Nuclear Exclusion of the Transcription Factor Max: A Novel Event Associated With Retinal Ganglion Cell Death

Abstract

Abstract: : Purpose: The necessity of protein synthesis for programmed ganglion cell death raises the idea of studying the participation of transcription factors in this process. Various transcription factors are associated with both differentiation and programmed cell death. The nucleocytoplasmic transport is essential for both of these activities. Previously we found alteration of subcellular localization of the transcription factor Max during programmed cell death in retinal ganglion cells. Healthy ganglion cells present Max inside the nucleus both in vivo an in vitro, and this immunoreactivity increases with development. Upon induction of cell death, Max was excluded from the nucleus and found in the cytoplasm of ganglion cells, a process that precedes nuclear condensation and is accelerated by thapsigargin. The control mechanisms of Max nuclear exclusion during programmed cell death were now studied using different approaches. Methods: Explants from retina of 13-day-old rats were maintained in vitro for various time periods. Apoptotic cells were detected by chromatin condensation and TUNEL. Max was detected by immunohistochemistry. The involvement of caspase activation with nuclear exclusion of Max were tested using the inhibitor of caspase activity BAF (100mM). Nucleo-cytoplasmic active transport was inhibited by low temperature (4°C) during 12h in vitro. The involvement of the nuclear export receptor CRM-1 with Max nuclear exclusion were also tested using Leptomycin B (3-30nM). Results: BAF did not block nuclear exclusion of Max. In low temperature, Max was kept inside the nucleus, showing that exclusion of Max was not a result of passive diffusion. The inhibitor of the CRM-1 receptor did not block the nuclear exclusion of Max. Conclusions:These data suggest that the nuclear exclusion of Max require active processes however independent of caspase activation. In addition, Max is not exported from nucleus to the cytoplasm by the CRM-1 receptor during programmed cell death. These results indicate that nuclear exclusion of Max is an integral event of programmed cell death in the central nervous system, and suggest that during programmed cell death Max does not act as a transcription factor.

Keywords: cell death/apoptosis • ganglion cells • transcription factors