Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer disease brain and in okadaic acid-treated SY5Y cells.

We investigated the role of neurofilament (NF) proteins in Alzheimer disease (AD) neurofibrillary degeneration. The levels and degree of phosphorylation of NF proteins in AD neocortex were determined by Western blots developed with a panel of phosphorylation-dependent NF antibodies. Levels of all three NF subunits and the degree of phosphorylation of NF-H and NF-M were significantly increased in AD as compared to Huntington disease brains used as control tissue. The increase in the levels of NF-H and NF-M was 1.7- and 1.5-fold (P<0.01) as determined by monoclonal antibody SMI33, and was 1.6-fold (P<0.01) in NF-L using antibody NR4. The phosphorylation of NF-H and NF-M in AD was increased respectively at the SMI31 epitope by 1.6- and 1.9-fold (P<0.05) and at the SMI33 epitope by 2.7- and 1.3-fold (P<0.01 and P<0.05). Essentially similar effects were observed in SY5Y human neuroblastoma cells when treated with okadaic acid, an inhibitor of protein phosphatase (PP)-2A and -1. This is the first biochemical evidence which unambiguously demonstrates the hyperphosphorylation and the accumulation of NF subunits in AD brain, and shows that the inhibition of PP-2A/PP-1 activities can lead to the hyperphosphorylation of NF-H and NF-M subunits.     

Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits

To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we generated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two- to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF-M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two-fold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and alpha-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- mice revealed enhanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined results demonstrate a requirement of the high-molecular-weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.     

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.     

Requirement of Heavy Neurofilament Subunit in the Development of Axons with Large Calibers

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.     

Identification of a neurofilament-like protein in the protocerebral tract of the crab <Emphasis Type="Italic">Ucides cordatus</Emphasis>

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

Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons

Neurofilaments (NFs) are composed of triplet proteins, NF-H, NF-M, and NF-L. To understand the dynamics of NFs in vivo, we studied the dynamics of NF-H and compared them to those of NF-L, using the combination of microinjection technique and fluorescence recovery after photobleaching. In the case of NF-L protein, the bleached zone gradually restored its fluorescence intensity with a recovery half time of approximately 35 min. On the other hand, recovery of the bleached zone of NF-H was considerably faster, taking place in approximately 19 min. However, in both cases the bleached zone was stationary. Thus, it was suggested that NF-H is the dynamic component of the NF array and is interchangeable, but that it assembles with the other neurofilament triplet proteins in a more exchangeable way, implying that the location of NF-H is in the periphery of the core NF array mainly composed of NF- L subunits. Immunoelectron microscopy investigations of the incorporation sites of NF-H labeled with biotin compounds also revealed the lateral insertion of NF-H subunits into the preexisting NF array, taking after the pattern seen in the case of NF-L. In summary, our results demonstrate that the dynamics of the L and H subunit proteins in situ are quite different from each other, suggesting different and separated mechanisms or structural specialization underlying the behavior of the two proteins.     

Assembly Properties of Amino- and Carboxyl-Terminally Truncated Neurofilament NF-H Proteins with NF-L and NF-M in the Presence and Absence of Vimentin

Abstract: To understand the assembly characteristics of the high-molecular-weight neurofilament protein (NF-H), carboxyl- and amino-terminally deleted NF-H proteins were examined by transiently cotransfecting mutant NF-H constructs with the other neurofilament triplet proteins, low- and middle-molecular-weight neurofilament protein (NF-L and NF-M, respectively), in the presence or absence of cytoplasmic vimentin. The results confirm that NF-H can coassemble with vimentin and NF-L but not with NF-M into filamentous networks. Deletions from the amino-terminus show that the N-terminal head is necessary for the coassembly of NF-H with vimentin, NF-L, or NF-M/vimentin. However, headless NF-H or NF-H from which the head and a part of the rod is removed can still incorporate into an NF-L/vimentin network. Deletion of the carboxyl-terminal tail of NF-H shows that this region is not essential for coassembly with vimentin but is important for coassembly with NF-L into an extensive filamentous network. Carboxyl-terminal deletion into the α-helical rod results in a dominant-negative mutant, which disrupts all the intermediate filament networks. These results indicate that NF-L is the preferred partner of NF-H over vimentin and NF-M, the head region of NF-H is important for the formation of NF-L/NF-H filaments, and the tail region of NF-H is important to form an extensive network of NF-L/NF-H filaments.     

