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.     

Effect of propionic and methylmalonic acids on the high molecular weight neurofilament subunit (NF-H) in rat cerebral cortex

Propionic and methylmalonic acidemias are inherited neurometabolic disorders biochemically characterized by tissue accumulation of propionic (PA) and methylmalonic (MMA) acids, respectively. Neurofilaments (NF) are important cytoskeletal proteins and phosphorylation/dephosphorylation of NF is important to stabilize the cytoskeleton. We investigated the effects of PA and MMA on the high molecular weight neurofilament subunit associated with the cytoskeletal fraction of rat cerebral cortex along development. Cortical slices from 9- to 60-day-old rats were incubated with 2.5 mM PA or MMA. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated or total NF-H was analyzed by immunoblotting using specific antibodies. Results showed that treatment of tissue slices with the acids induced an increased Triton-insoluble phosphorylated NF-H immunoreactivity in up to 17-day-old rats. Furthermore, treatments significantly increased the total amount of NF-H in 12-day-old rats. These findings indicate that PA and MMA alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction.     

Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain.

The degradation of phosphorylated and dephosphorylated neurofilament proteins by the Ca2+-activated neutral proteinase calpain was studied. Neurofilaments were isolated from bovine spinal cord, dephosphorylated by alkaline phosphatase (from Escherichia coli) and radioiodinated with [125I]-Bolton-Hunter reagent. The radioiodinated neurofilament proteins (untreated and dephosphorylated) were incubated in the presence and absence of calpain from rabbit skeletal muscle, and the degradation rates of large (NF-H), mid-sized (NF-M) and small (NF-L) neurofilament polypeptides were analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. The degradation of dephosphorylated neurofilament proteins occurred at a higher rate, and to a greater extent, than did that of the phosphorylated (untreated) neurofilament proteins. The dephosphorylated high-molecular-mass neurofilament (NF-HD) was proteolyzed 6 times more quickly than the untreated NF-H. The degradation rate of the NF-M and NF-L neurofilament proteins was also enhanced after dephosphorylation, but less than that of NF-H. This indicates that the dephosphorylation of neurofilament proteins can increase their sensitivity to calpain degradation.     

Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules

We sought the mammalian neurofilament tail domain-specific kinase. Several well known kinases including cAMP-dependent protein kinase, protein kinase C, Ca(2+)-calmodulin-dependent protein kinase II, casein kinase I, and casein kinase II phosphorylated the high (NF-H) and middle molecular mass subunit (NF-M) of bovine neurofilaments, but they did not reduced the electrophoretic mobility of the dephosphorylated form of NF-M and NF-H by phosphorylation nor was the amount of phosphorylation increased by dephosphorylation of NF proteins, indicating that the phosphorylation sites by these kinases are not major in vivo phosphorylation sites at the tail domain. In contrast, cdc2 kinase phosphorylated specifically the dephosphorylated form of NF-H. 4 mol of phosphates were incorporated per mol of NF-H and this phosphorylation returned the electrophoretic mobility of the dephosphorylated form of NF-H to the position of the isolated, fully phosphorylated form of NF-H. Furthermore, the phosphorylation by cdc2 kinase dissociated the binding of dephosphorylated NF-H to microtubules. Phosphorylation sites were located at the carboxyl-terminal tail domain. The KSPXK motif, but not KSPXX, in the repetitive sequence was suggested to be the phosphorylation site by using synthetic peptides.     

Phosphorylation of the head domain of neurofilament protein (NF-M): a factor regulating topographic phosphorylation of NF-M tail domain KSP sites in neurons

In neurons the phosphorylation of neurofilament (NF) proteins NF-M and NF-H is topographically regulated. Although kinases and NF subunits are synthesized in cell bodies, extensive phosphorylation of the KSP repeats in tail domains of NF-M and NF-H occurs primarily in axons. The nature of this regulation, however, is not understood. As obligate heteropolymers, NF assembly requires interactions between the core NF-L with NF-M or NF-H subunits, a process inhibited by NF head domain phosphorylation. Phosphorylation of head domains at protein kinase A (PKA)-specific sites seems to occur transiently in cell bodies after NF subunit synthesis. We have proposed that transient phosphorylation of head domains prevents NF assembly in the soma and inhibits tail domain phosphorylation; i.e. assembly and KSP phosphorylation in axons depends on prior dephosphorylation of head domain sites. Deregulation of this process leads to pathological accumulations of phosphorylated NFs in the soma as seen in some neurodegenerative disorders. To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites. In rat cortical neurons we showed that head domain phosphorylation of endogenous NF-M by forskolin-activated PKA inhibits NF-M tail domain phosphorylation. To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues. Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells. This is consistent with our hypothesis that transient phosphorylation of NF-M head domains inhibits tail domain phosphorylation and suggests this as one of several mechanisms underlying topographic regulation.     

