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Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo.

Ueki Y, Reh TA - PLoS ONE (2012)

Bottom Line: During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells.Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone.Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Structure, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.

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Retinal damage induces Smad phosphorylation in retinal ganglion cells and inner retinal cells, and increases BMP mRNA expression in the retina. A.Injection of 100 mM NMDA induced Smad1/5/8 activation (pSmad1/5/8) in the retinal ganglion cells and inner retinal cells. The peak of activation was observed at 2 days. Smad2/3 was also activated (pSmad2/3) in the inner retina to a lesser extent. Scale bar: 100 µm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganclion cell layer. B–C. While strong Smad1/5/8 activation was induced in Hes5-GFP+ Müller glia (green) 2 days after NMDA injection (B), Smad2/3 activation was observed in Hes5-GFP- cells in the INL (C). Scale bars: 30 µm. D. NMDA damage activated Smad1/5/8 in remaining retinal ganglion cells and displaced amacrine cells located in the GCL. Scale bars: 10 µm. E. Real-time qPCR data showing that NMDA damage induced significant increase in Bmp4 expression in the retina 2 days after NMDA damage. Expression of other ligands of BMP signaling, Bmp2 and −7, was also induced. *p<0.05 with paired t-test (n = 4). Images shown in A–D are representative of at least 3 animals per each treatment group.
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pone-0038690-g002: Retinal damage induces Smad phosphorylation in retinal ganglion cells and inner retinal cells, and increases BMP mRNA expression in the retina. A.Injection of 100 mM NMDA induced Smad1/5/8 activation (pSmad1/5/8) in the retinal ganglion cells and inner retinal cells. The peak of activation was observed at 2 days. Smad2/3 was also activated (pSmad2/3) in the inner retina to a lesser extent. Scale bar: 100 µm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganclion cell layer. B–C. While strong Smad1/5/8 activation was induced in Hes5-GFP+ Müller glia (green) 2 days after NMDA injection (B), Smad2/3 activation was observed in Hes5-GFP- cells in the INL (C). Scale bars: 30 µm. D. NMDA damage activated Smad1/5/8 in remaining retinal ganglion cells and displaced amacrine cells located in the GCL. Scale bars: 10 µm. E. Real-time qPCR data showing that NMDA damage induced significant increase in Bmp4 expression in the retina 2 days after NMDA damage. Expression of other ligands of BMP signaling, Bmp2 and −7, was also induced. *p<0.05 with paired t-test (n = 4). Images shown in A–D are representative of at least 3 animals per each treatment group.

Mentions: Previous studies have shown that growth factors, such as CNTF, LIF, FGF2, and BDNF are upregulated in the retina after damage [1], [2], [3], [4], [5]. However, there has been little characterization of BMP signaling after retinal damage. We therefore examined whether BMP signaling increases following NMDA-induced retinal damage, using antibodies against activated forms of Smad, key downstream components of the BMP and TGF-beta signaling pathways. As shown in Figure 2A, pSmad1/5/8 is barely detectable in the undamaged retina, but there is a substantial increase in the labeling throughout the inner retina at 1 and 2 days after NMDA treatment. Many of the cells in which the BMP signaling has been activated are Müller glia (Figure 2B), as shown by their co-expression of pSmad 1/5/8 and Hes5-GFP [24]. The majority of cells in the ganglion cell layer are also labeled with the pSmad1/5/8 antibody, and these were either displaced amacrine cells or surviving ganglion cells (Figure 2D). The activation of Smad1/5/8 was likely to be mediated by an increased expression of BMP ligands, BMP2, −4, and −7 in the retina following NMDA damage (Figure 2E). Bmp4 mRNA level was increased by 2.07±0.33 fold 2 days after NMDA damage compared to untreated retinas. Smad2/3 is also activated after damage, indicating that TGF-beta signaling is also increased after damage, though fewer cells are labeled with pSmad2/3 than for pSmad1/5/8. While most of the pSmad2/3 labeled cells in the INL, Hes5-GFP+ Müller glia were not labeled with the pSmad2/3 antibody (Figure 2C).


Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo.

