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Neurofilaments (NF) are classed as type IV intermediate filaments found in 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. Th ...
of
neurons A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...
. They are protein polymers measuring 10 nm in diameter and many micrometers in length. Together with
microtubule Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27  nm and have an inner diameter between 11 an ...
s (~25 nm) and
microfilament Microfilaments, also called actin filaments, are protein filaments in the cytoplasm of eukaryotic cells that form part of the cytoskeleton. They are primarily composed of polymers of actin, but are modified by and interact with numerous other p ...
s (7 nm), they form the neuronal cytoskeleton. They are believed to function primarily to provide structural support for
axon An axon (from Greek ἄξων ''áxōn'', axis), or nerve fiber (or nerve fibre: see spelling differences), is a long, slender projection of a nerve cell, or neuron, in vertebrates, that typically conducts electrical impulses known as action p ...
s and to regulate axon diameter, which influences
nerve conduction velocity In neuroscience, nerve conduction velocity (CV) is an important aspect of nerve conduction studies. It is the speed at which an electrochemical impulse propagates down a neural pathway. Conduction velocities are affected by a wide array of fact ...
. The proteins that form neurofilaments are members of the intermediate filament protein family, which is divided into six types based on their gene organization and protein structure. Types I and II are the
keratin Keratin () is one of a family of structural fibrous proteins also known as ''scleroproteins''. Alpha-keratin (α-keratin) is a type of keratin found in vertebrates. It is the key structural material making up scales, hair, nails, feathers, ho ...
s which are expressed in epithelia. Type III contains the proteins
vimentin Vimentin is a structural protein that in humans is encoded by the ''VIM'' gene. Its name comes from the Latin ''vimentum'' which refers to an array of flexible rods. Vimentin is a type III intermediate filament (IF) protein that is expresse ...
,
desmin Desmin is a protein that in humans is encoded by the ''DES'' gene. Desmin is a muscle-specific, type III intermediate filament that integrates the sarcolemma, Z disk, and nuclear membrane in sarcomeres and regulates sarcomere architecture. S ...
,
peripherin Peripherin is a type III intermediate filament protein expressed mainly in neurons of the peripheral nervous system. It is also found in neurons of the central nervous system that have projections toward peripheral structures, such as spinal moto ...
and
glial fibrillary acidic protein Glial fibrillary acidic protein (GFAP) is a protein that is encoded by the ''GFAP'' gene in humans. It is a type III intermediate filament (IF) protein that is expressed by numerous cell types of the central nervous system (CNS), including astroc ...
(GFAP). Type IV consists of the neurofilament proteins L, M, H and
internexin Internexin, alpha-internexin, is a Class IV intermediate filament approximately 66 KDa. The protein was originally purified from rat optic nerve and spinal cord.Levavasseur F, Zhu Q, and JP Julien. No requirement of alpha-internexin for nervous sys ...
. Type V consists of the nuclear lamins, and type VI consists of the protein nestin. The type IV intermediate filament genes all share two unique
introns An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene. ...
not found in other intermediate filament gene sequences, suggesting a common evolutionary origin from one primitive type IV gene. Any proteinaceous filament that extends in the cytoplasm of a nerve cell is also termed a ''neurofibril''. This name is used in the
neurofibrillary tangle Neurofibrillary tangles (NFTs) are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary biomarker of Alzheimer's disease. Their presence is also found in numerous other diseases known as tauopathies. Little is k ...
s of some
neurodegenerative disease A neurodegenerative disease is caused by the progressive loss of structure or function of neurons, in the process known as neurodegeneration. Such neuronal damage may ultimately involve cell death. Neurodegenerative diseases include amyotrophic ...
s.


