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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 vivo. Photomicrographs show retinal sections collected from postnatal rats 1 h, 2 h, and 1 day after intravitreal injection of BrdU-saline (Control) or BrdU-NMDA (NMDA). The retinal sections were labeled with antibodies against P-CREB or BrdU and counterstained with DAPI. Abbreviations: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL). Arrow indicates P-CREB immunopositive cells. Asterisk (*) denotes P-CREB-BrdU coimmunolabeled cells. Pound sign (#) marks BrdU immunopositive cells. Calibration bar denotes 50 μm in middle left panel.
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f5: NMDA induces CREB phosphorylation in vivo. Photomicrographs show retinal sections collected from postnatal rats 1 h, 2 h, and 1 day after intravitreal injection of BrdU-saline (Control) or BrdU-NMDA (NMDA). The retinal sections were labeled with antibodies against P-CREB or BrdU and counterstained with DAPI. Abbreviations: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL). Arrow indicates P-CREB immunopositive cells. Asterisk (*) denotes P-CREB-BrdU coimmunolabeled cells. Pound sign (#) marks BrdU immunopositive cells. Calibration bar denotes 50 μm in middle left panel.

Mentions: Consequently we wanted to analyze the immunoreactivity pattern of phosphorylated CREB after NMDA injection in vivo at various times after treatment. We injected solutions containing 2 M NMDA/10 mM BrdU intravitreally and retinal sections were collected 1 h, 2 h, or one day after injection and immunolabelled for BrdU and P-CREB. Both P-CREB and BrdU were detectable at 1 h, 2 h, and one day after injection, and a small number of P-CREB positive cells were proliferating as indicated by BrdU labeling (Figure 5). The number of P-CREB positive cells was increased in NMDA-treated retinas. These results indicate that NMDA receptor activation induced CREB phosphorylation signal transduction pathway also in vivo in the retina, suggesting a role for this event in retinal proliferation.


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 vivo. Photomicrographs show retinal sections collected from postnatal rats 1 h, 2 h, and 1 day after intravitreal injection of BrdU-saline (Control) or BrdU-NMDA (NMDA). The retinal sections were labeled with antibodies against P-CREB or BrdU and counterstained with DAPI. Abbreviations: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL). Arrow indicates P-CREB immunopositive cells. Asterisk (*) denotes P-CREB-BrdU coimmunolabeled cells. Pound sign (#) marks BrdU immunopositive cells. Calibration bar denotes 50 μm in middle left panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: NMDA induces CREB phosphorylation in vivo. Photomicrographs show retinal sections collected from postnatal rats 1 h, 2 h, and 1 day after intravitreal injection of BrdU-saline (Control) or BrdU-NMDA (NMDA). The retinal sections were labeled with antibodies against P-CREB or BrdU and counterstained with DAPI. Abbreviations: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL). Arrow indicates P-CREB immunopositive cells. Asterisk (*) denotes P-CREB-BrdU coimmunolabeled cells. Pound sign (#) marks BrdU immunopositive cells. Calibration bar denotes 50 μm in middle left panel.
Mentions: Consequently we wanted to analyze the immunoreactivity pattern of phosphorylated CREB after NMDA injection in vivo at various times after treatment. We injected solutions containing 2 M NMDA/10 mM BrdU intravitreally and retinal sections were collected 1 h, 2 h, or one day after injection and immunolabelled for BrdU and P-CREB. Both P-CREB and BrdU were detectable at 1 h, 2 h, and one day after injection, and a small number of P-CREB positive cells were proliferating as indicated by BrdU labeling (Figure 5). The number of P-CREB positive cells was increased in NMDA-treated retinas. These results indicate that NMDA receptor activation induced CREB phosphorylation signal transduction pathway also in vivo in the retina, suggesting a role for this event in retinal proliferation.

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