Antibodies against neurofilament subunits label retinal ganglion cells but not displaced amacrine cells of hamsters.

Although neurofilament (NF) antibodies have been used to visualize ganglion cells and their axons in the retina, it is not known, however, how many ganglion cells contain NF, and how the various NF subunits are distributed in the ganglion cells. Moreover, it is not known whether displaced amacrine cells in the ganglion cell layer are also labelled. In order to see whether NF antibodies can be used as a specific marker for ganglion cells, antibodies raised against the low (NF-L), middle (NF-M) and high (NF-H) molecular weight subunits of NF were employed to stain retinal whole-mounts of adult hamsters after pre-labelling the ganglion cells with Granular Blue. It was found that NF-L and NF-H antibodies labelled 38,777 and 17,750 cells in the ganglion cell layer respectively. By co-localization with GB-labelled cells, 88% of NF-L positive cells and 91% of NF-H positive cells were found to be ganglion cells. In contrast, the NF-M antibody labelled only very few ganglion cells (418 per retina) although robust staining of axonal bundles was observed. Thus, NF antibodies may prove useful in studying this population of ganglion cells.     

Neurofilaments are obligate heteropolymers in vivo

Neurofilaments (NFs), composed of three distinct subunits NF-L, NF-M, and NF-H, are neuron-specific intermediate filaments present in most mature neurons. Using DNA transfection and mice expressing NF transgenes, we find that despite the ability of NF-L alone to assemble into short filaments in vitro NF-L cannot form filament arrays in vivo after expression either in cultured cells or in transgenic oligodendrocytes that otherwise do not contain a cytoplasmic intermediate filament (IF) array. Instead, NF-L aggregates into punctate or sheet like structures. Similar nonfilamentous structures are also formed when NF-M or NF-H is expressed alone. The competence of NF-L to assemble into filaments is fully restored by coexpression of NF- M or NF-H to a level approximately 10% of that of NF-L. Deletion of the head or tail domain of NF-M or substitution of the NF-H tail onto an NF- L subunit reveals that restoration of in vivo NF-L assembly competence requires an interaction provided by the NF-M or NF-H head domains. We conclude that, contrary to the expectation drawn from earlier in vitro assembly studies, NF-L is not sufficient to assemble an extended filament network in an in vivo context and that neurofilaments are obligate heteropolymers requiring NF-L and NF-M or NF-H.     

Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha- internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.     

Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.

Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.     

Acrylamide Retards the Slow Axonal Transport of Neurofilaments in Rat Cultured Dorsal Root Ganglia Neurons and the Corresponding Mechanisms

Chronic acrylamide (ACR) exposure induces peripheral-central axonopathy in occupational workers and laboratory animals, but the underlying mechanisms remain unclear. In this study, we first investigated the effects of ACR on slow axonal transport of neurofilaments in cultured rat dorsal root ganglia (DRG) neurons through live-cell imaging approach. Then for the underlying mechanisms exploration, the protein level of neurofilament subunits, motor proteins kinesin and dynein, and dynamitin subunit of dynactin in DRG neurons were assessed by western blotting and the concentrations of ATP was detected using ATP Assay Kit. The results showed that ACR treatment results in a dose-dependent decrease of slow axonal transport of neurofilaments. Furthermore, ACR intoxication significantly increases the protein levels of the three neurofilament subunits (NF-L, NF-M, NF-H), kinesin, dynein, and dynamitin subunit of dynactin in DRG neurons. In addition, ATP level decreased significantly in ACR-treated DRG neurons. Our findings indicate that ACR exposure retards slow axonal transport of NF-M, and suggest that the increase of neurofilament cargoes, motor proteins, dynamitin of dynactin, and the inadequate ATP supply contribute to the ACR-induced retardation of slow axonal transport.     

