Changes in Neurofilament Protein NF-L and NF-H Immunoreactivity Following Kainic Acid-Induced Seizures

Abstract: Postlesion plasticity of neuronal processes might contribute to secondary spontaneous seizures after kainic acid administration. In this study, neurofilament (NF) proteins were examined following intraperitoneal injection of kainic acid, and special reference was given to temporal changes in quantity and quality of the NF light (NF-L) and heavy (NF-H) subunits. A pronounced decrease in phosphorylation-related immunoreactivity of NF-H occurred as early as 1 day after the injection in the amygdala/pyriform cortex, hippocampus, striatum, and dorsal cerebral cortex. A shift of NF-H from the phosphorylated to nonphosphorylated form was evident in immunoblots, suggesting dephosphorylation contributed to the decrease. Decreases in NF-L and phosphorylated NF-H contents in the limbic structure at 3 days were correlated with the increasing kainic acid doses from 2.5 to 10 mg/kg. The degradation pattern in immunoblots with antibodies against NF-L indicated that the decrease in NF-L was probably due to calcium-activated proteolysis. NF-L and phosphorylated NF-H contents secondarily increased from 9 days onward, with ∼20% above the control level of phosphorylated NF-H immunoreactivity at 27 days in the amygdala/pyriform cortex and ventral hippocampus. Immunohistochemical examination of the hippocampus revealed that an increase of NF staining in the mossy fiber system may contribute to the NF recovery in this region. Furthermore, the temporal changes of NF-L and phosphorylated NF-H contents were positively correlated with those of the neuronal cell adhesion molecule, a neuritic growth cone marker, substantiating postlesion regenerative reactions of NF proteins. Functional consequences of the NF plasticity remain to be identified.     

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.     

Neurofilament proteins NF-L, NF-M and NF-H in brain of patients with Down syndrome and Alzheimer's disease

Summary. Neurofilaments (NFs) are integral constituents of the neuron playing a major role in brain development, maintenance, regeneration and the pattern of expression for NFs suggests their contribution to plasticity of the neuronal cytoskeleton and creating and maintaining neuronal architecture. Using immune-histochemical techniques the altered expression of NFs in Down syndrome (DS) and Alzheimer's disease (AD) has been already published but as no corresponding systematic immune-chemical study has been reported yet, we decided to determine proteins levels of three NFs in several brain regions of DS and AD brain. We evaluated immunoreactive NF-H, NF-M and NF-L levels using Western blotting in brain regions temporal, occipital cortex and thalamus of patients with DS (n = 9), AD (n = 9) and controls (n = 12). We found significantly increased NF-H in temporal cortex (controls: means 0.74 ± 0.39 SD; DS: means 3.01 ± 2.18 SD) of DS patients and a significant decrease of NF-L in occipital cortex of DS and AD patients (controls: means 1.19 ± 0.86 SD; DS: means 0.35 ± 0.20; AD: 0.20 ± 0.11 SD). We propose that the increase of NF-H in temporal cortex of DS brain is due to neuritic sprouting as observed in immune-histochemical studies. The increase may not be caused by the known accumulation of NFs in plaques, tangles or Lewy bodies due to our solubilization protocol. The decrease of NF-L in occipital cortex of DS and AD patients may well be reflecting neuronal loss. Altogether, however, we suggest that NFs are not reliable markers for neuronal death, a hallmark of both neurodegenerative diseases, in DS or AD. The increase of NF-H in DS or the decrease of NF-L in DS and AD leaves the other NFs unchanged, which points to dysregulation in DS and AD and raises the question of impaired structural assembly of neurofilaments. Received July 19, 2000 Accepted July 28, 2000     

Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

A Rapid HPLC Method to Separate the Triplet Proteins of Neurofilament

In this article a fast HPLC technique to separate the individual neurofilament proteins is described. Highly pure fractions of the three neurofilament proteins can be obtained. As much as 50 mg of each neurofilament polypeptide can be separated from a crude neurofilament protein preparation in one step in less than 2 h. The short separation time is of importance in minimizing degradation, especially of the 150-kilodalton neurofilament polypeptide.     

