kDa Neurofilament Protein Research Articles

Phosphorylation modulates calpain-mediated proteolysis and calmodulin binding of the 200-kDa and 160-kDa neurofilament proteins.

The effects of enzymatic dephosphorylation on neurofilament interaction with two calcium-binding proteins, calpain and calmodulin, were examined. Dephosphorylation increased the rate and extent of 200-kDa neurofilament protein proteolysis by calpain. In contrast, dephosphorylation of the 160-kDa neurofilament protein did not alter the rate or extent of calpain proteolysis. However, the calpain-induced breakdown products of native and dephosphorylated 160-kDa neurofilament protein were different. Dephosphorylation did not change the proteolytic rate, extent, or breakdown products of the 68-kDa neurofilament protein. Calmodulin binding to the purified individual 160- and 200-kDa neurofilament proteins was increased following dephosphorylation. These results suggest that phosphorylation may regulate the metabolism and function of neurofilaments by modulating interactions with the calcium-activated proteins calpain and calmodulin.

Immunocytochemical localization of intermediate filaments in the guinea pig vestibular periphery with special reference to their alteration after ototoxic drug administration.

The present study examined the immunocytochemical localization of various intermediate filaments (IFs), 68 kDa, 160 kDa and 200 kDa neurofilament protein (NFP), cytokeratin (CK) 1, 8, 10 and 19, vimentin, and glial fibrillary acidic protein (GFAP) in the vestibular end-organs and ganglia of normal and streptomycin-treated guinea pigs. In normal animals, 68 kDa, 160 kDa and 200 kDa NFP were found in afferent nerve fibers and nerve terminals (probably nerve chalices). Fine nerve fibers (probably efferent and/or sympathetic nerve fibers) were also immunoreactive to NFP. In the vestibular ganglia, 68 kDa and 160 kDa NFP were predominantly distributed in larger cells, whereas 200 kDa NFP was also found in some small ganglion cells. Cytokeratin 8 and 19 were located in supporting cells, transitional cells, dark cells of vestibular end-organs, and the epithelial cell lining of the membranous labyrinth. Vimentin was observed in the hair cells distributed in the central region of the end organs, supporting cells, most connective tissue cells, and Schwann cells of the vestibular ganglion. Although GFAP-like immunoreactivity was evident in glial cells of the proximal vestibular nerve, no immunoreactivity was detected in the distal portion of the vestibular nerve, vestibular ganglion, or vestibular end-organs. These highly distinct staining patterns of IFs indicated that they may play different roles in the different cell types, and that they may serve as a specific marker for each cell type. In streptomycin-treated guinea pigs, immunoreactivities for NFP and vimentin (found in the hair cells) decreased after treatment, whereas immunoreactivities for the other IFs were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

De novo generation of neuronal cells from the adult mouse brain.

Cells of neuronal morphology, expressing the 150- and 200-kDa neurofilament proteins, were generated in vitro from populations of neural cells dissociated from adult (greater than 60-day-old) mouse brain. Most of these neurons arose from dividing precursors, as demonstrated by the incorporation of [3H]thymidine during the culture period and autoradiography. Neuronal production was optimal under the conditions in which precursors were initially stimulated with basic fibroblast growth factor and then exposed to medium conditioned by an astrocytic cell line, Ast-1, in serum-free medium. Few, if any, neurons arose in control cultures or in cultures kept in serum and fibroblast growth factor. These results suggest that neuronal precursors exist in the adult mammalian brain, but they require discrete epigenetic signals for their proliferation and differentiation.

Local radiolabeling of the 68 kDa neurofilament protein in rat sciatic nerves

Rat sciatic nerve segments, 4.5 cm distal to the dorsal root ganglion (DRG), were incubated in vivo with [ 35S]methionine. Fluorography of 2-D polyacrylamide gels of the nerve proteins demonstrated the labeling of the 68-kDa neurofilament protein, which was identified by immunoblotting. This experimental design excludes the dorsal root ganglion as the source of the radiolabeled neurofilament protein and suggests that this neuronspecific protein may be synthesized in axons.

Coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in mouse clonal hybrid neurons derived from the septal region.

Two clonal immortalized neurons designated SN6.1b and SN6.2a were isolated by limiting dilution from a mouse embryonic septal cholinergic neuronal hybrid cell line SN6 (Hammond et al., 1986). In the serum-containing medium without extra differentiating agents, one-third of SN6.1b cells stably exhibited a morphology of differentiated neurons with extensive elaborate neurites, while a majority of SN6.2a cells, along with the parent cell line SN6, were round in shape with poorly branched short processes. Neurochemical studies showed that both clones synthesized choline acetyltransferase (ChAT), dopamine, norepinephrine, serotonin, and glutamate. Immunocytochemically, they expressed a number of neuronal antigens, such as 200-kDa neurofilament protein, neuron-specific enolase, microtubule-associated protein 2, tau protein, tubulin, neural cell adhesion molecule, Thy-1.2, saxitoxin-binding sodium channel protein, ChAT, tyrosine hydroxylase, serotonin, and glutamate. The coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in the clonal hybrid septal neurons that express a variety of immunocytochemical properties of differentiated neurons suggests that embryonic septal cholinergic neurons are potentially multiphenotypic with respect to neurotransmitter synthesis.

IgG monoclonal proteins from patients with axonal peripheral neuropathies bind to different epitopes of the 68 kDa neurofilament protein

We describe three patients with a sensorimotor axonal polyneuropathy and an IgG M-protein that binds to a 68 kDa axonal protein identified as the low molecular weight neurofilament protein (NF-L). The immunological studies revealed that the M-proteins have different target epitopes: one is phosphorylated and the other two are nonphosphorylated. One of the nonphosphorylated epitopes is common to other intermediate filaments, such as desmin and vimentin.

Differentiation of embryonic chick brain cells in monolayer and reaggregate cultures: A potential model in vitro for neurotoxicity

In order to develop a model for potential neurotoxicity and teratogenicity, chick brain cells (embryonic day 7) were mechanically dissociated and cultured for up to several months. Differentiation of nerve and glial cells (monolayers in petri dishes and reaggregates in suspension cultures) were monitored with monoclonal antibodies against 68 kDa neurofilament protein (anti-NF) and glial fibrillary acidic protein (anti-GFAP). Anti-NF stains neurons in vitro as they differentiate morphologically; at day 1 many nerve processes already are feebly stained. In monolayer cultures extensive neural networks develop at day 6, which are strongly stained by anti-NF. At day 8 staining fades, days before deterioration becomes visible. In reaggregates a stable differentiation of nerve cells could be observed by intensive anti-NF staining for as long as 3 wk in culture; 5-wk-old cultures still showed substantial staining. The differentiation of GFAP-positive cells takes 3 days in vitro in monolayers as well as in reaggregate cultures. Small groups of marked cells continually increase in number; after 2 wk in culture they are present throughout the reaggregates. The differentiation of astrocytes as measured by anti-GFAP immunostaining is considerably faster in vitro than in vivo. It is suggested that the expression of NF in nerve cells could be used as a sensitive cytotoxicity endpoint, whereas GFAP expression could serve as an endpoint for monitoring differentiation of neural tissue.

Cholinergic fiber perturbations and neuritic outgrowth produced by intrafimbrial infusion of the neurofilament-disrupting agent 2,5-hexanedione

The compound 2,5-hexanedione (HD) produces axonopathies in peripheral nerves characterized by selective accumulation of neurofilaments. Its direct actions on neurotransmitter-specific neurons in the brain are unknown. In an attempt to address this latter issue, we infused HD into the fimbria and evaluated histochemically and immunohistochemically possible structural alterations in cholinergic neurons projecting from the basal nuclear complex to the hippocampus. Putative cholinergic fibers expressing nerve growth factor receptor and acetylcholinesterase showed increases in caliber and perturbations in trajectories 2–4 days following HD treatment. Similar morphologic changes were observed in neuronal elements processed for the 68 kDa neurofilament protein. At 7 days, short collateral ramifications appeared in many cholinergic axons that were suggestibe of neurite outgrowth. Correlated with these fiber alterations was a transient reduction in the number of medial septal and diagonal band somata expressing choline acetyltransferase, which returned to control levels within 6 weeks following HD treatment. These data support the view that neurofilaments play an important, perhaps cytoarchitecturally stabilizing, role in regulating axonal morphology in certain populations of cholinergic neurons.

