Identification of Neuronal Nuclei (NeuN) as Fox-3, a New Member of the Fox-1 Gene Family of Splicing Factors

Abstract

NeuN (neuronal nuclei) is a neuron-specific nuclear protein which is identified by immunoreactivity with a monoclonal antibody, anti-NeuN. Anti-NeuN has been used widely as a reliable tool to detect most postmitotic neuronal cell types in neuroscience, developmental biology, and stem cell research fields as well as diagnostic histopathology. To date, however, the identity of its antigen, NeuN itself, has been unknown. Here, we identify NeuN as the Fox-3 gene product by providing the following evidence: 1) Mass spectrometry analysis of anti-NeuN immunoreactive protein yields the Fox-3 amino acid sequence. 2) Recombinant Fox-3 is recognized by anti-NeuN. 3) Short hairpin RNAs targeting Fox-3 mRNA down-regulate NeuN expression. 4) Fox-3 expression is restricted to neural tissues. 5) Anti-Fox-3 immunostaining and anti-NeuN immunostaining overlap completely in neuronal nuclei. We also show that a protein crossreactive with anti-NeuN is the synaptic vesicle protein, synapsin I. Anti-NeuN recognizes synapsin I in immunoblots with one order of magnitude lower affinity than Fox-3, and does not recognize synapsin I using immunohistology. Fox-3 (also called hexaribonucleotide-binding protein 3 and D11Bwg0517e) contains an RNA recognition motif and is classified as a member of the Fox-1 gene family that binds specifically to an RNA element, UGCAUG. We demonstrate that Fox-3 functions as a splicing regulator using neural cell-specific alternative splicing of the non-muscle myosin heavy chain II-B pre-mRNA as a model. Identification of NeuN as Fox-3 clarifies an important element of neurobiology research. Mullen et al. (1) have reported a monoclonal antibody (mAb)2 , which was generated using brain cell nuclei as antigens. This mAb recognizes 2-3 protein bands with apparent molecular masses of 46 -48 kDa following SDS-PAGE that are expressed in neuronal tissues. Intensive immunohistochemical analyses using embryonic and adult murine tissues have demonstrated that this mAb stains exclusively neuronal cells in the central and peripheral nervous systems, especially postmitotic and differentiating neurons, as well as terminally differentiated neurons. The mAb staining is localized primarily to nuclei. Thus, the authors named the antigen recognized by this mAb NeuN for "Neuronal Nuclei." The original study and subsequent studies by others have shown that anti-NeuN stains most types of neurons throughout the nervous system with few exceptions (1, 2). In no case has NeuN expression been observed in glial cells. This mAb can detect the NeuN antigen in a wide range of vertebrate species, including mammals, birds, and amphibians. Because of its high specificity for postmitotic neurons, its broad specificity for most types of neuronal cells, and its cross-reactivity with multiple species, anti-NeuN has gained widespread acceptance as a reliable tool to detect neuronal cells in neuroscience and developmental biology research and in diagnostic histopathology for neural diseases and tumors (3-10). More recently, it has also been used to mo...

Neuronal Per Arnt Sim (PAS) Domain Protein 4 (NPAS4) Regulates Neurite Outgrowth and Phosphorylation of Synapsin I

“…Although NeuN was not identified as a target of NPAS4 in previous studies (13,45), NeuN has been identified as a transcriptional factor, FOX-3, and appears to be a nervous system-specific nuclear regulatory molecule and a splicing regulator (46,47). In our study, a ChIP assay showed that Npas4 binds the promoter of NeuN in Neuro2a cells.…”

Target selection of proprioceptive and motor axon synapses on neonatal V1‐derived Ia inhibitory interneurons and Renshaw cells

“…This monoclonal antibody recognizes a double band of 46-58 kDa in Western blots of mouse brain nuclear extracts (Mullen et al, 1992), which recently were shown to correspond to the splicing factor Fox-3 (Kim et al, 2009). In whole brain Western blots it additionally recognizes a band of 70 kDa, identified as synapsin I, but this crossreactivity disappears with paraformaldehyde fixation (Kim et al, 2009) and is not apparent in immunostained sections. Our immunostaining of neuronal nuclei and cell bodies in the spinal cord is identical to that reported previously by us and that reported in spinal cord atlases (Watson et al, 2009).…”

Alterations in sulfated chondroitin glycosaminoglycans following controlled cortical impact injury in mice

“…This antibody may crossreact with other protein that labels axonal structures in mouse brain section if section is not well permeated with triton. On the gel, it gives rise to 2 bands at around 38 and 50kDa (Kim et al, 2009). …”

“…The immunogen for this antibody is purified cell nuclei from mouse brain, but later has been shown to detects RNA spicing factor Fox-3 (Kim et al, 2009). In our hands, not all neuronal nuclei are labeled with this antibody in the brain.…”