Mammalian Neurofilaments Research Articles

Conformation of neurofilament heavy chain effect on neurofilament-neurofilament association and bundling.

Mammalian neurofilaments (NFs) are composed of Heavy (NF-H), Medium (NF-M), and Light (NF-L) subunits, named according to their molecular mass, along with α-internexin or peripherin (in central or peripheral nervous systems, respectively). Phosphorylated NF subunits assemble linearly, with C-terminal regions extending from the filament “backbone.” Phosphorylation of C-terminal “side arms” of NF-H promotes NF-NF interactions, providing stability to axons. Transport of NFs from the perikaryon into the axon and NF-NF interactions are dependent upon regulation of multiple kinases. The complete sequence of phosphorylation events does not occur until NFs have entered the axon. Sidearm phosphorylation fosters lateral extension away from the NF backbone. By contrast, sidearm conformation prior to phosphorylation remains unresolved. Scrutiny of the NF-H side-arm sequence revealed 4 proline-proline (PP) motifs; these motifs allow tremendous rotation of their peptide chains from a closed, folded-back hairpin (cis) to a fully extended, linear confirmation (trans). Notably, one PP motif is located within each of the consensus sequences for the NF kinases. We hypothesize that phosphorylation-induced alterations in the configuration of these PP motifs are critical for NF transport, NF-NF association within axons. Mutated NFs constructs with changed PP motifs were expressed (with or without PP motifs /Phosphorylated or unphosphorylated) and incubated with NFs medium chain then trapped using a his-tag column to demonstrate bundling. Our initial data were inconclusive. A different experimental approach, such as Localized Surface Plasma Resonance (LSPR) is necessary to analyze the role of PP motifs in NFs association. We predict that constructs lacking PP motifs will present lower association affinity to NF-M (even when phosphorylated. Understanding the mechanisms that allow NF-NF associations within neurites will help guide targeted treatments for NF-based neurodegenerative disorders.

How does neurofilament phosphorylation regulate axonal outgrowth and stabilization?

Neurofilaments (NFs) are tissue-specific intermediate filaments that maintain neuronal structural stability. Mammalian NFs are composed of Heavy (NF-H), Medium (NF-M), and Light (NF-L) subunits, named according to their molecular mass, along with α-internexin or peripherin (in central or peripheral nervous systems, respectively). NF subunits assemble linearly, with C-terminal regions extending from the filament “backbone.” Phosphorylation of C-terminal “side arms” of NF-H promotes NF-NF interactions, providing stability to axons.

Assembly properties of lamprey neurofilament subunits and their expression after spinal cord transection

In mammals neurofilaments (NF) are formed by coassembly of three subunits: NFL, NFM, and NFH (light, medium, and heavy). It had been believed that lampreys have only one subunit, NF180. However, a previous study showed that NF180 could not self-assemble but could coassemble with rat NFL, suggesting the existence of additional NF subunits in lamprey. More recently, we cloned three additional NF subunits. These new subunits and NF180 have now been transfected in combinations into SW13cl.2Vim(-) cells, which lack endogenous cytoplasmic intermediate filaments. None of the subunits could self-assemble. No combination of NF subunits could form filaments in the absence of lamprey NFL (L-NFL). Assembly occurred at 28°C, but not at 37°C. L-NFL could form thick NF bundles with NF180 but not with NF132 and NF95, which formed only fine filamentous arrays. To determine which parts of the NF subunits are required for filament or bundle formation, we constructed deletion mutants of NF180 and cotransfected them with L-NFL. As with mammalian NF, only constructs with intact head and core domains could form filaments with L-NFL. However, the full length of NF180 was required to form NF bundles. As with NF180, in situ hybridization indicated that mRNA for L-NFL and NF132 was downregulated in identified reticulospinal neurons by 5 weeks after spinal cord transection, but was reexpressed at 10 weeks selectively in those neurons whose axons have a high probability of regenerating. This is consistent with a possible role of NFs in the mechanism of axon regeneration.

