Simultaneous cyclin D1 overexpression and p27kip1 knockdown enable robust Müller glia cell cycle reactivation in uninjured mouse retina

Müller glia (MG) stem cells are dormant retinal stem cells. While teleost fish MG re-enter the cell cycle after retinal damage and regenerate lost retinal neurons through asymmetric cell division of MG-derived progenitors, mammalian MG lack such ability. Here, we show that forced p27 downregulation and Cyclin D1 overexpression in MG using a single adeno-associated virus cell cycle activator (CCA) vector had a strong synergistic effect to induce MG proliferation. We used transcriptome profiling at single-cell level to demonstrate that CCA could induce MG proliferation and reprogram MG into neurogenic and rod-like cells. In addition, retinal injury combined with histone deacetylase inhibitor facilitates CCA to induce more rod-like cells, but not new cell types. Our study suggests new and powerful strategies for awakening the proliferative and neurogenic potential of MG in adult mammalian retinas. Overall design: MG were labelled by Tdtomato in the Glast-CreER; Rosa26-tdTomato mice. MG proliferation was induced by the cell cycle activator (CCA) vector AAV7M8-GFAP-cyclin D1-p27sh. The CCA vector suppressed p27kip1 expression while overexpressed Cyclin D1 specifically in the MG. After receiving the CCA treatment, some mice were further injected with NMDA and TSA (CCA+NMDA+TSA, referred to as CCANT), as NMDA induced retinal injury and histone deacetylase inhibitor have been shown to improve MG reprogramming. The control group was injected with AAV7m8-GFAP-GFP-LacZsh only to control for any non-specific effect caused by virus injection and shRNA expression. Retinas were collected at around 3 weeks post AAV injection, and Tdtomato+ MG were sorted by FACS for scRNA-Seq analysis.