Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain,spinal cord,and sciatic nerve

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were invetigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by indentification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.     

Cross-Linking of Neurofilament Proteins of Rat Spinal Cord In Vivo After Administration of 2,5-Hexanedione

The aliphatic hexacarbons n-hexane, methyl-n-butyl ketone, and 2,5-hexanedione are known to produce a peripheral neuropathy that involves an accumulation of 10-nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. In this study, rats were treated with 0.5% 2,5-hexanedione in drinking water for 180 days, and their spinal cord neurofilaments were isolated after development of the neuropathy. Visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a significant reduction in content of the neurofilament triplet proteins in treated animals and the presence of bands migrating at 138K and 260K that were not present in control animals. Analysis of the lanes using immunoblotting procedures and anti-70K, anti-160K, and anti-210K neurofilament antibodies revealed many cross-linked peptides. The 138K band cross-reacted with the anti-160K neurofilament antibody. This suggests that the 138K band is an intramolecular cross-link of the 160K neurofilament subunit. In addition to this peptide, there were numerous high-molecular-weight peptides immunoreactive with all three neurofilament protein antibodies. In addition to cross-linking, there was also a diminished amount of immunoreactive breakdown product of all three neurofilament proteins. This report demonstrates direct evidence of 2,5-hexanedione-induced cross-linking of neurofilament proteins in vivo, which maybe responsible for the accumulation of neurofilament proteins pathognomic of this neuropathy.     

Neurofilament Proteins in Cultured Chromaffin Cells

Antibodies were raised against the 200-kd, 145-kd, and 68-kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross-react with any of the two other neurofilament polypeptides. Use of the three antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the three neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The three anti-neurofilament antibodies labelled the cells in a similar pattern: very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in our cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The three neurofilament antibodies were also used to immunoprecipitate specifically three proteins of molecular weights 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabelled with [35S]methionine. These proteins correspond in molecular weight to the neurofilament triplet found in bovine brain. Finally, the presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68-kd antibody. A 70-kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. It is concluded from these results that chromaffin cells contain completely assembled neurofilaments. This additional neuronal property again illustrates that chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.     

Study of the 10-nm-filament fraction isolated during the standard microtubule preparation.

The cold non-depolymerizable fractions obtained during the standard procedure for the isolation of microtubules from ox brain stem-cerebral hemispheres and spinal cord have been studied. The cerebral-hemisphere preparation was composed of 10-nm filaments but also contained large amounts of membranes. The polypeptide content included tubulin, microtubule-associated proteins and minor proteins corresponding to the neurofilament triplet of proteins of mol.wt. 210 000, 160 000 and 70 000 respectively. The brain-stem preparation contained more 10-nm filaments than membranes. The polypeptide content consisted of the neurofilament triplet (35%), tubulin (30%) and minor proteins. In contrast, the spinal-cord preparation was mainly composed of 10-nm filaments, free of membranes and containing essentially the neurofilament protein triplet (64%). These filaments appeared very similar to the peripheral-nervous-system neurofilaments described by several authors. Since the best neurofilament from the central nervous system often contained less than 15% of the neurofilament protein triplet, our spinal-cord preparation is an improvement on the usual neurofilament preparation. This simple and rapid method gave large amounts of 10-nm filaments (100 mg per 100 g of spinal cord) characterized by the absence of membranous material, a low content of tubulin and the 50 000-mol.wt.-protein component, and a high content of neurofilament peptides. Thus, the presence of tubulin in 10-nm filament preparations seems to be related to the contaminant membranous material and not to be linked to the interaction in vitro of tubulin or microtubules with neurofilaments, as has been suggested previously.     