Multiple neurofilament subunits are present in lamprey CNS

In mammals, there are three neurofilament (NF) subunits (NF-L, NF-M, and NF-H), but it was thought that only a single NF, NF180, exists in lamprey. However, NF180 lacked the ability to self-assemble, suggesting that like mammalian NFs, lamprey NFs are heteropolymers, and that additional NF subunits may exist. The present study provides evidence for the existence of a lamprey NF-L homolog (L-NFL). Genes encoding two new NF-M isoforms (NF132 and NF95) also have been isolated and characterized. With NF180, this makes three NF-M-like isoforms. In situ hybridization showed that all three newly cloned NFs are expressed in spinal cord neurons and in spinal-projecting neurons of the brainstem. Like NF180, there were no KSP multiphosphorylation repeat motifs in the tail regions of NF132 or NF95. NF95 was highly identical to homologous parts of NF180, sharing 2 common pieces of DNA with it. Northern blots suggested that NF95 may be expressed at very low levels in older larvae. The presence of L-NFL in lamprey CNS may support the hypothesis that as in mammals, NFs in lamprey are obligate heteropolymers, in which NF-L is a required subunit.

The glutamate-rich region of the larger lamprey neurofilament sidearm is essential for proper neurofilament architecture

The carboxyl terminal “tail” domains of the heavy and middle molecular weight mammalian neurofilament (NF) proteins regulate inter-NF spacing and formation of organized networks. The C-terminal region of the larger of the two lamprey NF subunits (NF-180) resembles these mammalian proteins in that it consists of a proximal glutamate-rich region and a distal region containing multiple phosphorylation sites. To investigate the role of these two sidearm domains in the organization of lamprey NFs, we generated plasmids lacking the glutamate-rich domain, the domain containing multiple phosphorylation sites, or both, and examined the impact of the resultant mutant proteins on the endogenous NF network in differentiated NB21/d1 neuroblastoma cells. We present evidence that, like mammalian NFs, the glutamate-rich region of NF-180 sidearm plays a critical role in NF architecture.

O-Glycosylation of the Tail Domain of Neurofilament Protein M in Human Neurons and in Spinal Cord Tissue of a Rat Model of Amyotrophic Lateral Sclerosis (ALS)

Mammalian neurofilaments (NFs) are modified by post-translational modifications that are thought to regulate NF assembly and organization. Whereas phosphorylation has been intensely studied, the role of another common modification, the attachment of O-linked N-acetylglucosamine (GlcNAc) to individual serine and threonine residues, is hardly understood. We generated a novel monoclonal antibody that specifically recognizes an O-glycosylated epitope in the tail domain of NF-M and allows determination of the glycosylation state at this residue. The antibody displays strong species preference for human NF-M, shows some reactivity with rat but not with mouse or bovine NF-M. By immunohistochemistry and Western blot analysis of biopsy-derived human temporal lobe tissue we show that immunoreactivity is highly enriched in axons parallel to hyperphosphorylated NFs. Treatment of cultured neurons with the GlcNAcase inhibitor PUGNAc causes a 40% increase in immunoreactivity within 1 h, which is completely reversible and parallels the total increase in cellular O-GlcNAc modification. Treatment with the mitogen-activated protein kinase kinase inhibitor PD-98059 leads to a similar increase in immunoreactivity. In spinal cord tissue of a transgenic rat model for amyotrophic lateral sclerosis, immunoreactivity is strongly decreased compared with wild-type animals while phosphorylation is increased. The data suggest that hyperphosphorylation and tail domain O-glycosylation of NFs are synchronously regulated in axons of human neurons in situ and that O-glycosylation of NF-M is highly dynamic and closely interweaved with phosphorylation cascades and may have a pathophysiological role.

Identification of a neurofilament-like protein in the protocerebral tract of the crab Ucides cordatus.