The 5'-flanking region of the synapsin I gene. A G+C-rich, TATA- and CAAT-less, phylogenetically conserved sequence with cell type-specific promoter function.

The 5'-terminal region of the rat gene for the neuron-specific phosphoprotein, synapsin I, was isolated and sequenced. It comprises 1472 nucleotides (nt) of 5'-flanking sequence, 507 nt of the first exon, and 242 nt of the first intron. A single transcription start site was mapped by primer extension and S1 nuclease analysis. A sequence of 340 nt upstream from the transcription start site and the first exon are G+C-rich and enriched in CpG dinucleotides, resembling a CpG island. The 5'-flanking sequence lacks TATA and CAAT consensus elements but contains a consensus motif for the cAMP-responsive element. Furthermore, we notice two potential consensus motifs which are also found in corresponding positions in the genes for the nerve growth factor receptor and the 68-kDa neurofilament protein. The 5'-terminal region of the human synapsin I gene was also cloned and sequenced. A high degree of sequence conservation between rat and human is found in the upstream 340 nt that coincides precisely with the G+C-rich domain and includes the consensus elements, and throughout the first exon including the untranslated sequence. Sequence conservation is also observed further upstream and at the beginning of the first intron. In a transient chloramphenicol acetyltransferase expression assay, 5'-flanking sequences of the rat synapsin I gene function as strong promoters in neuroblastoma cells, but not in fibroblastoid cells. 225 nt of 5'-flanking sequence and 105 nt of 5'-untranslated sequence are sufficient for cell-type specific transcription in this assay.

Transient expression of neurofilament protein during hair cell development in the mouse cochlea

A polyclonal antiserum raised against the 145 kDa neurofilament protein (NFM) has been used to study the distribution of neurofilaments both in organotypic cultures of the early postnatal mouse cochlea and during development of the mouse cochlea in vivo. In the cultures, both the inner hair cells and the outer hair cells are stained by the antibodies, as well as the innervating afferent fibres from the spiral ganglion. In cultures denervated at the time of preparation, neurofilament positive hair cells can still be detected after 7 days in vitro. NFM can also be detected by immunoblotting in such denervated cultures. Characteristic 10 nm diameter, cytoplasmic filaments can also be observed in cultured hair cells using transmission electron microscopy. Immunostaining of cryosections prepared from cochleas at embryonic days 17 and 19, and days 1, 2, 5, 10 and 21 post-partum reveals that hair cells transiently express NFM during their development in vivo. Expression of NFM in hair cells is first detected at embryonic day 19 in the basal region of the cochlea and, by 2 days post-partum, neurofilament positive hair cells are found throughout the entire length of the cochlea. By 10 days post-partum, staining of hair cells begins to diminish and, by 21 days post-partum, NFM can no longer be detected in hair cells.

Slow transport rates of cytoskeletal proteins change during regeneration of axotomized retinal neurons in adult rats

To investigate cytoskeletal changes associated with axonal regrowth from damaged nerve cells in the mammalian CNS, we examined the slow transport of axonal proteins during the regeneration of adult rat retinal ganglion cell (RGC) axons. Although normally such RGC axons do not regrow after injury in the CNS, they can extend several centimeters when their nonneuronal environment is changed by replacing the optic nerve (ON) with a grafted segment of peripheral nerve (PN). Proteins transported in axons of RGCs from intact control and PN-grafted animals were labeled by an intraocular injection of 35S-methionine and examined 4-60 days later by SDS PAGE. During RGC regeneration into PN grafts, the transport rate of tubulin and neurofilament increased twofold, whereas that of actin decreased to nearly one third of its normal rate. Thus, in these regenerating RGC axons, all three major cytoskeletal proteins were largely transported within a single rate component rather than in the two separate components (SCa and SCb) normally observed in the intact ON. Furthermore, the 200 kDa neurofilament protein (NF-H) was persistently detected in Western blots during periods of active regeneration, a finding that contrasts with the late appearance of the NF-H during the developmental growth of retinal axons. The changes in slow transport observed during RGC regeneration in adult rats may reflect growth-associated responses of mature CNS neurons during periods of active axonal extension.

