Cannabinoid signaling promotes the reprogramming of Muller glia into proliferating progenitor cells

Abstract

Endocannabinoids (eCB) are lipid-based neurotransmitters that are known to influence synaptic function in the visual system. eCBs are also known to suppress neuroinflammation in different pathological states. However, nothing is known about the roles of the eCB system during reprogramming of Müller glia (MG) into proliferating progenitor-like cells in the retina. Accordingly, we used the chick and mouse model to characterize expression patterns of eCB-related genes and applied pharmacological agents to examine how the eCB system impacts glial reactivity and the capacity of MG to become Müller glia-derived progenitor cells (MGPCs). We probed single cell RNA-seq libraries to identify eCB-related genes and identify cells with dynamic patterns of expression in damaged retinas. MG and inner retinal neurons expressed the eCB receptor CNR1, as well as enzymes involved in eCB metabolism. In the chick, intraocular injections of 2-Arachidonoylglycerol (2-AG) and Anandamide (AEA) potentiated the formation of MGPCs. Consistent with these findings, CNR1-agonists and MGLL-inhibitor promoted reprogramming, whereas CNR1-antagonist and inhibitors of eCB synthesis suppressed reprogramming. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB signaling in both chick and mouse models. However, eCB-signaling suppressed the activation of NFkB-reporter in MG in damaged mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and is important for regulating glial reactivity and the reprogramming of MG into proliferating MGPCs, but not for regulating the reactivity of immune cells in the retina.

Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas

“…2G: Fig. S2) known to influence the reprogramming of MG (Campbell et al, 2021b;Todd et al, 2017). In NMDA-damaged retinas, we identified LR-interactions between microglia and MG involving TGFB1/TGFBR3/SMAD3…”

“…We performed analyses on MG from each treatment group and compared changes across the most significant LRinteractions (Fig. 2A-D To identify putative paracrine LR-interactions between MG and microglia, we bioinformatically isolated MG and microglia from scRNA-seq libraries that we previously generated and described in detail (Campbell et al, 2021b;Campbell et al, 2022;Campbell et al, 2023). These libraries were generated from cells captured from normal and NMDA-damaged retinas at 3, 12 and 48hrs after treatment (Fig.…”

“…To gain a more comprehensive understanding of patterns of expression of Wntrelated genes, we probed a large aggregate scRNA-seq library of MG (>70,000 cells) generated as described in previous studies (Campbell et al, 2021a;Campbell et al, 2021b;El-Hodiri et al, 2022;Hoang et al, 2020). We bioinformatically isolated the MG from retinas treated with saline, 24/48/72hrs NMDA, 2 or 3 doses of insulin+FGF2 and 48hrs NMDA+insulin+FGF2 (Fig.…”

“…Pax2 and Pax6 are expressed at relatively low levels in resting MG, with Pax2 prominently expressed by MG in central regions of the retina, and levels of expression are increased in response to neuronal damage (Stanke et al, 2010). By contrast, GS is expressed at high levels by resting MG and is downregulated in response to neuronal damage Development • Accepted manuscript (Campbell et al, 2021a;Campbell et al, 2021b;Fischer et al, 2002). We found that GSK3β inhibitors had no significant effect on levels of Pax2, whereas levels of GS were significantly decreased in MG, and levels of Pax6 appeared increased in nuclei of delaminated cells (Fig.…”