Neurofilaments (NFs) have not been observed in crustaceans using conventional electron microscopy, and intermediate filaments have never been described in crustaceans and other arthropods by immunocytochemistry. Since polypeptides, labeled by the NN18-clone antibody, were revealed on microtubule side-arms of crayfish, we have tested, in this study, whether proteins similar to mammalian NFs are present in the protocerebral tract (PCT) of the crab Ucides cordatus. We used immunohistochemistry for light microscopy with monoclonal antibodies against three different NF subunits, high (NF-H), medium (NF-M), and light (NF-L). Labeling was observed with the NN18-clone, which recognizes NF-M. In order to confirm the results obtained with the immunohistochemical reactions, Western blotting, using the three primary antibodies, was performed and the presence of NF-M was confirmed. The NN18-clone monoclonal antibody recognized a protein of approximately 160 kDa, similar to the mammalian NF-M protein, but NF-L and NF-H were not recognized. Conventional transmission electron microscopy was used to observe the ultrastructural components of the axons and immunoelectron microscopy was used to show the distribution of the NF-M-like polypeptides along cytoskeletal elements of the PCT. Our results agree with previous studies on crustacean NF proteins that have reported negative immunoreactions against NF-H and NF-L subunits and positive immunoreactions against the mammalian NF-M subunit. However, the protein previously referred to as P600 and recognized by the NN18-clone, has a very high molecular weight, thus, being different from mammalian NF-M subunit and from the protein revealed now in our study.

Modulation of repulsive forces between neurofilaments by sidearm phosphorylation

Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured “sidearm” domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate–phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces.

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport.

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine–serine–proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.

Axonal Charcot-Marie-Tooth Disease and the Neurofilament Light Gene ( NF-L)

Axonal Charcot-Marie-Tooth Disease and the Neurofilament Light Gene ( NF-L)

The single neurofilament subunit of the lamprey forms filaments and regulates axonal caliber and neuronal size in vivo.

Neurofilaments (NFs) are composed of a heteropolymer of three related subunits in mammalian neurons, where they are a major component of the cytoskeleton in large neurons and are thought to regulate axonal diameter. NFs in the lamprey, while ultrastructurally and functionally indistinguishable from mammalian NFs, are polymers of a single subunit protein, NF180. In this study, we use the simplicity of lamprey NFs and the accessibility of the lamprey central nervous system (CNS) to examine the effects of overproducing NFs in an identified giant neuron in vivo, and thus to elucidate the role of NFs in regulating neuronal size and axonal caliber in the vertebrate CNS. We show that overexpression of NF180 tagged with a variant of Green Fluorescent Protein (EYFP) in identified lamprey neurons (ABCs) and in human neuroblastoma (NB2a) cells results in the assembly of exogenous NF180 into ultrastructurally normal NFs that are tightly packed and unphosphorylated. These accumulate in the somata of NB2a cells and produce somatic swelling by 3 days post-transfection. NF180 overexpression in lamprey ABCs in vivo causes exogenous NFs to accumulate in ABC axons, somata, and dendrites, and induces a significant increase in axonal diameter without increasing axonal NF packing density. Overexpression of EYFP alone has none of these effects. We conclude that NF180 normally plays a critical role in determining axonal caliber in ABCs and may influence neuronal size in situations where NFs accumulate in the soma, such as after axonal injury.

