Early Phosphorylation of Calcium / Calmodulin-Dependent Protein Kinase II in NMDA-Induced Retinal Damage

Abstract

Purpose:: To evaluate the penetration of CaMK II siRNA and determine the role of the phosphorylation of CaMK II in NMDA-induced neurotoxicity in the rat retina.

Methods:: 500 pmol of CaMK II siRNA or Cy3-labeled CaMK II siRNA was injected intravitreally 2 hr before NMDA injection. Penetration of Cy3-labeled CaMK II siRNA was examined by immunohistochemistry. Time course of p-CaMK II protein level after NMDA injection and the suppressive effect of CaMK II siRNA on p-CaMK II protein were evaluated by Western blot analysis. Retrograde labeling analysis was performed to determine the effect of CaMK II siRNA on retinal ganglion cells (RGCs) death after NMDA injection.

Results:: At 2 hr after Cy3-labeled CaMK II siRNA injection, an uptake of labeled siRNA was mainly observed in the RGC layer (RGCL). The uptake was also seen in the RGCL 1 hr after NMDA injection and this uptake appeared to be apparent not only in the RGCL but also the inner nuclear layer 6 hr after injection. Western analysis showed that the level of p-CaMK II protein was significantly increased from 30 min to 3 hr after NMDA injection. The early induction of p-CaMK II was significantly inhibited by pretreatment of CaMK II siRNA. Retrograde labeling analysis revealed that the pretreatment of CaMK II siRNA accelerated NMDA-induced RGCs losses.

Conclusions:: Early phosphorylation of CaMK II may occur as an endogenous protective function in NMDA-induced retinal neurotoxicity.

Keywords: calcium • ganglion cells • retina: neurochemistry