Abstract
Purpose :
Irreversible loss of neurons is the key pathological feature of retinal degenerative diseases that often result in permanent blindness. While there is currently no effective treatment, one of the most promising strategies is through direct reprogramming of endogenous Müller glia (MG) into retinal neurons. MG are progenitor-like radial glial cells that serve as cellular sources for retinal regeneration in cold-blooded vertebrates. In zebrafish, Müller glia respond to retinal injury by regenerating all types of retinal neurons. In mammals, however, MG undergo reactive gliosis and do not regenerate neurons following retinal injury. Despite successful in vitro reprogramming applications, 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.
Methods :
We inhibited Notch signaling by deleting its central mediator, Rbpj, in adult mouse in MG. We further generated combined loss of function of Rbpj and NFI factors. We used genetic lineage analysis, immunohistochemistry, and integrated analysis of multiome (scRNA-Seq, scATAC-Seq) and CUT&Tag to identify and characterize MG-derived neurons unambiguously.
Results :
We found that loss of Rbpj induces mature MG to reprogram into bipolar and amacrine neurons in uninjured adult mouse retinas, and observed that this effect was further enhanced by retinal injury. We also observe that Rbpj directly activates Notch effector genes and genes specific to mature Müller glia while also indirectly repressing 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 Müller glia to neurons. Finally, we demonstrated that inducing MG proliferation by AAV (adeno-associated virus)-mediated overexpression of dominant-active Yap supports efficient levels of MG-derived neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia.
Conclusions :
These findings demonstrate that, much like in zebrafish, Notch signaling actively represses neurogenic competence in mammalian MG. Inhibition of Notch signaling and Nfia/b/x in combination with overexpression of activated Yap could potentially serve as an effective component of regenerative therapies for degenerative retinal diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.