Mice with disrupted midsized and heavy neurofilament genes lack axonal neurofilaments but have unaltered numbers of axonal microtubules

Mammalian neurofilaments are assembled from the light (NF-L), midsized (NF-M), and heavy (NF-H) neurofilament proteins. While NF-M and NF-H cannot self-assemble into homopolymers, the data concerning NF-L has been more contradictory. In vitro bovine, porcine, and murine NF-L can homopolymerize in the absence of other subunits. However, in vivo studies suggest that neither rat nor mouse NF-L can form filaments when transfected alone into cells lacking endogenous intermediate filaments. By contrast, human NF-L forms homopolymers in similar cell lines. Recently we generated mice with null mutations in the NF-M and NF-H genes. To determine if mouse NF-L can homopolymerize in mouse axons, NF-M and NF-H null mutants were bred to create a line of double mutant animals. Here we show that axons in NF-M/H double mutant animals are largely devoid of 10-nm filaments. Instead, the axoplasm is transformed to a microtubule-based cytoskeleton—although the lack of any increase in tubulin levels per unit length of nerve or of increases in microtubule numbers relative to myelin sheath thickness argues that microtubules are not increased in response to the loss of neurofilaments. Thus in vivo rodent neurofilaments are obligate heteropolymers requiring NF-L plus either NF-M or NF-H to form a filamentous network. J. Neurosci. Res. 57:23–32, 1999. © 1999 Wiley-Liss, Inc.

Mice with disrupted midsized and heavy neurofilament genes lack axonal neurofilaments but have unaltered numbers of axonal microtubules.

Mammalian neurofilaments are assembled from the light (NF-L), midsized (NF-M), and heavy (NF-H) neurofilament proteins. While NF-M and NF-H cannot self-assemble into homopolymers, the data concerning NF-L has been more contradictory. In vitro bovine, porcine, and murine NF-L can homopolymerize in the absence of other subunits. However, in vivo studies suggest that neither rat nor mouse NF-L can form filaments when transfected alone into cells lacking endogenous intermediate filaments. By contrast, human NF-L forms homopolymers in similar cell lines. Recently we generated mice with null mutations in the NF-M and NF-H genes. To determine if mouse NF-L can homopolymerize in mouse axons, NF-M and NF-H null mutants were bred to create a line of double mutant animals. Here we show that axons in NF-M/H double mutant animals are largely devoid of 10-nm filaments. Instead, the axoplasm is transformed to a microtubule-based cytoskeleton-although the lack of any increase in tubulin levels per unit length of nerve or of increases in microtubule numbers relative to myelin sheath thickness argues that microtubules are not increased in response to the loss of neurofilaments. Thus in vivo rodent neurofilaments are obligate heteropolymers requiring NF-L plus either NF-M or NF-H to form a filamentous network.

Mitogen-Activated Protein Kinases (Erk1,2) Phosphorylate Lys-Ser-Pro (KSP) Repeats in Neurofilament Proteins NF-H and NF-M

Mammalian neurofilament proteins, particularly midsized (NF-M) and heavy (NF-H) molecular weight neurofilament proteins, are highly phosphorylated in axons. Neurofilament function depends on the state of phosphorylation of the numerous serine/threonine residues in these proteins. Most phosphorylation occurs in the lys-ser-pro (KSP) repeats in the C-terminal tail domains of NF-H and NF-M. In our previous study, cyclin-dependent kinase 5 (cdk5) was shown to phosphorylate specifically the KSPXK repeats in rat NF-H. Because 80% of the repeats are of the KSPXXXK type, it was of interest to determine which kinase phosphorylates these motifs. Using a synthetic KSPXXXK peptide to screen for a specific kinase, we fractionated rat brain extracts by column chromatography and identified extracellular signal-regulated kinase (Erk2) activated by an upstream activator, the mitogen-activated protein kinase kinase MAPKK (MEK), by Western blot analysis, sequence identification, and inhibition by a specific MEK inhibitor (PD 98059). The fraction containing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylated all types of KSP motifs in peptides (KSPXK, KSPXXK, KSPXXXK, and KSPXXXXK) derived from NF-M and NF-H. They also phosphorylated an expressed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as dephosphorylated native rat NF-H, and NF-M proteins with accompanying decreases in their respective electrophoretic mobilities. A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK peptides revealed that, in contrast to cdk5, which phosphorylated only the KSPXK peptide, Erk2 could phosphorylate both. The preferred substrate for Erk2 was KSPXXXK peptide. The MEK inhibitor PD 98059 also inhibited phosphorylation of NF-H, NF-M, and microtubule-associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neurite outgrowth, suggesting that Erk1,2 may play an important role in neurite growth and branching. These data suggest that neuronal Erk1 and Erk2 are capable of phosphorylating serine residues in diverse KSP repeat motifs in NF-M and NF-H.

