Identification of Cellular, Molecular and Genetic Factors Regulating RGC Regeneration

点击翻译按钮获取中文摘要

Project Summary Teleost fish have a natural capacity to regenerate lost retinal neurons. This is due to activation of endogenous retinal stem cells, Müller Glia (MG), that undergo reprogramming and divide asymmetrically in response to injury. In contrast, mammalian MG are reactive to retinal injury do not divide and replace lost cells in the absence of exogenous stimulation. Prior research has successfully identified factors such as Achaete-scute homolog 1 (ASCL1) and Lin-28 homologue A (LIN28A) as critical regulators of MG regenerative potential. Intriguingly, mouse MG can be stimulated to divide by inducing expression changes in ASCL1 and treatment with histone deacetylases, demonstrating that regenerative potential is intact. These studies almost exclusively induce broad retinal damage prior to investigating regenerative potential. Much less is known about how retinal regeneration is regulated following the loss of discrete cell-types that have clear disease relevance. Selective retinal ganglion cell (RGC) degeneration is implicated in several human diseases linked to vision loss. Glaucoma, one example of disease caused by optic nerve damage, is the leading causing of irreversible blindness in the world. To investigate RGC regeneration, we created a novel transgenic model enabling selective RGC ablation in zebrafish. These fish co-express a bacterial enzyme Nitroreductase (NTR) and a yellow fluorescent protein (YFP) reporter in RGCs. NTR converts prodrugs such as metronidazole (MTZ) into DNA damage inducing agents, resulting in rapid targeted ablation of RGCs. Recently, we used the NTR- prodrug ablation system to study rod photoreceptor regeneration. We identified a critical role for immune cells in rod cell regeneration and concluded a neuroprotective drug screen. Using our new model, we propose to identify novel factors regulating zebrafish RGC regeneration and compare function in regeneration-deficient mouse models. I hypothesize that large-scale discovery in zebrafish will reveal novel cellular, molecular, and/or genetic factors that regulate RGC regeneration, and that a subset of these factors will stimulate regenerative responses in mice. Such insights may lead to transformative therapeutics for RGC degeneration diseases. In addition to their regenerative competence, zebrafish are amenable to high-throughput screening (HTS), in vivo imaging, and rapid genomic manipulation. We will take advantage of these strengths by characterizing our regeneration model and determining key immune cell responders to RGC death (Aim 1), screening for drugs that enhance regeneration or protect RGCs from cell death and testing hit drugs in complementary mouse RGC degeneration models (Aim 2), and disrupting newly identified “regeneration-associated” genes for roles in RGC regeneration (Aim 3). These aims, and the comprehensive research plan behind them, are aligned with areas of emphasis articulated by the National Eye Institute (NEI): the emerging field of regeneration, the immune system’s role in visual disease, and connecting disease-associated genes to mechanisms.