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NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina.

Bai N, Aida T, Yanagisawa M, Katou S, Sakimura K, Mishina M, Tanaka K - Mol Brain (2013)

Bottom Line: Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A-D) are major determinants of the pharmacological and biophysical properties of NMDARs.However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. bai.ning.aud@mri.tmd.ac.jp

ABSTRACT

Background: Loss of retinal ganglion cells (RGCs) is a hallmark of various retinal diseases including glaucoma, retinal ischemia, and diabetic retinopathy. N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR)-mediated excitotoxicity is thought to be an important contributor to RGC death in these diseases. Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A-D) are major determinants of the pharmacological and biophysical properties of NMDARs. All NMDAR subunits are expressed in RGCs in the retina. However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.

Results: GluN2B- and GluN2D-deficiency protected RGCs from NMDA-induced excitotoxic retinal cell death. Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.

Conclusions: Our data suggest that GluN2B- and GluN2D-containing NMDARs play a critical role in NMDA-induced excitotoxic retinal cell death and RGC degeneration in glutamate aspartate transporter deficient mice. Inhibition of GluN2B and GluN2D activity is a potential therapeutic strategy for the treatment of several retinal diseases.

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Expression of NMDA receptor subunits in mouse retinal ganglion cell. (A-C) Immunohistochemical analysis of Brn3 (B red) in Thy1-CFP mice. CFP fluorescence (A green) was overlaid with Brn3 (C). Arrows in (C) indicate double-labeled cells. Scale bar, 20 μm. (D-E) After dissociation the fluorescent RGC was picked up from the cell suspension. CFP (green) and DIC pictures for the same isolated cell are superimposed (E). Arrowhead indicates CFP-expressing RGC. Scale bar, 20 μm. (F) Single-cell RT-PCR analysis for GluN1, four GluN2 subunits, Brn3 and β-actin. Distilled water was used for PCR negative control. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell.
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Figure 1: Expression of NMDA receptor subunits in mouse retinal ganglion cell. (A-C) Immunohistochemical analysis of Brn3 (B red) in Thy1-CFP mice. CFP fluorescence (A green) was overlaid with Brn3 (C). Arrows in (C) indicate double-labeled cells. Scale bar, 20 μm. (D-E) After dissociation the fluorescent RGC was picked up from the cell suspension. CFP (green) and DIC pictures for the same isolated cell are superimposed (E). Arrowhead indicates CFP-expressing RGC. Scale bar, 20 μm. (F) Single-cell RT-PCR analysis for GluN1, four GluN2 subunits, Brn3 and β-actin. Distilled water was used for PCR negative control. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell.

Mentions: To investigate the expression of NMDAR subunits in RGCs, we used a single-cell reverse transcriptase polymerase chain reaction (RT-PCR) method. After dissociation of the retina into single cells, RGCs can no longer be identified by their morphology. We therefore used dissociated retina from B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice (thy1-CFP mice), which express cyan fluorescent protein (CFP) in most RGCs [17]. We first confirmed that the CFP-containing cells in the thy1-CFP mouse retina were RGCs by immunostaining with Brn3, a neurochemical marker for RGCs [18]. CFP expression colocalized with Brn3 immunoreactivity in most somata in the ganglion cell layer (GCL) (Figure 1A-C). A single CFP-expressing cell was picked with a glass capillary from the dissociation mix and transferred to the reaction tube (Figure 1D, E), and was further identified as RGC by expression of Brn3 (Figure 1F). Typical results of single-cell RT-PCR on isolated RGCs are shown in Figure 1F. GluN1 and GluN2A–D could be amplified together with an internal control (β-actin) from a single RGC, as well as from whole retina. In our samples of 4 isolated RGCs, two cells express GluN1/GluN2A/GluN2B/GluN2C/GluN2D, whereas the other two cells express GluN1/GluN2A/GluN2B/GluN2D. These results indicate the presence of GluN1 and all GluN2 subunits (GluN2A–D) in the mouse RGCs.


NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina.