Association of Neurofilament Proteins with Neuronal Cdk5 Activator

Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35nck5a and other protein components (Lee, K.-Y., Rosales, J. L., Tang, D., and Wang, J.H. (1996) J. Biol. Chem. 271, 1538-1543). The yeast two-hybrid system was employed to identify p35nck5a-interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment revealed significant homology between the p35nck5a-binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35nck5a and the Cdk5/p35nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35nck5a.

Heterogeneous expression of neurofilament proteins in forebrain and cerebellum during development: clinical implications for spinocerebellar ataxia

Using quantitative immunoblotting, we have measured the level of two mammalian neurofilament proteins, the 68-kDa NF-L and the 66-kDa NF-66 (α-internexin), in the rat CNS during development. NF-66 is localized in neurons and neuronal processes in both embryonic and postnatal brain. Importantly, NF-66 is more abundant than NF-L in both forebrain and cerebellum during development. The prevalence of NF-66 over NF-L is most pronounced in brain gray matter. The expression of both NF-66 and NF-L increases continuously during the first month after birth. In situ hybridization demonstrated that NF-66, but not NF-L, is expressed in the cerebellar granule cells. Our findings suggest that the neurofilaments are heterogeneous in developmental expression, among neuronal subtypes and in composition. Human NF-66 neurofilament has recently been mapped to chromosome 10q24. Careful analysis of the human genome map indicates NF-66 gene lies within the critical region of infantile-onset spinocerebellar ataxia (IOSCA). The characteristic developmental expression and spatial localization of the NF-66 gene suggests it as a candidate gene for the disease.

Identification of Phosphorylation Sites on Neurofilament Proteins by Nanoelectrospray Mass Spectrometry

Neurofilament (NF) proteins are intermediate filaments found in the neuronal cytoskeleton. Phosphorylation of these proteins is considered an important factor in the assembly of filaments and determination of filament caliber and stability. Mammalian neurofilaments are composed of three polypeptide subunits, NF-L, NF-M, and NF-H, all of which are phosphorylated. Here we used techniques for the mass spectrometric sequencing of proteins from polyacrylamide gels to analyze in vivo phosphorylation sites on NF-M and NF-L. Neurofilaments were isolated from rat brain and enzymatically digested in gel. The resulting peptides were analyzed and sequence data obtained by nanoelectrospray mass spectrometry. Four phosphorylation sites have been found in the C-terminal domain of NF-M: serines 603, 608, 666, and 766. Two of these are found in lysine-serine-proline (KSP) motifs and two in the variant motifs, glutamic acid-serine-proline (ESP) and valine-serine-proline (VSP). Serine 55 in NF-L was not found to be phosphorylated, which confirms the possible role of phosphorylation and dephosphorylation of this site in early neurofilament assembly. The techniques used enable sequence data and characterization of posttranslational modifications to be obtained for each individual subunit directly from polyacrylamide gels.

Cytoplasmic O-GlcNAc Modification of the Head Domain and the KSP Repeat Motif of the Neurofilament Protein Neurofilament-H

