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Melina Morkin, Ephraim Trakhtenberg, Yan Wang, Stephanie Fernandez, Gregory Mlacker, Jeffrey Goldberg; Regulation of Set-β’s subcellular localization and posttranslational modifications affect axon growth and regeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2473.
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© ARVO (1962-2015); The Authors (2016-present)
Adult mammalian central nervous system (CNS) neurons are unable to regrow axons after injury, but immature CNS axons can regenerate. Manipulation of various cell-autonomous factors along with overcoming the inhibitory adult CNS environment only partially restores regeneration. However, regenerative capacity of CNS neurons themselves declines after birth. We found that an epigenetic factor Set-β, previously reported to be upregulated in Alzheimer’s disease patients’ neurons, is also postnatally upregulated in retinal ganglion cells’ nuclei. Here we investigated the role of Set-β in axon growth of RGCs.
Embryonic and postnatal rat retinal sections were co-stained against RGC marker Brn3b and Set-β; immunofluorescence intensity was analyzed with AxioVision. Cytoplasmic and nuclear fractions of P5 RGCs were separated and immunoblotted for Set-β. Wild-type Set-β, myristoylated-Set-β, Set-β serine-9 phospho-mutants and nuclear localization signal deletion mutants, or anti-Set-β shRNAs were overexpressed in purified P4 RGCs or E18 hippocampal neurons, which were then immunostained and imaged after 1-3 days in vitro. Neurite length was quantified using the Image J Neurite Tracer.
We found that in RGCs, full-length 39 kDa Set-β is predominantly nuclear whereas a shorter 25 kDa isoform is predominately cytoplasmic. Set-β overexpressed in postnatal RGCs or embryonic hippocampal neurons localized to the nucleus and suppressed axon growth. In contrast, experimentally increased myristoylated-Set-β cytoplasmic localization, or shRNA knockdown of Set-β, promoted axon growth. Serine-9 phosphorylation of Set-β blocks Set-β’s ability to suppress axon growth. Finally, we showed that in the CNS, Set-β binds selectively to PP2A-A-β but not α isoform; PP2A was recently shown to modulate axon growth in CNS.
Full-length Set-β is developmentally upregulated in CNS primary neurons’ nuclei whereas a shorter isoform is found in the cytoplasm. Set-β inhibits or promotes axon growth in the CNS neurons depending on its subcellular localization and serine-9 phosphorylation, and Set-β binds selectively to PP2A-A-β but not α isoform. Manipulation of Set-β in vivo may provide a strategy to enhance regeneration.
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