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Karan Patel, Ephraim Trakhtenberg, Yan Wang, Melina Isabel Morkin, Stephanie Fernandez, Gregory Mlacker, Kapil Gupta, Susan Dombrowski, Xiongfei Liu, Jeffrey L Goldberg; Set-β regulates retinal ganglion cells’ neurite growth and optic nerve regeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2387.
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© ARVO (1962-2015); The Authors (2016-present)
The failure of the adult mammalian central nervous system (CNS) neurons to regenerate axons is a major clinical problem associated with traumatic and ischemic optic neuropathies and glaucoma. Transcriptional regulators like Set-β are well-positioned to regulate intrinsic axon regeneration ability, which declines developmentally in maturing CNS neurons. Set-β also functions at cellular membranes and its subcellular localization is already known to be disrupted in Alzheimer’s disease, but many of its other biological mechanisms have not yet been explored in neurons. Here, we investigated Set-β’s role in retinal ganglion cells (RGC) neurite growth and axon regeneration.
Embryonic and postnatal rat retinal sections were immunostained against RGC marker Brn3b and Set-β. Immunofluorescence intensity was analyzed with AxioVision (Zeiss). Set-β mRNA expression in RGCs was analyzed with qRT-PCR at E19, P8, and P21. Set-β, myristoylated (myr)-Set-β, Set-β mutants, PP2A-A, and mCherry were overexpressed in purified P3-4 RGCs, and Neurite length was quantified using ImageJ Plugin Neurite Tracer. For in vivo, myr-Set-β or GFP were delivered intravitreally through AAV2 into anesthetized animals prior to the optic nerve crush. Analysis with ANOVA and post hoc test (SPSS).
We found that Set-β was upregulated postnatally in RGCs, and was primarily localized to the nucleus but was also detected adjacent to the plasma membrane. Remarkably, nuclear Set-β suppressed whereas cytoplasmic membrane Set-β promoted neurite growth in rodent RGC and hippocampal neurons. Mimicking serine 9 phosphorylation delayed nuclear import and furthermore blocked the ability of nuclear Set-β to suppress neurite growth. We also showed that nuclear Set-β regulates transcription of multiple genes in RGCs, identified Set-β’s cytoplasmic binding partner PP2A-A which suppressed neurite growth, and detected a novel isoform of Set-β transcript lacking the nuclear localization signal. Finally, we demonstrated that increasing recruitment of Set-β to cellular membranes promoted optic nerve axon regeneration after injury in vivo in adult rats.
Set-β differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation, and myr-Set-β gene therapy could be relevant for developing treatments for optic neuropathies.
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