Elsevier

Neuroscience

Volume 224, 8 November 2012, Pages 210-222
Neuroscience

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

https://doi.org/10.1016/j.neuroscience.2012.08.044Get rights and content

Abstract

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 βIII-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.

Highlights

GABA induces neuronal specification in rat Müller glia-derived retinal progenitors. ► Müller-derived progenitors’ response to GABA resembles that of developing neurons. ► This response involves intracellular Ca2+ dynamics and epigenetic alterations. ► Rapid and sustained CREB phosphorylation is required for NeuroD1 induction by GABA. ► GABA genetic/epigenetic effects could boost retinal regeneration from Müller glia.

Introduction

The neural retina is generated from multipotent precursors, which give rise, in a precise temporal order, to: ganglion cells, horizontal cells and cones, amacrine cells, rods, bipolar cells and Müller cells, a retinal-specific glia (Marquardt and Gruss, 2002). Throughout this process, neurotransmitter systems and extrinsic factors participate in the exquisite coordination of progenitor cell proliferation and differentiation to produce the appropriate number and proportion of the different retinal cell types to generate a functional retina (Young and Cepko, 2004, Martins and Pearson, 2008). It has been proposed that the progressive reduction of the intrinsic multipotency of retinal progenitors during development could be a consequence of changes in the expression of a variety of receptors that allow progenitors to respond to signaling pathways, including those elicited by neurotransmitter receptor activation (Cepko et al., 1996, Martins and Pearson, 2008).

Impelled by the characterization of the relevant function of the amino acid GABA in the proliferation, migration and neuronal maturation from neural precursors in the developing brain (reviewed in Salazar et al. (2008)), a number of studies have addressed the role of GABA during retinogenesis (reviewed in Martins and Pearson (2008)). Indeed, it has been shown that GABA and GABA transporters are expressed early during retinogenesis (Redburn and Madtes, 1986, Johnson et al., 2003), supporting the notion that GABA could exert trophic functions in synaptogenesis, growth and differentiation of retinal neurons. In addition, the expression of GABA A, B and C receptors has been found at different times during retinal development (Huang and Redburn, 1996, Mitchell and Redburn, 1996, Karne et al., 1997, Mitchell et al., 1999, Greka et al., 2000, Wu and Cutting, 2001, Martins and Pearson, 2008).

Although active neurogenesis has not been detected in the normal adult mammalian neural retina, several recent studies have demonstrated that Müller cells can acquire neurogenic potential in response to injury to the retina, thus acting as latent neural stem cells (Fischer and Reh, 2001, Fischer et al., 2002, Das et al., 2006, Bernardos et al., 2007, Karl et al., 2008, Takeda et al., 2008). Remarkably, the capacity of differentiated postnatal Müller cells to undergo a proliferative response could be evoked after intraocular injection of growth factors in the chicken (Fischer et al., 2002) and after glutamate treatment in mice and rats (Takeda et al., 2008, Ramirez and Lamas, 2009), suggesting that even in the postnatal retina these cells retain the ability to respond to signaling pathways that modulate retinal progenitor function.

It has been well established that the GABAA receptor mediates depolarization, the canonical activation of voltage-gated calcium channels (VGCCs) and intracellular Ca2+ increases in immature cells (Ben-Ari, 2002, Owens and Kriegstein, 2002). Through these mechanisms GABA promotes neuronal differentiation in adult hippocampal progenitor cells (Tozuka et al., 2005). Here, we wanted to examine the effect of GABA on the differentiation of postnatal retinal progenitors derived from primary cultures of glial Müller cells and found that GABA mediates the induction of the expression of early neuronal markers in postnatal Müller glia-derived retinal progenitors, similar to what occurs in adult hippocampal progenitor cells (Tozuka et al., 2005). Moreover, we show that this process involves the increase of intracellular calcium transients mediated by L-type VGCCs, the activation of transcription factor CREB, and an increased acetylation of histone H4. These results reinforce the current notion that progenitor cells derived from Müller glia can be stimulated to differentiate to neuronal lineages and represent a novel strategy to enhance their neural versus glial specification.

Section snippets

Experimental procedures

All experiments were conducted on laboratory animals treated and handled in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals for Ophthalmic and Vision Research and the guidelines of the internal animal care committees of our institutions.

GABA signaling induces early neuronal marker expression in postnatal retinal progenitor cells derived from Müller glia primary cultures

In order to determine the response of retinal progenitors derived from Müller primary cell cultures to GABA-activated signal transduction cascades, we assessed whether GABA receptor activation affected Müller-derived progenitor differentiation. Cells were allowed to differentiate in the absence of growth factors and treated either with GABA (100 μM) or with the specific GABAA receptor agonist muscimol (50 μm) for 5 days. We performed immunoblot analysis of total protein extracts with specific

Discussion

Accumulating evidence indicates that Müller glia can be stimulated to produce neurons in different types of vertebrates (Fischer and Bongini, 2010) and thus, these cells constitute a retina-intrinsic source of neural progenitors. Here we demonstrate that enriched cultures of retinal progenitor cells derived from Müller primary cell cultures, but not the original Müller primary cell culture, accumulate a neurogenic transcription factor, NeuroD1, in response to GABAA receptor activation. Our

Acknowledgements

The authors thank A. Huerta and A. Márquez-Ramos for technical assistance. This work was partially supported by grants from Conacyt and DGAPA-UNAM to M.L. and F.P.-O.; M.R., S.F. was recipient of a postdoctoral fellowship; H.Q. and S.L.-S.S. of a doctoral scholarship, and J.H.-M. of a MSc fellowship from Conacyt.

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