Immunoelectronmicroscopical localization of the three neurofilament triplet proteins along neurofilaments of cultured dorsal root ganglion neurones

Correlated immunofluorescence and electron microscopy was used to study neurofilament expression, organization and structure in cultured neurones of newborn rat dorsal root ganglia. The results extend previous immunofluorescent data subdividing the neurones into two main classes: neurones rich in neurofilaments, expressing all three triplet proteins and neurones without noticeable neurofilaments which cannot be stained positively for any of the triplet proteins. The two classes are identified as the large light cells and small dark cells characteristically found in adult dorsal root ganglia in situ. Further ultrastructural characterization identifies the various subclasses of each major class in the cultures used. Cytoskeletons of neurofilament-rich neurones decorated by antibodies specific for each triplet protein lead to the following model. All three triplet proteins are associated with each individual filament, although the antibodies show a different localization. Whereas the 68K protein seems to form the backbone of the filament, the 200K protein is periodically arranged (repeat approx. 100 nm) in a more peripheral position. The 145 K protein is revealed in a nearly continuous manner along the filament.     

Calcium-Activated Proteolysis of Neurofilament Proteins in the Squid Giant Neuron

The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium-activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [gamma-32P]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220-kilodalton (NF-220) and a high-molecular-weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium-activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 110K protein is the putative side arm of the NF-220. In contrast, neither the HMW or the NF-220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium-activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium-activated proteolysis generated characteristic fragments that could be labeled with the anti-intermediate filament antibody.     

Vimentin and 70K Neurofilament Protein Co-exist in Embryonic Neurones from Spinal Ganglia

Abstract: The mesenchymal intermediate filament protein vimentin and the 70K component of neurofilament were detected by two-dimensional gel electrophoresis in cultures of pure sensory and sympathetic neurones derived from chick embryos. The identities of these neuronal intermediate filament proteins were confirmed by comparison of their molecular weights, isoelectric points, and peptide patterns from limited papain digestions with those of the corresponding proteins from fibroblasts and brain, respectively. A specific antibody to vimentin stained filamenteous structures and colcemid-induced coils in both neurones and associated satellite cells. In contrast, a specific antibody to the 70K neurofilament protein stained these structures solely in neurones. This neurone-specific staining, as well as its molecular weight and isoelectric point, distinguishes the 70K neurofilament protein from the 68K neurofilament as sociated protein described by others, which has been claimed to resemble the tubulin assembly protein.     

Characterization of mammalian neurofilament subunits by circular dichroism

Critical steps in the disassembly and reassembly of neurofilaments, the intermediate filaments of neurons, have been investigated. Bovine neurofilament subunits (Mr 210 000, 160 000 and 70 000) were purified by urea-polyacrylamide gel electrophoresis and renatured by dialysis against several non-denaturing buffers. The quality of the protein renaturation was measured by circular dichroism. The spectra of renatured neurofilament subunits were interpreted in terms of secondary structure and this showed that the solubilization of proteins in guanidine-HCl buffers is more suitable than in urea buffer for a good recovery of a filamentous structure. Furthermore, it is shown that (i) the three neurofilament subunits exhibit specific CD spectra, with shapes reminiscent of those obtained for the alpha/beta class of proteins and that (ii) there is good correlation between CD spectra, the state of renaturation and the ability of the proteins to assemble into filamentous structures. We conclude that CD studies of neurofilament proteins should help in understanding the numerous variables affecting the disassembly and reassembly of neurofilaments.     

Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons

Studies on the axonal transport of neurofilament proteins in cultured neurons have shown they move at fast rates, but their overall rate of movement is slow because they spend most of their time not moving. Using correlative light and electron microscopy, we have shown that these proteins move in the form of assembled neurofilament polymers. However, the polypeptide composition of these moving polymers is not known. To address this, we visualized neurofilaments in cultured neonatal mouse sympathetic neurons using GFP-tagged neurofilament protein M and performed time-lapse fluorescence microscopy of naturally occurring gaps in the axonal neurofilament array. When neurofilaments entered the gaps, we stopped them in their tracks using a rapid perfusion and permeabilization technique and then processed them for immunofluorescence microscopy. To compare moving neurofilaments to the total neurofilament population, most of which are stationary at any point in time, we also performed immunofluorescence microscopy on neurofilaments in detergent-splayed axonal cytoskeletons. All neurofilaments, both moving and stationary, contained NFL, NFM, peripherin and alpha-internexin along>85% of their length. NFH was absent due to low expression levels in these neonatal neurons. These data indicate that peripherin and alpha-internexin are integral and abundant components of neurofilament polymers in these neurons and that both moving and stationary neurofilaments in these neurons are complex heteropolymers of at least four different neuronal intermediate filament proteins.     