Coding sequence and flanking regions of the mouse vimentin gene

Summary Using a polyclonal antibody, a cDNA clone coding for part of mouse vimentin was identified in a gt11 expression library. DNA from this clone was used to screen a genomic library from Ehrlich Ascites Tumor cells for the mouse vimentin gene. A clone was found which contained the whole coding sequence and a large part of the 5- and 3-untranslated sequences. It was used to prepare a construct equivalent to a full-length cDNA clone. Extensive homologies to the vimentin sequence from other species were found for the coding and 3-untranslated sequences and the promoter region.     

Isolation of the intermediate filament protein vimentin by chromatofocusing

A novel, simple and relatively rapid method is described for the isolation of the intermediate-sized filament protein vimentin from eye lens tissue. Chromatofocusing is applied as the sole purification step. The apparent isoelectric point of the protein in 6 M urea and at 22°C is 4.9. Electrophoretic mobility on one- and two-dimensional polyacrylamide gels, solubility in 6 M urea and amino acid composition were used for identification     

Analysis and Comparison of In Vitro Synthesized Glial Fibrillary Acidic Protein with Rat CNS Intermediate Filament Proteins

Intermediate filament (IF) proteins from rat spinal cord were analyzed by two-dimensional gel electrophoresis and compared with the in vitro translation products of a messenger RNA-dependent reticulocyte lysate system stimulated with 16-day-old rat brain polysomes. In two dimensions, the molecular weight 49,000 to 50,000 band of the IF preparation resolved to seven spots, whereas antiserum to glial fibrillary acidic (GFA) protein precipitated only two immediately adjacent radiolabeled in vitro synthesized products, with molecular weights of 49,000 to 50,000. Autoradiographs of two-dimensional gels of extracted IF proteins incubated with iodinated IgG fraction of GFA protein antiserum showed that all seven spots were recognized by the antiserum. These observations suggest that the primary gene product of GFA protein is modified either by post-translational processing or experimental artifact.     

Domain unfolding in neurofilament sidearms: effects of phosphorylation and ATP

Lateral projections of neurofilaments (NF) called sidearms (SA) affect axon stability and caliber. SA phosphorylation is thought to modulate inter-NF distance and interactions between NF and other subcellular organelles. SA were probed by atomic force microscopy (AFM) and dynamic light scattering (DLS) as a function of phosphorylation and ATP content. DLS shows SA are larger when phosphorylated, and AFM shows four unfoldable domains in SA regardless of phosphorylation state or the presence of ATP. However, the native phosphorylated SA requires three-fold higher force to unfold by AFM than dephosphorylated SA, suggesting a less pliant as well as larger structure when phosphorylated.     

Ultrastructural Studies of Carboxyl-Terminal Truncation Mutants of the Neuronal Intermediate Filament Protein Peripherin

Abstract: We have prepared carboxyl-terminal truncation mutants of the neuronal intermediate filament (IF) protein peripherin and examined the assembly characteristics of these mutant proteins in SW13 cells in the presence and absence of vimentin. In the absence of vimentin, tailless peripherin protein (Per-C424) self-assembles into bundles and clumps as observed by immunofluorescence, whereas a peripherin mutant that is missing the tail as well as a small portion of the rod (Per-C356) appears as spherical aggregates. Similar phenotypes are observed when vimentin-positive cells are transfected with Per-C424 or Per-C356. In these cells, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundles and clumps formed by Per-C424 at the electron microscopic level, we prepared stable cell lines expressing different levels of this mutant protein. By immunofluorescence, Per-C424 appears as either clumps or bundles of filaments depending on the expression level of the mutant protein. However, under electron microscopy, it is apparent that both clumps and bundles are composed of tightly packed IFs. We were unable to obtain stable cell lines expressing Per-C356, indicating that this mutant may prevent cell proliferation. Using a vector containing an internal ribosomal entry site, we prepared a construct that expresses Per-C356 and green fluorescent protein as a single mRNA, and we were able to isolate cells that expressed Per-C356 by fluorescence-activated cell sorting. Electron microscopic analysis of these cells showed that these aggregates are solid and contain no obvious filamentous structures.     

