title: Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization.
abstract: NeuN (neuronal nuclei) is an antigen used widely in research and diagnostics to identify postmitotic neurons. The present study aims at an initial understanding of the molecular nature and functional significance of this as yet ill-defined antigen. Using isoelectric focusing, both the 46- and 48-kDa isoforms of NeuN can be separated in multiple spots spanning a pH range of 8-10.5, suggesting that they might be phosphorylated. Enzymatic dephosphorylation abolishes NeuN immunoreactivity, confirming that NeuN is indeed a phosphoprotein, and establishing that binding of the defining antibody depends on its state of phosphorylation. Combined biochemical and immunohistochemical analysis show that both the 46- and the 48-kDa NeuN isoforms can be localized to the cell nucleus as well as in the neuronal cytoplasm. Their relative concentration in these compartments is distinct, however, with the 48-kDa isoform being the predominant isoform in the cytoplasm. Within the nucleus, NeuN is found preferentially in areas of low chromatin density and virtually excluded from areas containing densely packed DNA. The present identification of multiple differentially phosphorylated isoforms of NeuN, together with recent reports on the dependence of NeuN immunoreactivity levels on a variety of physiologic or pathologic signals, suggests a previously unappreciated level of complexity in the regulation of this enigmatic, neuron-specific antigen.
authors.affiliation: Anatomisches Institut, Anatomie und Zellbiologie, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Lind
authors.forename: Sebastian
authors.initials: S
authors.lastname: Franken
authors.forename: Joachim
authors.initials: J
authors.lastname: Kappler
authors.forename: Jakob
authors.initials: J
authors.lastname: Jankowski
authors.forename: Karl
authors.initials: K
authors.lastname: Schilling
mesh_terms: D000818:Animals; D000941:Antigens / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D001921:Brain / Q000378:metabolism*; D002451:Cell Compartmentation / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D005260:Female; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000378:metabolism*; D009687:Nuclear Proteins / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000378:metabolism
chemical_list: D000941:Antigens; D015415:Biomarkers; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C492380:NeuN protein, mouse; D009687:Nuclear Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms