Abstract
Purpose: :
During NMDA–induced cell death in the neural retina, there is an elevation of the nuclear isoform of CaMKIIα (CaMKIIαB). This result leads to the question of how CaMKIIαB might be involved in either a cell death or cell survival pathway, for example, in retinal ganglion cells (RGCs). The purpose of this study is to investigate if CaMKIIα regulates BDNF expression in RGCs.
Methods: :
Highly purified RGCs or dissociated retinal cells were obtained from P6–8 SD rat eyes and treated with glutamate (200–1000uM) for the indicated times. Glutamate cytotoxicity on RGCs and localization of CaMKIIα was determined by cell counting and immunostaining, respectively. To identify the role of CaMKIIα in regulating BDNF expression, CaMKIIαB expression vector was constructed and over–expressed in cells of the RGC–5 cell line, and cell viability was assayed. Specific siRNAs to knock down CaMKIIαB or CaMKIIα were then tested in CaMKIIαB–transfected or non–transfected RGC–5 cells. The siRNAs were also used to knock down endogenous CaMKIIα or CaMKIIαB in purified RGCs or in dissociated retinal cells. Expression levels of BDNF and CaMKIIα or CaMKIIαB were determined by real–time PCR, western blots and/or immunofluorescence microscopy.
Results: :
CaMKIIα is redistributed in RGCs after glutamate treatment such that more of the CaMKIIα was localized to the nucleus. In the CaMKIIαB transfected RGC–5 cells, CaMKIIαB located in the nucleus of the cells. BDNF expression was increased in these cells even in conditions without glutamate induced stress. Further, these cells were more resistant to glutamate treatment than non–transfected cells. Knockdown of CaMKIIα by RNAi resulted in decreased expression of BDNF in response to glutamate treatment both at mRNA and protein level in the RGC–5 cells. RNA interference using siRNA in highly purified RGCs or dissociated retinal cells was practicable. Application of specific siRNA for CaMKIIα or CaMKIIαB reduced the expression level of CaMKIIα in purified RGCs. Corresponding to the knockdown of CaMKIIα, BDNF was reduced and the cell death was enhanced.
Conclusions: :
Our data suggest that the nuclear isoform of CaMKIIα is involved in glutamate induced cell survival response in RGCs. CaMKIIαB seems to be required for glutamate–mediated enhanced BDNF expression.
Keywords: ganglion cells • cell survival • growth factors/growth factor receptors