Rapid purification of intact tonofilaments from newborn rats : Comparison with glial filaments and neurofilaments

A rapid, new procedure for the isolation of intact tonofilaments from newborn rat skins is described. The filament preparations show two major protein subunits on SDS-PAGE with molecular weights of 58000 and 66000 D. An antiserum prepared against the 58000 D protein reacted specifically with the tonofilament preparation, but not with the protein subunits of neurofilaments, glial filaments, tubulin or actin. This specificity is confirmed by indirect immunofluorescence: anti-P58 reacts with the epidermis, whereas antisera against the neurofilament or glial filament proteins and anti-tubulin do not. These data suggest that epidermal filaments represent a class of intermediate filaments distinct from either glial filaments or neurofilaments.     

The breakdown of the individual neurofilament proteins by cathepsin D

In a continuing study of proteolysis of CNS proteins by CNS enzymes, neurofilament proteins (210 K, 155 K, 70 K) and desmin were separated, and the breakdown of individual proteins by purified brain cathepsin D was measured and compared to breakdown by plasma thrombin. With both cathepsin D and thrombin, the rate of breakdown of the 70 K protein was the highest, followed by the 155 K, and that of the 210 K was the lowest. With each substrate cathepsin D breakdown was the highest at pH 3; small but significant breakdown could be seen at pH 6. The pattern of intermediate breakdown products depended on pH, with greater amounts of fragments detected at higher pH, and the patterns with the two enzymes were different. We showed that differences exist in cleavage sites and breakdown rates of the neurofilament proteins. The capacity of the cathepsin D present in the tissue to hydrolyze these substrates was high, even at pH close to neutral, and was greatly in excess of that needed for physiological neurofilament turnover.     

Neurofilament functions in health and disease.

Transgenic approaches have recently been used to investigate the functions of neuronal intermediate filaments. Gene knockout studies have demonstrated that neurofilaments are not required for axogenesis and that individual neurofilament proteins play distinct roles in filament assembly and in the radial growth of axons. The involvement of neurofilaments in disease is supported by the discovery of novel mutations in the neurofilament heavy gene from cases of amyotrophic lateral sclerosis and by reports of neuronal death in mouse models expressing neurofilament and alpha-internexin transgenes. However, mouse studies have shown that axonal neurofilaments are not required for pathogenesis caused by mutations in superoxide dismutase and that increasing perikaryal levels of neurofilament proteins may even confer protection in this disease.     

Metabolic studies in vitro of the CNS cytoskeletal proteins: Synthesis and degradation

General aspects of metabolic features of the most prominent CNS intermediate filament proteins, the 200,000 (200K), 150,000 (150K), and 70,000 (70K) dalton proteins of the neuron, and the glial fibrillary acidic protein (GFAP) have been explored using the incubated spinal cord slice from the rat. Measurement of shortterm uptake of³H-labeled amino acids into the individual proteins separated on polyacrylamide gels revealed that of the three neurofilament proteins, 200K was most metabolically active, 150K was less active, and 70K contained very little incorporated radioactivity. Glial fibrillary acidic protein based on Coomassie blue stain affinity showed less metabolic activity than any of the neurofilament proteins. Those relationships were constant at all ages, but the metabolic activity of all CNS intermediate filaments decreased with age. When Ca²⁺ was present in the medium of the incubated slices, the intermediate filaments were rapidly destroyed, but GFAP was more resistant to degradation than the neurofilament proteins. GFAP and probably the neurofilament proteins also were relatively resistant to Ca²⁺-activated degradative mechanisms in spinal cords of rats at younger ages (15 day) than in those of older animals (10–18 months). It is likely that the Ca²⁺ activated protease is less active in developing animals in which the nerve tracts are still elongating, than in adults. These results suggest that GFAP is less active metabolically and more resistant to degradation than the neurofilament proteins at all stages of maturation, but that metabolic activity of all CNS intermediate filaments decreases with age while the susceptibility to degradation increases.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Comparative studies of the neurofilament triplet protein peptide mapping by chemical cleavage

