Abstract
Purpose :
Retinal degeneration is a major cause of visual impairment and currently has no effective treatment to replace lost retinal neurons. Directed reprogramming of endogenous Müller glia (MG) into retinal neurons has been proposed as a potential strategy to achieve this, as MG are capable of regenerating neurons in cold-blooded vertebrates like zebrafish. However, in mammals, MG do not spontaneously regenerate neurons following injury and instead undergo reactive gliosis. Overexpression of the neurogenic factor Ascl1 or deletion of the glial quiescence-promoting factors Nfia/b/x has been shown to induce neurogenesis in MG in mice, but only a subset of MG become neurons with limited cell fate diversity, suggesting that there may be other factors that regulate neurogenic competence in these cells. It has been suggested that the activation of certain extrinsic signaling pathways, such as NF-κB, STAT, and TGFB/SMAD, may inhibit neurogenesis in MG, and inhibiting these pathways has been shown to enhance Ascl1-mediated MG reprogramming. However, it is not known whether these pathways play similar role by repressing neurogenesis in Nfia/b/x-deficient MG.
Methods :
To address this, we performed intravitreal injection of small molecule inhibitors of the NF-κB, STAT and TGF-β/SMAD signaling pathways and induced microglia ablation in Nfia/b/x-deficient mice MG. To overcome possible epigenetic barriers that prevent activation of genes that stimulate neurogenesis, we also tested inhibitors of histone deacetylase (HDAC) and DNA methylation.
Results :
Using immunohistochemical and single-cell RNA-seq analyses, we found a significant increase in Nfia/b/x-deficient MG-derived bipolar and amacrine neurons following inhibition of NF-κB, STAT, and Notch signaling as well as HDAC and DNA methylation. In contrast, ablation of microglia and inhibition of TGF-β/SMAD signaling had little to no effect on neurogenesis.
Conclusions :
These findings identify key negative regulators of MG reprogramming and provide insights into the molecular mechanisms controlling MG-derived neurogenesis in mammals, potentially leading to the development of cell-based therapies for retinal degenerative diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.