Expression of Major Histocompatibility Complex on Human Medulloblastoma Cells

Medulloblastoma is one of the most common malignant brain tumors in childhood. These cells are immature bipotential cells that could differentiate into both neuronal and glial cells. The authors established two human medulloblastoma cell lines. One was derived from a 2-year-old girl with cerebellar tumor (designated as ONS-76) and another was from a 9-year-old girl with metastatic tumor in the right frontal lobe (ONS-81). Immunohistochemical studies showed that both cell lines possessed 145 and 200 kDa neurofilament proteins and neuron-specific enolase, without glial fibrillary acidic protein and S-100 protein. It was shown that interferon gamma could enhance or induce the expression of the major histocompatibility complex (MHC) antigens which play a major role in immune response. Also shown for the first time was the expression of MHC class II antigens on human medulloblastoma (ONS-76 and 81) with neuronal differentiation.

Ultrastructural, Morphometric, and Immunocytochemical Study of Anterior Horn Cells in Mice with “Wasted” Mutation

Mice with the autosomal recessive gene "wasted" (wst/wst) manifest hindlimb paralysis and tremulousness, develop reduced secretory immune responses, and have abnormal DNA repair mechanisms. There is prominent vacuolar degeneration of neurons within anterior horns of the spinal cord and motor nuclei of the brainstem. A morphometric analysis of motor neurons in the spinal cord was performed on 2-hydroxyethyl methacrylate-embedded tissue from ten wst/wst mice, ten littermates (wst/+, +/+) without clinical deficits, and ten parental (+/+) control mice. Vacuolated neurons were present only in wst/wst mice (p = 0.0008). Fibrillary neurons were more numerous in the wst/wst mice than in littermates (p = 0.01) or controls (p = 0.007). The number of total or normal neurons did not differ significantly among the three groups. Volume measurements for normal, fibrillary, vacuolated, and total neurons were greater in wst/wst mice (p less than 0.008). Electron microscopic studies revealed vacuolar degeneration exclusively within neurons of wst/wst mice with the prominent accumulation of neurofilaments. Immunocytochemical staining of Araldite-embedded sections with monoclonal antibodies (MAb) to 68 kDa, 160 kDa, and 200 kDa neurofilament proteins showed prominent staining of vacuolated and fibrillary neurons in wst/wst mice exclusively with the MAb to 200 kDa neurofilaments. Dephosphorylation of tissue reduced the staining of 200 kDa neurofilaments in wst/wst mice. These studies suggest that phosphorylated neurofilaments may be important in events producing neuronal dysfunction. Therefore the "wasted" mutation may be an excellent model for the study of motor neuron disease.

Evidence that Formation of an Intermediate Filament-Protein Complex Plays a Primary Role in Aggregation of Neurofilaments, Glial Fibrillary Acidic Protein (GFAP)-Filaments and Vimentin-Filaments by 2,5-Hexanedione

