Set-beta protein plays different roles in neurons, but the diversity of Set-beta neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-beta are altered in Alzheimer disease, cleavage of Set-beta leads to neuronal death after stroke, and the full-length Set-beta regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-beta isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-beta-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-beta transcript lacking the nuclear localization signal and demonstrated that the full-length (similar to 39-kDa) Set-beta is localized predominantly in the nucleus, whereas a shorter (similar to 25-kDa) Set-beta isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-beta cleavage product can induce neuronal death.

The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-β are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-β also functions at cellular membranes and its subcellular localization is disrupted in Alzheimer's disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-β was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-β suppressed, whereas Set-β localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimer's disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-β to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-β in primary neurons, raising the hypothesis that nuclear Set-β may preferentially regulate gene expression whereas Set-β at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growthin vitro. Finally, increasing recruitment of Set-β to cellular membranes promoted adult rat optic nerve axon regeneration after injuryin vivo. Thus, Set-β differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation.