Robust reprogramming of glia into neurons by inhibition of Notch signaling and NFI factors in adult mammalian retina.

Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for neurodegenerative diseases. Despite successful applications in vitro, in vivo implementation has been hampered by low efficiency. In this study, we present a highly efficient strategy for reprogramming retinal glial cells into neurons by simultaneously inhibiting key negative regulators. By suppressing Notch signaling through the removal of its central mediator Rbpj, we induced mature Muller glial cells to reprogram into bipolar and amacrine neurons in uninjured adult mouse retinas, and observed that this effect was further enhanced by retinal injury. We found that specific loss of function of Notch1 and Notch2 receptors in Muller glia mimicked the effect of Rbpj deletion on Muller glia-derived neurogenesis. Integrated analysis of multiome (scRNA- and scATAC-seq) and CUT&Tag data revealed that Rbpj directly activates Notch effector genes and genes specific to mature Muller glia while also indirectly represses the expression of neurogenic bHLH factors. Furthermore, we found that combined loss of function of Rbpj and Nfia/b/x resulted in a robust conversion of nearly all Muller glia to neurons. Finally, we demonstrated that inducing Muller glial proliferation by AAV (adeno-associated virus)-mediated overexpression of dominant- active Yap supports efficient levels of Muller glia-derived neurogenesis in both Rbpj- and Nfia/b/x/Rbpj- deficient Muller glia. These findings demonstrate that, much like in zebrafish, Notch signaling actively represses neurogenic competence in mammalian Muller glia, and suggest that inhibition of Notch signaling and Nfia/b/x in combination with overexpression of activated Yap could serve as an effective component of regenerative therapies for degenerative retinal diseases.