Elevation of p-NR2AS1232 by Cdk5/p35 contributes to retinal ganglion cell apoptosis in a rat experimental glaucoma model
Research highlights
► Cdk5/p35 in the retina is activated by elevated IOP in a rat glaucoma model. ► Elevation of p-NR2AS1232 by activated Cdk5/p35 contributes to RGC apoptosis. ► Roscovitine ameliorates RGC apoptosis by inhibiting the activity of Cdk5/p35.
Introduction
Glaucoma, the second leading cause of blindness, is a neurodegenerative disease that is characterized by optic nerve degeneration resulting from apoptotic death of retinal ganglion cells (RGCs) (Guo et al., 2005, Hitchings, 2000, Resnikoff et al., 2004). Although elevated intraocular pressure (IOP) is commonly regarded as a hallmark risk factor (Quigley et al., 1995, Weinreb and Khaw, 2004), the pathogenesis of RGC death following intraocular hypertension is still poorly understood. The role of the excitotoxicity induced by glutamate (Glu), a major excitatory neurotransmitter in the retina (Thoreson and Witkovsky, 1999, Yang, 2004), in neurodegeneration in glaucoma models is still a controversial issue (Dreyer et al., 1996, Levkovitch-Verbin et al., 2002, Ullian et al., 2004). Nevertheless, lots of evidence suggest that the excess of extracellular Glu may be a potential risky factor for retinal malfunction in glaucoma (Guo et al., 2006, Levin, 2003, Salt and Cordeiro, 2006). Indeed, expression of Glu transporters is significantly reduced in rat glaucoma models, indicating frustration of the effective buffering of extracellular Glu (Martin et al., 2002, Vorwerk et al., 2000). Moreover, prolonged injection of Glu of low-concentration was shown to induce RGC death in rat (Nucci et al., 2005). Glu-induced apoptotic death of RGCs is known to be primarily mediated by the N-methyl-d-aspartate (NMDA) subtype receptor (Guo et al., 2006, Lipton, 2001, Seki and Lipton, 2008), and the NMDA channel blocker MK-801/memantine indeed prevents RGC death in experimental rat glaucoma models (Calzada et al., 2002, Chaudhary et al., 1998, Guo et al., 2006, Hare et al., 2004, Woldemussie et al., 2002).
Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, has multiple roles in neural development and synaptic plasticity by phosphorylating numerous synaptic substrates (Dhavan and Tsai, 2001, Ko et al., 2001, Lee et al., 2004, Morabito et al., 2004, Tomizawa et al., 2002). Cdk5 is expressed ubiquitously, but shows high activity exclusively in the central nervous system (CNS) due to the restricted distribution of its activators (Tang et al., 1995). P35, an essential activator of Cdk5 (Chae et al., 1997, Ko et al., 2001), restricts the expression of active Cdk5 primarily to post-mitotic neurons (Tang et al., 1995, Tsai et al., 1994). The dysregulation of Cdk5/p35 has been implicated in many neurological disorders (Borghi et al., 2002, Bu et al., 2002, Chen and Wang, 2010, Lee et al., 1999, Nguyen et al., 2003, Smith et al., 2003). There is accumulating evidence suggesting that Cdk5 may be a “Jekyll and Hyde” kinase in that it takes different responsibilities under different conditions (Cruz and Tsai, 2004). 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 (Wang et al., 2003). In contrast, Cdk5 is shown to prevent neuronal apoptosis through ERK-mediated upregulation of B-cell lymphoma protein 2 (Bcl-2) in vitro (Wang et al., 2006) and by directly phosphorylating Bcl-2 at Ser70 site in rat cortical neurons (Cheung et al., 2008), respectively. Furthermore, Cdk5 may exert a pro-survival role by negatively regulating c-Jun N-terminal kinase 3 (Bu et al., 2002) and/or by participating in the neuregulin-dependent activation of PI3K and Akt pathway (Li et al., 2003). In the present study we investigate the possible involvement of Cdk5/p35 in the pathogenesis of RGCs in a rat experimental glaucoma model. Our results demonstrate that activation of Cdk5/p35 causes an elevation of p-NR2AS1232 that likely contributes to RGC apoptotic death in this model and this defect is significantly ameliorated by roscovitine, an inhibitor of Cdk5/p35.
Section snippets
Animals and glaucoma model
All experimental procedures described here were carried out in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals and the guidelines of Fudan University on the ethical use of animals. During this study all efforts were made to minimize the number of animals used and their suffering. Male Sprague–Dawley rats weighing 100–300 g obtained from SLAC Laboratory Animal Co. Ltd (Shanghai, China) were housed on a 12 h light/dark schedule, with
Steady elevation of IOP and progressive apoptotic death of RGCs in rat glaucoma model
Changes of IOP in operated right eyes and contralateral left eyes of rats were monitored. As reported in previous studies, using glaucoma rats produced by similar methods (Naka et al., 2010, Wu et al., 2010, Yu et al., 2006), the average IOP in the operated eyes showed a sharp increase to 32.0 ± 0.4 mmHg on D1 after operation [vs 16.9 ± 0.2 mm Hg before operation (D0), n = 73, P all < 0.001 vs D0 and the contralateral left eyes, one-way ANOVA with Bonferroni post test and paired t test]. The IOP declined
Discussion
In our rat glaucoma model, RGC apoptosis was first detected on D14 (Fig. 2), which was in parallel with the decrease of RGC number, suggesting that apoptotic death may be a main cause for the RGC number decrease (Doh et al., 2010, Ju et al., 2008). It should be noted that blocking of the episcleral veins may unavoidably lead to retinal ischemia, in addition to elevating IOP, which would also result in RGC death (Chen et al., 2007, Schmidt et al., 2004), just as it occurred in all other IOP
Acknowledgments
We would like to thank Dr. Xiong-Li Yang for his helpful discussion and critical comments on the manuscript. We also thank Dr. Xiang-Tian Zhou at Wenzhou Medical College and Dr. Xiang-Ge He in Daping Hospital affiliated to the Third Military Medical University for their kind help in reproduction of the rat glaucoma model. This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2007CB512205; 2011CB504602), the
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