Neurofilament proteins

The protein composition of neurofilaments varies widely across different animal phyla. Most is known about mammalian neurofilaments. Historically, mammalian neurofilaments were originally thought to be composed of just three proteins called neurofilament protein L (low molecular weight;
NFL The National Football League (NFL) is a professional American football league that consists of 32 teams, divided equally between the American Football Conference (AFC) and the National Football Conference (NFC). The NFL is one of the maj ...
), M (medium molecular weight; NFM) and H (high molecular weight; NFH). These proteins were discovered from studies of
axonal transport Axonal transport, also called axoplasmic transport or axoplasmic flow, is a cellular process responsible for movement of mitochondria, lipids, synaptic vesicles, proteins, and other organelles to and from a neuron's cell body, through the cytoplas ...
and are often referred to as the "neurofilament triplet". However, it is now clear that neurofilaments also contain the protein internexin and that neurofilaments in the peripheral nervous system can also contain the protein peripherin. (this is different from
peripherin 2 Peripherin-2 is a protein, that in humans is encoded by the ''PRPH2'' gene. Peripherin-2 is found in the rod and cone cells of the retina of the eye. Defects in this protein result in one form of retinitis pigmentosa, an incurable blindness. Muta ...
that is expressed in the
retina The retina (from la, rete "net") is the innermost, light-sensitive layer of tissue of the eye of most vertebrates and some molluscs. The optics of the eye create a focused two-dimensional image of the visual world on the retina, which then p ...
). Thus mammalian neurofilaments are heteropolymers of up to five different proteins: NfL, NfM, NfH, internexin-alpha and peripherin. The five neurofilament proteins can co-assemble in different combinations in different nerve cell types and at different stages of development. The precise composition of neurofilaments in any given nerve cell depends on the relative expression levels of the neurofilament proteins in the cell at that time. For example, NfH expression is low in developing neurons and increases postnatally in neurons with myelinated axons. In the adult nervous system neurofilaments in small unmyelinated axons contain more peripherin and less NfH whereas neurofilaments in large myelinated axons contain more NfH and less peripherin. The type III intermediate filament subunit,
vimentin Vimentin is a structural protein that in humans is encoded by the ''VIM'' gene. Its name comes from the Latin ''vimentum'' which refers to an array of flexible rods. Vimentin is a type III intermediate filament (IF) protein that is expresse ...
, is expressed in developing neurons and a few very unusual neurons in the adult in association with type IV proteins, such as the horizontal neurons of the
retina The retina (from la, rete "net") is the innermost, light-sensitive layer of tissue of the eye of most vertebrates and some molluscs. The optics of the eye create a focused two-dimensional image of the visual world on the retina, which then p ...
. The triplet proteins are named based upon their relative size (low, medium, high). The apparent
molecular mass The molecular mass (''m'') is the mass of a given molecule: it is measured in daltons (Da or u). Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The related quanti ...
of each protein determined by
SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. ...
is greater than the mass predicted from the amino sequence. This is due to the anomalous electrophoretic migration of these proteins and is particularly extreme for neurofilament proteins M and H due to their high content of charged amino acids and extensive phosphorylation. All three neurofilament triplet proteins contain long stretches of polypeptide sequence rich in
glutamic acid Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...
and
lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −CO ...
residues, and NfM and especially NfH also contain multiple tandemly repeated
serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...
phosphorylation sites. These sites almost all contain the peptide lysine-serine-proline (KSP), and phosphorylation is normally found on axonal and not dendritic neurofilaments. Human NfM has 13 of these KSP sites, while human NF-H is expressed from two
allele An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution. ::"The chro ...
s one of which produces 44 and the other 45 KSP repeats.


Neurofilament assembly and structure

Like other intermediate filament proteins, the neurofilament proteins all share a common central
alpha helical The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ea ...
region, known as the rod domain because of its rod-like tertiary structure, flanked by amino terminal and carboxy terminal domains that are largely unstructured. The rod domains of two neurofilament proteins dimerize to form an alpha-helical
coiled coil A coiled coil is a structural motif in proteins in which 2–7 alpha-helices are coiled together like the strands of a rope. ( Dimers and trimers are the most common types.) Many coiled coil-type proteins are involved in important biological ...
. Two dimers associate in a staggered antiparallel manner to form a tetramer. This tetramer is believed to be the basic subunit (i.e. building block) of the neurofilament. Tetramer subunits associate side-to-side to form unit-length filaments, which then anneal end-to-end to form the mature neurofilament polymer, but the precise organization of these subunits within the polymer is not known, largely because of the heterogeneous protein composition and the inability to crystallize neurofilaments or neurofilament proteins. Structural models generally assume eight tetramers (32 neurofilament polypeptides) in a filament cross-section, but measurements of linear mass density suggest that this can vary. The amino terminal domains of the neurofilament proteins contain numerous phosphorylation sites and appear to be important for subunit interactions during filament assembly. The carboxy terminal domains appear to be intrinsically disordered domains that lack alpha helix or beta sheet. The different sizes of the neurofilament proteins are largely due to differences in the length of the carboxy terminal domains. These domains are rich in acidic and basic amino acid residues. The carboxy terminal domains of NFM and NFH are the longest and are modified extensively by post-translational modifications such as
phosphorylation In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...
and
glycosylation Glycosylation is the reaction in which a carbohydrate (or 'glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not a ...
in vivo. They project radially from the filament backbone to form a dense brush border of highly charged and unstructured domains analogous to the bristles on a bottle brush. These entropically flailing domains have been proposed to define a zone of exclusion around each filament, effectively spacing the filaments apart from their neighbors. In this way, the carboxy terminal projections maximize the space-filling properties of the neurofilament polymers. By electron microscopy, these domains appear as projections called sidearms that appear to contact neighboring filaments.


