Müller glia cell cycle re-activation by simultaneous cyclin D1 overexpression and p27^kip1knockdown promotes retinal regeneration in mice

Harnessing the regenerative potential of endogenous stem cells to restore lost neurons is a promising strategy for treating neurodegenerative disorders. Müller glia (MG), the primary glial cell type in the retina, exhibit remarkable regenerative abilities in lower vertebrate species, such as zebrafish and amphibians, where injury induces MG to proliferate and differentiate into various retinal neuron types. The regenerative potential of mammalian MG is constrained by their inherent inability to re-enter the cell cycle, likely due to high levels of the cell cycle inhibitor p27^Kip1and low levels of cyclin D1 observed in adult mouse MG. In this study, we found that adeno-associated virus (AAV)-mediated cyclin D1 overexpression and p27^Kip1knockdown exerts a strong synergistic effect on MG proliferation. MG proliferation induced by this treatment was potent but self-limiting, as MG did not undergo uncontrolled proliferation or lead to retinal neoplasia. Single-cell RNA sequencing (scRNA-seq) revealed that cell cycle reactivation leads to immunosuppression and dedifferentiation of MG. Notably, scRNA-seq analysis identified a new cluster of rod-like MG cells expressing both rod and MG genes, which was further validated by RNA in situ hybridization. Cell cycle reactivation also led to de novo genesis of bipolar- and amacrine-like cells from MG. Overall, our findings suggest that AAV- mediated cyclin D1 overexpression and p27^Kip1knockdown stimulate MG proliferation and promote MG reprogramming. This approach may be a promising strategy, especially when combined with other regeneration-promoting factors, to enhance MG-mediated retinal repair.