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

Abstract

SummaryGeneration of neurons through direct reprogramming has emerged as a promising therapeutic approach for neurodegenerative diseases. Despite successful applicationsin vitro,in vivoimplementation 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 mediatorRbpj,we induced mature Müller 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 ofNotch1andNotch2receptors in Müller glia mimicked the effect ofRbpjdeletion on Müller 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 Müller glia while also indirectly represses the expression of neurogenic bHLH factors. Furthermore, we found that combined loss of function ofRbpjandNfia/b/xresulted in a robust conversion of nearly all Müller glia to neurons. Finally, we demonstrated that inducing Müller glial proliferation by AAV (adeno-associated virus)-mediated overexpression of dominant- active Yap supports efficient levels of Müller glia-derived neurogenesis in bothRbpj- andNfia/b/x/Rbpj- deficient Müller glia. These findings demonstrate that, much like in zebrafish, Notch signaling actively represses neurogenic competence in mammalian Müller glia, and suggest that inhibition of Notch signaling andNfia/b/xin combination with overexpression of activated Yap could serve as an effective component of regenerative therapies for degenerative retinal diseases.