Assembly Properties of Neurofilament Light Chain Ser55 Mutants in Transfected Mammalian Cells

Abstract: Ser⁵⁵ within the head domain of neurofilament light chain (NF-L) is transiently phosphorylated by protein kinase A, and phosphorylation of this residue is thought to regulate assembly of neurofilaments. To understand how Ser⁵⁵ phosphorylation influences NF-L assembly, wild-type and mutant NF-L genes in which Ser⁵⁵ was mutated to alanine, so as to prevent phosphorylation, or to aspartate, so as to mimic permanent phosphorylation, were transfected into mammalian cells that contain or do not contain an endogenous intermediate filament network. Wild-type and mutant NF-Ls localised to the Triton X-100-insoluble fraction, which suggests that phosphorylation of Ser⁵⁵ does not inhibit assembly of NF-L and NF-L/vimentin polymers at or below the tetrameric stage. Immunofluorescence microscopy of transfected cells demonstrated that the wild-type and mutant NF-Ls all colocalised with vimentin to produce similar filamentous arrays. However, in cells lacking an endogenous intermediate filament network, the aspartate mutant produced a pattern of staining different from that of the wild-type or alanine mutant. These results suggest that phosphorylation of NF-L Ser⁵⁵ is not a mechanism that precludes assembly of neurofilaments from monomers into intermediate filament structures but that phosphorylation/dephosphorylation of this residue might confer more subtle characteristics on neurofilament assembly properties and architecture.     

Vmac: a novel protein associated with vimentin-type intermediate filament in podocytes of rat kidney

Vimentin-type intermediate filaments (IFs) play an important role in cytoskeletal organization and cell morphology. We identified here a novel protein associated with vimentin-type IFs and named it vimentin-type IF-associated coiled-coil protein (Vmac). Vmac consists of 171 amino acids with a calculated Mr of 18,844 and has a coiled-coil domain in its N-terminal region and the PDZ-binding tetrapeptide consensus motif in its C-terminal region. Northern blotting showed that the Vmac mRNA was expressed in many rat tissues examined but most abundantly expressed in the kidney. Immunofluorescence microscopy revealed that Vmac was highly concentrated at podocytes of renal glomeruli. Podocytes are highly specialized epithelial cells characterized by a large cell body and numerous foot processes, and express vimentin-type IFs that are distributed in the cell body and the major processes. Immunoelectron microscopy revealed that Vmac was associated with vimentin-type IFs of podocytes. These results indicate that Vmac is a novel protein associated with vimentin-type IF in podocytes of rat kidney.     

Review of the Multiple Aspects of Neurofilament Functions, and their Possible Contribution to Neurodegeneration

Neurofilaments (NF) are the most abundant cytoskeletal component of large myelinated axons from adult central and peripheral nervous system. Here, we provide an overview of the complementary approaches, including biochemistry, cell biology and transgenic technology that were used to investigate the assembly, axonal transport and functions of NF in normal and pathological situations. Following their synthesis and assembly in the cell body, NFs are transported along the axon. This process is finely regulated via phosphorylation of the carboxy-terminal part of the two high-molecular-weight subunits of NF. The correct formation of an axonal network of NF is crucial for the establishment and maintenance of axonal calibre and consequently for the optimisation of conduction velocity. The frequent disorganisation of NF network observed in several neuropathologies support their contribution. However, despite the presence of NF mutations found in some patients, the exact relations between these mutations, the abnormal NF organisation and the pathological process remain a challenging field of investigation.     

The Cytoskeleton of Primary Astrocytes in Culture Contains Actin, Glial Fibrillary Acidic Protein, and the Fibroblast-Type Filament Protein, Vimentin

Abstract: Primary astrocytes were cultured from the forebrains of 1-day-old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and >80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton-insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower-molecular-weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL-8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle actin. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.     

Proteolysis of the neurofilament 68 kDa protein explains several previously described brain proteins of unique composition and high acidity

Neurofilaments follow the structural principles of non-neuronal intermediate filaments but contain additional sequences which are carboxyterminally located and increase in length between triplet proteins (68 kDa, 160 kDa and 200 kDa). The tailpiece domain has been sequenced in the case of the porcine 68 kDa protein. It has a unique amino acid composition. Within 106 residues there are only 12 different amino acid types, and glutamic acid accounts for 46% of the sequence. Examination of the literature on highly acidic brain proteins leads us to the proposal that microglutamic acid-rich protein, Glu-50, macroglutamic protein, as well as some unusual components of the S100 class, are most likely proteolytic degradation products of the neurofilament 68 kDa protein.     