Using a variety of cell types in culture, we have investigated the relevance of intermolecular crosslinking involving intermediate filament proteins (IF-proteins) to the changes in distribution of intermediate filaments induced by the neurotoxicant, 2,5-hexanedione (2,5HD). Aggregation of vimentin-filaments (vimentin-IF), glial fibrillary acidic protein (GFAP)-IF and neurofilaments was preceded by appearance on immunoblots of a high molecular weight complex (IF-complex) labeled by antibodies against the IF-protein of the cell type: pV-170 from human skin fibroblasts and pV-130 from 3T3 mouse fibroblasts, which were labeled by anti-vimentin and, from cultures of dissociated mouse spinal cord and dorsal root ganglia, pNFH-300 labeled by antibody against the 200 kDa neurofilament protein (NF-200 or NF-H) and a doublet labeled by antiserum to GFAP (pGFAP-145).pV-170 was detected in human skin fibroblasts within one hour of exposure to 2,5HD and the amount of both pV-170 and pNFH-300 was related to the concentration of 2,5HD and the duration of exposure. Intermediate filament-complexes were not detected in PtK1 epithelial cells by labeling of transblots with anti-cytokeratin, nor are keratin-IF aggregated by 2,5HD. Intermediate filament-complexes were not detected in fibroblasts with IF-aggregates secondary to disruption of microtubules by colchicine or in fibroblasts from patients with giant axonal neuropathy.

On the role of the 200-kDa neurofilament protein at the developing neuromuscular junction

To examine whether the 200-kDa neurofilament protein (200K NFP) is involved in mechanically stabilizing axons, we studied the developmental appearance of immunoreactivity to nonphosphorylated and phosphorylated 200K NFP at the neuromuscular junction. Polyinnervated rat muscle fibers become singly innervated during the first 3 weeks of postnatal life through the process of synapse elimination. If production or post-translational modification of the 200K NFP is actively involved in imparting mechanical stability on neuromuscular synapses, then the selective presence of this protein in only one of several axons at each developing end plate region might make that one axon selectively resistant to elimination. The remaining axons would then be eliminated. Immunoreactivity to the 200K NFP is present on Gestational Day 14 and can be seen in more than one preterminal axon in the end plate region of a muscle fiber during the period of synapse elimination. These results suggest that the 200K NFP is present and phosphorylated early in development and, although the 200K NFP may increase the mechanical stability of axons, this increased stability does not determine the final outcome of synapse elimination.

Neurofilament and tubulin expression recapitulates the developmental program during axonal regeneration: induction of a specific beta-tubulin isotype.

We examined the differential expression of genes encoding three beta-tubulin isotypes (classes I, II, and IV) and the 68-kDa neurofilament protein (NF68) in rat sensory neurons during development, maturation, and axonal regeneration. Expression of the specific beta-tubulin gene encoding the class II isotype was induced to high levels during development and axonal regeneration, whereas the expression of genes encoding the two other isotypes (classes I and IV) remained comparable to mature levels. Conversely, expression of the NF68 gene was relatively low during development and regeneration. Thus, the developmental program for cytoskeletal gene expression is recapitulated during axonal regeneration. The high level of class II beta-tubulin expression found in developing and regenerating neurons occurs during the longitudinal growth of axons. In contrast, induction of NF68 gene expression is associated with the radial growth of axons in maturing neurons.

Neither laminin nor prior optic nerve section are essential for the regeneration of adult mammalian retinal ganglion cell axons in vitro.

Retinal explants obtained from normal adult rats and from operated animals in which the optic nerve had been sectioned 10 days previously were cultured in either serum-containing or serum-free medium on poly-L-lysine and laminin substrata. Regenerating ganglion cell axons growing from these explants have been identified using monoclonal antibodies against Thy-1.1 cell surface glycoprotein and the 200-kDa subunit neurofilament protein. Irrespective of substratum or medium composition, axons regenerated from 28-49% of normal rat retinal explants. This percentage increased to 60-84% of explants from operated rats. There were no significant differences in percentages of explants from normal or operated rats showing neurite outgrowth when substrata of either poly-L-lysine or laminin were compared in serum-free medium. In serum-containing medium the results were less easily interpreted due to the presence of an outgrowth of non-neuronal (glia and mesenchymal) 'flat cells', which served as a preferred axonal substratum in many cases. Thus we show that adult rat retinal ganglion cell axons will regrow in vitro, and that a 'priming' optic nerve section will increase this response. In neither case is the response laminin-dependent.