Bai N, Aida T, Yanagisawa M, Katou S, Sakimura K, Mishina M, Tanaka K - Mol Brain (2013)

Expression of NMDA receptor subunits in mouse retinal ganglion cell. (A-C) Immunohistochemical analysis of Brn3 (B red) in Thy1-CFP mice. CFP fluorescence (A green) was overlaid with Brn3 (C). Arrows in (C) indicate double-labeled cells. Scale bar, 20 μm. (D-E) After dissociation the fluorescent RGC was picked up from the cell suspension. CFP (green) and DIC pictures for the same isolated cell are superimposed (E). Arrowhead indicates CFP-expressing RGC. Scale bar, 20 μm. (F) Single-cell RT-PCR analysis for GluN1, four GluN2 subunits, Brn3 and β-actin. Distilled water was used for PCR negative control. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733768&req=5

Figure 1: Expression of NMDA receptor subunits in mouse retinal ganglion cell. (A-C) Immunohistochemical analysis of Brn3 (B red) in Thy1-CFP mice. CFP fluorescence (A green) was overlaid with Brn3 (C). Arrows in (C) indicate double-labeled cells. Scale bar, 20 μm. (D-E) After dissociation the fluorescent RGC was picked up from the cell suspension. CFP (green) and DIC pictures for the same isolated cell are superimposed (E). Arrowhead indicates CFP-expressing RGC. Scale bar, 20 μm. (F) Single-cell RT-PCR analysis for GluN1, four GluN2 subunits, Brn3 and β-actin. Distilled water was used for PCR negative control. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell.
Mentions: To investigate the expression of NMDAR subunits in RGCs, we used a single-cell reverse transcriptase polymerase chain reaction (RT-PCR) method. After dissociation of the retina into single cells, RGCs can no longer be identified by their morphology. We therefore used dissociated retina from B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice (thy1-CFP mice), which express cyan fluorescent protein (CFP) in most RGCs [17]. We first confirmed that the CFP-containing cells in the thy1-CFP mouse retina were RGCs by immunostaining with Brn3, a neurochemical marker for RGCs [18]. CFP expression colocalized with Brn3 immunoreactivity in most somata in the ganglion cell layer (GCL) (Figure 1A-C). A single CFP-expressing cell was picked with a glass capillary from the dissociation mix and transferred to the reaction tube (Figure 1D, E), and was further identified as RGC by expression of Brn3 (Figure 1F). Typical results of single-cell RT-PCR on isolated RGCs are shown in Figure 1F. GluN1 and GluN2A–D could be amplified together with an internal control (β-actin) from a single RGC, as well as from whole retina. In our samples of 4 isolated RGCs, two cells express GluN1/GluN2A/GluN2B/GluN2C/GluN2D, whereas the other two cells express GluN1/GluN2A/GluN2B/GluN2D. These results indicate the presence of GluN1 and all GluN2 subunits (GluN2A–D) in the mouse RGCs.

Bottom Line: Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A-D) are major determinants of the pharmacological and biophysical properties of NMDARs.However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. bai.ning.aud@mri.tmd.ac.jp

ABSTRACT

Background: Loss of retinal ganglion cells (RGCs) is a hallmark of various retinal diseases including glaucoma, retinal ischemia, and diabetic retinopathy. N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR)-mediated excitotoxicity is thought to be an important contributor to RGC death in these diseases. Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A-D) are major determinants of the pharmacological and biophysical properties of NMDARs. All NMDAR subunits are expressed in RGCs in the retina. However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.

Results: GluN2B- and GluN2D-deficiency protected RGCs from NMDA-induced excitotoxic retinal cell death. Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.

Conclusions: Our data suggest that GluN2B- and GluN2D-containing NMDARs play a critical role in NMDA-induced excitotoxic retinal cell death and RGC degeneration in glutamate aspartate transporter deficient mice. Inhibition of GluN2B and GluN2D activity is a potential therapeutic strategy for the treatment of several retinal diseases.

Show MeSH
Related in: MedlinePlus