The role of epigenetics in RPE heterogeneity with early AMD

总结 NEI AMD病理生物学小组得出结论，识别所有的致病信号，优先考虑他们的 贡献相对于彼此，并建立当这些信号是致病性的将导致有效的 每个AMD阶段的治疗。因为预防或治疗早期AMD将消除晚期AMD的负担， 并减轻AMD的财务负担，这一建议将重点放在AMD早期.细胞异质性是一个很好的- 公认的现象。有序的异质性是保护性的，而压力导致无序的异质性 即使由有限数量的细胞组成，也能通过侵袭性亚克隆诱导疾病。视网膜色素 视网膜上皮细胞（RPE）异质性在早期AMD中已被充分认识，但其致病作用尚未探讨。 表观基因组在机制上将环境暴露与转录组联系起来，以影响生物学特性。 过程和表观遗传变化在个体细胞中不同，导致致病性细胞异质性。 吸烟是AMD的最高环境风险因素，部分原因是吸烟是一种强大的表观遗传诱导剂， 染色质修饰酶的表达改变，可以重新编程转录组，以破坏多个 细胞保护途径，这是复杂疾病如AMD的基本特征。使用ATAC-seq和 RNA-seq，我们先前将基因启动子区域中染色质可及性降低与RPE 在早期AMD眼睛中的转录组，暗示RPE是早期AMD的驱动因素。iPSC-RPE细胞 用烟雾处理的眼睛具有与早期AMD眼睛的RPE相似的染色质可及性特征。而 有价值的是，这种全局方法没有定义RPE异质性或异质性对RPE的影响 与AMD病理生物学相关的功能。目的是定义早期AMD中的RPE异质性， 通过解决病理性RPE亚群 通过由表观遗传或遗传改变诱导的转录组变化来促进早期AMD。SA 1. 通过衰老中的区域定位和早期AMD通过染色质可及性定义致病性RPE异质性 和转录组修饰。ScRNA-seq和scATAC-seq将用于来自黄斑的RPE， 衰老和早期AMD基因分型球的外周，以及染色质可及性和病理途径 将通过ATAC-see和免疫组织化学观察来自细胞亚群的细胞。SA 2.确定在多大程度 RPE异质性i）由于染色质可及性和转录异质性随着衰老而发展 或吸烟，和ii）在早期AMD模型中影响RPE功能。将使用ScATAC-seq和scRNA-seq 为了评估野生型（同窝出生）中的染色质可及性和转录RPE异质性，HDAC 11-/-， 和暴露于慢性香烟烟雾的HDAC 11抑制剂Mocetinostat处理的WT小鼠。空间 RPE异质性的分布将使用细胞亚群的生物标志物的免疫荧光来鉴定 和ATAC-see，并评估对RPE功能的影响。

Summary The NEI AMD Pathobiology group concluded that identifying all of the pathogenic signals, prioritizing their contribution relative to one another, and establishing when these signals are pathogenic will lead to effective treatment for each AMD stage. Because preventing or curing early AMD will eliminate the burdens of advanced AMD and reduce its financial burden, this proposal will focus on early AMD. Cellular heterogeneity is a well- recognized phenomenon. While ordered heterogeneity is protective, stress causes disordered heterogeneity that induces disease by aggressive subclones, even if comprised of a limited number of cells. Retinal pigment epithelium (RPE) heterogeneity is well recognized in early AMD, but its pathogenic role is unexplored. The epigenome mechanistically links environmental exposures with the transcriptome to influence biological processes, and epigenetic changes vary across individual cells to cause pathogenic cellular heterogeneity. Smoking, the highest environmental risk factor for AMD, is a powerful epigenetic inducer in part, due to the altered expression of chromatin modification enzymes that can reprogram the transcriptome to disrupt multiple cytoprotective pathways, a fundamental characteristic of a complex disease like AMD. Using ATAC-seq and RNA-seq, we previously linked reduced chromatin accessibility in promoter regions of genes with the RPE transcriptome in early AMD eyes, implicating the RPE as a driver of early AMD. Furthermore, iPSC-RPE cells treated with smoke had chromatin accessibility profiles similar to the RPE from early AMD eyes. While valuable, this global approach does not define RPE heterogeneity or the impact of heterogeneity on RPE functions related to AMD pathobiology. The objective is to define RPE heterogeneity in early AMD due to reduced chromatin accessibility from smoking by addressing the hypothesis that pathologic RPE subsets contribute to early AMD through transcriptome changes induced by epigenetic or genetic alterations. SA1. Define pathogenic RPE heterogeneity by regional location in aging and early AMD by chromatin accessibility and transcriptome modifications. ScRNA-seq and scATAC-seq will be used on the RPE from the macula and periphery from aging and early AMD genotyped globes, and chromatin accessibility and pathologic pathways from cellular subsets will be visualized by ATAC-see and immunohistochemistry. SA2. Determine the extent that RPE heterogeneity i) develops due to chromatin accessibility and transcriptional heterogeneity with aging or smoking, and ii) impacts RPE function in a model of early AMD. ScATAC-seq and scRNA-seq will be used to assess chromatin accessibility and transcriptional RPE heterogeneity in wild-type (littermates), HDAC11-/-, and WT mice treated with HDAC11 inhibitor Mocetinostat exposed to chronic cigarette smoke. The spatial distribution of RPE heterogeneity will be identified using immunofluorescence of biomarkers of cellular subsets and ATAC-see and impact on RPE function will be assessed.