Temporal and topographic relationships between the phosphorylated and nonphosphorylated epitopes of the 200 kDa neurofilament protein during development in vitro

The ontogeny of the triplet of neurofilament proteins (NF), and the phosphorylated and nonphosphorylated derivatives of the 200 kDa neurofilament subunit (NF200P, NF200D) have been investigated in dissociated cultures prepared from gestational day 13 mouse spinal cord and dorsal root ganglia (DRG), using immunocytochemical methods. Neurofilament-like immunoreactivity (NF-LI), as detected with antiserum, occurred in the somata and processes of all neurons from day 1 in culture, and reached a maximum density and intensity at days 16-20. The first labeling of neurons by NF200D antibodies occurred at day 3, and was confined to DRG cells. Only a small, proximal portion of the axons from these cells exhibited NF200D-LI. At later stages, however, this immunoreactive region extended to include progressively more distal parts. Spinal cord neurons first became NF200D-positive at day 9; however, many NF200D-negative neurons still remained in mature cultures. Also at these later stages, some axons were stained for less than their full length with the NF200D antibody. NF200P-LI was first apparent at day 17, in smooth and varicose axons and only where NF-LI was also present. In contrast, NF200P- and NF200D-LI were usually localized in mutually exclusive populations of axons and other fibers. In some, predominantly thick axons, however, the proximal segment was NF200D-positive, whereas the distal part exhibited solely NF200P-LI. In contrast to NF70 and NF150, the 200 kDa neurofilament is dilatory in its appearance in most neurons in culture. The development of the nonphosphoderivative precedes that of the phosphoderivative, and the respective ontogenies are specific for different neuronal types. Posttranslational phosphorylation of NF200 seems therefore to occur at a later stage of development than the induction of NF200 itself, while there is a wide variation in its rates of phosphorylation during passage down different axons.

Anti-neurofilament protein antibodies in opsoclonus-myoclonus

Opsoclonus-myoclonus (OM) is a neurological disorder usually occurring in infancy, clinically manifested by various involuntary movements. The pathogenesis of OM is unknown, but since the disease often is associated with viral infection or with neuroblastoma, an immunologic basis for OM has been postulated. We have studied two children with OM whose serum contained antibodies directed against the 210 kDa neurofilament protein; these antibodies were not seen in the serum of 21 children with other neurological disorders. Neurofilament proteins, which are found only in neurons, may be of prime importance in neuronal function, especially during development of the nervous system. Our findings suggest that generation of antibodies to the neurofilament proteins can occur in patients with opsoclonusmyoclonus; the role of the anti-NF210K antibodies in the pathogenesis of OM, however, is uncertain.

Neurofilament gene expression: a major determinant of axonal caliber.

Within the wide spectrum of axonal diameters occurring in mammalian nerve fibers, each class of neurons has a relatively restricted range of axonal calibers. The control of caliber has functional significance because diameter is the principal determinant of conduction velocity in myelinated nerve fibers. Previous observations support the hypothesis that neurofilaments (NF) are major intrinsic determinants of axonal caliber in large myelinated nerve fibers. Following interruption of axons (axotomy) by crushing or cutting a peripheral nerve, caliber is reduced in the proximal axonal stumps, which extend from the cell bodies to the site of axotomy. (The distal axonal stumps, which are disconnected from the cell bodies, degenerate and are replaced by the outgrowth of regenerating axonal sprouts arising from the proximal stump). This reduction in axonal caliber in the proximal stumps is associated with a selective diminution in the amount of NF protein undergoing slow axonal transport in these axons, with a decrease in axonal NF content, and with reduced conduction velocity. The present report demonstrates that changes in axonal caliber after axotomy correlate with a selective alteration in NF gene expression. Hybridization with specific cDNAs was used to measure levels of mRNA encoding the 68-kDa neurofilament protein (NF68), beta-tubulin, and actin in lumbar sensory neurons of rat at various times after crushing the sciatic nerve. Between 4 and 42 days after axotomy by nerve crush, the levels of NF68 mRNA were reduced 2- to 3-fold. At the same times, the levels of tubulin and actin mRNAs were increased several-fold. These findings support the hypothesis that the expression of a single set of neuron-specific genes (encoding NF) directly determines axonal caliber, a feature of neuronal morphology with important consequences for physiology and behavior.