Increased Solubility of High-Molecular-Mass Neurofilament Subunit by Suppression of Dephosphorylation: Its Relation to Axonal Transport

Abstract: To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of l -[³⁵S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.     

The immunological homology between two filamentous cross-linker phosphoproteins, connectin and cross-bridge region of neurofilament-H, is not affected by the phosphorylation state

It has recently been shown that a monoclonal antibody SM 1-36-2 against connectin, an elastic filament of striated muscles, binds to the "elastic" domain of the molecule, and that the H subunit of neurofilament (NF-H), an intermediate filament of nerve cells, shares a homologous domain (Shimizu, T. et al. (1988) Biomed. Res. 9, 227-234 and Itoh, Y. et al. (1988) J. Biochem. 104, 504-508). In order to characterize (1) the intramolecular localization of the domain in the NF-H and (2) the effect of the phosphorylation state on the immunoreactivity, the homologous domain in the NF-H was analyzed by Western blotting after limited digestion with trypsin or alpha-chymotrypsin and dephosphorylation with E. coli alkaline phosphatase. It was found that (1) the epitope was located not in the core region but in the carboxyl-terminal peripheral (cross-bridge) region of NF-H and (2) the epitopes in connectin and NF-H were not affected by the phosphorylation state.     

Aluminum Alters the Electrophoretic Properties of Neurofilament Proteins: Role of Phosphorylation State

Exposure of each of the three neurofilament proteins (NFPs) to AlCl3 resulted in their failure to migrate into sodium dodecyl sulfate (SDS)-containing gels. This effect was dependent on length of incubation (minimum, 2 h) and AlCl3 concentrations (minimum, 50 microM) and was not reversed by 20% SDS, 6 M urea, freeze-thawing, boiling, or extensive dialysis. The migration of vimentin and glial fibrillary acidic protein was not affected by AlCl3. The high-molecular-weight neurofilament subunit (NF-H) entered SDS-containing gels after exposure to aluminum lactate but migrated aberrantly as a long high-molecular-weight streak. Migration of the 160-kDa alpha-chymotryptic cleavage product of NF-H, which contains the higher phosphorylated tail domain, was also prevented from migrating into SDS-containing gels by AlCl3. Dephosphorylation of NF-H and the middle-molecular-weight neurofilament subunit (NF-M) eliminated these effects on gel migration. EDTA, EGTA, MgCl2, CaCl2, or FeCl3 had no effect on NF-H or NF-M migration; furthermore, preincubation with, or simultaneous exposure to, CaCl2 or FeCl3 did not alter the effect of AlCl3. One interpretation of these results is that Al3+ interacts with phosphate groups on extensively phosphorylated C-terminal sidearms of NFPs, resulting in intermolecular cross-linking. These findings demonstrate a direct effect of aluminum on NFPs and provide a possible mechanism for neurofilament accumulation in perikarya during aluminum intoxication.     

Phosphorylation-Dependent Immunoreactivity of Neurofilaments Increases During Axonal Maturation and β,β''-Iminodipropionitrile Intoxication

The immunoreactivity of the high-molecular-weight neurofilament (NF) subunit toward antibodies that react with phosphorylation-related epitopes was determined at different anatomic sites in the PNS of rats during normal maturation and after intoxication with beta,beta'-iminodipropionitrile (IDPN). A maturational increase in the relative binding of phosphorylation-dependent antibodies compared to phosphorylation-inhibited antibodies occurred from age 3 to 12 weeks. An increase in phosphorylation-related immunoreactivity with increasing distance from the cell bodies was present in ventral and dorsal roots at all ages. The degree of phosphorylation-related immunoreactivity was greater for centrally directed axons in the dorsal roots of the L5 ganglion than for peripherally directed axons. IDPN, a toxin that impairs NF transport, caused a marked increase in reactivity toward the phosphorylation-dependent antibody. NFs from IDPN-treated rats also bound less of an antibody that is normally phosphorylation independent and this inhibition of binding was sensitive to phosphatase digestion. In each instance, greater degrees of phosphorylation-dependent immunoreactivity correlate with conditions known to exhibit slower net rates of axonal transport of NF proteins.     

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).     

Inhibition of Pin1 reduces glutamate-induced perikaryal accumulation of phosphorylated neurofilament-H in neurons

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.     

Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H

Summary 1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons.2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylatedin vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40^ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that:3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H.4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40^ERK.5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40^ERK catalyzes the incorporation of, respectively, 4.2±1.3 and 2.8±0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46±0.17.6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.     

Identification of Endogenously Phosphorylated KSP Sites in the High-Molecular-Weight Rat Neurofilament Protein

Abstract: The high-molecular-weight neurofilament protein (NF-H) is highly phosphorylated in vivo, with estimates as high as 16–51 mol of P_i/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy-terminal tail region of the molecule. Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, P_i determinations, microsequencing, and mass-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord. Twenty Ser residues in NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 "non-KSP" Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of P_i/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu-C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal-derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF-H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.     

Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments

The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one- dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton- insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.     

Hyperphosphorylated neurofilament NF-H is a serum biomarker of axonal injury

Several lines of reasoning suggest that the phosphorylated axonal form of the neurofilament subunit NF-H is likely to be released from damaged and diseased neurons in significant amounts. Detection of this protein in serum or CSF might therefore provide information about the presence and degree of neuronal loss. We therefore developed a sensitive NF-H ELISA capable of detecting picogram quantities of phosphorylated NF-H (pNF-H). This assay showed that soluble pNF-H immunoreactivity is readily detectable in the sera of adult rats following various types of experimental spinal cord injury (SCI) and traumatic brain injury (TBI), but is undetectable in the sera of normal animals. Here we describe details of the time course and extent of serum pNF-H expression following experimental SCI and TBI. Following SCI, serum pNF-H showed an initial peak of expression at 16h and a second, usually larger, peak at 3 days. Following TBI, lower levels of serum pNF-H were detected with a peak at 2 days post-injury. We also show that the higher levels of pNF-H released from injured spinal cord as compared to brain are in line with the approximately 20-fold higher levels of pNF-H present in spinal cord. These findings suggest that serum levels of pNF-H immunoreactivity may be used to conveniently monitor neuronal damage and degeneration in experimental and presumably clinical situations.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on differentiating mouse N2a neuroblastoma cells

The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) was investigated in mouse N2a neuroblastoma cells, induced to differentiate by serum withdrawal and addition of dibutyryl cyclic AMP, over a 24-h period. Addition of MPTP (10 microM) during differentiation caused a change in cell morphology characterised by an inhibition of axon outgrowth, in the absence of cell death. Biochemical characterisation by western blotting revealed that MPTP had no significant effects on the levels of actin, alpha-tubulin, or total heavy-chain neurofilament (NF-H). However, NF-H phosphorylation appeared to increase following MPTP treatment when blots were probed with the phosphorylation state-specific antibodies RMd09 and Ta51. In addition, indirect immunofluorescence analysis revealed an accumulation of phosphorylated NF-H in the cell perikaryon, suggesting that altered NF-H distribution was associated with the observed effects of MPTP on cell morphology. These changes may represent a useful in vitro marker of MPTP neurotoxicity within a simple differentiating neuronal cell model system.     

Distinctly Phosphorylated Neurofilaments in Different Classes of Neurons

Abstract: Recent immunohistochemical experiments revealed that specific anti-neurofilament monoclonal antibodies yield distinct patterns in different types of neurons. This led to the suggestion that neurofilaments are a family of heterogeneous molecules whose occurrence and distribution are a function of cell type. In the present study we examined the hypothesis that this heterogeneity is due to differences in the extent of phosphorylation of neurofilament proteins in distinct types of neurons. In view of the large number of potential phosphorylation sites on the heavy neurofilament protein (NF-H), we focused on this protein and examined its extent of phosphorylation in different types of neurons. This was performed using neurofilaments isolated from axons of the cholinergic bovine ventral root motor neurons and of the chemically heterogeneous bovine dorsal root neurons. Two-dimensional gel electrophoresis revealed that the isoelectric point of ventral root NF-H (pl 5.10) was ∼0.2 pl units more acidic than that of dorsal root NH-F. This difference was abolished by treating the neurofilaments with alkaline phosphatase, suggesting that the excess negative charge of ventral root NF-H is due to increased levels of phosphorylation. Amino acid analysis confirmed that the phosphoserine content of ventral root NF-H (27.2 ± 2.5% of the serines) is markedly higher than that of dorsal root NF-H (15.5 ± 6.2% of the serines). These findings provide a novel system for studying the biochemistry and function of distinctly phosphorylated neurofilaments in different types of neurons.     

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.     

Increasing neurofilament subunit NF-M expression reduces axonal NF-H, inhibits radial growth, and results in neurofilamentous accumulation in motor neurons

The carboxy-terminal tail domains of neurofilament subunits neurofilament NF-M and NF-H have been postulated to be responsible for the modulation of axonal caliber. To test how subunit composition affects caliber, transgenic mice were generated to increase axonal NF- M. Total neurofilament subunit content in motor and sensory axons remained essentially unchanged, but increases in NF-M were offset by proportionate decreases in both NF-H and axonal cross-sectional area. Increase in NF-M did not affect the level of phosphorylation of NF-H. This indicates that (a) in vivo NF-H and NF-M compete either for coassembly with a limiting amount of NF-L or as substrates for axonal transport, and (b) NF-H abundance is a primary determinant of axonal caliber. Despite inhibition of radial growth, increase in NF-M and reduction in axonal NF-H did not affect nearest neighbor spacing between neurofilaments, indicating that cross-bridging between nearest neighbors does not play a crucial role in radial growth. Increase in NF- M did not result in an overt phenotype or neuronal loss, although filamentous swellings in perikarya and proximal axons of motor neurons were frequently found.