Set-β Subcellular Localization-dependent Regulation Of Retinal Ganglion Cell Neurite Growth

Abstract

Purpose: : Adult mammalian central nervous system (CNS) axons are unable to regrow their axons after injury, but immature CNS axons grow robustly. Manipulation of various cell-autonomous factors along with overcoming the inhibitory adult CNS environment only partially restores regeneration. Recent evidence suggests that postnatally, CNS neurons themselves decrease their intrinsic regenerative capacity. Here we investigated whether Set-β oncoprotein is developmentally regulated, whether it affects axon growth, and whether subcellular localization and posttranslational modifications modulate its effect on axon growth.

Methods: : Embryoinc and postnatal rat retinal sections were immunostained against retinal ganglion cell (RGC) marker Brn3b and Set-β. Immunofluorescence intensity was analyzed with AxioVision (Zeiss). Set-β, myristoylated (myr)-Set-β, Set-β phospho-mutants, and mCherry were overexpressed in purified P3 RGCs, incubated for 24 hours, immunostained against transfection and neurite markers, and imaged. Neurite length was quantified using ImageJ Plugin Neurite Tracer. Data was analyzed with SPSS, by ANOVA with post hoc test.

Results: : We found that Set-β is developmentally upregulated in RGCs’ nuclei. Set-β overexpressed in RGCs in vitro localized to the nucleus and suppressed neurite growth. In contrast, experimentally increased myr-Set-β cytoplasmic localization enhanced axon growth. We also found that N-terminal phosphorylation of Set-β blocks nuclear Set-β’s ability to suppress neurite growth.

Conclusions: : Set-β inhibits or promotes axon growth in RGCs depending on its subcellular localization and N-terminal phosphorylation. Further examination of whether Set-β regulates RGC axon regeneration in vivo may be warranted.

Keywords: optic nerve • retinal development • regeneration