OVEREXPRESSION OF THE POU DOMAIN TRANSCRIPTION FACTOR, BRN3B CAUSES NEURITE OUTGROWTH IN CULTURED PC 12 CELLS UNDER CONDITION OF OXYGEN GLUCOSE DEPRIVATION

Loss of vision is either due to the injury or diseases of the eye like glaucoma. In glaucoma, retinal ganglion cells and optic nerve are affected, leading to decrease in vision. so in order to maintain or improve the vision in this condition, We need to find out different modalities of treatment from conventional glaucoma treatment. The protein, we are working has promising effect on retinal ganglion cell survival and outgrowth. We will be using neuronal cell line as a model for retinal ganglion cells. Oxygen glucose deprivation insult will mimic glaucomatous conditions. Purpose (a): Brn3b is a POU domain transcription factor shown to play a key role in regulating retinal ganglion cell axon outgrowth during development. Hypoxia is a contributing factor in many neurodegenerative diseases including glaucoma. The purpose of this study was to determine if overexpression of Brn3b could promote neurite outgrowth in cultured PC 12 cells during conditions of oxygen glucose deprivation (OGD). Methods (b): Rat Pheochromocytoma cells ( PC 12) were grown on poly-D-lysine coated 100 mm dishes and transfected either with pCMV6-Brn3b (an expression vector encoding Brn3b) or pCMV6-Empty (empty vector). Following 6 h of transfection, cells were maintained overnight in a differentiating medium containing NGF (100ng/ml). Subsequently, the cells were transferred to glucose free DMEM and maintained for 2 h in 0.5% O2 and 5% CO2 (for hypoxia) in an Invivo2 200 hypoxia chamber. For the normoxia controls, PC12 cells overexpressing Brn3b or Empty vector were maintained in differentiating medium for 2 h in 5% CO2 and 95% air in a standard incubator. Protein extracts were isolated from these cells and analyzed for Brn3b and GAP43, TUBA-1 protein expression by immunoblot analysis. In another set of experiments, PC 12 cells were seeded on Poly-D-Lysine coated 25mm cover slips and transfected with either pCMV6-Brn3b or pCMV6 -Empty and maintained in differentiating medium for 4 days. The cells were subjected to either hypoxia (2h) or normoxia. Brn3b, GAP43 and TUBA-1 expression were analyzed using immunocytochemistry. Morphological changes in PC 12 cells transfected with Brn3b were studied by using LSM 510 confocal microscopy. Results (c): Immunoblot analysis confirmed overexpression of Brn3b in PC12 cells transfected with Brn3b cDNA in normoxic as well as in OGD conditions. Interestingly, a marked upregulation of GAP-43 and ac-TUBA expression was observed in Brn3b overexpressing cells under conditions of both normoxia and OGD. Overexpression of transcription factor Brn3b in PC12 cells produced a statistically significant increase in maximum neurite length and number of neurites per cell under conditions of both normoxia and OGD. A marked increase in immunostaining for Brn3b and neurite-specific GAP-43, TUBA-1 were also observed in PC12 cells overexpressing Brn3b in condition of normoxia and OGD. Conclusions (d): The POU domain transcription factor, Brn3b, could promote neurite outgrowth in PC12 cells under conditions of normoxia and as well as OGD.