Abstract
Purpose :
Loss of vision is caused by irreversible death of retinal neurons. Müller glia (MG) are the primary support cell of the retina and are capable of reprogramming into progenitor cells with potential to regenerate neurons following retinal damage. The capacity for MG mediated retinal neuron regeneration varies widely across species. In lower vertebrate species, MG undergo a neurogenic reprogramming event that results in regeneration of functional neurons to restore loss of vision. However, in mammalian systems, MG undergo a “gliotic” response associated with activation of inflammatory gene networks, and fail to regenerate neurons. NFkB signaling is a master regulator of a pro-inflammatory response, and our previous work established that NFkB regulates MG reprogramming in the chick retina. The goal of this study was to investigate the role that NFkB signaling plays in regulating the neurogenic vs. gliotic response in MG after damage in the mammalian retina.
Methods :
We investigated patterns of NFkB activation in the mammalian retina after NMDA damage using an NFkB-eGFP reporter mouse line, as well as probing for NFkB signaling components in single cell RNA-seq (scRNAseq) libraries from damaged mouse retinas. Additionally, we utilized an Ascl1 overexpressing mouse line (GlastER-cre/LNL-tTA/tetO-Ascl1-GFP) to investigate neuron regeneration after NMDA damage. We performed intraocular injections of NMDA and a pharmacological inhibitor of NFkB signaling (PGJ2) followed by immunohistochemistry or scRNAseq library preparation to identify retinal neuron regeneration and transcriptomic networks underlying the response, respectively.
Results :
We found that NFkB signaling is rapidly activated in Müller glia cells after NMDA damage, and that this activation is dependent on the presence of microglia in the retina. Additionally, inhibition of NFkB signaling decreases macrophage populations within the retina (n=5, p=.03) and promotes Ascl1-mediated retinal neuron regeneration by 30% (n=8, p=.002). Further, scRNA-seq analysis showed that inhibition of NFkB signaling decreases pro-inflammatory and anti-neural gene regulatory networks via coordination with Tgfb signaling and Id factors.
Conclusions :
We conclude that NFkB is a key signaling hub that is activated in MG after damage, mediates the accumulation of immune cells, and suppresses the neurogenic potential of MG.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.