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.     

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.     

Absence of the Mid-sized Neurofilament Subunit Decreases Axonal Calibers,Levels of Light Neurofilament (NF-L), and Neurofilament Content

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.Neurofilaments (NFs)¹ are the most prominent cytoskeletal components in large myelinated axons and probably the most abundant and widely expressed of neuronal intermediate filament (IF) proteins. In mammals, NFs are composed of three proteins termed light (NF-L), mid-sized (NF-M), and heavy (NF-H) NFs. These proteins are encoded by separate genes (17, 21, 27) and have apparent molecular weights of ∼68,000, 150,000, and 200,000, respectively, when separated on SDS-PAGE gels.Like all IFs, NF proteins contain a relatively well-conserved α helical rod domain of ∼310 amino acids with variable NH₂-terminal and COOH-terminal regions (33). In NFs, the COOH-terminal domains are greatly extended relative to other IFs and contain a glutamic acid–rich region of unknown significance and in NF-M and NF-H a series of lysine-serine-proline-valine (KSPV) repeats (21, 27) which are major sites of phosphorylation in both proteins. In axons, NFs form bundles of 10-nm diameter “core filaments” with sidearms consisting of phosphorylated COOH-terminal tail sequences of NF-M and NF-H (12, 13, 26, 29) that have been thought to extend and maintain the spacing between filaments (4). Similar sidearm extensions are not found in IFs composed of other IF proteins such as desmin, glial fibrillary acidic protein, or vimentin. In NFs assembled in vitro, all three subunits appear to be incorporated into core filaments (12, 26). Thus, current models of NF assembly suggest that NF-M and NF-H are the major components of sidearm extensions and are anchored to a core of NF-L via their central rod domains.Although much is known about NF structure and assembly, questions remain concerning NF function. A primarily structural role for NFs is suggested by their prominence in large axons (41). Small unmyelinated axons contain few NFs (9) and some small neurons lack morphologically identifiable NFs (3, 32, 38). Most dendrites contain few NFs and only in dendrites of large neurons such as motor neurons are NFs numerous (41).A role for NFs as a major determinant of axonal diameter has long been suspected from the correlation between NF content in axonal cross sections and axonal caliber (16). This correlation persists during axonal degeneration and regeneration (14) and changes in NF transport correlate temporally with alterations in the caliber of axons in regenerating nerves (15). Additionally, fewer NFs occur at nodes of Ranvier where axonal diameter is reduced (1), and certain NF epitopes are found only in regions where maximal axonal caliber has developed (6).Several animal models have supported a role for NFs in establishing axonal diameter. One is a Japanese quail (Quiverer) with a spontaneous mutation in NF-L that generates a truncated protein incapable of forming NFs (31). Homozygous mutants contain no axonal NFs and exhibit a mild generalized quivering. In these animals, radial growth of myelinated axons is severely attenuated (44) with a consequent reduction in axonal conduction velocity (37). In transgenic mice, Eyer and Petersen (8) expressed an NF-H/β-galactosidase fusion protein in which the COOH terminus of NF-H was replaced by β-galactosidase. NF inclusions were found in the perikarya of neurons and the resulting NF aggregates blocked all NF transport into axons resulting in axons with reduced calibers. More recently, Zhu et al. (45) have shown that mice lacking NFs due to a targeted disruption of the NF-L gene have diminished axonal calibers and delayed maturation of regenerating myelinated axons.Although these models clearly suggest a role for NFs in establishing axonal diameter, they contribute only limited information concerning the roles of the individual NF subunits. During development, NF-L and NF-M are coexpressed initially whereas NF-H appears later (4). Studies in transgenic mice have found that overexpressing mouse NF-L leads to an increased density of NFs, but no increase in axonal caliber (25). More recently, Xu et al. (43) overexpressed each of the mouse NF subunits either individually or in various combinations. They found that only when NF-L was overexpressed in combination with either NF-M or NF-H was axonal growth significantly increased. Interestingly, when NF-M and NF-H were overexpressed alone or in combination with one another, radial axonal growth was inhibited.It also remains incompletely understood how NF stoichiometries are regulated and the degree to which any one NF subunit is dominant in this regulation. Recently, conflicting data has appeared concerning the role of NF-M in regulating NF stoichiometries. We found that overexpression of human NF-M in transgenic mice increases the levels of endogenous mouse NF-L protein and decreases the extent of phosphorylation of NF-H (39). These results imply that NF-M may play a dominant role in regulating the levels of NF-L protein, the relative stoichiometry of NF subunits, and the phosphorylation status of NF-H. However different results were obtained by Wong et al. (40) who found that overexpression of mouse NF-M in transgenic mice did not effect the levels of axonal NF-L, and although it reduced NF-H, it did not effect its phosphorylation status.To further address these issues we generated mice bearing a null mutation in the mouse NF-M gene. Here we describe the effects of this mutation on nervous system development with particular reference to the role of the NF-M subunit in specifying axonal diameter and its effect on levels of the remaining NF subunits.     

