Cannabinoid signaling promotes the de‐differentiation and proliferation of Müller glia‐derived 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 the transition 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 investigate how the eCB system impacts glial reactivity and the capacity of MG to become Müller glia‐derived progenitor cells (MGPCs). We queried 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 cannabinoids, 2‐Arachidonoylglycerol (2‐AG) and Anandamide (AEA), stimulated the formation of MGPCs. Cannabinoid Receptor 1 (CNR1)‐agonists and Monoglyceride Lipase‐inhibitor promoted the formation of MGPCs, whereas CNR1‐antagonist and inhibitors of eCB synthesis suppressed this process. In damaged mouse retinas where MG activate NFkB‐signaling, activation of CNR1 decreased and inhibition of CNR1 increased NFkB, whereas levels of neuronal cell death were unaffected. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB‐signaling in both chick and mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and the formation of proliferating MGPCs, but does not influence the reactivity of immune cells in the retina.

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

“…Observed labeling was not due to off-target labeling of secondary antibodies or tissue autofluorescence because sections incubated with secondary antibodies alone were devoid of fluorescence. Patterns of immunolabeling for all antibodies precisely match patterns of mRNA expression in scRNA-libraries of control and damaged retinas, similar previous descriptions (Campbell et al, 2019; Campbell et al, 2021c; Campbell et al, 2021d; Todd et al, 2019). Secondary antibodies utilized include donkey-anti-goat-Alexa488/568, goat-anti-rabbit-Alexa488/568/647, goat-anti-mouse-Alexa488/568/647, goat-anti-rat-Alexa488 (Life Technologies) diluted to 1:1000 in PBS and 0.2% Triton X-100.…”

“…To assess patterns of expression of the different components of the PRC2 complex we probed previously established scRNA-seq libraries (Campbell et al, 2019; Campbell et al, 2021b; Campbell et al, 2021c; Hoang et al, 2020). scRNA-seq libraries were generated and aggregated for retinal cells obtained from control and NMDA-damaged retinas at different times (24, 48 and 72 hrs) after treatment (Fig.…”

“…We analyzed scRNA-seq databases of control and NMDA-damaged chick retinas, for patterns of expression of AHCY , AHCYL1, AHCYL2 and HMTs. We probed libraries of chick retinas from undamaged retinas treated with saline or NMDA at 3, 12 and 48 hrs after treatment, as described previously (Campbell et al, 2021c; Campbell et al, 2022a). After excluding cells with low numbers of genes/cells, putative doublets (high numbers of genes/cells) and dying cells (high percentage of mitochondrial genes), we analyzed a total of 42,202 cells including a total of 4,700 MG (Fig.…”

GSK3 Is a Central Player in Retinal Degenerative Diseases but a Challenging Therapeutic Target

“…Therefore, a better strategy for more effective therapies could instead target specific GSK3 downstream targets. For instance, some promising treatments targeting GSK3-regulated pathways are combining anti-oxidant effects and anti-inflammatory effects, as is the case of flavonoids coming from fruits and vegetables [ 215 , 216 ] or cannabidiol [ 217 ]. To illustrate the interest in targeting GSK3 targets, one can cite NRF2 for its antioxidant and anti-inflammatory functions.…”

Glia Regulate the Development, Function, and Plasticity of the Visual System From Retina to Cortex

“…The interactions between microglia, the resident immune cell in the central nervous system, and Müller glia also appear to mediate important roles in retinal development and repair as the presence of microglia is required for the regenerative potential of Müller glia in the adult zebrafish retina (Conedera et al, 2019). Additionally, endocannabinoids, known to modulate glia throughout the CNS, have also been suggested to play a role in mediating the proliferative function of Müller glia as pharmacological activation of Müller glia endocannabinoid receptors promotes the formation of progenitor cells without having an observable impact on microglial responses in the damaged retina (Campbell et al, 2021).…”