The role of epigenetics in RPE heterogeneity with early AMD

摘要 NEI AMD病理生物学小组的结论是，识别所有致病信号，优先处理它们 相对于彼此的贡献，并确定这些信号何时致病将导致有效 对各AMD分期进行治疗。因为预防或治疗早期AMD将消除晚期AMD的负担 减少AMD的财务负担，这项建议将重点放在AMD的早期。细胞异质性是一口井- 公认的现象。虽然有序的异质性是保护性的，但压力会导致无序的异质性 这会通过侵略性的亚克隆诱发疾病，即使是由有限数量的细胞组成。视网膜色素 上皮细胞(RPE)的异质性在早期AMD中已被广泛认识，但其致病作用尚不清楚。 表观基因组机械地将环境暴露与转录组联系起来，以影响生物学。 过程和表观遗传变化在单个细胞中不同，导致致病细胞异质性。 吸烟是AMD的最高环境风险因素，是一个强大的表观遗传学诱因，部分原因是 染色质修饰酶的表达改变，可以重新编程转录组以扰乱多个 细胞保护途径，这是像AMD这样的复杂疾病的基本特征。使用atac-seq和 RNA-seq，我们先前将基因启动子区域染色质可及性降低与RPE联系起来 早期AMD眼的转录组，暗示RPE是早期AMD的驱动因素。此外，IPSC-RPE细胞 烟雾治疗后，染色质可及性轮廓与早期AMD眼的RPE相似。而当 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的RPE异质性，原因是 通过解决病理性RPE亚群的假设，减少吸烟对染色质的可及性 通过表观遗传或遗传改变引起的转录组改变，导致早期AMD。SA1. 用染色质可及性确定老年和早期AMD的致病RPE异质性 和转录组修饰。ScRNA-seq和scATAC-seq将用于黄斑RPE和 衰老和早期AMD的外周，以及染色质可及性和病理途径 将通过ATAC-SEE和免疫组织化学显示细胞亚群。SA2.确定范围 RPE异质性1)是由于染色质可及性和转录异质性随年龄增长而形成的 或者吸烟，以及ii)影响早期AMD模型的RPE功能。将使用scATAC-seq和scRNA-seq 为了评估野生型(窝种)的染色质可及性和转录RPE的异质性，HDAC11-/-， 用HDAC11抑制剂莫西替尼坦治疗的WT小鼠暴露在慢性香烟烟雾中。空间上的 RPE异质性的分布将使用细胞亚群生物标记物的免疫荧光来识别 将评估ATAC-SEE和对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.