May 2008
Volume 49, Issue 13
ARVO Annual Meeting Abstract  |   May 2008
Calpain-Mediated Degradation of G-Substrate Plays a Critical Role in Retinal Excitotoxicity
Author Affiliations & Notes
  • T. Nakazawa
    Ophthalmology, Tohoku Univ School of Medicine, Sendai, Japan
  • M. Shimura
    Ophthalmology, NTT East Japan Tohoku Hospital, Sendai, Japan
  • H. Takahashi
    Ophthalmology, Tohoku Univ School of Medicine, Sendai, Japan
  • S. Yokokura
    Ophthalmology, Tohoku Univ School of Medicine, Sendai, Japan
  • S. Endo
    Unit for Molecular Neurobiology of Learning and Memory, Okinawa Institute of Science and Technology (OIST), Uruma, Japan
  • K. Nishida
    Ophthalmology, Tohoku Univ School of Medicine, Sendai, Japan
  • Footnotes
    Commercial Relationships  T. Nakazawa, None; M. Shimura, None; H. Takahashi, None; S. Yokokura, None; S. Endo, None; K. Nishida, None.
  • Footnotes
    Support  Suda glaucoma research foundation (T.N.), Grant in aid for scientific research from the ministry of education science and culture of Japanese government (T.N. 19791255)
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2061. doi:
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      T. Nakazawa, M. Shimura, H. Takahashi, S. Yokokura, S. Endo, K. Nishida; Calpain-Mediated Degradation of G-Substrate Plays a Critical Role in Retinal Excitotoxicity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2061.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Retinal excitotoxicity is involved in various retinal ischemic diseases, such as diabetic retinopathy and retinal vessel occlusion. In the retinal neurons, amacrine cells are the most susceptible to excitotoxicity, however, the mechanisms underlying amacrine cell death remain unknown. To explore the mechanistic details, we investigate NMDA-elicited intracellular events in the retina.

Methods: : Retinal excitotoxicity was induced by an intravitreal injection of NMDA in Sprague-Dawley (n=60), C57BL/6 mice (n=64) and G-substrate knockout (G-substrate-/-) mice (n=36). All animals were maintained and handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Immunostaining with antibodies specific for the 145 kD fragment of α-fodrin, phosphorylated Akt, G-substrate was performed in NMDA-treated retina in the presence or absence of calpain inhibitors. Degeneration of amacrine cells was evaluated by TUNEL assay 24 hours after the injury or measuring retinal thickness 7 days after the injury.

Results: : NMDA induced the dephosphorylation of Akt in dying amacrine cells, and ALLN, a calpain inhibitor, prevented NMDA-induced Akt dephosphorylation. ALLN also prevented the death of TUNEL (+) amacrine cells (Control: 66.7±18.6 cells , ALLN: 26.8±12.9 cells, p<0.0001). G-substrate, an endogenous inhibitor of protein phosphatase, presents in amacrine cells. To elucidate physiological function of G-substrate was examined in NMDA-induced amacrine cell death. Immunohistochemical analysis revealed that NMDA treatment led to immediate reduction of G-substrate immunoreactivity and the reduction was blocked in the presence of ALLN. Moreover, 3nmol NMDA caused more TUNNE (+) cells in G-substrate-/- mice (81.9±19.2 cells) than in wild-type mice (2.7±8.2 cells; p<0.0001). ALLN did not prevent the appearance of TUNEL (+) cells and nor loss of amacrine cells in G-substrate-/- mice.

Conclusions: : The results strongly suggest that calpain-mediated loss of G-substrate may represent a critical mechanism for NMDA-induced amacrine cell death. These new findings shed light on the pathogenesis of retinal ischemic diseases.

Keywords: excitatory amino acid receptors • signal transduction • apoptosis/cell death 

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