Transcription Factor Brn-3b Overexpression Enhances Neurite Outgrowth in PC12 Cells Under Condition of Hypoxia

Abstract

Background Transcription factor Brn-3b plays a key role in retinal ganglion cell differentiation, survival and axon outgrowth during development. However, the precise role of Brn-3b in the normal adult retina as well as during neurodegeneration is unclear. In the current study, the effect of overexpression of Brn-3b was assessed in vitro, in PC12 cells under conditions of normoxia and hypoxia. Results Immunoblot analysis showed that overexpression of Brn-3b in PC12 cells as well as 661W cells produced significant increase in the growth cone marker, growth associated protein-43 (GAP-43) and acetylated-tubulin (ac-TUBA). In addition, an increased immunostaining for GAP-43 and ac-TUBA was observed in PC12 cells overexpressing Brn-3b, which was accompanied by a marked increase in neurite outgrowth, compared to PC12 cells overexpressing empty vector. In separate experiments, one set of PC12 cells transfected either with a Brn-3b expression vector or an empty vector was subjected to conditions of hypoxia for 2h, while another set of similarly transfected PC12 cells was maintained in normoxic conditions. It was found that the upregulation of GAP-43 and ac-TUBA in PC12 cells overexpressing Brn-3b under conditions of normoxia was sustained under conditions of hypoxia. Immunocytochemical analysis revealed not only an upregulation of GAP-43 and ac-TUBA, but also increased neurite outgrowth in PC12 cells transfected with Brn-3b as compared to PC12 cells transfected with empty vector in both normoxia and hypoxia. Conclusions The findings have implications for a potential role of Brn-3b in neurodegenerative diseases in which hypoxia/ischemia contribute to pathophysiology of the disease.

Analysis of axonal growth in organotypic neural cultures

“…This program, implemented as a plug-in for ImageJ software, markedly reduces the time needed in the manual analysis, improves the accuracy and increases the amount of information obtained from each sample. Therefore, the organotypic 3D cultures and the computed aided method constitute a powerful and useful tool to obtain valuable data of neurite growth under different conditions (Allodi et al, 2011(Allodi et al, , 2013(Allodi et al, , 2014Daud et al, 2012;Farrell et al, 2012;Auer et al, 2013;Forostyak et al, 2013;Jin et al, 2013;Parsons 2013;Riggio et al, 2013;Torres-Espín et al, 2013Gerardo-Nava et al, 2014;Schizas et al, 2014a,b;Assunção-Silva et al, 2015;Chitsaz et al, 2015;Geuna et al, 2015;Kraus et al, 2015;Mòdol et al, 2015;Park et al, 2015;Parra et al, 2015;Phatak et al, 2015;Tong et al, 2015;Gonzalez-Perez et al, 2016;. The objective of the present work is to provide the protocol of our DRG and SC slice cultures, from the animal to the image analysis, that will allow studying neurite outgrowth in reliable in vitro models.…”