Ueki Y, Reh TA - PLoS ONE (2012)

Retinal damage induces Smad phosphorylation in retinal ganglion cells and inner retinal cells, and increases BMP mRNA expression in the retina. A.Injection of 100 mM NMDA induced Smad1/5/8 activation (pSmad1/5/8) in the retinal ganglion cells and inner retinal cells. The peak of activation was observed at 2 days. Smad2/3 was also activated (pSmad2/3) in the inner retina to a lesser extent. Scale bar: 100 µm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganclion cell layer. B–C. While strong Smad1/5/8 activation was induced in Hes5-GFP+ Müller glia (green) 2 days after NMDA injection (B), Smad2/3 activation was observed in Hes5-GFP- cells in the INL (C). Scale bars: 30 µm. D. NMDA damage activated Smad1/5/8 in remaining retinal ganglion cells and displaced amacrine cells located in the GCL. Scale bars: 10 µm. E. Real-time qPCR data showing that NMDA damage induced significant increase in Bmp4 expression in the retina 2 days after NMDA damage. Expression of other ligands of BMP signaling, Bmp2 and −7, was also induced. *p<0.05 with paired t-test (n = 4). Images shown in A–D are representative of at least 3 animals per each treatment group.
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pone-0038690-g002: Retinal damage induces Smad phosphorylation in retinal ganglion cells and inner retinal cells, and increases BMP mRNA expression in the retina. A.Injection of 100 mM NMDA induced Smad1/5/8 activation (pSmad1/5/8) in the retinal ganglion cells and inner retinal cells. The peak of activation was observed at 2 days. Smad2/3 was also activated (pSmad2/3) in the inner retina to a lesser extent. Scale bar: 100 µm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganclion cell layer. B–C. While strong Smad1/5/8 activation was induced in Hes5-GFP+ Müller glia (green) 2 days after NMDA injection (B), Smad2/3 activation was observed in Hes5-GFP- cells in the INL (C). Scale bars: 30 µm. D. NMDA damage activated Smad1/5/8 in remaining retinal ganglion cells and displaced amacrine cells located in the GCL. Scale bars: 10 µm. E. Real-time qPCR data showing that NMDA damage induced significant increase in Bmp4 expression in the retina 2 days after NMDA damage. Expression of other ligands of BMP signaling, Bmp2 and −7, was also induced. *p<0.05 with paired t-test (n = 4). Images shown in A–D are representative of at least 3 animals per each treatment group.
Mentions: Previous studies have shown that growth factors, such as CNTF, LIF, FGF2, and BDNF are upregulated in the retina after damage [1], [2], [3], [4], [5]. However, there has been little characterization of BMP signaling after retinal damage. We therefore examined whether BMP signaling increases following NMDA-induced retinal damage, using antibodies against activated forms of Smad, key downstream components of the BMP and TGF-beta signaling pathways. As shown in Figure 2A, pSmad1/5/8 is barely detectable in the undamaged retina, but there is a substantial increase in the labeling throughout the inner retina at 1 and 2 days after NMDA treatment. Many of the cells in which the BMP signaling has been activated are Müller glia (Figure 2B), as shown by their co-expression of pSmad 1/5/8 and Hes5-GFP [24]. The majority of cells in the ganglion cell layer are also labeled with the pSmad1/5/8 antibody, and these were either displaced amacrine cells or surviving ganglion cells (Figure 2D). The activation of Smad1/5/8 was likely to be mediated by an increased expression of BMP ligands, BMP2, −4, and −7 in the retina following NMDA damage (Figure 2E). Bmp4 mRNA level was increased by 2.07±0.33 fold 2 days after NMDA damage compared to untreated retinas. Smad2/3 is also activated after damage, indicating that TGF-beta signaling is also increased after damage, though fewer cells are labeled with pSmad2/3 than for pSmad1/5/8. While most of the pSmad2/3 labeled cells in the INL, Hes5-GFP+ Müller glia were not labeled with the pSmad2/3 antibody (Figure 2C).

Bottom Line: During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells.Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone.Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Structure, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.

Show MeSH
Related in: MedlinePlus