Cigarette smoke and biological age induce degenerative heterogeneity in retinal pigment epithelium [scRNA-seq]
Environmental exposure such as cigarette smoke induces epigenetic changes that can induce degenerative heterogeneity and accelerate aging. In early age-related macular degeneration (AMD), the leading worldwide cause of blindness among the elderly, retinal pigment epithelial (RPE) cell heterogeneity is a key change. Since smoking is the strongest environmental risk factor for AMD, we hypothesized that cigarette smoke induces degenerative RPE heterogeneity through epigenetic changes that are distinct from aging, and that with aging, the RPE becomes vulnerable to cigarette smoke insult. We administered cigarette smoke condensate (CSC) intravitreally to young and aged mice and performed snRNA-seq and snATAC-seq on the RPE/choroid. This analysis identified separate cell clusters corresponding to healthy and abnormal, dedifferentiated RPE in both aged vehicletreated and young CSC-treated mice. The dedifferentiated RPE were characterized by a global decrease in chromatin accessibility and decreased expression of genes in functional categories that were linked to hallmarks of aging. Notably, young, dedifferentiated RPE also exhibited a compensatory upregulation of hallmarks of aging-related genes, specifically those related to mitochondrial function and proteostasis. In contrast, aged dedifferentiated RPE did not express these compensatory changes, and did not survive CSC treatment, as experimentally verified with TUNEL labeling. These changes are relevant to early AMD because we identified through scRNA-seq, similar dedifferentiated and healthy macular RPE clusters in a donor who smoked and another with early AMD, but not from a nonsmoker. Degenerative cellular heterogeneity can include an abnormal cluster that jeopardizes cell survival and may represent an additional hallmark of ocular aging. Overall design: The tenets of the Declaration for Helsinki for research involving human tissue were followed. Human eyes (n=3) were obtained from the Advancing Sight Network (Birmingham, AL). Preliminary experiments were conducted to determine the viability of RPE that was based on death to enucleation (D-E) time. Single cell RNA-seq on normal eyes from two different 86 yr old female donors with D-E of 3.3 and 6 hr with experimentation time of <24 hours and found that the 3.3 hr D-E interval globe exhibited superior viability (>70%), and nearly double the number of genes detected per cell (1301 vs 708 genes/cell). Therefore, globes with D-E times of < 6 hr and death to experimentation of <24 hr. Specifically, human globes from a 79 yr old female nonsmoker without AMD with a death to enucleation (D-E) time of 4.9 hr, a 67 yr old male smoker without AMD with a D-E of 3.8 hr, and a 67 yr old female smoker with early AMD (Minnesota Grade 2) with a D-E of 1.8 hr were immediately preserved on ice for overnight shipping to Hopkins, and dissected no later than 16 hr postmortem. After removing the anterior segment, the fundus was examined both with the retina and with the retina removed and classified using the Minnesota Grading Scale105. A 6 mm sample of the RPE/choroid in the macula was obtained using a 6 mm punch (Integra Lifesciences, Princeton, NJ) that was centered around the fovea. RPE/choroidal cells were dissociated using Papain Dissociation System (Worthington Biochemical, Lakewood, NJ) following the manufacturer's instructions. Dissociated cells were resuspended in ice-cold PBS, 0.04% BSA and 0.5 U/µl of RNAse inhibitors. Cells were then filtered through a 50µm filter and processed for single-cell RNA-sequencing. Cell viability (>90%) was confirmed by negative staining with trypan blue.