O‐GLcNAc post‐translational modifications regulate the entry of neurons into an axon branching program

Many neuronal cytosolic and nuclear proteins are post‐translationally modified by the reversible addition of O‐linked N‐acetylglucosamine (O‐GlcNAc) on serines and threonines. The cellular functions of O‐GlcNAc modifications in neuronal development are not known. We report that O‐GlcNAc‐modified proteins are distributed nonuniformly throughout cultured primary chicken forebrain neurons, with intense immunostaining of the cell body, punctuate immunostaining in axons and all processes, and localization in filopodia/lamellipodia. Overexpression of O‐GlcNAcase, the enzyme that removes O‐GlcNAc from proteins, increased the percentage of neurons exhibiting axon branching without altering the frequency of axon branches on a per neuron basis and increased the numbers of axonal filopodia. Conversely, pharmacologically increasing O‐GlcNAc levels on proteins through specific inhibition of O‐GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching. Treatment with an alternative O‐GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia. Furthermore, axon branching induced by the adenylyl cyclase activator forskolin was suppressed by pharmacological inhibition of O‐GlcNAcase. Western analysis revealed that O‐GlcNAc levels regulate the phosphorylation of some PKA substrates in response to forskolin. These data provide the first evidence of O‐GlcNAc modification‐specific influences in neuronal development in primary culture, and indicate specific roles for O‐GlcNAc in the regulation of axon morphology. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009