Elevation Of P-NR2A^S1232 By Cdk5/p35 Contributes To Retinal Ganglion Cell Apoptosis In Rat Chronic Ocular Hypertension Model

Glaucoma, the second leading cause of blindness, is characterized by optic nerve degeneration resulting from apoptotic death of retinal ganglion cells (RGCs). Although elevated intraocular pressure (IOP) is commonly regarded as a hallmark risk factor, the pathogenesis of RGC death following intraocular hypertension is still poorly understood. While the role of the excitotoxicity induced by glutamate, a major excitatory neurotransmitter in the mammalian retina, in neurodegeneration of glaucoma models is still a controversial issue, lots of evidence suggest that the excess of extracellular glutamate may be a potential risky factor for retinal injury in glaucoma. Many studies reported that the intraocular concentration of glutamate in the glaucoma patients and animal models are overly elevated, and excess glutamate may increase calcium influx and intracellular calcium overload through the activation of NMDA receptors in RGCs (but there are some reports showing that the concentration of glutamate is not increased in glaucoma models). Indeed, glutamate transporters were found to be significantly reduced in a rat glaucoma model, indicating frustration of the effective buffering of extracellular glutamate. Furthermore, prolonged injection of low-concentration glutamate was shown to induce RGC death in rats. On the other hand, the NMDA channel blocker MK-801 or memantine prevented RGC death in experimental rat models of glaucoma.Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is expressed with high activity exclusively in the central nervous system (CNS) and has multiple roles in neural development and synaptic plasticity by phosphorylating numerous synaptic substrates. p35, an essential activator of Cdk5, restricts the expression of active Cdk5 primarily to post-mitotic neurons. The deregulation of Cdk5/p35 has been implicated in many neurological disorders. There is accumulating evidence suggesting that Cdk5 may be a "Jekyll and Hyde" kinase in that it takes different responsibilities under different conditions. For instance, activation of Cdk5 induces hippocampal CA1 cell death by directly phosphorylating the NR2A subunit at S1232 site (p-NR2AS1232) in an ischemic rat model. In contrast, Cdk5 is shown to prevent neuronal apoptosis through ERK-mediated upregulation of B-cell lymphoma protein 2 (Bcl-2) in vitro or by directly phosphorylating Bcl-2 at Ser70 site in rat cortical neurons. Furthermore, Cdk5 may exert a pro-survival role by negatively regulating c-Jun N-terminal kinase 3 and/or by participating in the neuregulin-dependent activation of PI3K and Akt pathway. Cdk5 and p35 are also widely distributed in the retina. We wonder whether Cdk5 and p35 may contribute to the apoptotic death of RGCs in experimental glaucoma models.In the present work, we reproduced the rat chronic ocular hypertension (COH) model by ligating three trunks of episcleral veins and slightly cauterization. Hematoxylin & Eosin (HE) staining and TUNEL staining were applied to assess degenerative and apoptotic changes of the retina. Cholera Toxin subunit B (CTB)-retrogradely labeling and immunocytochemistry were used to identify RGCs and analyze the distribution of p-NR2AS1232. Western blot was used to explore changes in expression of NMDA receptors and Cdk5/p35, and possible correlation of Cdk5 and p-NR2AS1232 was examined by co-immunoprecipitation. The Cdk5 inhibitor roscovitine was injected intraperitoneally from day 17 after operation once daily till day 28 at a dose of 2.8 mg/kg to examine its effect on RGC survival.The IOPs of the operated eyes were at higher levels ranging from 23.6±0.4 to 26.6±0.3 mmHg on day 3-28 after surgery (versus 16.9±0.2 mmHg before operation). The images of H-E stained retinal sections showed that on day 28 the ganglion cell layer (GCL) and inner plexiform layer (IPL) slightly shrunk in thickness and cells in the GCL tended to become sparse. TUNEL staining further showed that TUNEL-positive signals were found in the GCL on days 21 and 28 after operation, indicating cell apoptosis. At the same time, a steady decrease in number of CTB-labeled RGCs with time was found on days 21 and 28, as compared to that in the normal eyes. The ratio (CTB-labeled RGCs/GCL cells) significantly decreased on days 21 and 28.In the operated eyes the expression level of Cdk5 decreased on day 7 and then significantly increased on day 21, whereas that of p35 markedly on days 14 and 21, as compared to the control eyes. Meanwhile, the ratio of p-NR2AS1232/NR2A first decreased on day 14 and then increased on days 21 and 28 after operation, which was in parallel with the expression change of Cdk5 and p35, suggesting that activated Cdk5/p35 may be involved in apoptotic death of RGCs by increasing the phosphorylation of NR2A at S1232. Co-immunoprecipitation experiments further confirmed the direct correlation between Cdk5 and p-NR2AS1232. The immunocytochemistry experiments clearly showed a redistribution of p-NR2AS1232 on day 21, i.e., a decrease of p-NR2AS1232 in the nuclei of GCL cells and an increase in the IPL, where RGC dendrites are located, suggesting that the increased functional NMDA receptors in RGC dendrites may accelerate apoptotic death of these cells in glaucoma.Administration of roscovitine almost eliminated TUNEL signals in the GCL on day 28, in contrast with that observed in the DMSO-injected rats, in which positive TUNEL signals clearly appeared in the GCL. Furthermore, roscovitine substantially inhibited the decrease of the number of CTB-positive RGCs in the COH model. Western blot results showed that roscovitine significantly reduced the expression of p35 and p-NR2AS1232 on day 28, suggesting that the neuroprotective effect of roscovitine in experimental glaucoma may be mediated by decreasing the production of p-NR2AS1232 through an inhibition of Cdk5/p35 activity.From the above work, we conclude that activation of Cdk5/p35 directly causes an elevation of p-NR2AS1232 that likely contributes to RGC apoptotic death in the COH rat model, and this defect is much ameliorated by roscovitine, an inhibitor of Cdk5/p35. Our results broaden our knowledge about the role of Cdk5 in neuronal survival/degeneration balance and also provide a potentially effective therapy for glaucoma.