Neurofilament function

Neurofilaments are found in
vertebrate Vertebrates () comprise all animal taxon, taxa within the subphylum Vertebrata () (chordates with vertebral column, backbones), including all mammals, birds, reptiles, amphibians, and fish. Vertebrates represent the overwhelming majority of the ...
neurons in especially high concentrations in axons, where they are all aligned in parallel along the long axis of the axon forming a continuously overlapping array. They have been proposed to function as space-filling structures that increase axonal diameter. Their contribution to axon diameter is determined by the number of neurofilaments in the axon and their packing density. The number of neurofilaments in the axon is thought to be determined by neurofilament gene expression and axonal transport. The packing density of the filaments is determined by their side-arms which define the spacing between neighboring filaments. Phosphorylation of the sidearms is thought to increase their extensibility, increasing the spacing between neighboring filaments by the binding of divalent cations between the sidearms of adjacent filaments Early in development, axons are narrow processes that contain relatively few neurofilaments. Those axons that become myelinated accumulate more neurofilaments, which drives the expansion of their caliber. After an axon has grown and connected with its target cell, the diameter of the axon may increase as much as fivefold. This is caused by an increase in the number of neurofilaments exported from the nerve cell body as well as a slowing of their rate of transport. In mature myelinated axons, neurofilaments can be the single most abundant cytoplasmic structure and can occupy most of the axonal cross-sectional area. For example, a large myelinated axon may contain thousands of neurofilaments in one cross-section


Neurofilament transport

In addition to their structural role in axons, neurofilaments are also cargoes of
axonal transport Axonal transport, also called axoplasmic transport or axoplasmic flow, is a cellular process responsible for movement of mitochondria, lipids, synaptic vesicles, proteins, and other organelles to and from a neuron's cell body, through the cytoplas ...
. Most of the neurofilament proteins in axons are synthesized in the nerve cell body, where they rapidly assemble into neurofilament polymers within about 30 minutes. These assembled neurofilament polymers are transported along the axon on
microtubule Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27  nm and have an inner diameter between 11 an ...
tracks powered by microtubule
motor protein Motor proteins are a class of molecular motors that can move along the cytoplasm of cells. They convert chemical energy into mechanical work by the hydrolysis of ATP. Flagellar rotation, however, is powered by a proton pump. Cellular functions ...
s. The filaments move bidirectionally, i.e. both towards the axon tip (anterograde) and towards the cell body (retrograde), but the net direction is anterograde. The filaments move at velocities of up to 8 µm/s on short time scales (seconds or minutes), with average velocities of approximately 1 µm/s. However, the average velocity on longer time scales (hours or days) is slow because the movements are very infrequent, consisting of brief sprints interrupted by long pauses. Thus on long time scales neurofilaments move in the slow component of axonal transport.


Clinical and research applications

Numerous specific
antibodies 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 ...
to neurofilament proteins have been developed and are commercially available. These antibodies can be used to detect neurofilament proteins in cells and tissues using
immunofluorescence Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to speci ...
microscopy or
immunohistochemistry Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to ant ...
. Such antibodies are widely used to identify neurons and their processes in
histological section Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
s and in
tissue culture Tissue culture is the growth of tissues or cells in an artificial medium separate from the parent organism. This technique is also called micropropagation. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, suc ...
. The type VI intermediate filament protein Nestin is expressed in developing neurons and glia. Nestin is considered a marker of neuronal stem cells, and the presence of this protein is widely used to define
neurogenesis Neurogenesis is the process by which nervous system cells, the neurons, are produced by neural stem cells (NSCs). It occurs in all species of animals except the porifera (sponges) and placozoans. Types of NSCs include neuroepithelial cells (NECs), ...
. This protein is lost as development proceeds. Neurofilament antibodies are also commonly used in diagnostic
neuropathology Neuropathology is the study of disease of nervous system tissue, usually in the form of either small surgical biopsies or whole-body autopsies. Neuropathologists usually work in a department of anatomic pathology, but work closely with the clini ...
. Staining with these antibodies can distinguish neurons (positive for neurofilament proteins) from
glia Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form mye ...
(negative for neurofilament proteins). There is also considerable clinical interest in the use of neurofilament proteins as
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, pa ...
s of axonal damage in diseases affecting the central nervous system When neurons or axons degenerate, neurofilament proteins are released into the blood or cerebrospinal fluid. Immunoassays of neurofilament proteins in cerebrospinal fluid and plasma can thus serve as indicators of axonal damage in neurological disorders. NfL is a useful marker for disease monitoring in
amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND) or Lou Gehrig's disease, is a neurodegenerative disease that results in the progressive loss of motor neurons that control voluntary muscles. ALS is the most comm ...
,
multiple sclerosis Multiple (cerebral) sclerosis (MS), also known as encephalomyelitis disseminata or disseminated sclerosis, is the most common demyelinating disease, in which the insulating covers of nerve cells in the brain and spinal cord are damaged. This ...
, and more recently
Huntington's disease Huntington's disease (HD), also known as Huntington's chorea, is a neurodegenerative disease that is mostly inherited. The earliest symptoms are often subtle problems with mood or mental abilities. A general lack of coordination and an unst ...
. It has also been evaluated as a prognostic marker for functional outcome following acute ischemic stroke
Mutant In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It ...
mice with neurofilament abnormalities have
phenotypes In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological prope ...
resembling
amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND) or Lou Gehrig's disease, is a neurodegenerative disease that results in the progressive loss of motor neurons that control voluntary muscles. ALS is the most comm ...
.


See also

* Bielschowsky stain


References

{{Cytoskeletal Proteins Cytoskeleton