NFkB-signaling promotes glial reactivity and suppresses Müller glia-mediated neuron regeneration in the mammalian retina

Abstract

Müller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification networks that regulate MG-mediated regeneration is key to harnessing the regenerative potential of MG. Here we study how NFkB-signaling influences glial responses to damage and reprogramming of MG into neurons in the rodent retina. We find activation of NFkB and dynamic expression of NFkB-associated genes in MG after damage, however NFkB activity is inhibited by microglia ablation. Knockout of NFkB in MG suppressed the accumulation of immune cells after damage. Inhibition of NFkB following NMDA-damage significantly enhanced the reprogramming of Ascl1-overexpressing MG into neuron-like cells. scRNA-seq of retinal glia following inhibition of NFkB reveals coordination with signaling via TGFb2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 or Id transcription factors increased numbers of neuron-like cells produced by Ascl1-overexpressing MG. 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.

Patterns of NFkB activation resulting from damage, reactive microglia, cytokines, and growth factors in the mouse retina

“…In the mouse retina, microglia repress the neurogenic reprogramming capacity of MG after damage (Todd et al, 2020), while microglia are necessary for initiation of MG reprogramming in the chick and fish (Fischer et al, 2014b;White et al, 2017). Consistent with this, we have shown that NFkB signaling is highly activated and suppresses neurogenic programs in mouse MG after damage (Palazzo et al, 2022).…”

Chromatin access regulates the formation of Müller glia-derived progenitor cells in the retina

“…7e-g). In addition, we identified significant differences in enriched access to motifs related to NFkB ( NFKB2, RELA ), Hedgehog ( GLI2 ), retinoic acid ( RARA ), glucocorticoid ( NR3C2 ) and Hippo-signaling pathways ( TEAD1 ), which are known to influence the formation of MGPCs (Gallina et al, 2014b; Palazzo et al, 2020; Palazzo et al, 2022; Rueda et al, 2019; Todd and Fischer, 2015; Todd et al, 2018; Young et al, 2000). We next analyzed levels of mRNA (scRNA-seq), chromatin access (scATAC-seq) and enriched motif access in MG for transcription factors that are known to be important for retinal development and the formation of MGPCs.…”

“…Mice were kept on a cycle of 12 hours light, 12 hours dark (lights on at 8:00 AM). The use of Ascl1 over-expressing mice ( Glast-CreER:LNL-tTA:tetO-mAscl1-ires-GFP) was as previously described (Jorstad et al, 2017; Palazzo et al, 2021).…”

“…The use of Ascl1 over-expressing mice (Glast-CreER:LNL-tTA:tetO-mAscl1-ires-GFP) was as previously described (Jorstad et al, 2017;Palazzo et al, 2021).…”

“…Post-hatch chicks were maintained in a regular diurnal cycle of 12 hours light, 12 hours dark (lights on at 8:00 AM). Chicks were housed in stainless-steel brooders at 25 o C and received water and Purina tm chick starter ad libitum.Mice were kept on a cycle of 12 hours light, 12 hours dark (lights on at 8:00 AM).The use of Ascl1 over-expressing mice (Glast-CreER:LNL-tTA:tetO-mAscl1-ires-GFP) was as previously described (Jorstad et al, 2017;Palazzo et al, 2021).…”