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.
Francisco
Herb
H
Department of Neurobiology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
Kollins
Katherine
K
Varghis
Neal
N
Vocadlo
David
D
Vosseller
Keith
K
Gallo
Gianluca
G
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D005576:Colforsin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020439:Growth Cones / Q000187:drug effects / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010091:Oximes / Q000494:pharmacology; D010590:Phalloidine / Q000378:metabolism; D048448:Phenylcarbamates / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D016548:Prosencephalon / Q000166:cytology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013997:Time Factors; D014162:Transfection / Q000379:methods
D004791:Enzyme Inhibitors; D010091:Oximes; D048448:Phenylcarbamates; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D010590:Phalloidine; D005576:Colforsin; D017351:N-Acetylglucosaminyltransferases; D000117:Acetylglucosamine