Peptide mapping of the three neurofilament protein subunits with apparent mol. weights of 210 kDa, 160 kDa and 70 kDa was performed with two different reagents: CNBr, BNPS-Skatole leading to the cleavage of methionyl and tryptophanyl bonds respectively. With BrCN we obtained two large fragments resistant to the cleavage, with mol. wts of 85 kDa for the 160 kDa and 135 kDa for the 210 kDa neurofilament proteins respectively. These fragments were located on the C-terminal part of the proteins (the tails) and correspond to specific regions responsible for their physiological identity. On the other hand, the cleavage with BNPS-Skatole at the tryptophanyl bonds gave similar patterns. The 210 kDa and 160 kDa neurofilament proteins gave a doublet of high mol. wt resistant to the cleavage, corresponding very likely to the C-terminal part and 4 fragments of mol. wt between 30 and 40 kDa corresponding to the N-terminal part. The neurofilament triplet share a common 30.5 kDa fragment located on the N-terminal part. From these peptide mapping studies, we conclude that the two neurofilament subunit proteins with mol. wts of 160 kDa and 210 kDa are different but related structures and that the CNBr characterized cleavage fragments of mol. wt 85,000 and 135 kDa are suitable polypeptides for sequence and immunological studies of the C-terminal part of these proteins.     

Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits.

Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of neurofilaments can have an impact on peripherin assembly in vivo.     

Survey of Intermediate Filament Proteins in Optic Nerve and Spinal Cord: Evidence for Differential Expression

The distribution of intermediate filament proteins in optic nerve and spinal cord from rat, hamster, goldfish, frog, and newt were analyzed by two-dimensional gel electrophoresis. General as well as specific monoclonal and polyclonal antibodies were reacted against putative intermediate filament proteins. In vitro incubations of excised optic nerve in the presence of [35S]methionine distinguished between neuronal and nonneuronal intermediate filament proteins. The proteins of the intermediate filament complex in the two tissues for rat and hamster were similar. The typical neurofilament triplet and glial fibrillary acidic protein (GFAP) were observed. Vimentin was more concentrated in the optic nerve than in the spinal cord. The goldfish, newt, and frog contained neurofilament proteins in the 145-150K range and in the 70-85K range. In addition, predominant neurofilament proteins in the 58-62K molecular-weight range were found in all three species. In contrast to mammalian species, the goldfish, newt, and frog displayed extensive heterogeneity between optic nerve and spinal cord in the expression of both neuronal and nonneuronal intermediate filament proteins. The distinctive presence of low-molecular-weight intermediate filament proteins and their high concentration in the optic nerve and spinal cord of these nonmammalian vertebrates is discussed in terms of neuronal development and regeneration.     

Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins

Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha-helical coiled-coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino-terminal region built analogously to non-neuronal intermediate filament proteins should allow a co-polymerization process via the interaction of coiled-coil domains. The extra mass of all triplet proteins is allocated to carboxy-terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence.     

Isolation and characterization of glial filaments from human brain

Intermediate (8--9 nm) filaments of human central nervous system astrocytes were isolated from the gliosed white matter of cases of adrenoleukodystrophy (ALD). This hereditary lipidosis is characterized pathologically by demyelination, loss of axons, and replacement of the white matter of the caudal cerebrum by a glial scar. Glial filaments were composed largely of a single protein component with a mol wt of about 49,000 daltons. Smaller components (44,000--39,000 daltons) were detected in some samples, and appear to represent degradation products of the filament protein. Human neurofilaments were isolated from the normal frontal white matter of ALD cases by the standard myelin-free axon technique. Isolated glial and neurofilament proteins comigrated during acrylamide gel electrophoresis in SDS. Polypeptides resulting from cyanogen bromide cleavage of the two filament proteins were the same. Both proteins reacted with rabbit antisera raised against isolated bovine neurofilament protein and human glial fibrillary acidic protein.