Phosphorylated IKBalpha is highly enriched in the axon initial segment

The transcription factor NF-KappaB (NF-KB) is present in the cytosol in an inactive state, complexed with the inhibitory protein IKappaBα. Constitutive activation of NF-KB occurs in neurons of the central nervous system via phosphorylation of IKappaBα at Ser32 and Ser36 and subsequent degradation of phospho-IKappaBα. We studied this constitutive neuronal activation of NF-KB in brain sections of several mammals (mice, rats, rhesus monkeys, humans) using immunostaining with polyclonal and monoclonal antibodies directed against IKappaBα phosphorylated at Ser32 and Ser36. Strikingly, a highly selective enrichment of phospho-IKappaBα was found in the axon initial segment of all species examined. Specificity of the immunostaining was demonstrated by preincubating the anti-phospho-IKappaBα antibodies with the corresponding phospho-IKappaBα blocking peptides which completely prevented immunolabeling. The selective axonal localization of phospho-IKappaBα was confirmed by double-immunostaining with antibodies directed against the axonal protein tau and the dendritic microtubule-associated protein 2 (MAP2). Distal segments of axons, dendrites and cell bodies were either negative for phospho-IKappaBα or only faintly labeled. Likewise, glial cells were phospho-IKappaBα-negative. A similiar selective localization of phospho-IKappaBα in the axon initial segment was found in organotypic slice cultures and dissociated primary neuron cultures prepared from rat hippocampi. Taken together, these findings implicate the axon initial segment as a cell compartment which is crucial for neuronal activation of NF-KB.