N-Methyl-D-Aspartic acid suppresses Akt activity through protein phosphatase in retinal ganglion cells.
OBJECTIVE
To investigate the relationship between Akt activity and retinal ganglion cell (RGC) death induced by N-Methyl-D-Aspartic acid (NMDA) in the rat retina.


METHODS
Two microlitres of 1, 10, 50, 100, or 200 mM NMDA, or vehicle was injected into the vitreous cavity of Sprague-Dawley (SD) rats (n=125). Retinal damage was estimated by counting ganglion cells labeled with fluorochrome and retinal apoptosis was detected by TUNEL. Akt activity was determined by immunohistochemical analysis with a specific antibody to the activated (phosphorylated) form of Akt. To investigate the mechanism of dephosphorylation of Akt, Okadaic acid, a potent protein phosphatase inhibitor, was injected 1 h before NMDA injury and accessed the number of phosphorylated Akt positive cells 1 h after NMDA injection. To stimulate Akt activity in the retina, brain derived neurotrophic factor (BDNF) was injected into the vitreous 15 min before NMDA injection.


RESULTS
Immunohistochemical analysis revealed a reduction in phosphorylated Akt in RGCs and amacrine cells one hour after NMDA injury. The RGCs and amacrine cells showed TUNEL positivity at 6 h and a decrease in cell number at 7 days after NMDA injury. No other cells in the retina stained positive with phosphorylated Akt antibody and TUNEL. Okadaic acid prevented the dephosphorylation of Akt by NMDA. The exogenous administration of BDNF prevented the dephosphorylation of Akt in N-Shc/ShcC-positive RGCs and significantly suppressed the NMDA-induced RGC death.


CONCLUSIONS
These observations suggest that Akt is one of the key signaling proteins in RGC death induced by NMDA, and that the presence of N-Shc/ShcC enhances BDNF-mediated neuroprotection via phosphorylated Akt. The regulation of phosphorylated Akt by growth factors and protein phosphatase activity may play an important role in cell fate following NMDA injury. Thus, an increase in phosphorylated Akt may have potential therapeutic implications in the treatment of glutamate-related disease.
Nakazawa
Toru
T
Department of Ophthalmology and Visual Science, Tohoku University Graduate School of Medicine, Seiryo, Aoba, Sendai, Miyagi, Japan. ntoru@fa2.so-net.ne.jp
Shimura
Masahiko
M
Endo
Shogo
S
Takahashi
Hidetoshi
H
Mori
Nozomu
N
Tamai
Makoto
M
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D016202:N-Methylaspartate / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013194:Staining and Labeling; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D019208:Brain-Derived Neurotrophic Factor; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016202:N-Methylaspartate; D051057:Proto-Oncogene Proteins c-akt; D010749:Phosphoprotein Phosphatases