Cyclin-Dependent Kinase Inhibitor 2b Attenuates Excitotoxicity-Induced Death of Retinal Ganglion Cells

Purpose: Glutamate excitotoxicity seems to contribute to retinal ganglion cell (RGC) death in various eye diseases, but the underlying molecular mechanisms are not fully understood. We studied the roles of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, known as cellular stress-related senescence markers, in N-methyl-d-aspartate (NMDA)-induced RGC death. Methods: Gene expression was analyzed using quantitative reverse transcription (qRT)-PCR, in situ hybridization, and immunochemistry. Cdkn2a and Cdkn2b gain- and loss-of-function experiments were performed using the adeno-associated virus type 2 (AAV2)-mediated gene delivery system. AAV2-CRISPR-Cas9-mediated knockout of Cdkn2a or Cdkn2b was validated using cultured cells by T7 endonuclease I assay and Western blot analysis. The effects of altered expression of Cdkn2a and Cdkn2b on NMDA-induced RGC death were evaluated by quantification of RNA binding protein with multiple splicing (Rbpms)-immunoreactive RGCs. Results: Intravitreal NMDA injection resulted in upregulation of Cdkn2a and Cdkn2b expression in RGCs of the mouse retina. AAV2-mediated overexpression of Cdkn2b led to increased expression of Cdkn2a in RGCs, but not vice versa. Overexpression of Cdkn2b, but not Cdkn2a, resulted in a further reduction in RGC viability in NMDA-injected retinas. However, excessive levels of Cdkn2a or Cdkn2b had no effect on RGC viability in healthy mice. AAV2-CRISPR-Cas9-mediated knockout of either Cdkn2a or Cdkn2b attenuated NMDA-induced RGC death. Conclusions: Cdkn2a and Cdkn2b have pivotal roles in the regulation of excitotoxic RGC degeneration under NMDA-induced pathologic conditions. Our findings imply that Cdkn2a and Cdkn2b are novel therapeutic targets for ocular diseases displaying excitotoxicity-induced neuronal degeneration.