury model that induces regional RPE atrophy, and in RPE specific aged Cfh heterozygous (Cfh+/-) mice fed with a high fat, high cholesterol diet, which have pathological changes mimicking early dry AMD. The results of these investigations will reveal the efficacy and viability of epigenetic reprogramming as a therapeutic strategy for RPE regeneration in vivo. My mentoring panel and the proposed training plan will equip me with new knowledge and skills to study age- related retinal diseases and will catalyze my transition to a career as an independent investigator.