mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish

Abstract

The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is necessary and sufficient for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity in the RPE is required for recruitment of macrophages/microglia to the injury site. In turn, these macrophages/microglia reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration. AUTHOR SUMMARYAge-related macular degeneration (AMD) is a leading cause of blindness world-wide, with incidences predicted to rise substantially over the next few decades. Cells of the retinal pigment epithelium (RPE) are affected in AMD and there are currently no effective therapies that slow RPE cell death or restore lost RPE cells in advanced-stage AMD. An exciting potential approach to treat many diseases of the eye, including AMD, is to stimulate endogenous regeneration to restore cells lost to disease. For this to become possible, we must first understand the molecular and cellular underpinnings of the regenerative response. In this study, we utilize zebrafish as a model system, which possess tremendous regenerative potential in multiple cell and tissue types, including the RPE. Our results identify the mTOR signaling pathway as a key regulator of RPE regeneration. We identify a link between mTOR signaling and immune responses, which are known to modulate regeneration of a variety of tissues and organs. Together, our results identify one of the first molecular mechanisms facilitating intrinsic RPE regeneration and these data could serve as a foundation for the development of new therapies aimed at stimulating the regeneration of RPE cells in the diseased eye.