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 ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a Hybrid word, blend of the Greek ''caeno-'' (recent), ''r ...

'', 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, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...

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, ...

for

neurons A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...

History

NeuN was first described in 1992 by Mullen et al., who raised a series of

monoclonal antibodies A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a Lineage (evolution), cell lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. Mon ...

to mouse

antigens In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. An ...

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 quantities of monoclonal antibodies by fusing antibody producing B cells with myeloma cells (cancerous B cells). This creates hybrid cells, ''hybridomas,'' that produce the antibody from their ...

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 The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...

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. Eventually Kim et al. used

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

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 sex determination in the

nematode The nematodes ( or ; ; ), roundworms or eelworms constitute the phylum Nematoda. Species in the phylum inhabit a broad range of environments. Most species are free-living, feeding on microorganisms, but many are parasitic. Parasitic worms (h ...

worm ''

'', see below.

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 detec ...

shows that mAb A60 binds to two bands of apparent

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

~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, alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to produce different splice variants. For example, some exons of a gene may be included ...

. 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 A nuclear localization signal ''or'' sequence (NLS) is an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines ...

, which presumably explains why NeuN/Fox-3 protein can be both nuclear and, in some cell types, also

cytoplasmic The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell and ...

. All forms are expressed only in neurons so the mAb A60

antibody An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as pathogenic bacteria, bacteria and viruses, includin ...

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 June 2024 Pubmed search using the keyword "NeuN" produced over 5000 hits. A few neuronal cell types are not recognized by NeuN antibodies, such as

Purkinje cells Purkinje cells or Purkinje neurons, named for Czech physiologist Jan Evangelista Purkyně who identified them in 1837, are a unique type of prominent, large neuron located in the cerebellar cortex of the brain. With their flask-shaped cell bo ...

stellate Stellate, meaning star-shaped, may be used to describe: Biology * Stellate cell (disambiguation) * Stellate ganglion * Stellate reticulum * Stellate veins * Stellate trichomes (hairs) Other * Stellate wounds from lacerations or incisions * S ...

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, 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 cell ...

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

Feminizing Locus on X Homologue

Fox-3 is one of a family of three mammalian homologues of the Fox-1 protein, originally discovered as the protein product of a gene involved in sex determination in the

worm ''

C. elegans ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' ( ...

''. "Fox" is an acronym of "Feminizing locus on X". The mammalian genome contains three genes homologous to ''C. elegans'' Fox-1 (a.k.a. RBFOX1, A2BP, A2BP1 and HRNBP1), Fox-2, (a.k.a. RBFOX2, RBM9, RTA and HRNBP2) and Fox-3 (a.k.a. RBFOX3 and HRNBP3). The

HGNC The HUGO Gene Nomenclature Committee (HGNC) is a committee of the Human Genome Organisation (HUGO) that sets the standardization, standards for human gene nomenclature. The HGNC approves a ''unique'' and ''meaningful'' name for every known human g ...

names for the three proteins are RBFOX1, RBFOX2 and RBFOX3 respectively. The Fox proteins are each 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 the DNA within each of the 23 distinct chromosomes in the cell nucleus. A small DNA molecule is found within individual Mitochondrial DNA, mitochondria. These ar ...

and are found in numerous proteins which bind

RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...

. The three mammalian 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 Protein biosynthesis, synthesizing a protein. mRNA is ...

splicing and bind specific RNA sequences. An alternate name the three proteins is hexaribonucleotide binding protein 1, 2 and 3, (HRNBP1, 2 and 3), since all three bind the hexaribonucleotide UGCAUG, this binding being involved in their regulation of mRNA splicing.

References

{{DEFAULTSORT:Neun Antibodies