Abstract
Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog, and Lin28, which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition.
Highlights
Müller glia (MG) is responsible for the maintenance of retinal homeostasis and neural nourishment (Pfeiffer-Guglielmi et al, 2005), protection against oxidative stress (Schütte and Werner, 1998), maintenance of the blood-retinal barrier (BRB; Bringmann et al, 2006), growth factor release (Eichler et al, 2001), and even light guidance to the photoreceptor layer (Agte et al, 2011).MG is remarkably capable of regenerating damaged neurons, albeit in a very restricted number of vertebrates, teleost fish, by dedifferentiating and acquiring a progenitor phenotype
We suggest a possible correlation between changes in these genes and in those related to DNA methylation and demethylation, whose blockage should enhance MG dedifferentiation
We demonstrated the excitotoxic effect of the NMDA injection by a Transferase dUTP Nick-End Labeling (TUNEL) assay, comparing injured retinas with positive controls
Summary
Müller glia (MG) is responsible for the maintenance of retinal homeostasis and neural nourishment (Pfeiffer-Guglielmi et al, 2005), protection against oxidative stress (Schütte and Werner, 1998), maintenance of the blood-retinal barrier (BRB; Bringmann et al, 2006), growth factor release (Eichler et al, 2001), and even light guidance to the photoreceptor layer (Agte et al, 2011). MG is remarkably capable of regenerating damaged neurons, albeit in a very restricted number of vertebrates, teleost fish, by dedifferentiating and acquiring a progenitor phenotype This is revealed by a drastic change in MG’s gene expression program, as they down-regulate and eventually lose the expression of its specific markers cellular retinaldehydebinding protein (CRALBP) and glutamine synthase (GS), while up-regulating progenitor-associated markers such as ascl1a, chx, and six, and genes usually expressed by pluripotent cells, like oct, lin, sox, klf, and c-myc (Raymond et al, 2006; Bernardos et al, 2007; Fischer and Bongini, 2010; Ramachandran et al, 2010). We suggest a possible correlation between changes in these genes and in those related to DNA methylation and demethylation, whose blockage should enhance MG dedifferentiation
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