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NMDA receptor mediates proliferation and CREB phosphorylation in postnatal Müller glia-derived retinal progenitors.

Ramírez M, Lamas M - Mol. Vis. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Farmacobiología, Cinvestav Sede Sur, México DF, México.

ABSTRACT

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.

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NMDA induces CREB phosphorylation in retinal progenitors. A-C: Photomicrographs show P-CREB labeled cells counterstained with DAPI after no treatment (A) and 100 μM NMDA (B), or glutamate (C). D: Graph shows the quantification of nestin-P-CREB double-positive cells in control, NMDA, or glutamate-treated cultures. Data represent mean±SEM of 100 cells counted on nonoverlapping fields in three experiments. Double asterisks (**) indicate p<0.05 compared to control. E: Graph shows the percentage of P-CREB positive cells after no treatment, 100 μM NMDA, or 100 μM NMDA plus 50 μM MK801. Data represent the percentage of P-CREB positive cells with respect to total number of cells counted on five nonoverlapping fields. F-G: Photomicrographs show NR1-P-CREB (F) and nestin-P-CREB double-labeled cells (G) counterstained with DAPI. Calibration bar equals 100 μm in A-C and 20 μm in F.
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f3: NMDA induces CREB phosphorylation in retinal progenitors. A-C: Photomicrographs show P-CREB labeled cells counterstained with DAPI after no treatment (A) and 100 μM NMDA (B), or glutamate (C). D: Graph shows the quantification of nestin-P-CREB double-positive cells in control, NMDA, or glutamate-treated cultures. Data represent mean±SEM of 100 cells counted on nonoverlapping fields in three experiments. Double asterisks (**) indicate p<0.05 compared to control. E: Graph shows the percentage of P-CREB positive cells after no treatment, 100 μM NMDA, or 100 μM NMDA plus 50 μM MK801. Data represent the percentage of P-CREB positive cells with respect to total number of cells counted on five nonoverlapping fields. F-G: Photomicrographs show NR1-P-CREB (F) and nestin-P-CREB double-labeled cells (G) counterstained with DAPI. Calibration bar equals 100 μm in A-C and 20 μm in F.

Mentions: Next we wanted to take advantage of our purified Müller-derived progenitor cell culture to analyze the molecular signal cascade induced by NMDA receptor activation in these cells. We had previously described that NMDA receptor activation induces CREB phosphorylation at early times in differentiated Müller glia [13]. To test whether CREB phosphorylation is also induced in Müller-derived progenitor cells in culture, we examined the frequency of P-CREB immunoreactivity in cells that had been treated with 100 μM NMDA or 100 μM glutamate for 1 h. In these conditions, we found that there was a 47±7% increase in P-CREB immunoreactivity in NMDA- or glutamate-treated cells (Figure 3A-D). Complementary experiments shown in Figure 3E indicate that treatment of the cells with the NMDA receptor antagonist MK801 prevents NMDA-induced CREB phosphorylation.


NMDA receptor mediates proliferation and CREB phosphorylation in postnatal Müller glia-derived retinal progenitors.

Ramírez M, Lamas M - Mol. Vis. (2009)

NMDA induces CREB phosphorylation in retinal progenitors. A-C: Photomicrographs show P-CREB labeled cells counterstained with DAPI after no treatment (A) and 100 μM NMDA (B), or glutamate (C). D: Graph shows the quantification of nestin-P-CREB double-positive cells in control, NMDA, or glutamate-treated cultures. Data represent mean±SEM of 100 cells counted on nonoverlapping fields in three experiments. Double asterisks (**) indicate p<0.05 compared to control. E: Graph shows the percentage of P-CREB positive cells after no treatment, 100 μM NMDA, or 100 μM NMDA plus 50 μM MK801. Data represent the percentage of P-CREB positive cells with respect to total number of cells counted on five nonoverlapping fields. F-G: Photomicrographs show NR1-P-CREB (F) and nestin-P-CREB double-labeled cells (G) counterstained with DAPI. Calibration bar equals 100 μm in A-C and 20 μm in F.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666774&req=5

f3: NMDA induces CREB phosphorylation in retinal progenitors. A-C: Photomicrographs show P-CREB labeled cells counterstained with DAPI after no treatment (A) and 100 μM NMDA (B), or glutamate (C). D: Graph shows the quantification of nestin-P-CREB double-positive cells in control, NMDA, or glutamate-treated cultures. Data represent mean±SEM of 100 cells counted on nonoverlapping fields in three experiments. Double asterisks (**) indicate p<0.05 compared to control. E: Graph shows the percentage of P-CREB positive cells after no treatment, 100 μM NMDA, or 100 μM NMDA plus 50 μM MK801. Data represent the percentage of P-CREB positive cells with respect to total number of cells counted on five nonoverlapping fields. F-G: Photomicrographs show NR1-P-CREB (F) and nestin-P-CREB double-labeled cells (G) counterstained with DAPI. Calibration bar equals 100 μm in A-C and 20 μm in F.
Mentions: Next we wanted to take advantage of our purified Müller-derived progenitor cell culture to analyze the molecular signal cascade induced by NMDA receptor activation in these cells. We had previously described that NMDA receptor activation induces CREB phosphorylation at early times in differentiated Müller glia [13]. To test whether CREB phosphorylation is also induced in Müller-derived progenitor cells in culture, we examined the frequency of P-CREB immunoreactivity in cells that had been treated with 100 μM NMDA or 100 μM glutamate for 1 h. In these conditions, we found that there was a 47±7% increase in P-CREB immunoreactivity in NMDA- or glutamate-treated cells (Figure 3A-D). Complementary experiments shown in Figure 3E indicate that treatment of the cells with the NMDA receptor antagonist MK801 prevents NMDA-induced CREB phosphorylation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Farmacobiología, Cinvestav Sede Sur, México DF, México.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus