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.

Changes in neuropeptide Y protein expression following photothrombotic brain infarction and epileptogenesis

“…NPY expression patterns were analyzed in conjunction with cellular markers that provided a clear demarcation of acute neuronal injury and neurodegeneration in and proximal to the infarct core as well as potential effects remote from the lesion. Although Nissl staining suggested that the hippocampus was spared direct injury by cortical photothrombosis and FJB-positive somata were not detected in the hippocampus at any time following photothrombosis, reduced NeuN IR in the hippocampus suggested some degree of neuronal dysfunction and/or a changed phosphorylation state of the NeuN protein (Lind et al, 2005). The distribution of increased GFAP IR was similar to that previously reported for the photothrombosis model (Bidmon et al, 2001b;Kharlamov et al, 2000;Oermann et al, 2004;Schroeter et al, 1995).…”

A morphologically distinct granule cell type in the dentate gyrus of the red fox correlates with adult hippocampal neurogenesis

“…Large granule cells contain scarce heterochromatin and stain more intensely for NeuN, whereas the evenly distributed fine heterochromatin granules in small granule cells stain less intensely for NeuN. This is in accordance with the observation that NeuN is expressed preferentially in areas of low chromatin density (Lind et al, 2005), indicating a higher cell activity in large granule cells. Thus, large granule cells may be also functionally related to adult born granule cells, as these cells have been shown to be more excitable than the resident granule cells (Schmidt-Hieber et al, 2004;Wang et al, 2000).…”

Heterogeneous Calretinin Expression in the Avian Cochlear Nucleus Angularis

“…Similar cellular expression profiles have been described in mouse and chicken, including a common lack of label in Purkinje cells of the cerebellum (Mezey et al 2012;Weyer and Schilling 2003). Specificity has been verified with Western blots using chicken protein extracted from whole brains which showed specific bands at estimated molecular weights of 46 and 48 kDa (Mezey et al 2012), similar to mouse (Lind et al 2005). NeuN has been used extensively to label postmitotic neurons in developmental studies in chick (Mezey et al 2012;Perez et al 1999;Zeng et al 2008) and in a study of cochlear nucleus neurons in mice (Mostafapour et al 2002).…”