Departamento de Farmacobiología, Cinvestav-Sede Sur, México, DF, Mexico.
Neuroscience. 2012 Nov 8;224:210-22. doi: 10.1016/j.neuroscience.2012.08.044. Epub 2012 Aug 28.
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.
视网膜发生是一个涉及视网膜祖细胞从神经元和神经胶质细胞顺序形成的发育过程。一旦视网膜发生完成，Müller 胶质细胞可以被刺激分化为神经元谱系，并构成视网膜内在的神经祖细胞来源。需要确定控制 Müller 细胞或视网膜祖细胞增殖和分化的内在和外在因素，以便充分定义它们在再生方法中的潜在治疗用途。在这里，我们确定了源自 Müller 胶质细胞原代细胞培养物的视网膜祖细胞对 GABA 激活的信号转导级联的反应。通过 Western blot 分析、免疫细胞化学和钙成像，我们发现 GABA 诱导祖细胞数量增加，这些祖细胞表现出自发的细胞内钙瞬变及其频率增加，涉及 L 型电压门控钙通道 (VGCC) 的参与。这个过程与转录因子 CREB 通过 Ser33 磷酸化的激活和早期神经元标志物 NeuroD1 和 βIII-微管蛋白的诱导表达相关。GABA 介导的 CREB 磷酸化迅速而持久，CREB 活性的药理学阻断抑制了 GABA 对 NeuroD1 表达的影响。此外，与 CREB 作为组蛋白乙酰转移酶募集物的作用一致，我们证明 GABA 诱导这些细胞中组蛋白 H4 乙酰化模式的修饰，表明表观遗传改变参与了分化过程。我们的结果支持这样的观点，即源自 Müller 胶质细胞原代细胞培养物的出生后视网膜祖细胞通过先前在海马祖细胞和发育神经元中表征的相同分子途径对 GABA 做出反应。我们推测，诱导 GABA 受体信号可能代表一种通过遗传和表观遗传机制增强这些细胞中神经与神经胶质特化的新策略。
