NMDA receptor mediates proliferation and CREB phosphorylation in postnatal Müller glia-derived retinal progenitors
NMDA receptor mediates proliferation and CREB phosphorylation in postnatal Müller glia-derived retinal progenitors
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发表时间：
2009-04
期刊：
影响因子：
2.2
通讯作者：
M. Ramírez;M. Lamas
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作者：
M. Ramírez;M. Lamas
目的出生后视网膜Müler胶质细胞被认为是视网膜祖细胞，因为它们在损伤后保留了去分化、增殖和分化为新的视网膜神经胶质细胞和神经元的能力。增殖和分化过程由几种外在因素和神经递质协调，包括谷氨酸。因此，在发育和损伤修复期间，不同类型的细胞会产生适当的数量和比例，以形成功能视网膜。在这里，我们分析了N-甲基-D-天冬氨酸(NMDA)谷氨酸受体激活后，出生后Müler神经胶质源性祖细胞增殖的变化。方法用抗NMDA受体NR1亚单位抗体和神经前体细胞标志物Nestin进行免疫细胞化学鉴定Müler神经胶质细胞来源的祖细胞。通过BrdU掺入或Ki67免疫染色、细胞计数和磷酸化cAMP反应元件结合蛋白(P-CREB)转录因子的免疫标记，分析谷氨酸受体激动剂和拮抗剂对细胞增殖的影响。免疫组织化学P-CREB法检测Long-Evans NMDA注射大鼠视网膜切片中NMDA受体的激活作用。结果NMDA受体激活可显著增加Müler-glia来源的祖细胞的增殖率，此作用可被NMDA受体拮抗剂阻断。此外，我们还发现，在培养的培养中，NMDA处理的Müler-glia来源的祖细胞可以诱导CREB的磷酸化，并且CREB磷酸化的特异性药物抑制会导致增殖细胞的数量减少。我们通过对体内注射NMDA后视网膜切片的分析证实了这些观察的相关性，治疗后视网膜切片对磷酸化CREB的免疫反应性也增加。结论在本研究中，在培养和体内实验中，NMDA受体激活均可诱导生后Müler胶质细胞来源的视网膜祖细胞的增殖和转录因子CREB的磷酸化。识别成熟的视网膜祖细胞的分子决定因素，如转录因子CREB和NMDA受体诱导的玩家，将有助于控制祖细胞培养的生长和操作，并可能识别涉及祖细胞自我更新的分子机制。
Purpose Postnatal retinal Müller glia are considered to be retinal progenitors as they retain the ability to dedifferentiate, proliferate, and differentiate to new retinal glia and neurons after injury. The proliferation and differentiation processes are coordinated by several extrinsic factors and neurotransmitters, including glutamate. Thus, the appropriate numbers and proportions of the different cell types are generated to form a functional retina during development and during injury repair. Here we analyze the changes in the proliferation of postnatal Müller glia-derived progenitors after activation of the N-methyl-D-aspartate (NMDA) glutamate receptors. Methods Müller glia-derived progenitor cell cultures were characterized by immunocytochemistry with antibodies against the NR1 subunit of the NMDA receptor and the progenitor cell marker nestin. The effect of glutamate receptor agonists and antagonists on cell proliferation was analyzed by BrdU incorporation or Ki67 immunostaining, cell counting, and by immunolabeling of phosphorylated cAMP response element binding protein (P-CREB) transcription factor. The effect of NMDA receptor activation was analyzed in vivo by P-CREB immunohistochemistry in retinal sections of Long-Evans NMDA injected rats. Results We show that NMDA receptor activation significantly increases the proliferation rate of Müller-glia derived progenitor cells and that this increase can be blocked by NMDA receptor antagonists. Furthermore, we show that CREB phosphorylation is induced in NMDA-treated Müller-glia derived progenitor cells in culture and that specific pharmacological inhibition of CREB phosphorylation results in a decreased number of proliferating cells. We confirmed the relevance of these observations by the analysis of retinal sections after NMDA injection in vivo where immunoreactivity to phosphorylated CREB is also increased after treatment. Conclusions In the present study we show that NMDA receptor activation induces postnatal Müller glia-derived retinal cell progenitor proliferation and transcription factor CREB phosphorylation both in culture and in vivo. The identification of the molecular determinants of mature retinal progenitors such as transcription factor CREB and NMDA receptor-induced players should facilitate the control of growth and manipulation of progenitor cell cultures and the possible identification of the molecular mechanisms involved in progenitor self-renewal.