Glutamate-induced epigenetic and morphological changes allow rat Müller cell dedifferentiation but not further acquisition of a photoreceptor phenotype.

Müller cells are not only the main glial cell type in the retina but also latent progenitor/stem cells, which in pathological conditions can transdifferentiate to a neuronal phenotype and regenerate the neurons lost in a mature retina. Several signal transduction pathways can induce the dedifferentiation of mature Müller cells to a progenitor-like state, including that stimulated by glutamate. However, the precise molecular mechanisms by which terminally differentiated cells are initially primed to acquire multipotency remain unclear. In the present study, we have characterized early genetic and epigenetic events that occur immediately after glutamate-induced dedifferentiation of fully differentiated Müller cells is initiated. Using Müller cell-enriched cultures from postnatal rats, we demonstrate that glutamate triggers a rapid dedifferentiation response characterized by changes in cell morphology coupled to the induction of progenitor cell marker gene expression (e.g., nestin, lin28 and sox2) within 1h. Dedifferentiation involved the activation of N-methyl-d-aspartate and type II metabotropic glutamate receptors, as well as global DNA demethylation (evident through the decrease in methyl-CpG-binding protein 2 immunoreactivity) and an increase in gadd45-ß gene expression; although, early progenitor gene expression was only partially inhibited by pharmacological impairment of DNA methylation. Importantly, the expression of Müller glia identity genes (i.e., glutamine synthetase; cellular retinaldehyde binding protein, CRALBP) is retained through the process. Dedifferentiated Müller cells held an early neuronal differentiation potential similar to that observed in retinal progenitor-enriched cultures but, contrary to the latter, dedifferentiated Müller cells failed to further differentiate into mature photoreceptor lineages. We speculate that, in spite of the initial triggering of the dedifferentiation pathways, these cells may exhibit a certain degree of epigenetic memory that precludes them from further differentiation.

GABA-mediated induction of early neuronal markers expression in postnatal rat progenitor cells in culture.

Retinogenesis is a developmental process that involves the sequential formation of neurons and glia from retinal progenitors. Once retinogenesis is completed, Müller glial cells can be stimulated to differentiate into neuronal lineages and constitute a retina-intrinsic source of neural progenitors. The identification of the intrinsic and extrinsic factors that control proliferation and differentiation of Müller cells or retinal progenitors is needed in order to fully define their potential therapeutic use in regenerative approaches. Here we determined the response of retinal progenitors derived from Müller glia primary cell cultures to GABA-activated signal transduction cascades. Using Western blot analysis, immunocytochemistry and calcium imaging we found that GABA induces an increase of the number of progenitor cells that present spontaneous intracellular calcium transients as well as their frequency, which involve the participation of L-type voltage-gated calcium channels (VGCCs). This process correlates with the activation of transcription factor CREB through Ser33 phosphorylation and the induction of expression of the early neuronal markers NeuroD1 and ßIII-tubulin. GABA-mediated CREB phosphorylation was rapid and sustained and the pharmacological blockade of CREB activity inhibited the effect of GABA on NeuroD1 expression. Furthermore, consistent with the role of CREB as a histone acetyltransferase recruiter, we demonstrate that GABA induces the modification of histone H4 acetylation pattern in these cells suggesting that epigenetic alterations participate in the differentiation process. Our results support the notion that postnatal retinal progenitors derived from Müller glia primary cell cultures respond to GABA through the same molecular pathway previously characterized in hippocampal progenitors and developing neurons. We speculate that the induction of GABA receptor signaling could represent a novel strategy to enhance neural versus glial specification from these cells through genetic and epigenetic mechanisms.

Repeated toluene exposure modifies the acetylation pattern of histones H3 and H4 in the rat brain.

Toluene is a volatile organic solvent with addictive potential that exhibits similarities in its physiological effects and modes of action to other addictive drugs. Despite its widespread abuse, the molecular mechanisms driving the response and adaptation of the organism to this drug are not fully understood. In recent years, different epigenetic mechanisms that modulate gene expression have been shown to be associated to cocaine, amphetamine and alcohol misuse-induced alterations in neuronal function. For example, it has been demonstrated that drug consumption induces variations in histone acetylation levels in brain reward regions and these play a relevant role on the abuse-associated behavioral plasticity. In order to decipher whether repeated toluene exposure could mediate epigenetic changes in the rat brain, we here analyzed the acetylation pattern of histones H3 and H4 in three brain areas that have been previously associated to substance abuse reward pathways: the Nucleus Accumbens (NAc), the Ventral Tegmental Area (VTA) and the Central Amygdala (CeA). Using immunofluorescence analysis of brain sections with specific antibodies that recognize the acetylated forms of histones H3 and H4, we demonstrate that chronic toluene inhalation differentially modifies histone H3 and H4 acetylation in the NAc and the VTA while no effect is observed in the CeA. Our results suggest that the activity of chromatin-modifying enzymes such as histone de-acetylases (HDACs) in certain brain areas are responsive to toluene inhalation and might be crucial mediators in the addictive response to toluene.