Lithium Chloride Inhibits the Phosphorylation of Newly Synthesized Neurofilament Protein, NF-M, in Cultured Chick Sensory Neurons

The middle and high molecular weight members of the neurofilament triplet, NF-M and NF-H, undergo extensive posttranslational polyphosphorylation, a process requiring 24 h or more for completion. We have investigated ways of perturbing this process in intact cells and have found that phosphorylation of newly synthesized NF-M in cultured chick sensory neurons is inhibited by Li+. [35S]Methionine pulse-chase experiments were carried out with pure neuronal cultures, and the phosphorylation of newly synthesized NF-M was monitored by following the accompanying change, with chase time, in apparent size and charge of the polypeptide. Addition of LiCl to the medium inhibited this mobility shift in a dose-dependent manner over concentrations between 2 and 25 mM. Incorporation of 32P into NF-M, as well as NF-H, was also inhibited, whereas incorporation into the low molecular weight neurofilament protein, beta-tubulin, and total protein was unaffected. Protein synthesis was not altered. Exposure to 25 mM LiCl for up to 72 h was not toxic, and the inhibition of NF-M phosphorylation was completely reversible. When 25 mM Li+ was added after NF-M had become partially phosphorylated, further progression was blocked, but there was no net dephosphorylation or degradation of NF-M. Additional experiments suggest that this action of Li+ is probably not due to effects on second messenger levels or to effects on tubulin metabolism and assembly state presented in our accompanying article, but rather to interference by Li+ itself, with the phosphorylation of NF-M and NF-H by specific neurofilament kinase(s).     

Serine-23 Is a Major Protein Kinase A Phosphorylation Site on the Amino-Terminal Head Domain of the Middle Molecular Mass Subunit of Neurofilament Proteins

Abstract : We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser²³ as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S₁ (¹⁴RRVPTETRSSF²⁴) and S₂ (²¹RSSFSRVSGSPSSGFRSQSWS⁴¹), localized within the amino-terminal head domain region. Ser²³ was identified as a phosphorylation site on the ³²P-labeled α-chymotryptic peptide that carried >80% of the ³²P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S₁ and S₂ were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser²³ was the major site of phosphorylation for protein kinase A on both S₁ and S₂ peptides. Low levels of incorporation of ³²P-phosphates into Ser²², Ser²⁸, and Ser³² by protein kinase A were also observed. Protein kinase C incorporated ³²P-phosphates into Ser²², Ser²³, Ser²⁵, Ser²⁸, Ser³², and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S₁ and that ions with up to four phosphates were detected on peptide S₂. Analysis of the data from the tandem ion trap mass spectrometry by using the computer program PEPSEARCH did not unequivocally identify the specific sites of phosphorylation on these serine-rich peptides. Our data suggest that Ser²³ is a major protein kinase A-specific phosphorylation site on the amino-terminal head region of the NF-M subunit. Phosphorylation of Ser²³ on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.     

Phosphorylation Modulates Calpain-Mediated Proteolysis and Calmodulin Binding of the 200-kDa and 160-kDa Neurofilament Proteins

Abstract: 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.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

Neurofilament protein triplet immunoreactivity in the dorsal root ganglia of the guinea-pig

Summary Immunoreactivity for the neurofilament protein triplet was investigated in neurons of the dorsal root ganglia of the guinea-pig by using a battery of antibodies. In unfixed tissue, nearly all neurons in these ganglia demonstrated some degree of neurofilament protein triplet immunoreactivity. Large neurons generally displayed intense immunoreactivity, whereas most small to medium-sized neurons showed faint to moderate immunoreactivity. Double-labelling immunofluorescence demonstrated that most antibodies to the individual subunits of the neurofilament protein triplet had the same distribution and intensity of labelling in sensory neurons. Increasing durations of tissue fixation in aldehyde solutions selectively diminished neurofilament protein triplet immunoreactivity in small to medium-sized neurons. Double-labelling with neurofilament protein triplet antibodies in combination with antibodies to other neuronal markers, such as neuron-specific enolase, substance P and tyrosine hydroxylase, showed that tissue processing conditions affect the degree of co-localization of immunoreactivity to the neurofilament protein triplet and to these other neuronal markers. These results indicate that, with a judicious manipulation of the duration of tissue fixation, neurofilament protein triplet immunoreactivity can be used in combination with other neuronal markers to distinguish groups of neurons according to their size and chemical coding.