NeuN (Fox-3, Rbfox3, or Hexaribonucleotide Binding Protein-3), a protein which is a homologue to the protein product of a sex-determining gene in '' Caenorhabditis elegans'', is a neuronal

nuclear Nuclear may refer to: Physics Relating to the nucleus of the atom: * Nuclear engineering *Nuclear physics *Nuclear power *Nuclear reactor *Nuclear weapon *Nuclear medicine *Radiation therapy *Nuclear warfare Mathematics *Nuclear space *Nuclear ...

antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...

that is commonly used as a

biomarker In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...

for neurons.

History

NeuN was first described in 1992 by Mullen et al., who raised a series of monoclonal antibodies to mouse antigens with the original intent of finding mouse species specific immunological markers for use in transplantation experiments. In the event they isolated a

hybridoma Hybridoma technology is a method for producing large numbers of identical antibodies (also called monoclonal antibodies). This process starts by injecting a mouse (or other mammal) with an antigen that provokes an immune response. A type of white ...

line which produced a monoclonal antibody called mAb A60, which proved to bind an antigen expressed only in neuronal nuclei and to a lesser extent the

cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...

of neuronal cells, and which appeared to work on all vertebrates. This antigen was therefore known as NeuN for "Neuronal Nuclei" though what the A60 antibody was binding to was unknown for the next 17 years. In 2009, Kim et al. used

proteomic Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...

methods to show that NeuN corresponds to a protein known as Fox-3, also known as Rbfox3, a mammalian homologue of Fox-1, a protein originally identified from genetic studies of the nematode worm ''C. elegans''.

Structure

Western blotting The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...

shows that mAb A60 binds to two bands of apparent

molecular weight A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...

~46kDa and ~48kDa on

SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...

. These two bands are generated from a single Fox-3 gene by

alternate splicing Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be ...

. There are in fact four protein products from the Fox-3 gene as a result of the presence or absence of two amino acid sequences coded by two exons. The inclusion or absence of 47 amino acids from exon 12 results in the ~46kDa and ~48kDa bands seen on SDS-PAGE gels, while the inclusion or absence of 14 amino acids from exon 15 produces two forms which are too similar in molecular size to be discerned on typical SDS-PAGE gels. Interestingly, the protein coded by exon 15 adds a C-terminal PY type nuclear localization sequence, which presumably explains why NeuN/Fox-3 protein can be both nuclear and, in some cell types, also cytoplasmic. All forms are expressed only in neurons so the mAb A60

antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

and other similar antibodies to NeuN/Fox-3 have become very widely used as robust markers of neurons.

Uses as a Neuronal Biomarker

NeuN antibodies are widely used to label neurons, despite some shortcomings, and a September 2018 Pubmed search using the keyword "NeuN" produced 3060 hits. A few neuronal cell types are not recognized by NeuN antibodies, such as

Purkinje cells Purkinje cells, or Purkinje neurons, are a class of GABAergic inhibitory neurons located in the cerebellum. They are named after their discoverer, Czech anatomist Jan Evangelista Purkyně, who characterized the cells in 1839. Structure T ...

, stellate and Golgi cells of the cerebellum, olfactory Mitral cells, retinal photoreceptors and spinal cord

gamma motor neurons A gamma motor neuron (γ motor neuron), also called gamma motoneuron, or fusimotor neuron, is a type of lower motor neuron that takes part in the process of muscle contraction, and represents about 30% of ( Aγ) fibers going to the muscle. Like ...

. However the vast majority of neurons are strongly NeuN positive, and NeuN immunoreactivity has been widely used to identify neurons in tissue culture and in sections and to measure the neuron/glia ratio in brain regions. NeuN immunoreactivity becomes obvious as neurons mature, typically after they have downregulated expression of

Doublecortin Neuronal migration protein doublecortin, also known as doublin or lissencephalin-X is a protein that in humans is encoded by the DCX gene. Function Doublecortin (DCX) is a microtubule-associated protein expressed by neuronal precursor cells an ...

, a marker seen in the earliest stages of neuronal development.

Feminizing Locus on X Homologue

Fox-3 is one of a family of mammalian homologues of the Fox-1 protein, originally discovered in the nematode worm ''C. elegans'' as the protein product of a gene involved in sex determination. Fox is, in fact, an acronym of "Feminizing locus on X". The mammalian genome contains three genes homologous to ''C. elegans'' Fox-1, called Fox-1, Fox-2 and Fox-3. The Fox proteins are all about 46kDa in size, and each includes a central, highly conserved ~70 amino acid RRM or

RNA recognition motif RNA recognition motif, RNP-1 is a putative RNA-binding domain of about 90 amino acids that are known to bind single-stranded RNAs. It was found in many eukaryotic proteins. The largest group of single strand RNA-binding protein is the eukaryot ...

. RRM domains are one of the most common in the

human genome The human genome is a complete set of nucleic acid sequences for humans, encoded as DNA within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual mitochondria. These are usually treated separately as the ...

and are found in numerous proteins which bind RNA molecules. NeuN/Fox-3 and the other Fox proteins function in the regulation of

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...

splicing and bind specific RNA sequences. For a review of the Fox family of proteins see this reference. An alternate name for Fox-3 is hexaribonucleotide binding protein 3 since Fox-3, like Fox-2 and Fox-1, bind the hexaribonucleotide UGCAUG, this binding being involved in the regulation of mRNA splicing.

References

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