Abstract
Purpose: :
Our previous work showed that the transcription factor Max is found essentially within the nucleus in healthy retinal ganglion cells (RGC), but when rat retinas were explanted in vitro, Max rapidly disappeared from the nucleus and was found in the cytoplasm of RGC, preceding nuclear condensation and the retrograde RGC degeneration caused by axon damage at explantation. This still ocurred under treatment with a pan-caspase inhibitor, which, nonetheless prevented programmed cell death (PCD). Here we further examined mechanisms of nuclear exclusion and tested for a neuroprotective role of Max upon RGC.
Methods: :
We used rat retinal explants in vitro and optic nerve crush in vivo, detected Max by both immunohistochemistry and Western Blot of nuclear and cytoplasmic fractions, and tested whether overexpression of Max following intravitreal injection of a recombinant adeno-associated viral vector (rAAV-Max) affects RGC resistance to cell death.
Results: :
Neither the calpain inhibitor calpeptin, nor the Crm1 exportin inhibitor leptomycin B prevented either nuclear exclusion of Max or PCD. Incubation at low temperature prevented nuclear exclusion of Max, whereas blockade of protein translation prevented both nuclear exclusion and PCD. The proteasome inhibitor lactacystin prevented both the loss of nuclear Max and PCD, while newly-synthesized Max still accumulated in the cytoplasm of the axon-damaged RGC. The rAAV-Max increased nuclear Max content and prevented RGC degeneration in the explants. Furthermore, overexpression of Max produced by intravitreal injection of rAAV-Max prevented RGC degeneration in vivo following optic nerve crush in adult rats.
Conclusions: :
Nuclear exclusion of Max is an integral event of PCD following axon damage in RGCs, and is due to nuclear proteasomal degradation coupled with blockade of nucleo-cytoplasmic transport, upstream of caspase activation. In addition, overexpressed Max is neuroprotective for axon damaged RGCs, suggesting that rAAV-Max may lead to a novel gene therapeutic approach to glaucoma.
Keywords: cell survival • gene transfer/gene therapy • retinal degenerations: cell biology