Acrylamide Alters Cytoskeletal Protein Level in Rat Sciatic Nerves

Occupational exposure and experimental intoxication with acrylamide (ACR) produce neuropathy characterized by nerve degeneration. To investigate the mechanism of ACR-induced neuropathy, male adult Wistar rats were given ACR (20, 40 mg/kg i.p. 3 days/week) for 8 weeks. Sciatic nerves were Triton-extracted and centrifuged at a high speed (100,000 × g) to yield pellet and supernatant fractions. The contents of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results showed that the three neurofilament (NF) subunits (NF-L, NF-M, NF-H) in both the pellet and the supernatant fraction decreased significantly (P < 0.01) in the high-dosing group, except for NF-M in the pellet. α-tubulin, β-tubulin, and β-actin increased significantly in the supernatant (P < 0.01), whereas both α-tubulin and β-tubulin decreased significantly in the pellet (P < 0.01). However, β-actin was not altered significantly in the sciatic nerves pellet. These findings suggest that ACR altered the cytoskeletal protein level in sciatic nerve, which may be one of the molecular mechanisms of ACR-induced peripheral neuropathy.     

Aggregation of Neurofilaments in NF-L Transfected Neuronal Cells: Regeneration of the Filamentous Network by a Protein Kinase C Inhibitor

Abstract: Cytoplasmic inclusion bodies that are accumulations of neurofilaments are the pathological hallmark of many neurodegenerative diseases and have been produced in transgenic mice by overexpression of mouse (NF-L and NF-M; light and medium chains, respectively) and human (NF-M and NF-H; medium and heavy chains, respectively) neurofilament subunits. This report describes a neuronal culture model in which human NF-L was overexpressed to produce cytoplasmic accumulations of neurofilaments within cell bodies concomitant with the collapse of the endogenous neurofilament network. Electron microscopy showed that, within accumulations, neurofilaments retained a filamentous structure. The culture model thus provides a novel system in which the effect on neurofilament accumulations of manipulating protein phosphorylation can be studied. Treatment of cells containing neurofilament accumulations with bisindolylmaleimide, a specific protein kinase C inhibitor, resulted in regeneration of the filamentous network; this effect was not due to a change in the level of transfected NF-L expression. These findings lend support to the suggestion that an impairment in the regulation of protein phosphorylation may lead to the accumulation of neurofilaments seen in neurodegenerative disease.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.