Histone deficiency and hypoacetylation in the aging retinal pigment epithelium.

Histones serve as a major carrier of epigenetic information in the form of post-translational modifications which are vital for controlling gene expression, maintaining cell identity, and ensuring proper cellular function. Loss of histones in the aging genome can drastically impact the epigenetic landscape of the cell leading to altered chromatin structure and changes in gene expression profiles. In this study, we investigated the impact of age-related changes on histone levels and histone acetylation in the retinal pigment epithelium (RPE) and retina of mice. We observed a global reduction of histones H1, H2A, H2B, H3, and H4 in aged RPE/choroid but not in the neural retina. Transcriptomic analyses revealed significant downregulation of histones in aged RPE/choroid including crucial elements of the histone locus body (HLB) complex involved in histone pre-mRNA processing. Knockdown of HINFP, a key HLB component, in human RPE cells induced histone loss, senescence, and the upregulation of senescence-associated secretory phenotype (SASP) markers. Replicative senescence and chronological aging in human RPE cells similarly resulted in progressive histone loss and acquisition of the SASP. Immunostaining of human retina sections revealed histone loss in RPE with age. Acetyl-histone profiling in aged mouse RPE/choroid revealed a specific molecular signature with loss of global acetyl-histone levels, including H3K14ac, H3K56ac, and H4K16ac marks. These findings strongly demonstrate histone loss as a unique feature of RPE aging and provide critical insights into the potential mechanisms linking histone dynamics, cellular senescence, and aging.

Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice.

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.