Neurofilaments, the major intermediate filaments in large myelinated neurons, are essential for specifying proper axonal caliber. Mammalian neurofilaments are obligate heteropolymers assembled from three polypeptides, neurofilament (NF)-H, NF-M, and NF-L, each of which undergoes phosphorylation at multiple sites. NF-M and NF-L are known to be modified by O-linked N-acetylglucosamine (O-GlcNAc) (Dong, D. L.-Y., Xu, Z.-S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993) J. Biol. Chem. 268, 16679-16687). Here we further report that NF-H is extensively modified by O-GlcNAc at Thr53, Ser54, and Ser56 in the head domain and, somewhat surprisingly, at multiple sites within the Lys-Ser-Pro repeat motif in the tail domain, a region in assembled neurofilaments known to be nearly stoichiometrically phosphorylated on each of the approximately 50 KSP repeats. Beyond the earlier identified sites on NF-M and NF-L, O-GlcNAc sites on Thr19 and Ser34 of NF-M and Ser34 and Ser48 of NF-L are also determined here, all of which are localized in head domain sequences critical for filament assembly. The proximity of O-GlcNAc and phosphorylation sites in both head and tail domains of each subunit indicates that these modifications may influence one another and play a role in filament assembly and network formation.

The structure of the neurofilament cytoskeleton in the squid giant axon and synapse.

Stereo views of thick sections (< 0.5 micron), or freeze etch replicas taken from selected regions of the squid giant axon and giant synapse, using an electron energy loss spectroscopic ultrastructural method, revealed a meshwork of anastomosing neurofilaments intersecting at a wide range of angles. This type of architectural organisation differs significantly from the ladder-like construction of the mammalian neurofilament cytoskeleton, in which longitudinally oriented core filaments are interconnected by lateral crossbridges. The deviant organization of squid neurofilaments is consistent with recent evidence that squid neurofilament proteins more closely resemble nuclear lamin rather than mammalian neurofilament proteins. A proximo-distal gradient of increasing width of the neurofilament meshes along the giant axon correlated well with the previously described gradient of increasing neurofilament phosphorylation. In these thick sections the presynaptic terminal of the giant synapse exhibited a mature neurofilament cytoskeleton that extended to the active zones without detectable signs of degradation. The observations are discussed in the context of current hypotheses concerned with the function of phosphorylation of neurofilaments, and with the steady state maintenance of the cytoskeleton in the squid axon and presynaptic terminal.

Expression of neurofilament proteins during retinoic acid-induced differentiation of P19 embryonal carcinoma cells.

Retinoic acid (RA) induces P19 embryonal carcinoma cells to differentiate into neurons with the extension of neuritic processes. We used the P19 cell as a model system to elucidate the regulation of neurofilament (NF) expression. Four mammalian NF proteins, NF-66 (alpha-internexin), peripherin, NF-L and NF-M, and the neural-specific, growth-associated gene, GAP-43, were studied during the RA treatment of P19 cells in vitro. As controls, untreated P19 cells were maintained in parallel. Indirect immunofluorescent staining showed that in RA-treated, morphologically differentiated P19 cells NF-66 was expressed in neuron-like cells characterized by phase bright cell bodies and long neuritic processes. At various times P19 cells were harvested for protein analysis by immunoblotting with antibodies to individual NF proteins or for total RNA extraction and Northern blotting with cDNA probes for NF-66, -L, -M, peripherin and GAP-43. During induction, both NF-66 and NF-L were expressed but in distinct patterns. NF-66 mRNA and protein were detected after 6 days of induction. In contrast, NF-L mRNA, but not protein, was expressed in both induced and control cells. Neither NF-M nor peripherin were expressed during induction. During differentiation of P19 cells, NF-66 mRNA levels rose markedly by the 1st day, reached a plateau between the 3rd-5th days and declined by the 7th day. NF-66 protein accumulation lagged slightly, reaching maximum abundance about the 5th day. The kinetics of NF-66 expression were similar to that of GAP-43. However, the pattern of NF-L expression was distinct from that of NF-66. NF-L mRNA, and some protein, was expressed in both RA-treated and control cells within 6 h after plating, but was down-regulated to baseline level thereafter in both populations. Neither NF-M or peripherin expression was detected during the differentiation. In summary, NF-66 was up-regulated most robustly among the four NF proteins during differentiation in P19 cells and was the major NF protein correlated with neurite extension.